key: cord- -v r d a authors: chan, jasper fuk-woo; zhang, anna jinxia; chan, chris chung-sing; yip, cyril chik-yan; mak, winger wing-nga; zhu, houshun; poon, vincent kwok-man; tee, kah-meng; zhu, zheng; cai, jian-piao; tsang, jessica oi-ling; chik, kenn ka-heng; yin, feifei; chan, kwok-hung; kok, kin-hang; jin, dong-yan; au-yeung, rex kwok-him; yuen, kwok-yung title: zika virus infection in dexamethasone-immunosuppressed mice demonstrating disseminated infection with multi-organ involvement including orchitis effectively treated by recombinant type i interferons date: - - journal: ebiomedicine doi: . /j.ebiom. . . sha: doc_id: cord_uid: v r d a background: disseminated or fatal zika virus (zikv) infections were reported in immunosuppressed patients. existing interferon-signaling/receptor-deficient mouse models may not be suitable for evaluating treatment effects of recombinant interferons. methods: we developed a novel mouse model for zikv infection by immunosuppressing balb/c mice with dexamethasone. results: dexamethasone-immunosuppressed male mice ( – weeks) developed disseminated infection as evidenced by the detection of zikv-ns protein expression and high viral loads in multiple organs. they had ≥ % weight loss and high clinical scores soon after dexamethasone withdrawal ( dpi), which warranted euthanasia at dpi. viral loads in blood and most tissues at dpi were significantly higher than those at dpi (p < . ). histological examination revealed prominent inflammatory infiltrates in multiple organs, and cd + and cd + inflammatory cells were seen in the testis. these findings suggested that clinical deterioration occurred during viral clearance by host immune response. type i interferon treatments improved clinical outcome of mice ( % vs % survival). conclusions: besides virus dissemination, inflammation of various tissues, especially orchitis, may be potential complications of zikv infection with significant implications on disease transmission and male fertility. interferon treatment should be considered in patients at high risks for zikv-associated complications when the potential benefits outweigh the side effects of treatment. zika virus (zikv) is an emerging flavivirus that has been largely neglected for n years after its discovery due to its restricted geographical distribution and its presumed low clinical significance (chan et al., a) . since , large-scale outbreaks of zikv infection have occurred in the pacific islands, latin america, and most recently, usa and southeast asia (duffy et al., ; musso and gubler, ; zhu et al., ) . as of october , n countries/territories have ebiomedicine ( ) - reported continuing mosquito-borne transmission of zikv (world health organization. zika situation report. october , ) . in addition to mosquito-borne transmission, sexual and transplacental transmissions of zikv have also been reported (chan et al., a; musso et al., a; foy et al., ; calvet et al., ) . these non-vectorborne transmission routes render the control of the continuing epidemic more complicated. zikv was not considered as an important human pathogen in the past as most infected adult patients were asymptomatic or developed a self-limiting acute febrile illness which resolved within - weeks (chan et al., a; duffy et al., ). however, it has been recently recognized that infected mothers may transmit the virus transplacentally to developing fetuses, leading to congenital malformations, including microcephaly, cerebral malformations, ophthalmological and hearing defects, and arthrogryposis (chan et al., a; mlakar et al., ; de paula et al., ; leal et al., ) . some infected adults may also develop severe neurological complications, such as guillain-barré syndrome, meningoencephalitis, and myelitis (cao-lormeau et al., ; carteaux et al., ; mecharles et al., ) . moreover, zikv-related fatalities have been increasingly recognized. most of the patients with fatal infection had underlying medical conditions and some were markedly immunosuppressed, including a patient with systemic lupus erythematosus and rheumatoid arthritis who was on corticosteroid therapy and died of disseminated infection with detectable zikv rna in blood, brain, spleen, liver, kidney, lung, and heart obtained at postmortem examination (pan american health organization/world health oganization (paho/who), ; sarmiento-ospina et al., ; arzuza-ortega et al., ) . a number of animal models have been developed for studying the pathogenesis and evaluating countermeasures for zikv infection. rhesus macaques with subcutaneous zikv inoculation develop mild clinical signs that resemble the self-limiting illness in most infected immunocompetent adults (dudley et al., ) . this non-human primate model provides a robust platform for the evaluation of vaccines and host immune response (abbink et al., ) . however, the mild clinical disease in these primates is suboptimal for antiviral treatment evaluation. moreover, expertise and facilities for working with non-human primates are not available in most research laboratories. wild-type adult balb/c mice are not susceptible to intraperitoneal zikv inoculation (dick, ) . suckling and young mice with intracerebral zikv inoculation develop disease that is localized to the central nervous system (dick, ; way et al., ; weinbren and williams, ; bell et al., ) . pregnant mice and fetal mice with partially intact type i interferon signaling response (fetuses of female mice deficienct in type i interferon signaling response crossed to wild-type male mice) were used to study pathogenesis in pregnancy and maternalfetal transmission, but these models are technically more demanding (miner et al., ; cugola et al., ) . type i/ii interferon-signaling-/ receptor-deficient mice with intraperitoneal or subcutaneous zikv inoculation develop fatal, disseminated infection (lazear et al., ; dowall et al., ; aliota et al., ; rossi et al., ) . these models are useful for the evaluation of countermeasures for zikv infection as the protective effects of antivirals drugs and vaccines are more easily observed in treated mice. however, such models have complete/nearcomplete deficiency in interferon response and do not resemble the real clinical situation in immunosuppressed humans. moreover, these mice are suboptimal for the study of host immune response and may be too expensive for laboratories in resource-limited areas. because of these limitations and knowledge gaps, we developed and characterized a more readily available mouse model which resembles immunosuppressed hosts with disseminated infection. we showed that these mice developed inflammation in multiple organs, including the testes, which may have important implications on zikv's longterm outcome and effects on fertility. we also utilized this novel animal model to show that early treatment with clinically approved recombinant type i interferons improved the clinical outcome of these mice. a clinical isolate of zikv (puerto rico strain prvabc ) was kindly provided by brandy russell and barbara johnson, centers for disease control and prevention, usa. the virus was amplified by three additional passages in vero cells (atcc) in minimum essential medium (mem) supplemented with % fetal calf serum and units/ml penicillin plus μg/ml streptomycin to make working stocks of the virus. for virus titration, aliquots of zikv were applied on confluent vero cells in well plates for % tissue culture infectious dose (tcid ) assay as we previously described with slight modifications (zhou et al., ) . briefly, serial -fold dilutions of zikv were inoculated in a vero cell monolayer in quadruplicate and cultured in penicillin/streptomycinsupplemented mem. the plates were observed for cytopathic effect for days. viral titer was calculated with the reed and münch endpoint method. one tcid was interpreted as the amount of virus that causes cytopathic effect in % of inoculated wells. approval was obtained from the committee on the use of live animals in teaching and research of the university of hong kong. male and female balb/c mice, - weeks old, were obtained from the laboratory animal unit of the university of hong kong. the mice were kept in biosafety level- housing and given access to standard pellet feed and water ad libitum. virus inoculation experiments were performed in a biosafety level- animal facility according to the standard operating procedures approved by the committee on the use of live animals in teaching and research of the university of hong kong as we described previously (zhang et al., ) . the mice were randomly divided into groups and given different regimens of virus inoculation, dexamethasone, and recombinant interferon treatment (table ) . phosphate-buffered saline (pbs) was used to dilute the virus stocks to the desired concentration, and inocula were back-titrated to verify the dose given. on the day of virus inoculation, a dose of the virus equivalent to × tcid ( . × plaque forming units) in μl of pbs was inoculated via the intraperitoneal route into mice under ketamine ( mg/kg) and xylazine ( mg/kg) anesthesia. mice in the negative-control groups (groups to ) were injected with the same volume of pbs. mice were monitored three times each day for clinical signs of disease and a numerical score was assigned at each observation as previously described (dowall et al., ; graham et al., ) . their body weight and survival were monitored for days post-inoculation (dpi) or until euthanasia. three mice in each group (except groups and which included mock-infected control mice without dexamethasone immunosuppression and group which included zikv-inoculated, dexamethasone-immunosuppressed mice without dexamethasone withdrawal) were sacrificed at dpi for virological, histological, and immunohistochemistry analyses. the remaining mice were sacrificed at dpi or euthanized when there was a % weight loss or % weight loss with ≥ clinical sign (dowall et al., ) . samples of brain, testis/ epididymis (male), prostate (male), ovary/uterus (female), kidney, urinary bladder, spleen, liver, pancreas, intestine, heart, lung, and salivary gland were collected at necropsy. the specimens were separated into two parts, one immediately fixed in % pbs-buffered formalin, the other immediately frozen at − °c until further experiments. blood samples were also collected for rna extraction and real-time pcr analysis. paraffin-embedded tissues were cut into - μm sections, mounted on slides, and stained with hematoxylin and eosin (h&e) for light microscopy examination as we previously described (zheng et al., ) . for immunohistochemical staining of zikv-ns antigen, mouse antiserum against zikv-ns protein prepared as we previously described was used as primary antibody (chan et al., b) . de-paraffinized and rehydrated tissue sections were treated with antigen unmasking solution according to manufacturer's instructions (vector laboratories inc., burlingame, ca, usa) and then stained with mouse on mouse polymer ihc kit (abcam, cambridge, united kingdom). the primary antibody mouse anti-zikv-ns antiserum ( : dilution with % bsa/pbs) was incubated at °c overnight. this was followed by mouse on mouse hrp polymer kit (abcam) with horseradish peroxidase-conjugated secondary antibody for min. color development was performed using , ′-diaminobenzidine (dab) (vector laboratories, burlingame, ca, usa). for immunohistochemical staining of cd and cd , the sections were incubated at °c for overnight with primary antibody (rabbit anti-mouse cd , or rat anti-mouse cd α (abcam) after antigen unmasking and blocking. this was then followed by incubation with biotin-conjugated goat anti-rabbit igg or goat anti-rat igg (calbiochem, darmstadt, germany) for min at room temperature. streptavidin/peroxidase complex reagent (vector laboratories) was then added and incubated at room temperature for min. color development was done with dab (vector laboratories). all tissue sections were examined microscopically by two pathologists in an operatorblinded manner. images were captured with nikon i imaging system equipped with spot-advance computer software. total nucleic acid (tna) was extracted from the blood and necropsied tissues using ez virus mini kit v . and qiasymphony dsp virus/pathogen mini kit (qiagen, hilden, germany), respectively, as we previously described (zheng et al., ; chan et al., b; chan et al., a) . zikv envelope gene was measured by using quantinova probe rt-pcr kit (qiagen) in lightcycler real-time pcr system (roche diagnostics, basel, switzerland). μl of purified tna was amplified in a μl-reaction containing μl of × quantinova probe rt-pcr master mix, . μl qn probe rt-mix, . μm forward primer, . μm reverse primer, and nm probe. forward primer ( ′-cgytgcccaacacaagg- ′), reverse primer ( ′-ccacyaaygttcttttgcabaca- ′), and probe ( ′-hex-agcctaccttgayaagcartcagacactc-iabkfq- ′) targeting the zikv envelope gene as we previously described were used (chan et al., b) . reactions were incubated at °c for min, followed by °c for min, and then thermal cycled for cycles ( °c for s, °c for s). internal control β-actin gene was measured by using quantinova sybr green rt-pcr kit (qiagen) in lightcycler real-time pcr system. μl of purified tna was amplified in a μl-reaction containing μl of × quantinova sybr green rt-pcr master mix, . μl qn sybr green rt-mix, . μm forward primer ( ′-acggccaggtcatcactattg- ′) and . μm reverse primer ( ′-caagaaggaaggctggaaaag- ′) for the β-actin gene. reactions were incubated at °c for min, followed by °c for min, and then thermal cycled for cycles ( °c for s, °c for s). a series of -fold dilutions equivalent to × to × copies/reaction mixture were prepared to generate standard curves and run in parallel with the test samples. all data were analyzed with graphpad prism software (graphpad software, inc). kaplan-meier survival curves were analyzed by the log rank test, and weight losses were compared using two-way anova. student's t-test was used to determine significant differences in virus titers, and tukey-kramer post hoc tests were used to discern differences among individual treatment groups as previously reported rossi et al., ) . p-values b . were considered statistically significant. to establish a novel mouse model for zikv infection, we compared the clinical, histological, and virological findings of male (group ) and female (group ) mice with dexamethasone immunosuppression and zikv inoculation with those of the appropriate controls (groups to ) (table ). in terms of the clinical parameters, the dexamethasone-immunosuppressed mice developed mild (~ %) weight loss (fig. a) and no mortality at dpi ( fig. b and c) . the weight losses of the dexamethasone-immunosuppressed mice with zikv inoculation (groups and ) table eleven groups of mice receiving different regimens of virus inoculation, dexamethasone, and antiviral treatment in this study a . group gender routes and inoculum of zikv ( dpi) dexamethasone antiviral treatment b date of sacrifice/euthanasia m ip × tcid mg/kg q h ip, from days before to dpi inclusively no (n = ) and (n = ) dpi c f ip × tcid mg/kg q h ip, from days before to dpi inclusively no (n = ) and (n = ) dpi m ip × tcid no no (n = ) and (n = ) dpi f ip × tcid no no (n = ) and (n = ) dpi m no mg/kg q h ip, from days before to dpi inclusively no (n = ) and (n = ) dpi f no mg/kg q h ip, from days before to dpi inclusively no (n = ) and (n = ) dpi no (n = ) dpi m ip × tcid mg/kg q h ip, from days before to dpi inclusively no (n = ) dpi m ip × tcid mg/kg q h ip, from days before to dpi inclusively pegylated ifn-α b iu/dose q h sc at , , and dpi (n = ) and (n = ) dpi m ip × tcid mg/kg q h ip, from days before to dpi inclusively ifn-β b , iu/dose q h ip at , , , , and dpi (n = ) and (n = ) dpi abbreviations: dpi, days post-inoculation; f, female; ifn, interferon; ip, intraperitoneal; m, male; sc, subcutaneous. a - week-old balb/c mice were used in all the groups. b the preparations of pegylated ifn-α b and ifn-β b used in this study were pegintron (merck & co., inc., whitehouse station, nj, usa) and betaferon (bayer schering pharma ag, berlin, germany), respectively. c the mice in group were euthanized at dpi as they had ≥ % weight loss and ≥ clinical symptom. were consistently more significant than those of their comparators, including the zikv-inoculated mice without dexamethasone immunosuppression (groups and ) and mock-infected mice without dexamethasone immunosuppression (groups and ) starting at dpi (p b . ). minimal histological changes and inflammatory infiltrates were seen in the tissues of the male and female mice with dexamethasone immunosuppression and zikv inoculation (groups and ). on the other hand, zikv-ns protein expression was detected by immunohistochemical staining in most tissues of these mice, but not in dexamethasone-immunosuppressed mice with mock infection (groups and ), suggesting that the viral protein expression was specific and not related to dexamethasone effects (fig. ) . the dexamethasone-immunosuppressed mice with zikv inoculation (groups and ) also had high mean viral loads in blood and most tissues at dpi, especially in the testis/epididymis, ovary/uterus, prostate, spleen, and pancreas (figs. a and b and s a and b). these findings at dpi were suggestive of disseminated but non-lethal zikv infection involving different organs with minimal inflammatory response due to dexamethasone immunosuppression. to investigate the possible effects of immune reconstitution in the dexamethasone-immunosuppressed mice, dexamethasone was stopped after dpi. this led to prominent weight loss and increased symptoms in the dexamethasone-immunosuppressed mice (groups and ). the most prominent body weight loss was observed in the male mice with dexamethasone immunosuppression and zikv inoculation (group ), with all mice having weight loss of ≥ % at dpi (fig. a) . all of the female mice with dexamethasone immunosuppression and zikv inoculation (group ) also had progressive weight loss and / ( . %) of them had ≥ % weight loss at dpi. in contrast, none of the mice with dexamethasone immunosuppression from days before to days post-infection (group ) developed abrupt weight loss between dpi and dpi (fig. a) . the weight loss of mice in groups and became consistently more than those of their comparators in the other control groups (groups to ), including those in the dexamethasone-immunosuppressed mice with mock infection (groups and ) since dpi (p b . ). together, these findings suggested that the combination of immune reconstitution after dexamethasone withdrawal and disseminated virus infection were responsible for the abrupt clinical deterioration. all of the mice in groups and developed rapid breathing, lethargy, and/or ruffled fur since dpi (group ) or dpi (group ), shortly after dexamethasone was stopped (fig. b) . the reasons for the earlier onset of weight loss and symptoms in the male mice were not fully understood, but might be related to the higher cumulative dose of dexamethasone because of their higher baseline body weights and/or possible effects of androgen on virus replication (tian et al., ) . based on the predefined criteria, all ( %) male and / ( . %) female mice were euthanized at dpi and dpi, respectively (fig. c) . comparatively, all the mice in the other control groups (groups to ) had either gained weight or had b % weight loss with spontaneous recovery at dpi (fig. a) , remained asymptomatic (fig. b) , and survived through the study period (fig. c) . at euthanasia ( - dpi), h&e staining of the necropsied tissues of these mice showed prominent acute inflammatory reactions with predominantly lymphocytic infiltrates. the most prominent inflammatory changes were seen in the brain (cortical parenchymal and perivascular lymphocytic infiltrates) (fig. a to c), kidney (acute tubulitis and interstitial inflammation) (fig. d to f), and testis (necrotic and hemorrhagic seminiferous tubules with marked lymphocytic infiltration in the perimeter of the tubules and the interstitium) (fig. a to d) . zikv-ns protein expression was still visible, but to a lesser degree, in the immunohistochemical staining of the testis/epididymis, ovary/uterus, kidney, spleen, small intestine, pancreas, and salivary gland of the dexamethasone-immunosuppressed mice with zikv inoculation at - dpi compared with dpi. in contrast, no inflammatory reaction and viral protein expression were seen in any organ of the control mice with zikv inoculation alone (groups and ) or dexamethasone immunosuppression alone (groups and ) ( fig. c and d) . these findings confirmed that mice with zikv inoculation but no dexamethasone immunosuppression were not susceptible to infection as previously reported, and that the histological changes in the model mice (groups and ) were unrelated to dexamethasone-induced effects such as drug-induced testicular toxicity (lazear et al., ; dowall et al., ; aliota et al., ; rossi et al., ; khorsandi et al., ) . the absence of inflammatory infiltrates in zikv-inoculated, dexamethasone-immunosuppressed mice without dexamethasone withdrawal (group ) supported the role of the host immune response in eliciting the clinical and histological changes in the zikv-inoculated mice with dexamethasone withdrawal (groups and ). to further confirm the presence of inflammatory infiltrates and characterize the cell types involved in the host immune response, we stained the necropsied testis of the dexamethasone-immunosuppressed mice with zikv inoculation and those of the dexamethasoneimmunosuppressed mock-infected control mice with cd (pan-leukocyte) and cd (cytotoxic t lymphocyte) antibodies. corroborative to the histological findings, only the testis of the dexamethasone-immunosuppressed mice with zikv inoculation, but not those of the control mice, stained positive for cd ( fig. e and f) and cd antibodies ( fig. g and h). these findings confirmed the presence of inflammatory infiltrates and especially cd + t lymphocytes in the testis of the zikv-infected mice. the dexamethasone-immunosuppressed mice with zikv inoculation (groups and ) also had significantly lower mean viral loads in blood and most tissues at euthanasia at - dpi as compared with those collected at dpi (↓ - log copies/ β-actin at - dpi) (figs. a and b and s a and b). at euthanasia ( - dpi), viral rna was still detectable in most tissues of the male mice (up to log copies/ β-actin), but viremia was absent. the control mice with zikv inoculation but no dexamethasone immunosuppression had undetectable viral rna in blood and most tissues, which was consistent with previous reports (lazear et al., ; dowall et al., ; aliota et al., ; rossi et al., ) . overall, these findings were suggestive of multi-organ inflammation upon immune reconstitution with partial viral clearance in the mice after withdrawal of dexamethasone immunosuppression. we next evaluated the effects of recombinant type i interferon treatment in our mouse model. we used male mice as they had earlier onset of weight loss and clinical symptoms requiring necropsy at dpi. the mice were treated with pegylated interferon-α b (pegintron®, merck & co., inc., whitehouse station, nj, usa) iu/dose every h subcutaneously at dpi, dpi, and dpi (group ) or interferon-β b (betaferon®, bayer schering pharma ag, berlin, germany) , iu/dose every h intraperitoneally at dpi, dpi, dpi, dpi, and dpi (group ). as shown in fig. a , the mice treated with pegylated interferon-α b (group ) or interferon-β b (group ) had b % weight loss with spontaneous recovery at dpi. the weight loss of the untreated group became significantly more than those of the mice treated with either interferon-α or interferon-β b starting at dpi (p b . ). all of these mice remained asymptomatic and survived through the study period ( fig. b and c) . none of their tissues showed prominent inflammatory reactions in h&e staining at dpi or dpi. zikv-ns protein expression was only rarely seen in the immunohistochemical staining of the testis/epididymis, kidney, spleen, small intestine, lung, and pancreas collected at dpi, and testis, epididymis, kidney, and spleen at dpi. they had reduced mean viral loads in blood and all the tissues (↓ - log copies/ β-actin) as compared with those of the untreated mice at dpi and dpi ( fig. a and b) . the reductions were most significant in the tissues with high viral loads, such as the spleen, testis, pancreas, and prostate (p b . ). overall, these findings suggested that early use of systemic recombinant type i interferons improved the clinical, histological, and virological parameters of mice with disseminated zikv infection. the full spectrum of clinical manifestations and complications of zikv infection remains incompletely understood as of today. the previous assumption that zikv infection is an entirely self-limiting disease without severe or long-lasting sequelae has been overturned by the increasing recognition of congenital malformations, neurological complications, immune-mediated thrombocytopenia, and even fatality in some immunosuppressed patients (pan american health organization/ a b fig. . viral loads in the blood and major organs of dexamethasone-immunosuppressed balb/c male and female mice with zikv inoculation. mice ((a) male ( dpi, n = from two independent experiments; dpi, n = from two independent experiments) and (b) female ( dpi, n = from two independent experiments; dpi, n = from two independent experiments)) with dexamethasone immunosuppression had higher blood and tissue viral loads at dpi while they were on dexamethasone than at euthanasia ( dpi for male mice and dpi for female mice) after dexamethasone was stopped ( dpi). zikv rna copies in the blood and tissues of the mice were determined by real-time rt-pcr and normalized by β-actin as described in the text. * denotes p-values of b . . *** denotes p-values b . . error bars represent standard error of the mean. world health oganization (paho/who), ; sarmiento-ospina et al., ; arzuza-ortega et al., ; duijster et al., ) . notably, patients with severe non-pregnancy-related complications of zikv often deteriorated suddenly after an initially mild disease phase as the viral load began to decrease (cao-lormeau et al., ; mecharles et al., ; sarmiento-ospina et al., ) . this has led us to hypothesize that, like many other flavivirus infections, including yellow fever, dengue, and west nile virus infection, the host immune response may also play a role in these zikv-associated complications, especially during the viral clearance phase by the host immune system (quaresma et al., ; screaton et al., ; wang et al., ) . in this study, we characterized a novel and readily available mouse model for severe zikv infection which attempts to provide an alternative venue for studying the host immune response of and evaluating countermeasures for zikv infection. the findings in our study have important implications on the pathogenesis, potential complications, and treatment of zikv infection. the dexamethasone-immunosuppressed mice with zikv inoculation in our study developed disseminated infection with viremia and multi-organ involvement, including the brain, urogenital tract, intestine, liver, spleen, pancreas, heart, lung, and salivary gland as evident by zikv-ns protein expression on immunohistochemical staining and/or detectable viral load in these tissues. immunohistochemistry staining of the testis confirmed the presence of inflammatory cell infiltrate (pan-leukocyte marker cd +) with predominantly cd + t lymphocytes. clinically, the male mice developed earlier onset of disease than the female mice, with ≥ % weight loss and ≥ clinical sign, which warranted euthanasia at dpi. their weight loss, clinical scores, and histological evidence of inflammatory reactions were most severe soon after dexamethasone withdrawal, when viral loads had already decreased by about - log copies/ β-actin. overall, these findings suggested that, like the other related flaviviruses, the host immune response might have led to marked clinical deterioration in the face of disseminated zikv infection at the time when immune-mediated clearance of the virus began. our findings provided an additional explanation for the pathogenesis of fatal zikv infection, which has been proposed to be related to uncontrolled virus dissemination in previously described mouse models utilizing types i/ii interferon-signaling-/receptor-deficient mice that were unable to mount a robust host innate immune response. our mouse model is also useful for studying zikv's tissue tropism and potential complications of severe zikv infection. in addition to the reported findings of detectable virus particles and/or rna in the brain, spinal cord, kidney, spleen, liver, testis, ovary, heart, lung, muscle, and blood of types i/ii interferon-signaling-/receptor-deficient mice with zikv infection, our study identified intestine, pancreas, and salivary gland as other possible tissues and anatomical sites for virus infection (dick, ; lazear et al., ; dowall et al., ; aliota et al., ; rossi et al., ) . this tissue tropism of zikv in our mouse model concurs with the in-vitro observation that zikv efficiently replicates in diverse cell types of neuronal, testicular, prostatic, renal, intestinal, hepatic, and placental origin (chan et al., b; brault et al., ; hughes et al., ) . such degree of virus dissemination and multiorgan involvement is also compatible with the clinical findings in patients with severe and/or fatal zikv infection, in whom viral particles and/or rna were detected in multiple organs at post-mortem examination (pan american health organization/world health oganization (paho/who), ; sarmiento-ospina et al., ; arzuza-ortega et al., ) . while inflammatory neurological complications, such as guillain-barré syndrome, meningoencephalitis, and myelitis, have been recently reported in patients with zikv infection, inflammatory disorders of the other non-neuronal tissues were not well recognized (cao-lormeau et al., ; carteaux et al., ; mecharles et al., ) . our findings showed that inflammation could be observed in multiple organs including the testis, kidney, spleen, liver, intestine, pancreas, lung, and salivary gland outside the nervous system. among these non-neuronal tissues, the inflammatory reactions were most prominent in the testis of our model mice. some patients with zikv infection reported hematospermia, pelvic pain, and dysuria with detectable viral particles and/or rna in their semen (chan et al., a; musso et al., a; foy et al., ) . histological evidence of orchitis has not been reported due to the difficulty in obtaining the patients' testicular tissues for histological examination. previous mouse fig. . representative histological findings in the brain and kidney of dexamethasone-immunosuppressed balb/c mice with zikv inoculation and dexamethasone-immunosuppressed mock-infected control mice. the brain and kidneys of all sacrificed mice were examined. each organ was entirely embedded in one paraffin block, and one full-face paraffin section at the maximum diameter of each organ was examined per block. sections of the brain ( dpi) of a dexamethasone-immunosuppressed mouse with zikv inoculation showing (a) moderate degree of perivascular lymphocytic infiltrate and (b) marked lymphocytic infiltration in the cortical parenchyma (h&e, original magnification × ). section of the brain ( dpi) of dexamethasone-immunosuppressed mock-infected mouse showing normal architecture in the parenchyma (c) (h&e, original magnification × ). sections of the kidney ( dpi) showing (d) acute tubulitis with a large amount of inflammatory exudation in tubular lumens and (e) moderate degree of interstitial inflammation (h&e, original magnification × ). (f) section of the kidney ( dpi) of a dexamethasone-immunosuppressed mock-infected mouse showing normal architecture (h&e, original magnification × ). models for zikv infection utilizing types i/ii interferon-signaling-/receptor-deficient mice have also showed that viral particles and rna could be detected in the mice's testes, but histological analysis were not reported (lazear et al., ) . the markedly necrotic and hemorrhagic seminiferous tubules observed in our mice are highly alarming as orchitis may have long-term effects on fertility. these changes were not accountable by dexamethasone-induced testicular toxicity, as they were morphologically different from the latter, and were not present in any of the testes of the control mice with dexamethasone treatment and mock infection (khorsandi et al., ) . during revision of this work, similar findings were reported in the testes of c bl/ mice treated with anti-ifna blocking monoclonal antibody and inoculated with zikv . clinical studies to confirm the presence of orchitis and to assess the fertility of convalescent male patients should be conducted to ascertain the long-term consequences of zikv infection regarding reproductive and hormonal derangements. inflammation of other organs, such as acute tubulitis, interstitial nephritis, sialadenitis, hepatitis, enteritis, and acute pancreatitis have been reported in patients with zikv or other flavivirus infections (chan et al., a; sarmiento-ospina et al., ; arzuza-ortega et al., ; duijster et al., ; bonaldo et al., ; gourinat et al., ; musso et al., b; mercado et al., ; bhagat et al., ; torres et al., ; macnamara, ; chatterjee et al., ) . these potential complications may be increasingly recognized as the zikv epidemic continues to expand into developed countries with a large ageing and immunosuppressed population. finally, our mouse model also provided a novel avenue for the evaluation of anti-zikv treatment. type i interferons have broad-spectrum antiviral activities including those against zikv, but type i interferonsignaling-/receptor-deficient mice were not suitable for evaluation of the effects of recombinant type i interferons. we therefore evaluated the antiviral effects of two commercially available preparations of recombinant type i interferons in this new mouse model (hamel et al., ; zumla et al., ; chan et al., ; chan et al., b) . we showed that the early use of either drug was associated with improved clinical outcome with no fatality ( % fatality in untreated mice), markedly decreased inflammatory response after dexamethasone withdrawal, and reduced viral loads in various tissues of the mice as compared to those of the untreated mice. the viral load reductions were especially significant in the early phase of the disease ( dpi), when the mice were on dexamethasone. these findings suggested that the early use of recombinant interferons might help to control viral replication during the initial phase of infection, and prevent the subsequent development of severe complications related to an exaggerated immune response in the presence of high viral loads as seen in the untreated mice. it is important to further confirm these results in our mouse model using different zikv strains and in clinical trials because zikv antagonizes mouse stat less efficiently than human stat , and thus may be more susceptible to type i interferons in mice (grant et al., ) . while most patients with mild zikv infection may not require systemic interferon treatment, clinical trials should be considered to evaluate the benefits of the early use of interferon treatment in patients at risk of developing severe zikv-associated complications, such as those with underlying comorbidities (sarmiento-ospina et al., ) . the increased risk of fetal loss and low birth weight associated with interferon therapy in the first trimester of pregnancy may be outweighed by the risk of congenital malformations due to zikv infection. the optimal timing of treatment commencement should be further investigated as late commencement of interferon treatment may be useless or deleterious and should be avoided (solomon et al., ) . in summary, this novel mouse model is useful for investigating host immune response-associated damage of and evaluating countermeasures for zikv infection. inflammation of different visceral organs may be important complications of zikv that should be further studied in infected humans. long-term monitoring of the testicular function of zikvinfected male patients should be considered. clinical trials should be , inc., whitehouse station, nj, usa) iu/dose every h subcutaneously at dpi, dpi, and dpi or interferon-β b (betaferon®, bayer schering pharma ag, berlin, germany) , iu/dose every h intraperitoneally at dpi, dpi, dpi, dpi, and dpi. no interferon treatment group (n = ), interferon-α b treatment group (n = ), and interferon-β b treatment group (n = ). results were combined from two independent experiments. p-values b . are indicated by (no interferon treatment versus interferon-α b treatment) and * (no interferon treatment versus interferon-β b treatment). abbreviation: ifn, interferon. clinical scores: normal = ; ruffled fur = ; lethargy, pinched, hunched, wasp waisted = ; labored breathing, rapid breathing, inactive, neurological = ; and immobile = . considered for evaluating the effects of recombinant interferon treatments in patients at high risk for zikv-associated complications when the potential benefits may outweight the side effects of treatment. supplementary data to this article can be found online at http://dx. doi.org/ . /j.ebiom. . . . jfwc, ajz, ccsc, and kyy designed the study. ajz, ccsc, hz, and vkmp performed infections. ccyy, jolt, kkhc, and khc prepared virus stocks and viral titrations. ajz, wwnm, vkmp, and rkhay prepared histology and immunohistochemistry slides. ccyy, kmt, zz, and jpc performed viral load studies. jfwc, ajz, rkhay, vkmp, and kyy acquired images at the microscope, analyzed, and quantified the data. fy, khk, dyj provided technical assistance and edited the manuscript. jfw, ajz, and kyy wrote the manuscript. jasper f.w. chan has received travel grants from pfizer corporation hong kong and astellas pharma hong kong corporation limited, and was an invited speaker for gilead sciences hong kong limited. the other authors declared no conflict of interests. the funding sources had no role in study design, data collection, analysis or interpretation or writing of the report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. we are grateful to can li, andrew chak-yiu lee, and shuofeng yuan for their facilitation of the study. this work was partly supported by the donations of larry chi-kin yung, and hui hoy and chow sin lan charity fund limited; and funding from the consultancy service for enhancing α β α β a b fig. . viral loads in the blood and major organs of dexamethasone-immunosuppressed balb/c mice with zikv inoculation with or without recombinant type i interferon treatment. male mice with interferon-α b or -β b treatment had reduced zikv blood and tissue viral loads as compared to untreated mice at (a) dpi (n = per group from two independent experiments) and (b) dpi (n = - per group from two independent experiments). zikv rna copies in the blood and tissues of the mice were determined by real-time rt-pcr and normalized by β-actin as described in the text. * denotes p-values of b . . error bars represent standard error of the mean. protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys characterization of lethal zika virus infection in ag mice fatal sickle cell disease and zika virus infection in girl from colombia zika virus infection of the central nervous system of mice acute glomerulonephritis in dengue haemorrhagic fever in the absence of shock, sepsis, haemolysis or rhabdomyolysis isolation of infective zika virus from urine and saliva of patients in brazil comparative analysis between flaviviruses reveals specific neural stem cell tropism for zika virus in the mouse developing neocortex detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study zika virus associated with meningoencephalitis broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus development and evaluation of novel realtime reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset zika fever and congenital zika syndrome: an unexpected emerging arboviral disease differential cell line susceptibility to the emerging zika virus: implications for disease pathogenesis, non-vector-borne human transmission and animal reservoirs an observational study of dengue fever in a tertiary care hospital of eastern india the brazilian zika virus strain causes birth defects in experimental models ocular findings in infants with microcephaly associated with presumed zika virus congenital infection in salvador, brazil zika virus. ii. pathogenicity and physical properties a susceptible mouse model for zika virus infection a rhesus macaque model of asian-lineage zika virus infection zika virus outbreak on yap island, federated states of micronesia zika virus infection in travellers returning from surinam and the dominican republic probable non-vector-borne transmission of zika virus detection of zika virus in urine zika virus infection damages the testes in mice genetic diversity in the collaborative cross model recapitulates human west nile virus disease outcomes zika virus targets human stat to inhibit type i interferon signaling biology of zika virus infection in human skin cells infectivity of immature neurons to zika virus: a link to congenital zika syndrome effect of curcumin on dexamethasone-induced testicular toxicity in mice a mouse model of zika virus pathogenesis hearing loss in infants with microcephaly and evidence of congenital zika virus infection -brazil zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria acute myelitis due to zika virus infection clinical and histopathological features of fatal cases with dengue and chikungunya virus co-infection in colombia zika virus infection during pregnancy in mice causes placental damage and fetal demise zika virus associated with microcephaly zika virus potential sexual transmission of zika virus detection of zika virus in saliva epidemiological alert -neurological syndrome, congenital malformations, and zika virus infeciton. implications for public health in the americas immunity and immune response, pathology and pathologic changes: progress and challenges in the immunopathology of yellow fever characterization of a novel murine model to study zika virus zika virus associated deaths in colombia new insights into the immunopathology and control of dengue virus infection interferon alfa- a in japanese encephalitis: a randomised double-blind placebo-controlled trial enhancement of hepatitis b virus replication by androgen and its receptor in mice acute parotitis due to dengue virus cd + t cells mediate recovery and immunopathology in west nile virus encephalitis comparative studies of some african arboviruses in cell culture and in mice zika virus: further isolations in the zika area, and some studies on the strains isolated zika situation report toll-like receptor agonist imiquimod in combination with influenza vaccine expedites and augments humoral immune responses against influenza a(h n )pdm virus infection in balb/c mice delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/ h n virus active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis comparative genomic analysis of pre-epidemic and epidemic zika virus strains for virological factors potentially associated with the rapidly expanding epidemic coronaviruses -drug discovery and therapeutic options key: cord- -oqc h lu authors: kumar, ashutosh; singh, himanshu n.; pareek, vikas; raza, khursheed; dantham, subrahamanyam; kumar, pavan; mochan, sankat; faiq, muneeb a. title: a possible mechanism of zika virus associated microcephaly: imperative role of retinoic acid response element (rare) consensus sequence repeats in the viral genome date: - - journal: front hum neurosci doi: . /fnhum. . sha: doc_id: cord_uid: oqc h lu owing to the reports of microcephaly as a consistent outcome in the fetuses of pregnant women infected with zikv in brazil, zika virus (zikv)—microcephaly etiomechanistic relationship has recently been implicated. researchers, however, are still struggling to establish an embryological basis for this interesting causal handcuff. the present study reveals robust evidence in favor of a plausible zikv-microcephaly cause-effect liaison. the rationale is based on: ( ) sequence homology between zikv genome and the response element of an early neural tube developmental marker “retinoic acid” in human dna and ( ) comprehensive similarities between the details of brain defects in zikv-microcephaly and retinoic acid embryopathy. retinoic acid is considered as the earliest factor for regulating anteroposterior axis of neural tube and positioning of structures in developing brain through retinoic acid response elements (rare) consensus sequence ( ′–aggtca– ′) in promoter regions of retinoic acid-dependent genes. we screened genomic sequences of already reported virulent zikv strains (including those linked to microcephaly) and other viruses available in national institute of health genetic sequence database (genbank) for the rare consensus repeats and obtained results strongly bolstering our hypothesis that zikv strains associated with microcephaly may act through precipitation of dysregulation in retinoic acid-dependent genes by introducing extra stretches of rare consensus sequence repeats in the genome of developing brain cells. additional support to our hypothesis comes from our findings that screening of other viruses for rare consensus sequence repeats is positive only for those known to display neurotropism and cause fetal brain defects (for which maternal-fetal transmission during developing stage may be required). the numbers of rare sequence repeats appeared to match with the virulence of screened positive viruses. although, bioinformatic evidence and embryological features are in favor of our hypothesis, additional studies including animal models are warranted to validate our proposition. such studies are likely to unfold zikv-microcephaly association and may help in devising methods to combat it. owing to the reports of microcephaly as a consistent outcome in the fetuses of pregnant women infected with zikv in brazil, zika virus (zikv)-microcephaly etiomechanistic relationship has recently been implicated. researchers, however, are still struggling to establish an embryological basis for this interesting causal handcuff. the present study reveals robust evidence in favor of a plausible zikv-microcephaly cause-effect liaison. the rationale is based on: ( ) sequence homology between zikv genome and the response element of an early neural tube developmental marker "retinoic acid" in human dna and ( ) comprehensive similarities between the details of brain defects in zikv-microcephaly and retinoic acid embryopathy. retinoic acid is considered as the earliest factor for regulating anteroposterior axis of neural tube and positioning of structures in developing brain through retinoic acid response elements (rare) consensus sequence ( ′ -aggtca- ′ ) in promoter regions of retinoic acid-dependent genes. we screened genomic sequences of already reported virulent zikv strains (including those linked to microcephaly) and other viruses available in national institute of health genetic sequence database (genbank) for the rare consensus repeats and obtained results strongly bolstering our hypothesis that zikv strains associated with microcephaly may act through precipitation of dysregulation in retinoic acid-dependent genes by introducing extra stretches of rare consensus sequence repeats in the genome of developing brain cells. additional support to our hypothesis comes from our findings that screening of other viruses for rare consensus sequence repeats is positive only for those known to display neurotropism and cause fetal brain defects (for which maternal-fetal transmission during developing stage may be required). the numbers of rare sequence repeats appeared to match with the virulence of screened positive viruses. although, bioinformatic evidence introduction zikv has recently been a hot topic among researchers as well as general public due to its extensive geographical distribution and perceived health related threats; though most of the cases, as of now, are being reported from brazil (cipriano and monteiro, ; samarasekera and triunfol, ) . one of the main health intimidations by zikv has been microcephaly in the fetuses born of the infected women. there is division of opinion among researchers regarding zikv-microcephaly association (rasmussen et al., ) . this is despite the fact that zikv has been detected in the placenta and amniotic fluid of fetuses born with microcephaly (calvet et al., ; mlakar et al., ; schuler-faccini et al., ) . it has also been detected in the brain of a fetus died of severe brain defects. there is strong epidemiological evidence of zikvmicrocephaly association (schuler-faccini et al., ; who | zika situation report, ) . it would, as is obvious, be hard to justify that a true cause-effect relationship exists between zikv and microcephaly until its neuro-embryological basis is established. the present study has made an attempt to identify and explain a plausible embryological basis of zikvmicrocephaly association. we chose retinoic acid for exploring its involvement in zikv-microcephaly relationship because of the wide-ranging similarities between brain malformations caused by retinoic acid signaling dysregulation and the brain defects observed in zikv infected fetuses (as provided in many important reports; aragao et al., ; calvet et al., ; hazin et al., ; mlakar et al., ) . retinoic acid is a non-peptide small lipophilic molecule derived from retinol-an active ingredient of vitamin a. retinol gets converted to retinal and further into retinoic acid by the action of dehydrogenases which includes cyp b . retinoic acid is taken to the nucleus by the cellular retinoic acid binding protein (crabp) where it binds to retinoic acid receptors (rar and rxr) on the promoter regions of specific genes (balmer and blomhoff, ; rhinn and dollé, ) . rar and rxr are important transcription factors (rhinn and dollé, ) and actively influence rare consensus sequences in promoter regions of a plethora of genes involved in neural tube development in addition to serving other pertinent embryological functions (balmer and blomhoff, ; rhinn and dollé, ) . these genes further activate a cascade of regulatory molecules involved in neural tube formation and brain development. any dysregulation of this intricate molecular process at any step can lead to various degrees of neural tube defects and brain malformations (grapin-botton et al., ; kam et al., ; rhinn and dollé, ) . the rare sequence is also spread all over human genome through commonly found alu repeats which contain rare sequence (vansant and reynolds, ) . retinoic acid enjoys the stature of being the initial-most molecular factor involved in the determination of the neural axis and, is the prime determinant of the anteroposterior axis owing to its interplay with nodal gene in developing neural tube (durston et al., ; kam et al., ) . together with fibroblast growth factor (fgf), it is known to set a code for establishing the anterio-posterior axis (instituted by opposing gradients of retinoic acid and fgf along the neural tube) which is followed by other molecular factors (martínez-morales et al., ; shimozono et al., ) . nodal, which is thought to be the master regulator gene for deciding the axes of the developing neural tube has rare consensus sequence in its intron- . retinoic acid is also involved in deciding dorsoventral axis by intricate crosstalk with nodal (kam et al., ) . a plethora of literature suggests that retinoic acid is involved in fgf repression during body axis extension and regulation of the homeobox (hox) genes involved in determining the position of the neural segments (grapin-botton et al., ) . more than deciding the axes and positioning of the neural segments at the time of neural tube formation, retinoic acid (at a later stage) also has imperative role in the development of midline, posterio-dorsal brain structures, and forebrain components in addition to corticogenesis. disruptions in the retinoic acid signaling pathway have been implicated in ensuing brain defects in developing fetus involving all the above mentioned structures and may, arguably, present as microcephaly (wilson et al., ; siegenthaler et al., ; crandall et al., ; gupta and sen, ) . retinoic acid is also a teratogen and can cause the neural tube defect and other brain malformations if provided in abnormal doses to developing fetus (yamamoto et al., ) . zikv is a positive sense rna flavivirus (baronti et al., ) . when an rna virus infects a host cell, its genetic material is reverse-transcribed into cdna by reverse transcriptase which, in turn, synthesizes complementary sense dna strand, and finally, the so composed dna gets integrated into the host dna. we proceed with an important and coherent line of arguments that in acute viremia heavy doses of viral dna (reverse-transcribed from rna) will be integrating into the host dna, and if the inserted dna sequences match the regulatory regions of certain host genes then they may influence their expressions. by the transitive property of similarities, it can then be justified that, the rare consensus sequence repeats (if present) in the genomic sequences of zikv strains would be inserted into the promoter regions of rare dependent genes of the host dna and, therefore, may influence their expression in a way that the developing fetus manifests with brain malformation like microcephaly (figure ) . current understanding of the zikv-microcephaly association is improving as more and more relevant reports are coming up and many recent studies strongly agree to the notion that zikv-microcephaly may be a developmental brain malformation rather than a mere brain destruction by the virus (hazin et al., ; mlakar et al., ) . so, in the light of comprehensive similarities between fetal brain defects in zikv infection and brain malformations caused by retinoic acid dysregulation in developing fetus, we considered it a plausible rationale to search for a possible retinoic acid-mediated mechanism involved in zikv-microcephaly association. we, for this important reason, searched for the rare consensus sequence ( ′ -aggtca- ′ ) repeats in the genomic sequences of the zikv strains with a hypothesis that the virus might act through disruption of normal retinoic acid signaling mediated by incorporation of these sequences into the dna of developing host brain cells. to validate this hypothesis robustly and to establish the cogency of our study, we also screened some other viruses (including members of the flaviviridae family and those viruses which are known to have maternal-fetal/perinatal transmission either causing congenital brain malformations or showing neurotropism) and subjected them to the same analysis. the genome sequence information of strains of zikv (including "brazil zkiv- " which was recently implicated in causing microcephaly in brazilian fetuses; calvet et al., ; mlakar et al., ) , and many other viruses were retrieved from the ncbi/genbank database (http://www.ncbi.nlm.nih.gov/genbank/) ( table ) and screened for the presence of rare consensus sequence ( ′ -aggtca- ′ ). among the viruses other than zikv, selection was made under four groups viz: ( ) members of the flaviviridae family other than zikv (to check if rare consensus sequence repeats were essentially present in the family) ( ) viruses showing maternal-fetal/perinatal transmission and causing congenital brain malformations ( ) viruses showing neurotropism but not known to cause fetal brain defects ( ) viruses chosen randomly from the rna/dna viruses not fitting in any of the above three groups (to account for the selection bias and to establish coherence and validity of the screening process). to identify the enrichment of rare sequence ( ′ -aggtca- ′ ) repeats present in the viruses, a perl script was written and executed. in order to find and count the rare sequences from the zika virus genome, a perl script was generated entitled "rare_seq_finder.pl." the perl script was based on the window shift algorithm. genome sequence information of zika virus isolates was downloaded from various genome databases. as input, the perl script requires only the list file carrying names of the genome sequence files. it searches the rare sequences globally in the genome, and lists all genome file names along with number of rare sequences present in the genome. a complete flow-chart with all the steps involved in the counting the rare sequences is shown in figure . the complete perl script for this study is available as supplementary material. the zikv strains associated with microcephaly contained rare consensus sequence repeats. the number of repeats corresponds to the virulence of each strain i.e., greater the number of rare repeats higher the chances of microcephaly. complete details of the number of rare sequence repeats in each zikv strain are given in table . for viruses other than zikv, the rare consensus sequence repeats were found to be present only in those viruses known for maternal-fetal/perinatal transmission, involved in fetal brain defects and displaying neurotropism. the numbers of rare sequence repeats was directly proportional to the virulence of screened viruses. the number of rare consensus sequences repeats in the checked strains of the zikv and other zikv viruses are provided in tables , respectively. the rare consensus sequence repeats were found in most of the zikv strains but the frequency of the repeats varied among the strains. surprisingly, the zikv strain (ku . , table ) which was implicated in the recent cases of the microcephaly in brazil (calvet et al., ) , and other strains which shared close homology with it contained the highest number of rare sequence repeats ( table ). the genomic sequence of the brazilian zikv strain was found to share near % homology to the polynesian strain (kj , table ) which was taken as a reference in a recent study (calvet et al., ) . another study (mlakar et al., ) which had retrieved the virus from the brain tissue of a dead fetus with microcephaly had similar interpretation that the retrieved strain had a very close homology to the polynesian strain ( . %) and two other strains (eu - %, jn - . %). all the three strains were amongst the highest number of rare sequence containing strains as revealed in our study (table ) . although, it would still be a far-fetched conjecture to consider an association of these zikv strains (with the highest number of rare sequence repeats) to microcephaly unless zikv-microcephaly link is well established. from analysis of the data from all the viruses other than zikv (table ) , it is clear that only those viruses contain the rare consensus sequence repeats which are either known to cause fetal brain malformations or show neurotropism. different strains of the same genus vary in the presence of these sequence repeats and it is absent altogether in some strains. a virus with comparatively higher rare sequence repeats seems to be a more potent teratogen (in terms of causing congenital brain malformation) or displays correspondingly robust neurotropism as suggested by the analysis of table . some of these viruses are well known to cause fetal microcephaly also ( the evidence of its neurotropism in literature (salazar et al., ) . the presence of the rare consensus sequence repeats revealed in the known neurotropic viruses (table ) could be an indication that retinoic acid signaling may have an important role to play in neurotropism, although we couldn't find sufficient support in literature for this claim due to paucity of research over this issue. there is, nevertheless, substantial evidence that retinoic acid inducible gene (rig)- which is upregulated by retinoic acid, functions as an intra-cellular pattern recognition receptor for the known neurotrophic receptor viruses (furr and marriott, ; carty et al., ) . a recent study reports the involvement of a similar viral pattern recognition receptor tlr- in zikv caused growth restriction and depletion of neural stem cell progenitors in human cerebral organoids (dang et al., ) . there are additional reports suggesting a necessary rig- and tlr- interaction to surge innate immunity in response of viral infections (liu et al., ; slater et al., ) . these reports indicate the involvement of retinoic acid signaling in proper brain development. further research is required to establish the nature of retinoic acid-neurotropism relationship but based on our observations on the zikv strains and various other viruses, it seems coherent to consider the presence of rare consensus repeats as the reason for the obligatory neurotropic character of the zikv strains implicated in microcephaly (calvet et al., ; hazin et al., ; mlakar et al., ) . the high frequency of rare consensus repeats in some double stranded dna viruses may be because of larger reading frame length and probably a rare sequence index value (which balances number of repeats with total reading frame length of genomic sequence). this index could be an appropriate measure for representing potency of a virus for causing congenital brain malformation or neurotropism. a further detailed study on the presence of rare consensus sequence repeats in zikv vs. other viruses may be desired to confirm if retinoic acid signaling dysregulation is a common mechanism used by such viruses to cause congenital brain malformations. fetal microcephaly has always been known as a developmental anomaly caused by multiple etiologies (mochida and walsh, ) . retinoic acid signaling dysregulation has also been implicated as a common mechanism in many environmental or developmental reasons of microcephaly (lammer et al., ; kot-leibovich and fainsod, ; paganelli et al., ) . the anatomical details of the brain of the fetuses suspected of zikv infection in recent reports (aragao et al., ; calvet et al., ; hazin et al., ; mlakar et al., ) closely resemble to those caused by retinoic acid signaling dysregulation and have many features in common i.e., cerebellar and brain stem hypoplasia (yamamoto et al., ) , ventriculomegaly (micucci et al., ) , hypoplasia of forebrain derivatives like frontal or parietal lobe of the brain (halilagic et al., ) , and hypogyration of cerebral cortex (siegenthaler et al., ) . the reason behind these striking resemblances could be that the retinoic acid signaling is crucially involved in development of the dorsal brain structures (wilson et al., ; including cerebellum and brain stem), midline forebrain structures (gupta and sen, ; including choroid plexus which synthesizes csf and a malformation of it may result in hydrocephalus and in turn ventriculomegaly), (siegenthaler et al., ; crandall et al., ) . so it is an obviously plausible concept in the light of our hypothesis as to why retinoic acid signaling dysregulation may result in malformations similar to that noted in zikv caused microcephaly (calvet et al., ; hazin et al., ; mlakar et al., ) . the presentation of the brain defects in retinoic acid dysregulation varies depending on the stage of embryonic development. if the signaling disruption ensues at late gastrulation or early neurulation stage, the defect should be occurring in rostrocaudal sequencing of the brain structures in the developing neural tube i.e., there may be a larger hindbrain at the expense of anterior brain structures. but if the disruption is at late developmental stages that will affect dorsalization of the brain or hindbrain structures i.e., may cause cerebellar or brain stem hypoplasia or absence of some hind brain components (wilson et al., ) . the presence of microcephaly or other brain malformations in the fetuses born of the zikv infected mothers (similar to the other viruses causing congenital brain malformations) may also depend upon the time-point of the fetal infection during neural tube formation, and also on the total viral load (adams waldorf and mcadams, ) . in some recent reports of the cases with zikv-microcephaly, pregnant women presented with clinical features of zikv infection at th week of gestation or beyond and microcephalic features were detected on ultrasound beyond th week (calvet et al., ; mlakar et al., ) . this matches with the retinoic acid signaling dysregulation at the late developmental stages when dorsal brain structures and hindbrain brain structures are still in development and cortical gyration still in progress. a disruption at this stage would essentially result in the features typically seen in zikv microcephaly (calvet et al., ; hazin et al., ; mlakar et al., ) as has been noted above. due to striking resemblance to the embryological malformations, the recent reports on zikv-microcephaly (calvet et al., ; hazin et al., ; mlakar et al., ) have stressed upon considering the fetal brain damage as a developmental problem rather than the brain destruction caused by viral invasion of neuronal cells. one important study has found substantial evidence of presence of zikv in the neurons through immuno-histological and microbiology investigations of the fetal brain tissue died of the severe microcephaly (mlakar et al., ) . interstingly, other probable infectious causes of the microcephaly were successfully ruled out in this study. on an interesting note, our data also suggests that a virus which contains rare consensus sequence repeats also shows some degree of neurotropism but its causal handcuff to microcephaly or any other type of fetal brain defect essentially depends upon its ability for maternal-fetal transmission. chikungunya virus ((+)ssrna, linear, bp: table ) seems to be a representative example. it contains four rare consensus sequence repeats and shows neurotropism (koyuncu et al., ) , maternal-fetal transmission in humans has also been sufficiently demonstrated for this virus (fritel et al., ; gérardin et al., ) , and is also known to cause microcephaly and/or other fetal brain defects (gérardin et al., ) . on the other hand japanese encephalitis virus ((+)ssrna, linear, bp: table ) which has no known evidence of maternal-fetal transmission in humans, although having shorter frame length and more number of rare consensus sequence repeats (numbering to six) is known to show neurotropism (kimura-kuroda et al., ) but do not cause fetal brain defect in humans. the obligatory nature of ability of maternal-fetal transmission in a virus to cause fetal brain damage is empirically coherent and further suggests that presence of rare consensus sequence repeats in a virus genome may be limited to confer it neurovirulence only, and may require additional contributing factors toward its maternal-fetal transmission ability. another evidence in favor of the retinoic acid signaling dysregulation as a probable mechanism in zikv-microcephaly is that the mouse model study of a well-known fetal anomaly syndrome called "charge" (in which dysregulation of retinoic acid signaling has been implicated) reported some anomalous features very similar to that found in fetal brains suspected of zikv microcephaly (calvet et al., ; hazin et al., ; mlakar et al., ) such as cerebellar hypoplasia and ventriculomegaly (micucci et al., ) . in addition to that, hydrocephalus and malformation of corpus callosum noted in some cases of zikv microcephaly (aragao et al., ) are also present in "charge" syndrome. furthermore, the widespread cortical anomalies in zikv microcephaly get explained by an interesting study involving retinoic acid (siegenthaler et al., ). these investigators reported cranial meninges as a source of retinoic acid to the developing cortex (radial glial cells carrying migrating progenitor cells to the upper cortical layers attach their end feet to the covering meninx, from where they get retinoic acid as a diffusible morphogen and transfer to other cells involved in corticogenesis). retinoic acid signaling has been extensively implicated in cortical genesis (harrison-uy et al., ) , especially in the migration of progenitor cells to the upper cortical layers riding on the radial glial cells (siegenthaler et al., ; crandall et al., ) . many contemporary reports have confirmed that zikv has detrimental effects on human cortical progenitor cells in neural stem cell cultures (crandall et al., ) and neurospheres or brain organoids developed from human induced pluripotent cells (garcez et al., ) . zikv has also been shown to induce cell death and attenuate growth mediated through vast transcriptomic alterations (crandall et al., ) involved in apoptosis (crandall et al., ; garcez et al., ) and cell cycle regulations (crandall et al., ) . the disruption of the normal retinoic acid signaling in developing forebrain has been shown to induce death of neural precursor cells and attenuate their growth with somewhat similar mechanisms (rajaii et al., ) , raising a possibility that zikv might be disrupting retinoic acid signaling pathway. our hypothesis, hence derives support from known literature with multiple justifications. it explains many mysteries and helps in understanding the embryological basis of zikv mediated microcephaly. additionally, the white matter hypodensity noted in zikv mediated microcephaly (hazin et al., ) can also be explained by dysregulation of retinoic acid signaling due to its noted involvement in myelination in the developing brain. to augment this, adequate retinoid receptor expression on oligodendrocyte precursor cells is necessary which later myelinate cns (huang et al., ) . dysregulation of retinoic acid signaling has also been implicated in some demyelinating pathologies (okuda and prados, ; könig et al., ) . also, there is sufficient evidence that the retinal or other ophthalmic lesions, a unique finding observed in some zikv infected fetuses (de paula freitas et al., ; ventura et al., ) may precipitate through disruption retinoic acid signaling (mic et al., ; micucci et al., ) . our claim that zikv-microcephaly may involve retinoic acid signaling dysregulation is further bolstered by the fact that many of the toxins that involve retinoic acid also have microcephaly as a presenting feature (lammer et al., ; mochida and walsh, ; kot-leibovich and fainsod, ; paganelli et al., ) and may share other malformations too. this is especially relevant to malformations arising in fetus with exposure of pregnant women to the isotretinoin (an analog of retinoic acid known to cause retinoic acid embryopathy). these malformations match to almost all the features described under zikv-microcephaly spectrum including enlarged cistern magna and calcifications in brain parenchyma (lammer et al., ; irving et al., ) . with the above-described pieces of evidence, a plausible mosaic of a justified picture comes up supporting our proposition that zikv interferes with retinoic acid signaling in the developing brain. in view of ( ) the bioinformatic evidence of the presence of rare sequence repeats in genome sequences of zikv strains and in other viruses known to cause congenital brain malformation (table ) and ( ) confirmed role of retinoic acid in neural tube formation and further brain development mediated through rare sequences, it is scientifically coherent to assume that a rare sequence dependent mechanism could be the underlying mechanism of microcephaly seen in zikv infected fetuses. we, however stress that there is a need of confirming the proposed hypothesis on animal models or neural cell cultures. why embryonic defects are limited to the central nervous system only, and no anomaly of the other organs or limbs are reported in zikv if it causes an embryopathy is quite surprising. it could probably be due to strong neurotropic nature of the virus as it has been reported to be completely absent in organs other than the brain. a recent study performed the autopsy on zikv infected fetuses with severe microcephaly confirmed the absence of this virus in organs other than brain (mlakar et al., ) . zikv infection has also been observed to be less prevalent in human precursor cells other than the neural precursors (crandall et al., ) . again, why did usg studies in zikv infected fetuses show no abnormality of brain structures almost until th weeks of gestation is a mystery and needs to be explained. a probable explanation for the late involvement of the brain structures has been indicated in the recent study based on immune-histological features of affected brain tissue. it indicates that time is consumed in virus invasion of the neurones and accumulation of approximate lesions capable of arresting cortical growth (mlakar et al., ) . we chose retinoic acid for its probable role in zikvmediated microcephaly but there is obvious need of exploring other molecular factors which may be involved in neural tube axis determination and its further development. also, retinoic acid signaling dysregulation is not able to explain multifocal calcifications widespread in the basal ganglia, thalamus and cortical regions as noted in histological and radiological studies of zikv-microcephaly cases (aragao et al., ; calvet et al., ; hazin et al., ; mlakar et al., ) . it is, however, important to mention that a rare presentation of an embryological anomaly syndrome ("charge" syndrome, in which retinoic acid signaling dysregulation; micucci et al., ) has been noted and reported with bilateral basal ganglia calcifications (jain et al., ) . calcifications in brain parenchyma have also been mentioned in retinoic acid embryopathy (lammer et al., ) . as the calcifications found in the zikv affected fetal brain are punctiform and appear as destroyed neuronal structure on histological examination (mlakar et al., ) , an alternative explanation for their formation could be that calcifications are due to the deposition of the calcium compounds in the accumulations of viral invaded dead neurons (mlakar et al., ) as the process of degeneration. to test our hypothesis, it was is essential to ascertain that the zikv strains which contain either equal or less number of rare sequences than brazilian strain (table ) in their genome can also cause microcephaly or not. as we discussed above based the zikv strain which was recently implicated in microcephaly in brazil has close similarity with the polynesian and other two strains (mlakar et al., ; schuler-faccini et al., ; table , iv, viii, xiv), and all three contained the highest numbers of rare consensus sequences in our study. but to make a valid claim to relate number of rare consensus sequences with virulence of the strains, further availability of knowledge on the zikv strains implicated in the microcephaly is indispensable. the immediate application of our study are immense. if it is established that zikv caused microcephaly in infected fetus is a developmental condition arising out of retinoic acid signaling dysregulation it would not only help to understand the zikvmicrocephaly pathogenesis but also may justify retinoic acid as a therapeutic target for preventing this condition. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fnhum. . influence of infection during pregnancy on fetal development clinical features and neuroimaging (ct and mri) findings in presumed zika virus related congenital infection and microcephaly: retrospective case series study gene expression regulation by retinoic acid complete coding sequence of zika virus from a french polynesia outbreak in detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study innate antiviral signalling in the central nervous system reporting on zika virus in brazil neurologic complications of hiv infection in children retinoic acid influences neuronal migration from the ganglionic eminence to the cerebral cortex zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor tlr ocular findings in infants with microcephaly associated with presumed zika virus congenital infection in salvador, brazil retinoic acid causes an anteroposterior transformation in the developing central nervous system the essential neurotropism of the yellow fever virus evidence for a cerebral effect of the hepatitis c virus chikungunya virus infection during pregnancy viral cns infections: role of glial pattern recognition receptors in neuroinflammation zika virus impairs growth in human neurospheres and brain organoids multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of la reunion neurocognitive outcome of children exposed to perinatal motherto-child chikungunya virus infection: the chimere cohort study on reunion island defined concentrations of a posteriorizing signal are critical for mafb/kreisler segmental expression in the hindbrain cerebral microglial activation in patients with hepatitis c: in vivo evidence of neuroinflammation incidence of yellow fever vaccine-associated neurotropic disease retinoic acid signaling regulates development of the dorsal forebrain midline and the choroid plexus in the chick retinoids control anterior and dorsal properties in the developing forebrain couptfi interacts with retinoic acid signaling during cortical development computed tomographic findings in microcephaly associated with zika virus retinoid x receptor gamma signaling accelerates cns remyelination morphogenesis of isotretinoin-induced microcephaly and micrognathia studied by scanning electron microscopy unique phenotype in a patient with charge syndrome retinoic acid synthesis and functions in early embryonic development specific tropism of japanese encephalitis virus for developing neurons in primary rat brain culture expression of retinoid x receptor beta is induced in astrocytes during corpus callosum demyelination ethanol induces embryonic malformations by competing for retinaldehyde dehydrogenase activity during vertebrate gastrulation virus infections in the nervous system retinoic acid embryopathy. new engl coronavirus hku and other coronavirus infections in hong kong retinoic acid-inducible gene i mediates early antiviral response and toll-like receptor expression in respiratory syncytial virus-infected airway epithelial cells neurotropic virus infections as the cause of immediate and delayed neuropathology fgf and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk raldh expression in optic vesicle generates a retinoic acid signal needed for invagination of retina during optic cup formation chd and retinoic acid signaling cooperate to regulate neural stem cell and inner ear development in mouse models of charge syndrome zika virus associated with microcephaly molecular genetics of human microcephaly ebola hemorrhagic fever and pregnancy recurrent asymptomatic demyelinating disease following -cis-retinoic acid exposure glyphosate-based herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling expression of the dominant negative retinoid receptor, rar , alters telencephalic progenitor proliferation, survival, and cell fate specification zika virus and birth defects-reviewing the evidence for causality retinoic acid signalling during development changes in mumps virus gene sequence associated with variability in neurovirulent phenotype dengue virus type : protein binding and active replication in human central nervous system cells concern over zika virus grips the world axonal transport mediates west nile virus entry into the central nervous system and induces acute flaccid paralysis the congenital varicella syndrome severe microcephaly associated with congenital varicella infection possible association between zika virus infection and microcephaly-brazil visualization of an endogenous retinoic acid gradient across embryonic development retinoic acid from the meninges regulates cortical neuron generation co-ordinated role of tlr , rig-i and mda in the innate response to rhinovirus in bronchial epithelium herpes simplex virus infection in pregnancy experimental mumps virus-induced hydrocephalus: viral neurotropism and neuronal maturity cytomegalovirus-lnduced brain malformations in fetuses neurotropism of rabies virus: an in vitro study phenotypic characterization of patient dengue virus isolates in balb/c mice differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome the consensus sequence of a major alu subfamily contains a functional retinoic acid response element zika virus in brazil and macular atrophy in a child with microcephaly who | zika situation report retinoic acid is a potential dorsalising signal in the late embryonic chick hindbrain hiv- encephalitis: case report and literature review detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis a critical period for retinoic acid teratogenesis and loss of neurophilic migration of pontine nuclei neurons fetal and neonatal abnormalities due to congenital rubella syndrome: a review of literature key: cord- -e z ypii authors: squires, raynal c; konings, frank title: preparedness for zika virus testing in the world health organization western pacific region date: - - journal: western pac surveill response j doi: . /wpsar. . . . sha: doc_id: cord_uid: e z ypii on february , the world health organization (who) declared that clusters of microcephaly cases and other neurological disorders occurring in zika virus (zikv)-affected areas constituted a public health emergency of international concern. increased surveillance of the virus, including the requirement for laboratory confirmation of infection, was recommended. the who regional office for the western pacific therefore initiated a rapid survey among national-level public health laboratories in countries and areas to determine regional capacity for zikv detection. the survey indicated that / ( %) countries had capacity for molecular detection of zikv while others facilitated testing through referral. these results suggest that robust laboratory capacity is in place to support zikv surveillance in the western pacific region. i nitially identified in a rhesus monkey from uganda's zika forest in and subsequently isolated from humans in in nigeria, zika virus (zikv) is a flavivirus transmitted by aedes mosquitoes, the same vector transmitting other arboviruses of public health impact such as yellow fever virus, dengue virus (denv) and chikungunya virus (chikv). the first known zikv outbreak occurred in in yap state of the federated states of micronesia in the world health organization (who) western pacific region followed by a - outbreak in french polynesia with an estimated cases. the virus has gone on to cause outbreaks in multiple pacific island countries and has spread throughout the americas. in november , brazil began reporting substantial increases in the number of children born with microcephaly in zikv-affected areas. that evidence, coupled with reports of guillain-barré syndrome cases in other zikv outbreaks, particularly in french polynesia, led who on february to declare that the cluster of microcephaly cases and other neurological disorders constituted a public health emergency of international concern (pheic). among the recommendations from that meeting of the international health regulations ( ) emergency committee were that "surveillance for zikv infection should be enhanced, with the dissemination of standard case definitions and diagnostics to at-risk areas". laboratory testing is a critical component of surveillance for zikv infection due to co-circulation of denv and chikv that cause similar symptoms. , to determine regional capacity for zikv detection, the who regional office for the western pacific initiated a voluntary, rapid survey among national-level public health laboratories in its countries and areas (areas are non-sovereign jurisdictions within a who region; countries and areas are together referred to as "countries" in this article). the survey sought to assess preparedness for zikv testing in the context of co-circulating denv and chikv. questions primarily addressed in-country capacity for molecular and serological detection of the three arboviruses, additional laboratory capacities specific for zikv and testing-related services to other countries. the -question, email-based survey was administered between and february , immediately following the pheic declaration. a total of surveys to national-level laboratories likely to be tasked with zikv testing were distributed to countries in the region (omitting resource-limited countries with zika virus testing preparedness in the western pacific region squires & konings reporting having this capacity. twelve countries indicated that they were willing to accept international specimens to supplement the capacity in other countries or for confirmation testing (data not shown). given the similarity of disease presentation, co-circulation and increasing prevalence of infection, - differential diagnosis for denv, chikv and zikv is crucial. molecular detection of denv and chikv was in place in / ( . %) countries, and a similarly large majority could perform serological diagnosis of denv ( / , . %) and chikv ( / , . %) infection by igm and/or igg detection. the algorithm followed for differential diagnosis should take into consideration the endemic circulation of denv, chikv and zikv. among countries detailing their algorithm, ( %) indicated they tested suspected samples for all three arboviruses concurrently, similar to the algorithm recommended by the united states centers for disease control and prevention; / ( . %) attempted to rule out each virus sequentially as outlined in the who regional office basic laboratory capacity known to rely on specimen referral). the survey was completed by laboratories in countries. for the country not responding, information from other sources such as recent peerreviewed publications was used where possible to augment the data set and cover all countries. of the remaining three, two were using specimen referral to neighbouring countries (similar to pacific island countries without pcr capacity), while the other has been working closely with the who regional office for the western pacific to obtain materials and reagents to enable in-country testing. of the countries with pcr test capacity for zikv, could additionally sequence the virus and isolate it in culture. serological diagnosis of zikv infection by immunoglobulin m (igm) and/or immunoglobulin g (igg) detection was also surveyed in the countries, with less than one third ( / ) table rely mainly on specimen referral, the study's geographic coverage only included countries of the asian sub-region and larger countries or referral hubs of the pacific subregion such as australia and french polynesia. the laboratory plays an important role in improving our understanding of zikv epidemiology. while this survey reveals a broad availability of molecular diagnostics to support surveillance of zikv in the western pacific region, further key roles remain for laboratories in helping to unravel the pathogenicity of the virus and its potential causal role in the observed cases of microcephaly and other neurological disorders. none declared. none. for the americas guidance. the remaining country indicated that the epidemiological circumstances of each case drove the specific algorithm followed. this survey, conducted immediately following the who declaration of a pheic surrounding clusters of microcephaly and neurological disorders in the context of zikv infection, suggests that robust coverage for molecular detection of priority arboviruses is in place in the region. molecular detection by pcr is the critical differential diagnostic tool in this public health event as serology is problematic due to antibody cross-reactivity in regions with multiple circulating flaviviruses and/or use of vaccines against those viruses (for example, japanese encephalitis virus). , other methodologies, such as the plaque reduction neutralization test (prnt), exist for the specific serological discrimination among the flaviviruses but require significant technical expertise for accurate execution and would not be practical for large-scale surveillance. only three of the countries responding to the survey reported they could perform prnt (data not shown). as in our previous survey of regional pcr testing capacity for middle east respiratory syndrome coronavirus, most ( / ) of the national-level public health laboratories supporting pcr testing of zikv in their countries functioned as national influenza centres in the global influenza surveillance and response system, showing the versatility of this network. a similar proportion ( / ) participated in the external quality assessment (eqa) for denv and chikv diagnostics, which has a substantial pcr-based component. this eqa, conducted by the who regional office for the western pacific , in and , showed robust proficiency for diagnosis of these viruses in the region. while the region seems prepared overall for testing of zikv, it is important to continue strengthening the apparatus for detection particularly through ensuring testing proficiency by eqa participation and enhancing referral mechanisms and international air transport association certification where needed. it should be noted that while the eqa for denv and chikv diagnostics gives confidence about regional testing proficiency for those arboviruses, proficiency of zikv diagnostics remains untested. note also that by omitting countries known to zika: the origin and spread of a mosquitoborne virus genetic diversity and phylogeny of aedes aegypti, the main arbovirus vector in the pacific emergence du virus zika en polynésie française, novembre -avril world health organization regional office for the americas. increase of microcephaly in the northeast of brazil zika virus, microcephaly, and guillain-barré syndrome world health organization regional office for the preparedness for molecular testing of middle east respiratory syndrome coronavirus among laboratories in the western pacific region first round of external quality assessment of dengue diagnostics in the who western pacific region external quality assessment of dengue and chikungunya diagnostics in the who south-east asia and western pacific regions who director-general summarizes the outcome of the emergency committee regarding clusters of microcephaly and guillain-barré syndrome ihr ) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations memorandum: revised diagnostic testing for zika, chikungunya, and dengue viruses in us public health laboratories laboratory testing for zika virus infection, interim guidance who western pacific region countries and areas. manila, world health organization regional office for the western pacific countries and territories where chikungunya cases have been reported (as of october the authors are grateful to the participating laboratories and countries that shared their information and took the time to complete the survey. we would also like to thank the other members of the zika incident management team, including: christopher lowbridge, katherine russell, may chiew, cong ze, joy caminade, jun nakagawa, thierry cordier-lassalle, janet mina, meg dichoso, takuya yamagishi, rabindra abeyasinghe, babatunde olowokure and li ailan. key: cord- -lrkscs authors: kurosaki, yohei; martins, danyelly bruneska gondim; kimura, mayuko; catena, andriu dos santos; borba, maria amélia carlos souto maior; mattos, sandra da silva; abe, haruka; yoshikawa, rokusuke; de lima filho, josé luiz; yasuda, jiro title: development and evaluation of a rapid molecular diagnostic test for zika virus infection by reverse transcription loop-mediated isothermal amplification date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: lrkscs the recent outbreak of zika virus (zikv) disease caused an enormous number of infections in central and south america, and the unusual increase in the number of infants born with microcephaly associated with zikv infection aroused global concern. here, we developed a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay using a portable device for the detection of zikv. the assay specifically detected zikv strains of both asian and african genotypes without cross-reactivity with other arboviruses, including dengue and chikungunya viruses. the assay detected viral rna at . tcid( )/ml in virus-spiked serum or urine samples within min, although it was slightly less sensitive than reference real time rt-pcr assay. we then evaluated the utility of this assay as a molecular diagnostic test using plasma or serum samples and urine samples collected from suspected cases of arbovirus infection in the states of paraíba and pernambuco, brazil in . the results of this assay were consistent with those of the reference rt-pcr test. this portable rt-lamp assay was highly specific for zikv, and enable rapid diagnosis of the virus infection. our results provide new insights into zikv molecular diagnostics and may improve preparedness for future outbreaks. zikv is a positive-stranded rna virus belonging to the genus flavivirus in the family flaviviridae. zikv shares its vector, the aedes mosquito, with other flaviviruses, including denv, yellow fever virus (yfv), and chikv . zikv has been isolated from humans in east and west africa and in southeast asia and polynesian countries where the host mosquitoes, a. aegypti and a. albopictus, are found , . based on phylogenetic analyses, these isolates can be categorised into two genotypes, african and asian. epidemiological studies have revealed that the recent outbreak of zikv in brazil occurred via the introduction of a virus from french polynesia, where an outbreak of the disease occurred in . all of the viruses isolated in brazil and other countries on the american continent belong to the asian genotype . in patients with zikv infection, the virus can be detected in several sample types, including blood, urine, saliva, and other body fluids [ ] [ ] [ ] [ ] [ ] . the viral load in blood reaches a peak at to days after the onset of illness, but decreases rapidly thereafter. therefore, it is difficult to detect zikv in blood samples from patients after the acute phase of infection, even with sensitive molecular diagnostic methods, such as reverse transcription-polymerase chain reaction (rt-pcr) , , . the virus can be detected in urine samples for longer durations (> - days after the onset of symptoms) than in those for blood samples . currently, blood and urine samples are typically used for the molecular diagnosis of zikv. zikv infection is diagnosed in the laboratory by nucleic acid amplification tests (naats) to detect viral rna [ ] [ ] [ ] [ ] [ ] or by elisa to detect igm or igg antibodies , . the naats such as rt-pcr and other technologies (e.g. recombinase polymerase amplification) are highly accurate, and rt-pcr is considered the gold standard to confirm zikv infection , . rt-pcr, however, requires a step for viral rna extraction prior to the assay and the use of expensive equipment, such as thermal cycler, to conduct the test. moreover, there is a risk of reduced sample quality due to rna degradation during transportation to the laboratory. for elisa, serological cross-reaction between zikv and other circulating flaviviruses like denv makes accurate diagnosis with serology difficult , . therefore, novel diagnostic technologies that can be conducted at the point-of-care or in regional laboratories are greatly needed to control zikv infections. reverse transcription loop-mediated isothermal amplification (rt-lamp) is a rapid, sensitive rna detection method performed under isothermal conditions using four or six unique oligonucleotide primers , . since lamp reactions can be performed with simple inexpensive equipment, rt-lamp assays can be conducted in the field and by under-funded laboratories . we previously developed a rt-lamp assay using a portable isothermal amplification and detection device for ebola virus in response to the recent outbreak of ebola virus disease in west africa, and the assay has been deployed for field surveillance in guinea , . here, we developed a rt-lamp assay for the detection of zikv with a portable battery-powered device. then, we evaluated the utility of this assay for molecular diagnosis using clinical specimens collected from the recent zikv outbreak in brazil. sensitivity. we designed zikv genotype-specific lamp primers that targeted conserved sequences in the e protein-coding region (table ) . each genotype-specific primer recognised the same genomic position. to detect all known zikv strains, we used a mixture of primers specific for each genotype in a single reaction. first, we examined the sensitivity of the assay using serial -fold dilutions of in vitro synthesised standard rnas from strain uganda, which was isolated from a rhesus macaque in uganda, and strain prvabc , which was isolated at the centres for disease control and prevention (cdc) from a patient who travelled to puerto rico in . ten copies of the rna standards were detected from both strains in quadruplicate reactions (fig. a) . the times to obtain positive results (tp) for rna standards ranging from to copies were mostly less than min, and within this range, tp was correlated with the number of rna copies ( fig. b and c) . single copies of the standard rnas from the uganda and prvabc strains were detected with % and % positivity, respectively, and tp values were dispersed. these results suggested that the rt-lamp assay could be used as a rapid, sensitive diagnostic test for zikv, the tp value (i.e., less than min) can be used as an indicator of the number of rna copies in each reaction. we evaluated the specificity of each primer in silico using zikv strain sequences available in genbank as of november . twenty-seven sequences of african genotype isolates collected in - and sequences of asian genotype isolates collected in southeast asia and polynesia in - as well as from the current outbreak in the americas were used for this analysis. the lamp primers consist of nucleotides in total length and recognise eight separate sites on zikv genome (fig. a) . we determined the proportion of sequences that had identical nucleotides at each position for either the asian or african genotype primers (fig. b) . african genotype zikv sequences had identical residues at out of positions ( . %) in the african or asian genotype-specific primers. at positions in the primer recognition sites, more than % of african genotype zikv sequences had mismatched nucleotides. at six positions, scattered in the f , f , lf, and b sites in the asian and african genotype primers, more than % of the african genotype zikv sequences had nucleotide differences (fig. b, upper panel) . asian genotype sequences showed greater identity than african genotype sequences to the lamp primers. asian genotype sequences had identical residues at out of positions ( . %) in the asian or african genotype primers. for one residue at the ′ terminus of the f site of the fip primers, . % of the asian genotype sequences had nucleotide differences (fig. b, lower panel) . to assess the primer specificity for zikv strains, we synthesised rnas with the partial genome sequences of two african genotype zikv strains, -dak and ard , and two asian genotype zikv strains, p - and cpc- , which had more mismatched nucleotides against the primer sequences compared with the average for all strains. these rna sequences were also detected using the rt-lamp assay, in addition to the sequences in the uganda and prvabc strains (table ) . furthermore, no cross-reactions with other tested arboviruses, including denv, yfv, west nile virus (wnv), chikv, and rift valley fever virus (rvfv), and plasmodium falciparum were observed. these results suggested that the rt-lamp assay developed here was highly specific for detecting zikv strains of both african and asian genotypes. and/or urine is used, since viral rna can be detected in these clinical specimens during the acute phase of infection. the feasibility of using the rt-lamp assay for clinical specimens was evaluated using zikv-spiked human serum and urine samples. we prepared human serum and urine spiked with four-fold serially diluted zikv strain uganda, and obtained samples with titres of . - . tcid /ml. the sensitivity of the rt-lamp assay was compared to that of the real time rt-pcr (rrt-pcr) assay developed by the cdc . using the rt-lamp assay, we detected viral rna in both serum and urine samples at a titre of . tcid /ml in quadruplicate reactions. the ct values in the rrt-pcr were . ± . and . ± . for serum and urine samples, respectively, which corresponded to . and . genome equivalents (geq) per reaction, respectively (table ). using the rrt-pcr assay, we detected viruses in both serum and urine samples at a titre of . tcid /ml, which corresponded to . and . geq per reaction, respectively; however, the rt-lamp assay failed for these samples, suggesting that the rt-lamp assay was less sensitive than the cdc rrt-pcr assay for zikv detection. together with the results table ). the rt-lamp assay did not show any false-positive results, even for six confirmed denv samples (data not shown). the ct values of these eight zikv-positive samples were . - . , and the viral loads were estimated to be . × - . × geq/ml using the viral rna standards (table ) . these viral titres were higher than those reported in previous studies. to examine whether the assay can detect viral rna in samples with lower titres, we randomly selected two zikv-positive samples confirmed in this study, mrl and mrl , and conducted a dilution test (table ). while the rt-lamp failed to detect samples with the ct value > , however, it detected viral rna at the ct < , consistent with our earlier results obtained using the virus-spiked serum and urine samples (table ). these results show that the rrt-lamp assay had sufficient specificity for the detection of zikv as a molecular diagnostic test. the assay can be used to detect an amount of viral rna equivalent to that yielding ct values of - in the reference rrt-pcr test. we developed a rapid molecular detection assay for zikv in response to the recent outbreak in south america. lamp assays and modified diagnostic methods for zikv have been reported; however, these molecular techniques have never been evaluated for clinical use [ ] [ ] [ ] [ ] . this is the first evaluation of the clinical usage of a lamp assay for molecular diagnostic testing in the recent outbreak of zikv infections. since zikv shares a vector with denv and chikv, these viral diseases can occur simultaneously, and northeast brazil is an endemic area for dengue and chikungunya . numerous severe mosquito-borne diseases, including arbovirus infections as well as malaria, share clinical symptoms during the acute phase. however, zikv infection is generally associated with mild symptoms. a major concern with respect to molecular diagnostic testing for zikv is the potential for cross-reactivity with other flaviviruses, especially dnev, which have close antigenic relation with zikv , , , . in contrast, our assay showed no cross-reactions with other arboviruses or p. falciparum, and did not show false-positive results when applied to zikv-negative samples. these results indicated that the rt-lamp assay is specific for the detection of zikv and is a reliable molecular diagnostic test. another potential limitation of molecular diagnostic testing is that zikv-infected samples often have low titres after the acute or early phase of infection due to rapid clearance by the host immune system. this makes it difficult to identify zikv cases, even using rt-pcr-based tests. the limit of detection for this assay was copies table . detection of zikv by rt-lamp and rrt-pcr using diluted zikv-confirmed samples. . for both genotypes. the assay was slightly less sensitive than the cdc rrt-pcr test, which was commonly used to confirm zikv infection during the recent outbreak. zikv-infected clinical samples often show high ct values (> ) , . however, the zikv-positive samples detected in this evaluation showed ct values of less than . (more than . × geq/ml), which was a higher titre than that reported in other studies. to confirm its clinical utility, this assay should be tested using samples with lower titres or borderline zikv infections. it has been reported that viral rna can be detected for longer periods in urine than in blood , . therefore, we considered urine to be one of the best sample types for detecting zikv infections. recently, paz-bailey et al. reported contradictory results for the persistence of viral rna in blood samples of zikv patients; rna can be detected or weeks after the onset of illness . in some cases, viral rna can also be detected at higher titres in saliva than in blood, but persists for shorter periods , . it is necessary to determine the sample types suitable for the rt-lamp assay and to establish a standardised rna extraction protocol adjusted to each clinical specimen type in order to improve the sensitivity of this assay. owing to the sequence diversity among zikv isolates, we designed lamp primers specific for each genotype and used a mixture of these primers to detect all known isolates of both african and asian genotypes. as shown in fig. , we conducted an in silico evaluation of each primer using available zikv sequences. african genotype strains supposedly have a longer history of circulation in african mosquitos and humans than that of asian genotype strains , and african genotype sequences showed a lower identity at some positions in the lamp primers. the lamp primers designed here showed high identities at most positions against the sequences of strains involved in the recent outbreak on the american continent, as well as its ancestral southeast asian and polynesian isolates. during the outbreak of zikv in americas, confirmed or probable zikv-infected cases has been continuously reported in southeast asia . our assay will be useful for virus detection and may contribute to preparedness for future outbreaks in these zikv endemic countries as well as in asia and africa. however, the evolution of zikv sequences must be constantly monitored to guarantee primer specificity. using samples obtained from subjects with suspected arbovirus infection, we did not find any zikv-positive samples in paraíba in march or july by rrt-pcr or our rt-lamp test. these samples were collected from patients within or weeks after the onset of arbovirus infection-like symptoms as part of an education and follow-up campaign for cardiovascular diseases. many samples might have been collected after the acute or early stage of infection. in addition, when this campaign was conducted, the prevalence of zikv infection may have been low, since most cases were reported from november to march , which is closely linked to the ecology of the vector aedes mosquito. the main advantages of this assay are its speed (positive results can be obtained within min) and the use of a battery-operated portable device. since the device has a user-friendly interface, training is not necessary to conduct the assay and interpret the results. recently, freeze-dried reagents for lamp assays have been made available, making cold-chain-free lamp assays a possibility. our assay is suitable for use in field surveillance or remote areas where it is difficult to implement laboratory diagnostic tests. the assay should be evaluated in a prospective study to confirm its utility for molecular diagnostic testing, especially under limited resources and by field laboratories in zikv endemic countries. in this paper, we successfully developed a rt-lamp assay for the detection of zikv by designing asian and african genotype-specific primers. the assay showed results consistent with those of the reference rrt-pcr assay in diagnostic tests with suspected cases of zikv infection. our results provide a potential new molecular diagnostic test for zikv and may serve as a basis for the development of alternative rapid diagnostic techniques to prepare for potential outbreaks. and were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % penicillin/streptomycin and % foetal bovine serum (fbs). zikv strain uganda was kindly provided by dr. shigeru tajima (national institute of infectious diseases; niid). the virus was propagated in vero cells grown in dmem supplemented with % fbs. two days after infection, culture supernatants were harvested, clarified by low-speed centrifugation, and then stored as virus stock at − °c until use. the infectious titre of the virus stock was determined by the % tissue culture infective dose (tcid ) using vero cells; titres are expressed as tcid / ml. viral rna was extracted from μl of infected culture supernatant using the qiaamp viral rna mini kit according to the manufacturer's protocol. the rna was eluted in μl of elution buffer and stored at − °c until use. viral rna from zikv strain prabc was kindly provided by dr. shigeru tajima (niid). viral rnas from other arboviruses, including denv serotype - , yfv, wnv, chikv, and rvfv, as well as genomic dna from p.falciparum strain d were kindly provided by dr. kouichi morita and dr. osamu kaneko (institute of tropical medicine, nagasaki university). preparation of rna standards. rna standards, consisting of partial genome sequences of zikv strains uganda and prvab c , were amplified by rt-pcr using for ward ( ′-ggagtcaggatggtacttgtacc- ′) and reverse ( ′-aaaattggatattcaggaacc- ′) primers with the primescriptii high fidelity one step rt-pcr kit (takara bio, shiga, japan). the reactions were performed using the takara pcr thermal cycler dice with the following program: °c for min, °c for min, followed by cycles of °c for s, °c for s, and °c for s. amplified pcr fragments were cloned into the pcr . vector using the topo-ta-cloning kit (invitrogen, carlsbad, ca, usa). the plasmids were digested with bamhi, purified from the agarose gel slice using a column purification kit (qiagen, hilden, germany), and used as templates for rna synthesis. the partial genomic rnas of each zikv strain were synthesised in vitro using t rna polymerase (promega, madison, wi, usa) and purified using the rneasy mini kit (qiagen). the rna concentration was determined by measuring the optical density at nm (od ) with scientific reports | : | doi: . /s - - - a nanodrop (thermo fisher scientific, waltham, ma, usa), and the rnas were diluted in depc-treated water to achieve the desired concentrations. primer design. lamp primers for zikv detection were designed based on the coding sequences for the e protein. the zikv sequences available in genbank were aligned using clustalx to identify conserved regions. a consensus sequence for a region in the e gene was used to design lamp primers using lamp designer (optigene; http://www.optigene.co.uk/lamp-designer/). primers specific for asian genotype viruses were designed first, and then african genotype-specific primers were designed by adapting each position to the african genotype consensus sequence. the rt-lamp assay required a set of six primers, two outer primers (f and b ), a forward inner primer (fip), a reverse inner primer (bip), a forward loop primer (lf), and a reverse loop primer (lb). the fip consisted of the f c sequence, which was complementary to the f and f sequences. the bip consisted of the b c sequence, which was complementary to the b and b sequences . the lb primer was designed to detect both asian and african genotype sequences. the sequences and locations of the oligonucleotide primers are shown in table . rt-lamp was performed with isothermal master mix reagent (optigene, west sussex, uk) using the genelyzer fiii real-time fluorescence detection platform (toshiba medical systems, otawara, japan). the reaction mixture (total volume, µl) contained µl of isothermal master mix; µl of warmstart rtx reverse transcriptase ( u; new england biolabs, ipswich, ma, usa); µl of the lamp primer mix consisting of pmol f and b , pmol fip and bip, pmol lf and lb; and µl of rna sample (template). the assay was carried out using a mixture of primers specific for the asian and african genotypes. all primers were cartridge-purified oligonucleotides purchased from hokkaido system science (sapporo, japan). the reaction was performed at °c for min, followed by a dissociation analysis at °c- °c. depc-treated distilled water and rna synthesised from uganda or prvabc were used for the negative and positive controls, respectively. nonspecific amplification was excluded by comparing the melting temperature to that of the positive control . real time rt-pcr. real time rt-pcr for zikv was performed using the quantitect probe rt-pcr kit (qiagen) as reported previously . the reaction mixture (total volume, µl) contained . µl of × quantitect probe rt-pcr master mix, . µl of quantitect rt mix, pmol each of primers and c, and pmol fam-labelled probe for zikv. then, aliquots of the rna samples ( µl) were added to the -µl reaction mixtures. each reaction was performed using the real-time pcr system (applied biosystems, tokyo, japan) with a thermal cycle profile consisting of °c for min, °c for min, followed by cycles of °c for s and °c for min. cut-off values were set at ct . . to quantify viral rna, a standard curve, generated with -fold serial dilutions of synthesised standard rna from uganda or prvabc , was used. table except zikv was quantified by droplet digital pcr (ddpcr). the complementary dna (cdna) of each arbovirus rna was synthesised from an extracted rna stock using the superscript iii first-strand synthesis system (invitrogen) with forward primer for rvfv and reverse primers for denv, wnv, yfv, and chikv, respectively (supplementary table ). the primers used for ddpcr were designed using primer (supplementary table ). all -μl ddpcr mixtures contained × evagreen ddpcr supermix (bio-rad, hercules, ca, usa), . μm forward and reverse primers, and μl of cdna. each oil compartment of the droplet generator dg cartridge (bio-rad) was filled with μl of droplet generation oil for evagreen (bio-rad), and approximately , droplets were generated in each well by the qx droplet generator (bio-rad). the reactions were performed in a -μl droplet emulsion using a geneamp pcr system (applied biosystems) under the following thermal cycling conditions: °c for min, followed by cycles of °c for s and °c for min, with a final step at °c for min. controls without the template were used to monitor for signals from contamination or primer-dimer formation. the cycled droplets were read individually using the qx droplet reader (bio-rad) and analysed with quantasoft droplet reader software (bio-rad). clinical specimens. peripheral blood and urine samples were obtained from patients between and year old with suspected arbovirus infection, who presented with fever, rash, and/or arthralgia symptoms. venous whole blood samples were collected in one vacuette ® z serum separator clot activator and two vacuette ® edta tubes (greiner bio-one, kremsmünster, austria). to one edta tube, rnalater (thermo fisher scientific) was added at half the volume of the collected blood samples to prevent rna degradation during transport. in total, plasma/serum and urine samples from patients with suspected arbovirus infection, including paired samples from cases, were used in this study. the separated plasma or serum samples and urine samples were stored at − °c until use. rnas were extracted from sera and urine using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions. rna samples were eluted with µl of elution buffer and stored at − °c until use. ethical declaration. this study was approved by the ccs-ufpe ethical committee (caae: . . . ) and all patients gave informed consent. whole blood and urine samples were collected as part of an education and follow-up campaign for arboviruses and cardiovascular diseases conducted by lika in the states of paraíba and pernambuco, brazil in february-july . all experiments were performed in accordance with relevant guidelines and regulations. zika virus: history of a newly emerging arbovirus zika virus outbreak, bahia, brazil zika virus associated with microcephaly possible association between zika virus infection and microcephaly -brazil association between zika virus infection and microcephaly in brazil congenital zika virus syndrome in brazil: a case series of the first livebirths with complete investigation probable transfusion-transmitted zika virus in brazil transmission of zika virus through sexual contact with travelers to areas of ongoing transmission -continental united states sexually acquired zika virus: a systematic review molecular evolution of zika virus during its emergence in the (th) century zika virus transmission from french polynesia to brazil zika virus in the americas: early epidemiological and genetic findings detection of zika virus in urine detection of zika virus in saliva comparison of test results for zika virus rna in urine, serum, and saliva specimens from persons with travel-associated zika virus disease -florida persistence of zika virus in body fluids -preliminary report zika virus testing considerations: lessons learned from the first real-time reverse transcription-pcr-positive cases diagnosed in new york state viral load kinetics of zika virus in plasma, urine and saliva in a couple returning from martinique, french west indies quantitative real-time pcr detection of zika virus and evaluation with field-caught mosquitoes genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia zika virus: diagnostics for an emerging pandemic threat prolonged detection of zika virus rna in urine samples during the ongoing zika virus epidemic in brazil rapid molecular detection of zika virus in acute-phase urine samples using the recombinase polymerase amplification assay first case of laboratory-confirmed zika virus infection imported into europe cross reactivity of commercial anti-dengue immunoassays in patients with acute zika virus infection loop-mediated isothermal amplification of dna development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus loop-mediated isothermal amplification (lamp): principle, features, and future prospects deployment of a reverse transcription loop-mediated isothermal amplification test for ebola virus surveillance in remote areas in guinea development and evaluation of reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay coupled with a portable device for rapid diagnosis of ebola virus disease in guinea complete genome sequences of three historically important, spatiotemporally distinct, and genetically divergent strains of zika virus: mr- , p - , and prvabc- rapid and sensitive detection of zika virus by reverse transcription loop-mediated isothermal amplification attomolar zika virus oligonucleotide detection based on loop-mediated isothermal amplification and ac susceptometry instrument-free point-of-care molecular detection of zika virus simple and highly sensitive molecular diagnosis of zika virus by lateral flow assays investigation into an outbreak of dengue-like illness in pernambuco, brazil, revealed a cocirculation of zika, chikungunya, and dengue virus type zika virus infections imported to italy: clinical, immunological and virological findings, and public health implications zika virus in asia pan american health organization and world health organization. zika -epidemiological update the authors would like to thank sayaka okada, shota koyano and olamide k. oloniniyi for technical assistance with the experiments at nagasaki university, renato p. melo neto and carlos henrique m. castelletti for bioinformatics support at lika, and all members of the staff for their hospitality during the visit when the main results of this paper were obtained. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -qdbcdn j authors: bassi, maria rosaria; sempere, raquel navarro; meyn, prashansa; polacek, charlotta; arias, armando title: extinction of zika virus and usutu virus by lethal mutagenesis reveals different patterns of sensitivity to three mutagenic drugs date: - - journal: antimicrob agents chemother doi: . /aac. - sha: doc_id: cord_uid: qdbcdn j flaviviruses constitute an increasing source of public health concern, with growing numbers of pathogens causing disease and geographic spread to temperate climates. despite a large body of evidence supporting mutagenesis as a conceivable antiviral strategy, there are currently no data on the sensitivity to increased mutagenesis for zika virus (zikv) and usutu virus (usuv), two emerging flaviviral threats. in this study, we demonstrate that both viruses are sensitive to three ribonucleosides, favipiravir, ribavirin, and -fluorouracil, that have shown mutagenic activity against other rna viruses while remaining unaffected by a mutagenic deoxyribonucleoside. serial cell culture passages of zikv in the presence of these compounds resulted in the rapid extinction of infectivity, suggesting elevated sensitivity to mutagenesis. usuv extinction was achieved when a -fold dilution was applied between every passage, but not in experiments involving undiluted virus, indicating an overall lower susceptibility than zikv. although the two viruses are inhibited by the same three drugs, zikv is relatively more susceptive to serial passage in the presence of purine analogues (favipiravir and ribavirin), while usuv replication is suppressed more efficiently by -fluorouracil. these differences in sensitivity typically correlate with the increases in the mutation frequencies observed in each nucleoside treatment. these results are relevant to the development of efficient therapies based on lethal mutagenesis and support the rational selection of different mutagenic nucleosides for each pathogen. we will discuss the implications of these results to the fidelity of flavivirus replication and the design of antiviral therapies based on lethal mutagenesis. h uman disease caused by flaviviruses represents a growing source of global health concern, with elevated numbers of deaths and cases of severe disease ( , ) . the incidence of flavivirus-related disease has increased during recent years. this is possibly related to multiple environmental and socioeconomic factors, such as long-distance spread of pathogenic flaviviruses (e.g., introduction to a different continent) and broader dissemination in temperate climate regions ( , , ) . despite having had limited relevance to public health prior to (only cases reported), recent large epidemics of zika virus (zikv) in asia and the americas have had a major socioeconomic impact. it is estimated that there have been over million cases of infection, leading to several thousand people suffering from severe disease ( , ) . in addition to severe neurological conditions, such as guillain-barré syndrome and congenital microcephaly, a wide range of disorders linked to the establishment of persistent infection in different tissues have been documented ( - ). without attracting the same level of attention as zikv, other emerging flaviviruses are affecting an increasing number of people. in particular, viruses infecting birds, such as west nile virus (wnv) and usutu virus (usuv), have been related to recent cases of neurologic disease in temperate countries ( ) ( ) ( ) ( ) . increased incidence of flaviviral disease seems to be connected to climate change and its impact on the migratory dynamics of birds and the geographic spread of mosquito vectors ( ) ( ) ( ) ( ) . usuv has caused recent epidemics in birds across europe, with elevated mortality in some species, such as blackbirds, owls, and other wild and captive animals ( , ) . recent sporadic cases of human disease geographically connected to these outbreaks are raising concerns of usuv becoming a potential threat to global health ( , , , , ( ) ( ) ( ) ( ) ( ) . lethal mutagenesis has long been proposed as a broad-spectrum strategy to control viral infections, with recent data supporting its feasibility and efficacy in vivo ( , ) . the rationale for antiviral therapies based on mutagenesis stems from theoretical studies by eigen and colleagues ( , ) . these investigations led to the proposal that the elevated error frequencies during rna virus replication are in the proximity of a maximum tolerated value for viability, namely, the error threshold ( ) ( ) ( ) . mutation rates beyond this value would be incompatible with maintaining meaningful genetic information, and thus virus propagation, leading to the extinction of the population in a process known as error catastrophe ( ) ( ) ( ) . the theory was empirically proven using mutagenic agents that induce increased mutation frequencies in viruses ( ) ( ) ( ) ( ) . a vast repertoire of molecules that exert broad-spectrum antiviral activities linked to viral mutagenesis have been identified, including nucleoside and nonnucleoside compounds ( , , ( ) ( ) ( ) ( ) ( ) ( ) . mutagenic nucleosides can be incorporated into newly synthesized viral rna genomes after their intracellular conversion into phosphorylated nucleoside analogues ( , ( ) ( ) ( ) ( ) ( ) ( ) . some of these compounds are currently used at the clinical level; e.g., ribavirin has been extensively used for the treatment of hepatitis c virus (hcv) infection, and favipiravir (also known as t- and commercialized as avigan), has been trialed against influenza virus and ebola virus disease ( , ( ) ( ) ( ) . several recent studies have indicated an association between mutagenesis and antiviral activity in vivo. we have demonstrated that the ribonucleoside favipiravir can cure persistent murine norovirus infection in the mouse intestine. this antiviral activity is accompanied by increased mutation frequency and decreased specific infectivity, both signatures of error catastrophe, in samples isolated preceding complete viral clearance ( ) . additional evidence of antiviral mutagenesis in vivo has been obtained from the analysis of hcvinfected patients treated with ribavirin ( ) . larger mutation frequencies accompanied by decreased specific infectivity were also observed in hantaan virus recovered from infected mice treated with ribavirin ( ) . several other studies have provided additional indirect proof of antiviral mutagenesis in vivo, further stimulating the development of therapies based on this strategy ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . several nucleoside analogues have demonstrated antiviral activities against a broad number of flaviviruses, which include zikv and wnv ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in particular, ribavirin and favipiravir efficiently inhibit zikv infection in different cell culture systems, including human neuronal progenitor cells ( ) . a correlation between the antiviral activity elicited by these molecules and larger mutation frequencies has been observed for some flaviviruses ( , , , ) . however, the possible mutagenic activity of these molecules on zikv and usuv has not been yet investigated. it also remains unclear whether two different although related pathogens can have different responses to the treatment with the same mutagenic compounds or whether they show distinct sensitivity to them. this information could be relevant in the design of broad-spectrum antiviral therapies against the flaviviruses based on lethal mutagenesis. here, we examine the antiviral activities displayed by three nucleoside analogues, all licensed for human use, in cell culture infection with zikv and usuv. we observe that ribavirin, favipiravir, and -fluorouracil are all inhibitors of both zikv and usuv, and that consecutive passage of virus in the presence of these drugs can lead to the complete extinction of infectivity. notably, the efficacies of these drugs vary depending on the virus. the molecules exhibiting better antiviral efficacies are typically associated with higher mutagenicity of the corresponding virus. however, the relative increases in mutation frequency observed for each drug treatment differ in usuv and zikv. we observed the highest mutation frequencies in zikv when treated with ribavirin and favipiravir, and in usuv when treated with -fluorouracil. the relevance of these results to a better understanding of flavivirus replication, genetic diversity, and the development of prospective antiviral therapies will be discussed. zika virus replication is suppressed by different ribonucleoside analogues. to investigate whether zikv replication could be affected by increased mutagenesis, we tested four different compounds known to be mutagenic for diverse viruses, -fluorouracil, ribavirin, favipiravir, and decitabine ( , , , ( ) ( ) ( ) . decitabine is an inhibitor of human immunodeficiency virus ( , ) . its antiviral activity has been associated with lethal mutagenesis, and it is possibly related to the incorporation of the phosphorylated deoxyribonucleoside derivative into viral cdna during reverse transcription ( , ) . we have included this analogue as a negative control for antiviral mutagenesis in our rna viral targets, as it is not predicted to be a substrate of rna polymerases. we first investigated the toxicities of these compounds on vero cells (fig. ) . only prolonged treatments with -fluorouracil ( h) led to cell death rates above %. the remaining drugs only showed modest or no effect on cellular viability even after prolonged exposures to the highest concentration tested ( m). treatment of infected cells with all the ribonucleosides, i.e., favipiravir, ribavirin, and -fluorouracil, led to significant inhibition of zikv replication (fig. ) . these molecules antimicrobial agents and chemotherapy exhibited similar antiviral activities when using an epidemic strain isolated in the americas (asian lineage; fig. a ) and an african isolate (fig. b ). as predicted, decitabine showed no effect on zikv replication, further supporting the idea that the antiviral activity elicited by the ribonucleosides is directly related to their incorporation by the viral rna polymerase (fig. c ). further analysis of zikv replication kinetics in the presence of different concentrations of each drug suggests that these molecules exhibit a similar inhibitory capacity. however, favipiravir seems to elicit a stronger inhibitory activity than ribavirin and -fluorouracil when higher concentrations are used ( fig. d to f). favipiravir, ribavirin, and -fluorouracil cause effective zikv extinction after serial passage in vero cells. we further analyzed the antiviral efficacies of these drugs against zikv during prolonged treatment by testing their capacity to abolish infectivity during consecutive viral passages in cell culture. sequential infections of zikv in the presence of these drugs resulted in a total loss of infectivity when a final concentration of m was used (except for decitabine; fig. ). the complete extinction of zikv infectivity was replicated in three independent lineages of passages in each drug ( fig. d and g). favipiravir eliminated zikv in a faster manner (undetectable viral levels reported after passages for all the three independent lineages) than ribavirin and -fluorouracil (all three lineages were extinct after passages). the extinction of zikv populations in these samples was confirmed by performing an additional blind passage in cell culture in the absence of mutagens (data not shown). we did not observe any detectable infectivity or viral rna in the samples recovered, confirming that these three nucleosides completely eliminate zikv infectivity. serial passage of zikv in cells treated with ribavirin or favipiravir at lower concentrations ( to m) also resulted in a gradual decrease in infectivity, with the two drugs presenting similar efficacies ( fig. a to c, e, and f). unlike favipiravir and ribavirin, zikv exhibited lower susceptibility to passages in the presence of -fluorouracil, suggesting that this molecule is a weaker inhibitor or mutagen for this virus. as anticipated, decitabine had no effect on infectivity, as the viral titers measured during serial transfers were similar to those found in passages of virus in the absence of drug ( fig. d and g). a typical signature of lethal mutagenesis is that viral populations isolated in passages preceding extinction show reduced specific infectivity ( , ) . to investigate whether zikv populations treated with these compounds manifested any alteration in their specific infectivity values, we determined the proportion of infectious particles relative to the total number of viral rna molecules in the sample. we confirmed that viral populations rescued after treatment with all three drugs exhibited lower specific infectivity than the untreated viruses, and most significantly those treated with favipiravir ( fig. ) . usuv shows a different sensitivity pattern to nucleoside analogues from that with zikv. to elucidate whether these drugs are also broadly effective against other flaviviruses, we analyzed their antiviral activity on vero cells infected with usuv. all three nucleosides (favipiravir, ribavirin, and -fluorouracil) that inhibited zikv replication also manifested antiviral activity on usuv (fig. ) . likewise, treatment with decitabine exhibited no effect on usuv replication ( fig. b and c). in contrast to what we had observed with zikv, -fluorouracil was the most effective compound against usuv (fig. ). single-cycle infection kinetics experiments with drugs at m revealed that -fluorouracil antiviral activity becomes more prominent at later replication time points (fig. a ). this is also observed when the same drugs are tested against usuv during multiple cycles of infection (infections at low multiplicity of infection [moi]). the . three independent lineages of passages were performed for each drug and concentration tested. serial passages were carried out with l of the cell culture supernatant recovered from the previous infection passage (corresponding to / of the total volume collected). the different graphs show the virus titers determined by tcid assay (a to d) and the genome copy equivalents obtained by quantitative pcr (qpcr) assays (e to g) that were found along serial passages of zikv. a black diamond represents zikv titers found in the supernatants of cultures treated with decitabine (dec); dark-gray squares illustrate -fluorouracil (fu)-treated series; light gray triangles, ribavirin (rbv); and white inverted triangles, favipiravir (fav). every value represents the average of virus titrations or viral genome copy equivalents (grna eq) from at least three biological replicas obtained from independent series of passages (Ϯ sem). in panel d, individual values obtained from each lineage are represented to better illustrate independent events of virus extinction. relative activity of -fluorouracil compared to those of ribavirin and favipiravir is substantially larger at h than at h ( fig. b and c) . this observation may be in agreement with a larger accumulation of mutations during larger number of replication cycles taking place during h. serial passage of usuv in the presence of each ribonucleoside drug led to sustained decreases in virus titers but not to complete viral extinction (fig. a) . virus lineages treated with ribonucleosides showed significantly lower titers ( to log on average) than during passages in decitabine or in the absence of drug. the lowest values were observed in the presence of -fluorouracil, further suggesting that this compound is most effective against usuv during serial passage in cell culture (fig. a) . the apparent lower susceptibility to nucleoside analogues in usuv than in zikv could be related to generally larger virus yields in infections with usuv. previous studies on unrelated foot-and-mouth disease virus (fmdv) suggested that the efficacy of lethal mutagenesis can be affected by the viral load; hence, extinction will be favored when the infection is initiated with a lower viral dose ( ) . thus, we decided to investigate whether passages at a lower infectious dose may also facilitate usuv extinction. we found that all three ribonucleoside analogues ( m) can reproducibly drive usuv to extinction in four independent replicas when a -fold dilution was applied before each transfer (fig. b ). viral sample dilution during sequential passages in the absence of drugs or in the presence of decitabine did not affect infectivity (virus titers in the range of to % tissue culture infective dose [tcid ] per ml; fig. b ). when drugs were used at a lower concentration ( m), the complete elimination of usuv was only observed in some sporadic cases. both ribavirin and -fluorouracil caused usuv extinction in two out of three lineages, while favipiravir never led to the complete loss of infectivity in any of three series analyzed (not shown). further analysis revealed that treatment with -fluorouracil resulted in significantly larger decreases in virus titers than passages in the presence of other drugs at m ( fig. c ; at passage , p Ͻ . for ribavirin versus -fluorouracil, and p Ͻ . for ribavirin versus favipiravir, -way analysis of variance [anova]). differences in sensitivities to nucleoside analogues in usuv and zikv are associated with alterations in their mutational patterns. to investigate whether the antiviral activities observed during treatment with favipiravir, ribavirin, and -fluorouracil are connected to their predicted mutagenic activity, we analyzed the mutation frequencies of both zikv and usuv rescued after passages in the presence of these compounds. since treatment at high concentrations ( and m) resulted in a rapid loss of zikv infectivity, we isolated and analyzed viral rna from the supernatant of infected cells after consecutive passages during exposure to drugs at m (fig. and ) . both favipiravir-and ribavirin-treated virus populations displayed significantly higher mutation frequencies (p Ͻ . ; mann-whitney test for the distribution of mutations per clone) than those untreated or isolated after treatment with -fluorouracil or decitabine. these results are in agreement with the relative antiviral efficacy of each nucleoside drug ( fig. and a ). the highest mutation frequencies were observed in the ribavirin-and favipiravir-treated zikv population ( -and -fold higher than in untreated virus, respectively). this positively links antiviral activity with mutagenesis, as the largest decreases in zikv titers observed during serial passages in the and e and a). in the -fluorouracil-treated population, we only found a modest ( -fold) increase in the mutation frequency (fig. a) , reflecting the milder antiviral behavior of this compound. treatment with decitabine did not significantly alter the mutation frequency (fig. a) , further suggesting that this compound is not affecting rna replication. similarly, we found that the largest mutation frequencies in usuv were observed in populations collected after serial passage in the presence of -fluorouracil (fig. . and b). viral populations recovered after passages in the presence of -fluorouracil ( m) showed -fold larger mutation frequencies than usuv passaged in the absence of drug. ribavirin and favipiravir led to modest increases in the mutational loads, also in agreement with their milder antiviral activities against usuv ( and -fold, respectively; fig. and b). further analysis revealed the expected transition biases typically found in viruses treated with the same drugs ( , , , , ( ) ( ) ( ) ( ) . thus, we observed slightly higher rates of g-to-a and c-to-u transitions in viruses treated with favipiravir, while the opposite changes (a-to-g and u-to-c) were identified in -fluorouracil-treated viruses (tables and ). the only exception to these typical transition mutational patterns was obtained in zikv populations treated with ribavirin (table ) , with higher frequencies in a-to-g and u-to-c nucleotide substitutions (when the [a-to-g ϩ u-to-c]/[g-to-a ϩ c-to-u] ratio is ). in contrast, usuv treated with ribavirin exhibited the expected mutational bias, with larger numbers of g-to-a and c-to-u transitions ( table ) . lethal mutagenesis has been posited as an alternative strategy to the current therapies based on classical antiviral drugs. several lines of evidence sustain that the antiviral properties of ribavirin and favipiravir in vivo can be, at least in part, connected to their mutagenic activity ( , , ) . these data encourage further study on the development of antiviral compounds with reduced toxicity, improved pharmacokinetics, and higher specificity for the viral polymerases ( , , ) . in this study, we have investigated the sensitivity to mutagenesis of two flaviviruses that have recently emerged as serious threats to public health, zikv and usuv. both pathogens were highly sensitive to the exposure of three mutagenic nucleosides, i.e., -fluorouracil, favipiravir, and ribavirin, although they exhibited different degrees of susceptibility to them. zikv was inhibited more efficiently by ribavirin and favipiravir, while usuv replication was affected to a greater extent by -fluorouracil. these inhibition profiles correlate with the relative increase in the mutation frequencies observed in populations rescued after treatment, supporting the idea that their antiviral efficacy is closely associated with their mutagenic activity. a possible explanation for the different sensitivities of usuv and zikv to mutagenic drugs is that the molecular determinants that regulate nucleotide recognition and discrimination in their respective polymerases vary. as for other rna viruses, flavivirus genome replication is an error-prone process catalyzed by the viral polymerase contained in the nonstructural protein (ns ) ( , ( ) ( ) ( ) ( ) ( ) . even though further analysis is needed to confirm this possibility, our data suggest that zikv is more prone to misincorporate purine analogues, such as ribavirin and favipiravir, while usuv shows a preference for pyrimidine substrates, like -fluorouracil. if certain, this information could be instrumental in the rational screening of nucleoside analogues to effectively control each flavivirus. many recent studies have contributed to the better understanding of the molecular biology of zikv replication, including the structural and biochemical characterization of the viral polymerase ns ( ) ( ) ( ) ( ) ( ) . however, there is limited knowledge on usuv replication, with no molecular data on its viral polymerase. future studies are thus needed to elucidate the molecular determinants responsible for the different sensitivities of these flaviviruses to mutagenic nucleosides. additional evidence hinting at molecular differences in zikv and usuv polymerase fidelity can be inferred from the mutation patterns found in viral populations after treatment with ribavirin (tables and ) . we observed the typical dominance of g-to-a and c-to-u transitions in usuv and an unexpected prevalence of the opposite transition types in zikv (the [g-to-a ϩ c-to-u]/[a-to-g ϩ u-to-c] ratios were and . for usuv and zikv, respectively). it has been suggested that the larger proportion of g-to-a and c-to-u transitions in viruses treated with ribavirin (included usuv) is possibly connected to the additive effect of mutagenesis by incorporation of ribavirintriphosphate into viral rna and depleted intracellular gtp pools as a consequence of inosine- =-monophosphate dehydrogenase (impdh) inhibition by ribavirin ( , , ) . although ribavirin-triphosphate is efficiently incorporated during viral rna synthesis opposite to u and c, in such a low intracellular gtp concentration scenario, it may be preferentially incorporated opposite to c, hence leading to those biases ( ) . the dominance of a-to-g and u-to-c mutations in ribavirin-treated zikv populations suggests that its polymerase may be favoring a different base pairing behavior of ribavirin than in other viruses with independence on intracellular nucleotide pools. other plausible scenarios may include differences in host cell rearrangements that indirectly affect virus mutability by the same drugs (e.g., alterations in the expression of cellular proteins associated with nucleotide uptake and its metabolism). further investigations are needed to clarify the mechanism underlying these differences. we deem that this study can stimulate an additional investigation on the therapeutic value of mutagenic drugs against flaviviruses and, in particular, for the treatment of persistent flaviviral disease ( , ( ) ( ) ( ) . mutagenic drugs seem to be especially effective against persistent infections, with major evidences of lethal mutagenesis obtained during treatment of chronically infected hosts, i.e., hcv-infected patients treated with ribavirin, and mice persistently infected with norovirus and treated with favipiravir ( , ) . a tentative explanation for the better efficacy of mutagenic drugs in this context may be linked to potentially larger accumulation of mutations during longer periods of exposure to drugs ( , ) . a vast range of flavivirus-associated disorders have been connected with persistent infection, including zikv ( , ( ) ( ) ( ) ) . zikv infects a broad range of cell types and tissues, including the reproductive tract and the central nervous system ( , , ) . persistent replication in these tissues has been connected to diverse severe conditions, such as end-organ disease, platelet-related illness, and potentially blinding uveitis ( ) . persistent zikv in the reproductive tissue is possibly responsible for a growing number of cases of sexually transmitted disease, with some studies predicting that this may become a major route of infection in the future ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . vaginal mucosa infection has also been related to fetal infection, further highlighting the impact of zikv persistence in the genital tract ( , ) . it is therefore tempting to investigate the potential therapeutic value of lethal mutagenesis in the treatment of persistent infection in the reproductive tract mucosa. it remains unclear whether ribavirin and favipiravir will elicit efficient antiviral mutagenesis in the central nervous system (cns), with recent data showing different efficacies of these drugs in neuron-derived cell culture systems. while some studies demonstrate that zikv replication remains unaffected by these drugs in stem cellderived neurons, other investigations have proven their inhibitory activity during the treatment of neural progenitor cell lines ( , ) . the different efficacies of these molecules could be linked to possible divergences in the cellular uptake or metabolism of nucleoside drugs in these cell lines. thus, for the control of neurotropic disease, it would be desirable to identify mutagenic compounds with improved pharmacological properties in the cns. alternatively, a combination of mutagenic drugs that are effective in controlling the infection outside the cns (favipiravir, ribavirin, and -fluorouracil), together with effective inhibitors in the neuronal tissue ( , ( ) ( ) ( ) , could lead to improved approaches to control zikv hidden in different body compartments. our study also encourages the investigation of -fluorouracil as a therapeutic drug for the control of usuv infection in an eventual epidemic spillover to humans. although severe disease in humans is rare, there is growing evidence that cases of neurologic disorders associated with usuv have been historically misdiagnosed as wnv ( , ) . the diagnosis of disease caused by usuv is further complicated by the resemblance of pathology to wnv cases and the serological cross-reactivity in diagnostic tests ( , , , - , , ) . a mouse model for usuv infection has been recently established, and it represents a valuable tool to test the in vivo antiviral effect of -fluorouracil or novel antivirals ( , ) . these studies can be extended to the potential treatment of severe disease caused by closely related flaviviruses, such as wnv and japanese encephalitis virus, for which antiviral therapies are not available. cells, viruses, and protocols for infections. we used two different zikv strains purchased from the american type cell culture (atcc). the majority of the experiments described in this paper were performed with an isolate from the asian lineage, collected during the recent epidemics in the americas (strain prvabc , puerto rico, , atcc reference number vr- ). in addition, for some experiments indicated here, we used the first zikv strain ever isolated as our reference african lineage virus (uganda, , strain mr , atcc reference number vr- ). the usuv strain ( / ) used in this study was initially isolated from infected birds in austria in and was kindly provided by giovanni savini, istituto g. caporale, italy ( ). we used african green monkey kidney epithelial cells (kindly provided by sylvie lecollinet, anses, france), namely, vero cells, for zikv and usuv propagation, titration, and viral infections in the presence or absence of mutagenic compounds. viral infections were performed as follows: on the day preceding virus inoculation, we seeded -well plates with ϫ cells per well in the presence of ml of complete medium containing % (vol/vol) fetal bovine serum (fbs; sigma), units/ml penicillin-streptomycin (thermo fisher), and mm hepes in high-glucose dulbecco's modified eagle medium (dmem; thermo fisher). cells were incubated overnight at °c with a % co concentration. on the following day, the supernatant was removed from each plate and replaced with l of fresh medium containing % fbs. then, l of virus sample (zikv or usuv) was applied to the cell monolayer at the multiplicity of infection (moi) indicated, and virus adsorption was allowed for h at °c with % co . the inoculum was removed and the cells washed with complete medium to eliminate unattached virus. cells were then covered in ml of medium containing % fbs, and supernatants from infected cultures collected at different time points. virus titration. virus samples were titrated by % tissue culture infectious dose (tcid ) assays. to this aim, vero cells in l were seeded in -well plates in the presence of medium containing % fbs. on the following day, l of -fold serial dilutions of each sample was applied to each well, reaching a final volume of l. the virus titers were determined by scoring the number of infected wells showing apparent cytopathic effect at to days postinfection, and using the spearman & kärber algorithm ( ) . cell culture infections in the presence of antiviral compounds. for infections in the presence of drugs, cell culture supernatants were removed, and l of % fbs complete medium containing to m decitabine ( -aza- =-deoxycytidine; selleckchem), -fluorouracil ( , -dihydroxy- -fluoropyrimidine; sigma-aldrich), ribavirin [ -(␤-d-ribofuranosyl)- h- , , -triazole- -carboxamide; sigma-aldrich], or favipiravir ( -fluoro- -hydroxy- -pyrazinecarboxamide; atomax) was added to each well. cells were then inoculated with l of virus at the moi indicated for each experiment in the corresponding section and incubated for h at °c and % co . supernatants were removed, cells were washed to eliminate unattached virus, and ml of fresh medium ( % fbs) containing each drug at the desired concentration was added. cell culture supernatants were collected at to h postinfection for subsequent analyses. for experiments involving serial passage of viruses in the presence of nucleoside analogues, the first infection with zikv or usuv was carried out at an moi of . tcid /cell. in sequential passages, l of neat virus from the supernatant of the previous passage (which represents / of the total volume collected) was applied to a new monolayer of cells. for experiments involving transfers of diluted usuv, we used in each transfer l of a preparation containing a -fold dilution of the viral sample collected in the previous passage, as described in previous work ( ) . viral rna extraction, reverse transcription-pcr amplification, and mutation frequency analysis. viral rna was extracted from l of viral sample supernatants using the genejet rna purification kit (thermo fisher). for the calculation of mutation frequency values, we followed previously described protocols ( , , ) . briefly, l of purified rna was reverse transcribed in l final volume using superscript iii (roche), as indicated by the manufacturer. three microliters of cdna was then pcr amplified using high-fidelity kod polymerase (toyobo). to this aim, we used primers spanning genomic positions to and to in usuv and to and to in zikv. pcr products were gel purified using the purelink quick gel extraction kit (invitrogen) and then ligated into plasmid pcr blunt using the zero blunt pcr cloning kit (thermo fisher). positive e. coli colonies were identified by pcr screening with primers flanking the vector-cloning site and dreamtaq dna polymerase (thermo fisher). the resulting pcr products, corresponding to individual zikv or usuv cdna clones, were sanger sequenced and the mutation frequency in each population calculated. quantitative pcr analysis of virus populations. the number of zikv genomic rna molecules in different biological samples was quantified using primers and a fam-tamra ( -carboxyfluorescein- carboxytetramethylrhodamine) probe targeting the zikv e gene (positions to ), following protocols previously described ( ) . for the amplification protocol, we used taqman fast virus -step master mix (thermo fisher) and one-step reaction reverse transcription-pcr (rt-pcr) amplification conditions, with a reverse transcription step ( min at °c), followed by min of incubation at °c and amplification cycles of s at °c and min at °c. for the quantification of usuv rna, we used a fam-tamra probe targeting the ns gene (positions to ) and primers previously described ( ) . the same rt-pcr cycle amplification protocol described above for the detection of zikv was used for usuv. statistical analysis. statistical significance was assessed using graphpad prism , as specified in the figure legends. for the statistical analysis of mutation frequencies, we employed a mann-whitney test that compares the ranked scores of the number of mutations found in individual clones grouped by population, as previously described ( ) . we are indebted to lars larsen (dtu vet) and his group for support in establishing our laboratory. we especially value hue thi thanh tran for helping with multiple technical challenges during our study. thomas bruun rasmussen (dtu vet) provided us with expertise in establishing qpcr methods for the detection of usuv and with constructive feedback on our research. we also thank antonio mas (uclm) and marta sanz-ramos for their critical reading and suggestions on the manuscript. this work is supported by funding procured by independent research fund denmark (technology and production sciences, dff-ftp) to a.a., application number - a. the funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. epidemiology of dengue: past, present and future prospects the global threat of zika virus to pregnancy: epidemiology, clinical perspectives, mechanisms, and impact emerging and reemerging arboviral diseases in southeast asia potential for zika virus introduction and transmission in resource-limited countries in africa and the asia-pacific region: a modelling study zika virus zika virus in the female genital tract persistence of zika virus in body fluids-preliminary report zika virus pathogenesis and tissue tropism vaginal exposure to zika virus during pregnancy leads to fetal brain infection evidence of sexual transmission of zika virus -raising awareness for diagnostic challenges initial and long-term costs of patients hospitalized with west nile virus disease usutu virus infections in humans: a retrospective analysis in the municipality of modena west nile virus: an historical overview widespread activity of multiple lineages of usutu virus, western europe a review of the epidemiological and clinical aspects of west nile virus usutu virus: an emerging flavivirus in europe is usutu virus ready for prime time? epizootic emergence of usutu virus in wild and captive birds in germany the next zika widespread usutu virus outbreak in birds in the netherlands blood donor screening for west nile virus (wnv) revealed acute usutu virus (usuv) infection first human case of usutu virus neuroinvasive infection first cases of human usutu virus neuroinvasive infection in croatia favipiravir elicits antiviral mutagenesis during virus replication in vivo the murine model for hantaan virusinduced lethal disease shows two distinct paths in viral evolutionary trajectory with and without ribavirin treatment the error threshold error catastrophe and antiviral strategy viruses at the edge of adaptation antiviral strategies based on lethal mutagenesis and error threshold molecular indetermination in the transition to error catastrophe: systematic elimination of lymphocytic choriomeningitis virus through mutagenesis does not correlate linearly with large increases in mutant spectrum complexity the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity lethal mutagenesis of hiv with mutagenic nucleoside analogs t- (favipiravir) induces lethal mutagenesis in influenza a h n viruses in vitro fidelity variants of rna dependent rna polymerases uncover an indirect, mutagenic activity of amiloride compounds effective lethal mutagenesis of influenza virus by three nucleoside analogs -fluorouracil in lethal mutagenesis of foot-and-mouth disease virus characterization of permeability, stability and anti-hiv- activity of decitabine and gemcitabine divalerate prodrugs kp- / , a nucleoside designed for the treatment of hiv by viral mutagenesis the ambiguous base-pairing and high substrate efficiency of t- (favipiravir) ribofuranosyl =-triphosphate towards influenza a virus polymerase biochemical evaluation of the inhibition properties of favipiravir and =-c-methyl-cytidine triphosphates against human and mouse norovirus rna polymerases molecular characterization of a dual inhibitory and mutagenic activity of -fluorouridine triphosphate on viral rna synthesis. implications for lethal mutagenesis determinants of rna-dependent rna polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-andmouth disease virus mutant with reduced sensitivity to ribavirin favipiravir (t- ), a novel viral rna polymerase inhibitor inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate ribavirin-current status of a broad spectrum antiviral agent mechanisms of action of ribavirin against distinct viruses experimental treatment with favipiravir for ebola virus disease (the jiki trial): a historically controlled, single-arm proof-of-concept trial in guinea deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis c virus genotype -infected patients lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis virus (lcmv) synergistic combinations of favipiravir and oseltamivir against wild-type pandemic and oseltamivir-resistant influenza a virus infections in mice broad spectrum antiviral activity of favipiravir (t- ): protection from highly lethal inhalational rift valley fever evaluation of antiviral efficacy of ribavirin, arbidol, and t- (favipiravir) in a mouse model for crimean-congo hemorrhagic fever successful treatment of advanced ebola virus infection with t- (favipiravir) in a small animal model vitro and in vivo activities of t- against arenavirus and bunyavirus infections mutagenesismediated decrease of pathogenicity as a feature of the mutant spectrum of a viral population chronic norovirus infection and common variable immunodeficiency favipiravir and ribavirin inhibit replication of asian and african strains of zika virus in different cell models replication of the zika virus in different ipsc-derived neuronal cells and implications to assess efficacy of antivirals zika virus replication is substantially inhibited by novel favipiravir and interferon alpha combination regimens mutagen resistance and mutation restriction of st. louis encephalitis virus error-prone replication of west nile virus caused by ribavirin sequence-specific fidelity alterations associated with west nile virus attenuation in mosquitoes extinction of west nile virus by favipiravir through lethal mutagenesis fitness peaks of dengue virus populations lack of mutational hot spots during decitabine-mediated hiv- mutagenesis preextinction viral rna can interfere with infectivity foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics lethal mutagenesis of hepatitis c virus induced by favipiravir favipiravir can evoke lethal mutagenesis and extinction of foot-and-mouth disease virus role of the mutant spectrum in adaptation and replication of west nile virus structure and function of the zika virus full-length ns protein the structure of zika virus ns reveals a conserved domain conformation the crystal structure of zika virus ns reveals conserved drug targets crystal structure of zika virus ns rna-dependent rna polymerase analysis of ribonucleotide =-triphosphate analogs as potential inhibitors of zika virus rnadependent rna polymerase using non-radioactive polymerase assays curing of foot-and-mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis mode of action of ribavirin: effect of nucleotide pool alterations on influenza virus ribonucleoprotein synthesis persistence of west nile virus zika virus persistence in the central nervous system and lymph nodes of rhesus monkeys the role of viral persistence in flavivirus biology mutagenesis-induced, large fitness variations with an invariant arenavirus consensus genomic nucleotide sequence zika virus infection of rhesus macaques leads to viral persistence in multiple tissues late sexual transmission of zika virus related to persistence in the semen sexually acquired zika virus: a systematic review update: interim guidance for prevention of sexual transmission of zika virus-united states persistent detection of zika virus rna in semen for six months after symptom onset in a traveller returning from haiti to italy, february mosquito-borne and sexual transmission of zika virus: recent developments and future directions prevention and control of zika as a mosquito-borne and sexually transmitted disease: a mathematical modeling analysis the risk of sustained sexual transmission of zika is underestimated zika virus infects human placental macrophages efficacy of the broad-spectrum antiviral compound bcx against zika virus in cell culture and in a mouse model the viral polymerase inhibitor -deaza- =-c-methyladenosine is a potent inhibitor of in vitro zika virus replication and delays disease progression in a robust mouse infection model the race to find antivirals for zika virus first evidence of simultaneous occurrence of west nile virus and usutu virus neuroinvasive disease in humans in croatia during the outbreak limited susceptibility of mice to usutu virus (usuv) infection and induction of flavivirus cross-protective immunity a recombinant dna vaccine protects mice deficient in the alpha/beta interferon receptor against lethal challenge with usutu virus emergence of usutu virus, an african mosquito-borne flavivirus of the japanese encephalitis virus group virus isolation and quantitation isolation of fidelity variants of rna viruses and characterization of virus mutation frequency norovirus polymerase fidelity contributes to viral transmission in vivo a rapid and specific real-time rt-pcr assay to identify usutu virus in human plasma, serum, and cerebrospinal fluid coxsackievirus b mutator strains are attenuated in vivo key: cord- -r mskri authors: magnani, diogo m.; silveira, cassia g. t.; rosen, brandon c.; ricciardi, michael j.; pedreño-lopez, núria; gutman, martin j.; bailey, varian k.; maxwell, helen s.; domingues, aline; gonzalez-nieto, lucas; avelino-silva, vivian i.; trindade, mateus; nogueira, juliana; oliveira, consuelo s.; maestri, alvino; felix, alvina clara; levi, josé eduardo; nogueira, mauricio l.; martins, mauricio a.; martinez-navio, josé m.; fuchs, sebastian p.; whitehead, stephen s.; burton, dennis r.; desrosiers, ronald c.; kallas, esper g.; watkins, david i. title: a human inferred germline antibody binds to an immunodominant epitope and neutralizes zika virus date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: r mskri the isolation of neutralizing monoclonal antibodies (nmabs) against the zika virus (zikv) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. here we describe the characterization of human mabs from the plasmablasts of an acutely infected patient. one of the mabs had the unusual feature of binding to and neutralizing zikv despite not appearing to have been diversified by affinity maturation. this mab neutralized zikv (neut( ) ~ μg/ml) but did not react with any of the four dengue virus serotypes. except for the expected junctional diversity created by the joining of the v-(d)-j genes, there was no deviation from immunoglobulin germline genes. this is a rare example of a human mab with neutralizing activity in the absence of detectable somatic hypermutation. importantly, binding of this mab to zikv was specifically inhibited by human plasma from zikv-exposed individuals, suggesting that it may be of value in a diagnostic setting. zika virus (zikv) belongs to the genus flavivirus of the flaviviridae family and is related to dengue virus (denv), yellow fever virus (yfv), japanese encephalitis virus (jev), and west nile virus (wnv) [ ] . the globally distributed mosquito species of the aedes genus, vectors for many flavivirus, can also transmit zikv [ , ] . however, zikv remained a relatively minor and obscure cause of human disease for most of the second half of the th century and was featured in a limited number of scientific reports. in fact, it was not until that autochthonous human infection was described outside africa and continental asia-in the federated states of micronesia [ ] [ ] [ ] . since then, reports from brazil have chronicled a rapidly spreading epidemic that co-exists with denv and chikungunya virus (chikv). the epidemic has spread north with mosquito-borne transmission being reported in many nations of the americas as far north as mexico and southern florida [ ] [ ] [ ] . more ominously, zikv has been implicated as the causative agent in fetal developmental problems [ , ] . there are reports of zikv-associated birth defects, primarily brain abnormalities and microcephaly in infants born to mothers infected with zikv [ ] . virus has been recovered from amniotic fluid, placental, and brain tissues [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . zikv infection has been classified as an ongoing threat by the world health organization. in the united states, the centers for disease control and prevention has issued guidance for the management of the infection in the general population, pregnant women, and infants [ ] [ ] [ ] . due to recent reports of sexually transmitted zikv infection, the cdc has also developed guidelines for prevention of this mode of transmission [ ] [ ] [ ] [ ] [ ] [ ] . more recently, zikv transmission has also been described in miami, florida [ ] , suggesting that autochthonous spread could occur in any region of the u.s. inhabited by aedes spp. treatment of a variety of human ailments using mabs is revolutionizing our ability to ameliorate human suffering. for infectious disease, the ebola epidemic highlighted the potential utility of a cocktail of three neutralizing (n)mabs that block infection by the ebola virus [ ] . most convincingly, the administration of a single nmab up to five days post infectious virus exposure prevents the development of disease in ebola-infected macaques [ ] . because mabs can be engineered to prevent antibody-dependent enhancement by incorporating the l a and l a (lala) mutations which reduce fcγr binding [ ] , they are a promising intervention in flaviviral therapies. our long-term goal is to use a cocktail of lala-mutated nmabs to prevent zikv infection in at-risk individuals, primarily pregnant women. therapeutic nmabs must be potent in order to be clinically viable, and most nmab isolation strategies are based on the identification of high-titer, antigen-selected repertoires. somatic hypermutation (shm) in germinal center (gc) b cells provides the basis for selection of b cells producing abs with increased affinity-a hallmark of the adaptive humoral response. this feature is conserved among mammals, highlighting the importance of ab affinity enhancement for evolutionary fitness [ ] . thus, it is unsurprising that the vast majority of human abs in the memory immunoglobulin (ig)g pool have undergone affinity maturation and have, on average, - nucleotide substitutions from precursor genes [ ] . the contribution of shm to ab-mediated viral neutralization is particularly clear for the chronicallyinduced broadly neutralizing antibodies to hiv [ ] [ ] [ ] [ ] . reversion of these anti-hiv nmabs to precursor germline antibodies results in a drastic reduction or complete loss of viral neutralization [ ] [ ] [ ] [ ] . although mutated mabs are found after secondary denv infection, the role of these mutations in acute virus-neutralization and clearance is less clear [ ] [ ] [ ] [ ] . still, the prevalent thought is that antiviral ab response involves the engagement of poor-or non-neutralizing germline clones generated by v(d)j rearrangement, followed by shm-mediated refinement in germinal centers to enhance neutralization potency. here we describe the isolation of plasmablast-derived human mabs, sorted days post onset of symptoms from a zikv-patient in são paulo, brazil. the patient reported a previous history of dengue infection and yellow fever vaccination (table ) . a few of the isolated abs neutralized zikv, most of them at relatively high concentrations. interestingly, one of these mabs (p f ) exhibited no nucleotide mutations when compared to its corresponding germline sequences, but still recognized a zikv immunodominant epitope and neutralized the virus. these results suggest unforeseen roles for gc-independent responses against zikv and possibly other viruses. blood samples were collected from volunteer , a -year-old woman who reported a pruriginous skin rash that started six days prior to the beginning of acute neurological deficits suggestive of gbs. zikv infection was confirmed by a positive real-time reverse-transcriptase pcr assay for zikv rna in urine samples collected at days and after the onset of the first rash symptoms. blood and cerebrospinal fluid were negative for zikv rna. previous history of a single dengue infection and yellow fever immunization were also reported. peripheral blood mononuclear cells (pbmcs) were obtained from blood samples collected days post onset of symptoms. blood samples from patient were obtained after signing a written consent form approved by the university of são paulo's institutional review board (cappesq / ). anonymized plasma samples from volunteers in brazil and us were obtained from naïve and convalescent subjects with rt-pcr-confirmed zikv or denv infection (s table) . four volunteers donated samples post yellow fever vaccination. we conducted reverse transcription followed by a nested pcr to amplify the variable region of the immunoglobulin (ig) chains using described protocols with minor modifications [ ] . briefly, cdna was synthesized in a μl reaction using the original sort plates. each reaction contained μl of ng random hexamer (idt), μl of mm dntp (life technologies), μl of superscript iii reverse transcriptase (life technologies), μl molecular biology grade water, and μl of single sorted cell sample in lysis buffer (described above). the reverse transcription reaction was performed at ˚c for min, ˚c for min, ˚c for min, c for min. after the reaction was completed, cdna was stored at - ˚c. heavy and light chains were amplified in three different nested pcr reactions, using a mix of ' v-specific primers with matching ' primers to the constant regions of igg, igl, and igk. pcr reactions were conducted using hotstartaq plus dna polymerase (qiagen). the second set of pcr reactions was carried out with primers redesigned to incorporate restriction sites compatible with subcloning into rhesus igg expression vectors, instead of the original human vectors [ ] . we sequenced the amplified and cloned products using primers complementary to the ig constant regions. sequences were analyzed using igblast and imgt/v-quest to identify v (d) j gene rearrangements, as well as shm levels [ , ] . we expressed mabs in expi f (thermofisher) human cell lines. the plasmids encoding heavy and light chains were co-transfected using the expifectamine transfection kit (a , thermofisher). after - days, we harvested the secreted mab in the supernatant. ig concentration in the supernatant was determined by an anti-rhesus igg elisa, before we proceeded with the functional assays. for the experiments with purified mabs, we used protein a plus (pierce)-containing columns to remove the impurities. the concentration of purified protein was determined by measurement of absorbance at nm (nanodrop, thermo scientific). virus capture assay and recombinant e protein elisa p f binding was determined by both virus capture assay (vca) and recombinant (r)e elisas. the vca plates were coated overnight with the mouse-anti-flavivirus monoclonal antibody g (clone d - g - - , emd millipore) followed by incubation with viral stocks (zikv or denv). the re elisa plates were coated with zikv e protein (mybiosource, mbs ) diluted to μg / ml in pbs. after the coating step, the plates were washed with pbs and mab samples diluted to μg / ml were added to designated wells and incubated for h at ˚c. subsequently, the plates were washed and detection was carried out using a goat anti-human igg hrp secondary ab (southern biotech), which was added to all wells at a dilution of : , . following a h incubation at c, the plates were washed and developed with tmb substrate at room temperature for - min. the plates were developed with tmb substrate at room temperature for - min. the reaction was stopped with tmb solution and absorbance was read at nm. the neutralizing potency of the mabs was measured using a flow cytometry-based assay [ , ] . in brief, recombinant mabs (transfection supernatant or purified) were diluted and preincubated with zikv (paraiba) or the reference denv serotypes in a final volume of μl for h at ˚c. the virus and mab mixture ( μl) was added onto wells of a -well plate of % confluent vero cell monolayers in duplicate. a new seed of vero cells (ccl- tm) was obtained from the american type culture collection (atcc) repository for this study. the inoculum was incubated in a ˚c incubator at % co for one hour with agitation of the plates every min. after one hour, the virus and mab-containing supernatants were aspirated and the wells were washed with media. fresh media was then added and the plates were incubated for a total of hours. cells were trypsinized with . % trypsin (life technologies), fixed (bd cytofix), and permeabilized (bd cytoperm). viral infection was detected with the g antibody (millipore) recognizing zikv or denv, followed by staining with an anti-mouse igg a apc fluorophore-conjugated secondary reagent (biolegend). the concentration to achieve half-maximal neutralization (neut ) was calculated using a nonlinear regression analysis with prnts were conducted as previously described [ ] . briefly, purified p f was serially diluted in optimem supplemented with % human serum albumin (vwr), % fetal bovine serum, and gentamicin. zikv paraiba was diluted to a final concentration of~ - pfu / ml in the same diluent added to equal volumes of the diluted ab. the virus/mab mixture was incubated at ˚c for min. cell culture medium was removed from % confluent monolayer cultures of vero cells on -well plates and μl of the virus/ab mixture was transferred onto duplicate cell monolayers. cell monolayers were incubated for min at ˚c and overlaid with % methylcellulose in optimem supplemented with % fbs mm glutamine + μg / ml gentamicin. samples were incubated at ˚c for four days after which plaques were visualized by immunoperoxidase staining, and a % plaque-reduction neutralization titer was calculated. inhibition of p f mab binding was determined by elisa. to begin, the elisa plate was coated with mouse anti-flavivirus monoclonal antibody g (emd millipore) diluted : , in carbonate binding buffer and incubated overnight at ˚c. the next day, the plate was washed five times with pbs-tween and wells were blocked with % skim milk in pbs for h at ˚c. after the block step, the plate was washed and virus samples were added to designated wells for h incubation at room temperature. subsequently, the plate was washed with pbs only and corresponding blocking plasma samples were added for h at ˚c. following the plasma block, the plate was washed and p f was added to corresponding wells for h at c. p f was detected using a rhesus igg-specific antibody (mouse anti-monkey igg-hrp clone sb a; southern biotech). thereafter, the plate was washed and wells were developed with tmb substrate at room temperature for - min before the reaction was stopped with tmb stop solution. absorbance was determined at nm. we isolated plasmablasts from patient who presented with suspected guillain-barré syndrome (gbs) ( table ) (first day of symptoms = d ). the patient had a previous history of dengue infection and yellow fever vaccination ( table ). the previously healthy -year-old woman presented to the emergency room (d ) reporting a progressive paresthesia mainly in the extremities of her hands, along with acute, intermittent pain in her left forearm during the previous four days. at physical examination, the patient presented with a grade iv asymmetric muscular weakness and hypoesthesia in her left limbs, with abolished deep tendon reflexes in the lower limbs. a mild weakness of her left facial muscles was also noted. the patient reported no respiratory disorders and no hoarseness, and no signs of dysautonomia were detected at the clinical evaluation. fever, conjunctivitis, and myalgia or joint pain were absent during the illness. afterwards, the patient was hospitalized with a clinical diagnosis of gbs, for which an intravenous human ig (ivig) treatment was initiated at a dosage of . g / kg / day for days. cerebrospinal fluid analysis and an electroneuromyogram were performed on fourth (d ) and fifth (d ) days after neurological symptom onset, respectively; the results were within normal limits. the electroneuromyogram was repeated on the th day of neurological symptoms, but no significant abnormalities were noted despite the persisting weakness in the patient's left leg and arm. during the treatment with ivig, the patient presented with transient worsening of her hemiparesis, but progressively recovered over the course of weeks after discharge from the hospital. at days post-neurological symptom onset (d ), a physical exam revealed significant improvement of muscular strength and abolished deep tendon reflexes in the lower limbs. the remittent skin rash cleared completely days after its initial emergence. blood, cerebrospinal fluid and urine samples were collected on the th day of neurological symptoms (d ) for detection of zikv by rt-pcr. the urine sample was zikv-positive by pcr, while blood and cerebrospinal fluid were negative. a saliva sample collected on d was negative for zikv. we isolated plasmablasts from peripheral blood mononuclear cells (pbmcs) collected on day (table ) . from wells containing single-sorted cells, we amplified, cloned, and sequenced heavy and light ab chains using ' primers complementary to the v gene segments and a ' primer annealing to the constant igg region [ ] . this resulted in paired heavy and light chains (s table) . eight of the mabs bound to zikv (fig ) . seven of these mabs exhibited cross-reactivity to one or more of the denv serotypes, and a single mab-p f -bound exclusively to zikv. interestingly, two mabs bound to denv but not zikv. we tested the neutralization potency of the zikv-specific p f mab in a flow-based neutralization assay and a plaque reduction neutralization test (prnt) and found that it neutralized zikv at approximately μg / ml (prnt ) (fig ) . analysis of the isolated antibody variable (v) domain sequences revealed five mabs with average gene mutation levels ( - nucleotide modifications), two mabs with over nucleotide substitutions, and mabs with unusually low levels of shm for isotype-switched mabs (lower than changes) (s table) . the most highly mutated mabs (p b and p c ) were not zikv-specific by binding (s table) . in fact, the eight zikv-binding mabs had the lowest shm levels, including four mabs lacking clearly recognizable mutations when compared with putative heavy and light chain germline precursors (s table, fig ) . except for junctional diversity, the zikv-neutralizer p f mab heavy chain did not exhibit signs of antigenselected ig diversification. p f had an identical sequence to the ig heavy chain variable (ighv) genes segment ighv - à up to the amino acid c , prior to the cdr-h (international immunogenetics information system [imgt]) [ ] . however, position g -the site of the junction between ighv and the igh diversity (ighd) genes-differed from the germline reference. interestingly, this region is part of a segment (n ) with non-germline nucleotides corresponding to six amino acids identified between the ighv and ighd genes (fig c) . this segment is likely the result of n nucleotide additions generated during b cell ig gene rearrangement, prior to antigen selection. because of the lack of mutations elsewhere in the sequences, it is likely that the r g substitution was also generated during this developmental step. the downstream sequence corresponding to the junction between ighd - à and the igh joining (ighj) ighj à genes also revealed similar nucleotide insertions. likewise, the kappa (k) chain junction between the igkv - à and igkj à genes also contained one insertion. although we cannot rule out the possibility of shm-mediated nucleotide changes in the n insertion regions, no mutation was identified in the remainder of the regions of the heavy and light chains. thus, the p f mab is likely very close or identical to the original v-(d)-j gene rearrangement in the naïve b cell before antigen contact. to investigate whether p f recognizes an immunodominant zikv epitope, we used a serological blocking assay. in brief, this assay detects the presence of competing abs that can inhibit the p f mab binding to its epitope. because p f did not bind to recombinant e protein (fig ) we used whole virus in our binding assays. we captured zikv on the plate using the g mab (pan-flavivirus), and incubated zikv with plasma from patients with diverse histories of denv and zikv exposure (s table) . we added unlabeled p f (engineered with rhesus igg constant regions) and detected binding of the mab using a hrplabeled mouse anti-rhesus mab (fig ) . nine of ten plasma samples from individuals that had been infected with zikv blocked the binding of p f in a blinded test (fig , s table) . similar blocking activity was observed regardless of whether individuals had been previously infected with denv or had been vaccinated for yellow fever. in contrast, little or no blocking activity was observed by denv+ plasma in the absence of prior zikv exposure (fig ) . furthermore, this recognition was specific in that it was not observed in of denv-only infected individuals. thus, the p f serological blocking assay accurately predicted previous zikv exposure, as confirmed by rt-pcr, in all but one of the patient plasma samples tested. although this patient, donor , had a positive urine rt-pcr result for zikv, plasma from did not block p f binding to zikv (s table) . interestingly, the plasma did not exhibit detectable zikv-neutralizing activity, suggesting that this patient did not mount a measurable antibody response against zikv. in conclusion, only the plasma that inhibited zikv infection of vero cells contained p f -blocking antibodies. here we show that a igg mab with no detectable shm was generated against zikv early in infection. remarkably, despite being germline-encoded, this mab is zikv-specific and does not bind to any of the four denv serotypes. furthermore, this mab not only neutralizes zikv, but it also binds to an immunodominant epitope on the virus. remarkably, despite being germline-encoded, p f binds specifically to zikv and does not cross-react with any of the four denv serotypes. our results also suggest that p f recognizes a unique epitope on zikv. it is unclear how this ab developed such specificity without shm. finally, these findings suggest that affinity maturation is not necessary for the generation of isotype switched virus-neutralizing abs. low levels of shm in abs possessing neutralizing activity have been previously reported in mice and humans [ ] [ ] [ ] , supporting the idea that germline-encoded mabs can indeed neutralize. abs with low levels of shm have also been reported during the acute phase of human denv infection, but it was not clear that these abs contributed to the antiviral neutralization activity [ ] . in studies in mice, vsv-specific mabs lacking shm have been isolated previously [ ] . interestingly, secondary, but not primary, mouse abs against vsv had mutations [ ] . furthermore, the reversion of these mutated abs to non-mutated precursors reduced, but did not abrogate, vsv binding and neutralizing activity. the binding differences between the mutated and germline abs were much less pronounced than might be expected [ ] . additionally, mice that cannot conduct shm due to aid knockout still mounted neutralizing ab responses against friend virus, a strain of murine leukemia virus [ ] . it has been suggested that these abs lacking extensive shm undergo a gc-independent developmental pathway [ ] , although the mechanistic basis for this phenomenon remains to be elucidated. rapid, gc-independent responses might be particularly relevant in the control of acute cytopathic viruses [ , , ] . the gc-independent abs would arise quickly after infection and then curtail viral replication, preventing virus-mediated damage [ ] . even more provocatively, hangartner et al. have argued that cytopathic viruses specifically evolved to retain binding to these germline sequences to decrease host lethality and increase fitness. on the other hand, chronic viruses may have evolved to avoid germline-binding and development of neutralizing responses to persist [ ] . so far, these hypotheses remain unsubstantiated by the lack of evidence for strictly germline neutralizing ab responses in humans. while our experiments were not specifically designed to detect gc-independent responses, it seems likely that the isotype-switched p f originated directly from a germline precursor. we isolated p f from a zikv-infected individual that developed neurological complications compatible with gbs and was treated with ivig. underlying factors that influence the potential association of gbs and zikv infection might involve an autoimmune process, which could influence the development of immune responses [ ] . additionally, ivig may have had a role in the selection of the ab responses mounted by peripheral b cell repertoires [ ] . this is unlikely, however, since the patient initiated ivig treatment on the same day that the plasmablasts were isolated. it is possible, then, that gbs or ivig-treatment influenced the development of p f . these potential associations are difficult to determine and were outside the scope of this study. it is clear, however, that these responses were not exclusive to volunteer , as p f binding can be blocked by the serum of most zikv-infected individuals (fig ) . recently [ ] . these mabs were isolated from memory cells sorted with soluble and monomeric zikv e proteins and, in contrast to p f , bind to the recombinant protein [ ] . in contrast, we isolated zikv-specific mabs from circulating plasmablasts at d . the peak recall of memory b-cell derived plasmablasts is thought to occur within the first week post-secondary infection [ , ] . thus, it is probable that most of the isolated mabs did not have a memory-b cell origin, and it remains possible that some of the plasmablasts were sorted from the basal population that circulate in low frequencies in the blood. in conclusion, the isolation of mabs using different b cell methods suggest that anti-zikv mabs with germline characteristics are not limited to specific b cell subtypes [ , ] . notably, the anti-zikv mabs isolated to date are less mutated than the mabs isolated after related denv infections [ ] [ ] [ ] [ ] . together, these findings suggest possible differences in the development of ab responses against zikv. unfortunately, despite our efforts, we were unable to map p f 's binding site. we first employed an in vitro escape assay [ ] , which did not result in a single mutated consensus sequence. also, p f did not bind to the prm/e proteins expressed in cells, precluding our ability to map this interaction using an ala-mutated envelope panel [ , ] . characterizing this interaction will, thus, require a significant effort that is beyond the scope of the current manuscript. because the p f mab retains the ability to bind virions, our conclusion is that it binds to a conformational epitope. based on the cohort of human plasma samples tested in this study, it appears that most zikv-infected individuals mount ab responses against the epitope recognized by p f . this epitope is recognized by abs in individuals previously infected by zikv, thereby preventing the binding of p f . by contrast, abs in the plasma from individuals previously infected by any of the denv serotypes, do not prevent binding of p f . p f may, therefore have potential as a diagnostic. several diagnostic options for testing for zikv exposure exist, including rt-pcr, igm elisa, and prnt methods [ , ] . while it is relatively straightforward to detect zikv nucleic acid during the acute phase in blood, urine, saliva, and semen, it has proven more difficult to design rapid and effective diagnostics for zikv exposure in the chronic phase. for samples collected after the first week of symptoms, the initial test is an anti-zikv, anti-denv, anti-chikv virus igm elisa [ ] . however, in patients who have received a flaviviral vaccine (denv, yfv, or jev) and/or have been infected with any flaviviruses in the past, these assays may be difficult to interpret due to the cross-reactivity of the abs [ ] [ ] [ ] [ ] [ ] [ ] . thus, a positive igm test needs to be confirmed with a laborious prnt assay. igm antibodies persist for - weeks in serum, and sera from individuals previously infected for more than weeks would also have to be confirmed with a virus neutralization-based method [ ] . our plasma inhibition assay may, perhaps, provide an alternative to these other techniques. in this study, we isolated plasmablast-derived abs from a zikv-infected individual with unusual characteristics. the human igg p f has no or limited shm yet binds to an immunodominant zikv epitope that is not present on any of the four denv serotypes. furthermore, this mab can neutralize the virus with a neut of approximately μg / ml. our results suggest that shm-independent pathways may generate neutralizing abs in the responses against zikv. molecular evolution of zika virus during its emergence in the (th) century a simple technique for infection of mosquitoes with viruses; transmission of zika virus twelve isolations of zika virus from aedes (stegomyia) africanus (theobald) taken in and above a uganda forest zika virus outbreak on yap island, federated states of micronesia zika virus outside africa genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia zika virus spreads across americas as concerns mount over birth defects zika virus: a previously slow pandemic spreads rapidly through the americas zika virus spreads to new areas-region of the americas zika virus infection in pregnant women in rio de janeiro. the new england journal of medicine zika virus outbreak in rio de janeiro, brazil: clinical characterization, epidemiological and virological aspects birth defects among fetuses and infants of us women with evidence of possible zika virus infection during pregnancy detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study. the lancet infectious diseases ocular findings in infants with microcephaly associated with presumed zika virus congenital infection in salvador, brazil pathology of congenital zika syndrome in brazil: a case series notes from the field: evidence of zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses-brazil zika virus associated with microcephaly. the new england journal of medicine placental inflammatory response to zika virus may affect fetal brain development zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? ultrasound zika virus infection and stillbirths: a case of hydrops fetalis, hydranencephaly and fetal demise possible association between zika virus infection and microcephaly-brazil interim guidelines for the evaluation and testing of infants with possible congenital zika virus infection-united states interim guidelines for pregnant women during a zika virus outbreak-united states human antibody responses after dengue virus infection are highly cross-reactive to zika virus a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus molecular determinants of human neutralizing antibodies isolated from a patient infected with zika virus efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning igblast: an immunoglobulin variable domain sequence analysis tool imgt/v-quest: imgt standardized analysis of the immunoglobulin (ig) and t cell receptor (tr) nucleotide sequences measuring antibody neutralization of dengue virus (denv) using a flow cytometry-based technique comparison of plaque-and flow cytometry-based methods for measuring dengue virus neutralization attenuation and immunogenicity in humans of a live dengue virus type- vaccine candidate with a nucleotide deletion in its '-untranslated region. the american journal of tropical medicine and hygiene imgt unique numbering for immunoglobulin and t cell receptor variable domains and ig superfamily v-like domains the role of somatic mutation in the generation of the protective humoral immune response against vesicular stomatitis virus h n influenza virus neutralizing antibodies that possess few somatic mutations class switch recombination and somatic hypermutation of virus-neutralizing antibodies are not essential for control of friend retrovirus infection lower igg somatic hypermutation rates during acute dengue virus infection is compatible with a germinal center-independent b cell response virus neutralization by germline vs. hypermutated antibodies generation of memory b cells inside and outside germinal centers early high-affinity neutralizing anti-viral igg responses without further overall improvements of affinity antiviral antibody responses: the two extremes of a wide spectrum guillain-barre and miller fisher syndromes-new diagnostic classification intravenous immunoglobulins (ivig) in the treatment of autoimmune diseases rapid and massive virus-specific plasmablast responses during acute dengue virus infection in humans origin and function of circulating plasmablasts during acute viral infections structure and function analysis of therapeutic monoclonal antibodies against dengue virus type a high-throughput shotgun mutagenesis approach to mapping b-cell antibody epitopes neutralizing human antibodies prevent zika virus replication and fetal disease in mice interim guidance for interpretation of zika virus antibody test results fields virology. th ed. philadelphia: wolters kluwer health/lippincott williams & wilkins structural basis of potent zika-dengue virus antibody cross-neutralization cross-reactivity and function of antibodies elicited by zika virus infection high specificity of a novel zika virus elisa in european patients after exposure to different flaviviruses diagnostics for zika virus on the horizon we thank the patients for their volunteer donations. we also thank the laboratory and clinical personnel responsible for collecting the plasma samples used in this study. conceptualization: dmm diw egk. key: cord- -xlk xpv authors: mögling, ramona; zeller, hervé; revez, joana; koopmans, marion; reusken, chantal title: status, quality and specific needs of zika virus (zikv) diagnostic capacity and capability in national reference laboratories for arboviruses in eu/eea countries, may date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: xlk xpv with international travel, zika virus (zikv) is introduced to europe regularly. a country's ability to robustly detect zikv introduction and local transmission is important to minimise the risk for a zikv outbreak. therefore, sufficient expertise and diagnostic capacity and capability are required in european laboratories. to assess the capacity, quality, operational specifics (guidelines and algorithms), technical and interpretation issues and other possible difficulties that were related to zikv diagnostics in european countries, a questionnaire was conducted among national reference laboratories in countries in the european union/european economic area (eu/eea) in may . while the coverage and capacity of zikv diagnostics in the eu/eea national reference laboratories were found to be adequate, the assessment of the quality and needs indicated several crucial points of improvement that will need support at national and eu/eea level to improve zikv preparedness, response and eu/eea zikv surveillance activities. zika virus (zikv) infections were historically not considered a significant public health concern [ ] . however, in the year following its first autochthonous transmission in the americas in , zikv has been linked to severe congenital anomalies in newborns and to other neurological disorders such as guillain-barré syndrome (gbs) [ ] . the world health organization (who) declared the cluster of microcephaly cases and other neurological disorders possibly associated with zikv a public health emergency of international concern (pheic) on february [ ] . by the end of , the unprecedented zikv outbreak affected countries and territories in the americas with more than half a million human cases [ ] . the pheic was declared over on november with the argument that zikvassociated congenital syndrome was considered to be a long-term public health challenge requiring a longterm commitment to control and prevent [ , ] . zikv is a mosquito-borne virus which belongs to the genus flavivirus, family flaviviridae [ ] . its genome can be detected in biological samples by reverse transcription pcr (rt-pcr) and the virus can be isolated in cell culture. however, viraemia is typically short-lived ( - days after onset of symptoms) [ ] [ ] [ ] , although an increased window of detection has been observed using urine (up to days after onset of symptoms) [ ] or whole blood (up to days after onset of symptoms) [ ] . consequently, the full spectrum of diagnostics includes serology, which is complex owing to extensive cross-reactivity with antibodies triggered by other flaviviral infections and/or vaccination [ ] . the majority of zikv infections are asymptomatic which complicates retracing the course of infection and increases the dependency on serology to confirm an infection [ ] . this is in particular an issue for pregnant women for whom correct diagnosis of a zikv infection, even if asymptomatic, is imperative [ , , ] . international travel and outbreaks of zikv in european overseas countries and territories are responsible for the regular introduction of zikv to europe [ ] [ ] [ ] [ ] . a risk assessment by the who regional office for europe (who/europe) indicated that the risk for an outbreak with zikv in europe should not be underestimated, in particular in countries with established presence of the vectors aedes aegypti and ae. albopictus [ , ] . a country's ability to robustly detect zikv introduction and local transmission is important to minimise the risk for a zikv outbreak. therefore sufficient expertise and diagnostic capacity and capability in european laboratories are required [ ] . to map zikv expertise and identify diagnostic capacity and capability gaps in europe during the initial phase of the pheic in february , the european commission (ec) asked the european centre for disease prevention and control (ecdc) for a rapid assessment of the capacity of laboratories in europe to detect zikv infections and the specific needs for support. the assessment was done by an in-depth questionnaire in may . here, the outcomes of the assessment are presented and discussed to identify knowledge and technical gaps to strengthen laboratory capacity and quality for zikv diagnostics in the european union/european economic area (eu/eea) and to support zikv preparedness and response as well as zikv surveillance activities in the eu/eea. a questionnaire was designed to address the capacity, quality, operational specifics (guidelines and algorithms), technical and interpretation issues and other possible difficulties related to zikv diagnostics. national reference laboratories for zikv diagnostics were asked to complete the online questionnaire through the eu/eea ecdc national focal points for microbiology (nmfps) [ ] . the questionnaire was sent to the nmfps on may and closed on may . as most questions in the questionnaire where not obligatory to answer, the sums in the presented data may vary as non-replies are not listed. in total, laboratories from eu/eea countries completed the questionnaire, of which laboratories in countries indicated that they were conducting zikv diagnostics by may (figure ). zikv infection was made notifiable in countries by may , and mandatory disease notification was planned for the near future in nine countries. for one country, the notification procedure was not specified. in agreement with the eu classification [ , ] , laboratories indicated that their operational biosafety level (bsl) for zikv diagnostics was bsl . eight laboratories indicated bsl as their operational biosafety level, while two laboratories performed zikv diagnostics at bsl . reasons given by laboratories to deviate from the official level bsl were (i) downgrading because diagnosis did not involve high titre virus culture (one laboratory), and (ii) upgrading based on inhouse assessment of biosafety and biosecurity issues or for logistical reasons such as aligning zikv diagnostics with other flavivirus diagnostic serology and virus isolation (three laboratories). the remaining six laboratories did not indicate reasons for the deviation. because of, at the time putative [ ] , teratogenic effects of zikv infection in pregnant women, laboratories conducting zikv diagnostics were asked whether special regulations for pregnant employees were in place. in six of the laboratories that answered this question, pregnant employees were not allowed to perform zikv diagnostic tests. in three additional laboratories, a restriction for pregnant employees was limited to handling zikv virus culture. the remaining laboratories did not have specific guidelines for pregnant employees. all laboratories conducting zikv diagnostics (n = , in countries) performed molecular testing, while laboratories in countries also conducted serology and one laboratory performed antigen detection. the laboratories accepted different types of patient samples, including plasma/serum, urine, semen, amniotic fluid, placenta, saliva and nasopharyngeal swabs. all the number of laboratories that participated in the questionnaire on invitation by their country nmfp in the period - may are indicated per country. one country indicated in january that they had implemented zikv diagnostics since may . two countries without implemented zikv diagnostics indicated to have access to diagnostics through an agreement with a laboratory in another country. laboratories received serum samples for primary diagnostics, and in addition, clinicians often provided urine as a second sample for testing ( / ). semen ( / ), saliva ( / ) and nasopharyngeal swabs ( / ) were received to a lesser extent. for an adequate choice of diagnostic tests to run and for a correct interpretation of results, a minimum of information on the patient history is required [ ] [ ] [ ] . laboratories were asked to indicate on a likert scale with - range ( = never, = always) how often certain essential background information about their patient samples was provided with the diagnostic requests ( figure ). overall, the provision of the date of sampling was scored the highest ( / laboratories scored this in category 'often' or 'always'), while the flavivirus vaccination status scored the lowest ( / scored it as 'often' or 'always'). other data essential for choice of testing algorithm and test interpretation such as 'first day of illness', 'country and dates of travel', 'clinical symptoms' and (in case of women) 'pregnancy status' were provided with requests in to of the laboratories. most laboratories ( / ) indicated that they could directly contact physicians to ask for more information about the requested samples or look up record sheets and laboratory reports. however, in practice the feasibility of this strongly depended on the daily sample load. twenty-one laboratories that conducted zikv molecular detection only used commercially available tests, laboratories only in-house tests, while nine laboratories indicated using both. the realstar zika virus [ ] . primary rt-pcrs were mostly carried out with a single zikv genome target ( / ). however, two laboratories performed a multiplex pcr targeting multiple viruses which were not specified further. positive results were confirmed independently in of laboratories. this was done either by a second rt-pcr targeting a different genome segment ( / laboratories) or through sequencing ( / ). forty-three of laboratories used a positive control, which was obtained through the european virus archive (https:// www.european-virus-archive.com) in six, through the robert koch institute (via former enivd expert laboratory network) in , from own patient materials in , or by using synthetic rna in four of laboratories. twenty-two laboratories indicated that they used another positive control, but not the specific source. eighteen of laboratories used zikv strains of the current outbreak in the americas as a control, while used strains belonging to the original (pre- ) asian lineage. african lineage strains were used in laboratories, had access to more than one zikv lineage as control. on both zikv igm and igg ( / ). ten laboratories in countries were able to assess the presence of zikv neutralising antibodies by plaque reduction neutralisation (prnt) and/or virus neutralisation (vnt) tests. thirty-one of laboratories carried out commercial serology assays. laboratories either used the euroimmun ag anti-zika virus elisa igm/igg ( / ), euroimmun arboviral fever mosaic if kit ( / ), both assays ( / ) or did not specify which assay they used ( / ). thirteen of laboratories used in-house assays, including elisa ( / ) and/or immunofluorescence assays (ifa) ( / ), as well as vnt ( / ) and prnt ( / ). the majority of these laboratories ( / ) indicated that they obtained their positive control material from own patient samples. four of laboratories were provided with zikv igm/igg positive control samples by collaborators. to gain insight in the level of quality control at the laboratories that performed zikv diagnostics, the laboratories were asked to specify their level(s) of laboratory accreditation. analysis at the laboratory level showed that international organization for standardization (iso) ('medical laboratories -requirements for quality and competence' [ ] another quality aspect concerns the extent of validation of the implemented diagnostics, in particular the serology in view of extensive cross-reactivity. the median size of in-house validation panels of confirmed zikv and wnv patients was small (table ) and serum samples from pregnant women and population panels from zikv-endemic regions were lacking in most laboratories (table ) . thirty-seven of laboratories indicated that they were willing to share validation data with other laboratories. however, of the zikv diagnostic laboratories indicated that their accreditation scheme did not accept validation done elsewhere, while this would be a possible option for of laboratories. laboratories were asked to indicate how many diagnostic samples they could process per week for the different types of test that they run ( figure ). diagnostic capacities of individual laboratories differed depending on the type of diagnostic test. in addition, the laboratories were asked how many samples they had processed and determined positive for molecular, igm, igg and neutralising antibody testing since january (table ). for of laboratories offering molecular testing, the cumulative number of total requests in the -week period covered in the questionnaire (including requests in support of other laboratories) remained below their indicated capacity per week. for of laboratories, the cumulative number of requests was approximately two or three times the indicated weekly capacity, while for three laboratories, it exceeded their capacity (five to -fold). for serology, a vast majority of the laboratories had a cumulative number of requests for the -week period that was two to three times their weekly capacity. fourteen of laboratories indicated that they supported other laboratories by performing molecular tests for them. fifteen laboratories supported others with serological testing and laboratories with provision of control materials. the majority of the laboratories ( / ) that had not offered laboratory support laboratories mainly implemented the zikv testing algorithms either as advised by ecdc ( / ) [ ] or by their national public health institutes ( / ). the remaining eight laboratories followed, among other algorithms, the zikv diagnostic algorithms advised by the pan american health organization (paho) [ ] . in case necessary background information such as first day of illness was not known, ten of the diagnostic laboratories performed both molecular and serological tests on the available samples, three always carried out serology, two always performed rt-pcr, while three only conducted the tests that were asked for by the clinician. ten laboratories either asked for more information or an additional sample from the patient for an interpretation based on kinetics. the remaining did not provide that answer. laboratories were also asked if there was a different testing algorithm for asymptomatic pregnant women with putative zikv exposure. ten laboratories indicated that they asked for paired serum samples of asymptomatic pregnant women for serology, while laboratories did both molecular and serological testing on the available samples. eight laboratories referred to their national zikv diagnostic algorithm and one laboratory indicated that asymptomatic patients were never tested regardless of pregnancy status. laboratories were asked to describe the main challenges they were faced with during the implementation of zikv molecular and/or serological diagnostics. the main indicated obstacles were the availability of a positive control and validation materials for molecular and serological tests, the availability of commercial serological tests and personnel capacity (figure ). in may , there was an eu/eea-wide coverage for zikv molecular diagnostics. only three countries were without in-country zikv molecular diagnostics but all had access to diagnostics through an agreement with a laboratory in another country and two had plans to implement zikv diagnostics in the near future. of these, one country had implemented zikv molecular diagnostics by january (figure ). in comparison to a february snapshot for the ec (data not shown), four more eu/eea countries had implemented zikv molecular diagnostics in may. the coverage for zikv serology increased from eu/eea countries to . access to zikv serology is particularly important because the confirmation or ruling out of a zikv infection during pregnancy is essential for medical followup regarding the teratogenicity of zikv [ , , ] . as an estimated % of zikv infections are asymptomatic and the genome detection window in serum is short [ ] , zikv diagnosis in pregnant women will often rely on zikv antibody detection in paired serum samples. an important aspect of conducting zikv serology is expertise about flaviviruses because the interpretation is complex [ , , ] . all laboratories conducting zikv serology indicated that they had experience with serodiagnostics for at least one other flavivirus. in addition, insight in the specificity and sensitivity of the serology tests used is required for proper test interpretation, but this appeared limited at the time of the questionnaire because adequate validation panels were lacking. validation data provided by commercial entities typically need to be confirmed in order to be acceptable for an accreditation scheme [ ] . similarly, validation of such assays performed in another laboratory may be insufficient to meet with accreditation requirements. indeed, the availability of validation panels for serology was indicated as the biggest challenge for implementation of zikv diagnostics by of zikv diagnostic laboratories. five of the zikv diagnostic laboratories did not have any kind of iso accreditation, while of laboratories did not have the most relevant iso accreditation [ ] . another concern is the broad reliance for zikv serology on one or two commercial tests, although it should be noted that in may , the commercial market for zikv serology offered very limited options. in december , no commercial serology test had been accepted for procurement through the who emergency use assessment and listing procedure, which requires extensive validation [ ] . this illustrates the importance of reference laboratory capacity. virus neutralisation is still considered the most specific flavivirus serology test, although cross-reactivity can be observed in patients with other flavivirus infections and research is ongoing to develop more specific assays [ , , , ] . broad implementation of this confirmatory test in eu/eea national reference laboratories is, however, expected to increase the reliability of serology results in returning travellers as the flavivirus background (in particular dengue) in european travellers is likely to be low. molecular testing, having capacity/capability to diagnose zikv, relied in of laboratories only on commercial assays, in laboratories only on in-house testing and in nine laboratories on both commercial and in-house testing. only one (lanciotti et al. [ ] ) of the two in-house tests that were most frequently used, was recommended based on in silico analysis [ ] and in an independent comparative study of zikv molecular tests [ ] , raising possible concerns about the performance of diagnostics in some laboratories. in november , the ecdc-funded emerging viral disease laboratory expert network (evd-labnet [ ] ) organised an external quality assessment (eqa) for member laboratories which included the majority of the national reference laboratories with zikv diagnostics that participated in this questionnaire. the results from such an eqa will provide points of improvement to the individual laboratories [ ] . for an adequate choice of which type of test to use and for correct interpretation of test results, a defined set of information on the patient history is essential. as shown for other diagnostics, the lack of information provided by clinicians requesting the diagnostics proved to be a major gap [ ] . although the date of sampling was given most often ( / scored 'often' or 'always'), this information is hardly meaningful without an indication of the first day of illness (only scored in the highest categories by laboratories). reliable interpretation of zikv serology is impeded without information on previous flavivirus infections or vaccinations that was often/always available in only of laboratories. reimbursements rules for the diagnostic tests differ between countries (data not shown). if the state covers the costs for the diagnostic tests, provision of necessary background information can be mandatory. the overall zikv diagnostic capacity in the eu/eea countries appeared to be sufficient, given the total number of reported requests vs the indicated capacities. at country level, the extent of under/overcapacities varied and were complicated by the fact that laboratories with certain iso accreditations are bound by strict regulations when sending a surplus of samples to a backup laboratory. the availability of validation materials, positive controls and personnel were indicated as the main challenges for implementation of zikv diagnostics in the reference laboratories of the eu/eea countries. that of laboratories indicated the cost of commercial tests as an obstacle for test implementation and the large dependency of the laboratories on commercial assays, while five of laboratories did not receive funding for development and/or implementation of in-house tests, illustrate an achilles' heel in the preparedness for emerging infections and needs careful consideration when defining strategies to strengthen laboratory preparedness and response for future outbreak situations. while the coverage and capacity of zikv diagnostics in eu/eea national reference laboratories were observed to suffice in may , the assessment of the quality and needs indicated several crucial points of improvement that will need support at national and eu/eea level. all reference laboratories should seek to have a relevant iso accreditation. awareness and facilitation of (temporary) acceptance of laboratory accreditation for novel diagnostics implemented in emerging situations is required, although it is currently hampered by lack of availability of well-defined validation panels. improved access is required to controls and validation panels for both molecular and serological tests. pending the development of more specific serology tests, a broader implementation of the current most specific neutralisation tests (prnt, vnt) is desirable, ideally in a comparative setting with other relevant flaviviruses. increased awareness is needed among clinicians to provide all necessary background information, and systems should be implemented that assure provision of necessary interpretation data. national and/ or eu contingency funding should be established to ensure adequate and robust laboratory preparedness and response. zika virus outside africa zika virus infection as a cause of congenital brain abnormalities and guillain-barré syndrome: systematic review who statement on the first meeting of the international health regulations : the year zika evolved from an emergency into a long-term public health challenge. washington: paho fifth meeting of the emergency commitee under the international health regulations ( ) regarding microcephaly, other neurological disorders and zika virus background review for diagnostic test development for zika virus infection detection of zika virus in saliva detection of zika virus in urine zika virus infection and prolonged viremia in whole-blood specimens zika virus: a new threat to human reproduction cdc guidelines for pregnant women during the zika virus outbreak travel-associated zika virus disease acquired in the americas through february : a geosentinel analysis assessing seasonal risks for the introduction and mosquito-borne spread of zika virus in rapid risk assessment. zika virus disease epidemic, tenth update rapid risk assessment. zika virus disease epidemic. ninth update world health organization regional office for europe (who europe). zika virus european centre for disease prevention and control (ecdc) coordinating competent bodies: structures, interactions and terms of reference on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of article l / the approved list of biological agents rapid risk assessment: zika virus disease epidemic: potential association with microcephaly and guillain-barré syndrome. fifth update mers coronavirus: data gaps for laboratory preparedness come fly with me: review of clinically important arboviruses for global travelers using routine diagnostic data as a method of surveillance of arboviral infection in travellers: a comparative analysis with a focus on dengue genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia quantitative real-time pcr detection of zika virus and evaluation with field-caught mosquitoes #iso:std:iso-iec: :ed- :v :en . international organization for standardization (iso) ecdc technical document. zika virus disease epidemic: interim guidance for healthcare providers and zika virus laboratory diagnosis zika virus (zikv) surveillance in the americas: laboratory detection and diagnosis. algorithm for detecting zika virus (zikv) laboratory testing for zika virus infection emergency use assessment and listing (eual) update on submission of applications to the who eual for zika virus ivds european union invests € million into research to combat the zika disease assay optimization for molecular detection of zika virus emerging viral diseases-expert laboratory network (evd labnet). ecdc. stockholm: ecdc variable sensitivity in molecular detection of zika virus in european expert laboratories; external quality assessment this study was conducted with ecdc-funding srs- - -rs. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indicate if changes were made. key: cord- -v uevz l authors: zukor, katherine; wang, hong; siddharthan, venkatraman; julander, justin g.; morrey, john d. title: zika virus-induced acute myelitis and motor deficits in adult interferon αβ/γ receptor knockout mice date: - - journal: j neurovirol doi: . /s - - -z sha: doc_id: cord_uid: v uevz l zika virus (zikv) has received widespread attention because of its effect on the developing fetus. it is becoming apparent, however, that severe neurological sequelae, such as guillian-barrë syndrome (gbs), myelitis, encephalitis, and seizures can occur after infection of adults. this study demonstrates that a contemporary strain of zikv can widely infect astrocytes and neurons in the brain and spinal cord of adult, interferon α/β receptor knockout mice (ag strain) and cause progressive hindlimb paralysis, as well as severe seizure-like activity during the acute phase of disease. the severity of hindlimb motor deficits correlated with increased numbers of zikv-infected lumbosacral spinal motor neurons and decreased numbers of spinal motor neurons. electrophysiological compound muscle action potential (cmap) amplitudes in response to stimulation of the lumbosacral spinal cord were reduced when obvious motor deficits were present. zikv immunoreactivity was high, intense, and obvious in tissue sections of the brain and spinal cord. infection in the brain and spinal cord was also associated with astrogliosis as well as t cell and neutrophil infiltration. cmap and histological analysis indicated that peripheral nerve and muscle functions were intact. consequently, motor deficits in these circumstances appear to be primarily due to myelitis and possibly encephalitis as opposed to a peripheral neuropathy or a gbs-like syndrome. thus, acute zikv infection of adult ag mice may be a useful model for zikv-induced myelitis, encephalitis, and seizure activity. electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. abstract zika virus (zikv) has received widespread attention because of its effect on the developing fetus. it is becoming apparent, however, that severe neurological sequelae, such as guillian-barrë syndrome (gbs), myelitis, encephalitis, and seizures can occur after infection of adults. this study demonstrates that a contemporary strain of zikv can widely infect astrocytes and neurons in the brain and spinal cord of adult, interferon α/β receptor knockout mice (ag strain) and cause progressive hindlimb paralysis, as well as severe seizurelike activity during the acute phase of disease. the severity of hindlimb motor deficits correlated with increased numbers of zikv-infected lumbosacral spinal motor neurons and decreased numbers of spinal motor neurons. electrophysiological compound muscle action potential (cmap) amplitudes in response to stimulation of the lumbosacral spinal cord were reduced when obvious motor deficits were present. zikv immunoreactivity was high, intense, and obvious in tissue sections of the brain and spinal cord. infection in the brain and spinal cord was also associated with astrogliosis as well as t cell and neutrophil infiltration. cmap and histological analysis indicated that peripheral nerve and muscle func-tions were intact. consequently, motor deficits in these circumstances appear to be primarily due to myelitis and possibly encephalitis as opposed to a peripheral neuropathy or a gbs-like syndrome. thus, acute zikv infection of adult ag mice may be a useful model for zikv-induced myelitis, encephalitis, and seizure activity. zika virus (zikv) is an emerging flavivirus that has received widespread attention because of its effect on the developing fetus. in utero infections cause congenital defects, most notably microcephaly along with other deformities (schuler-faccini et al. ) . while zikv infection of adults generally produces only a mild disease, it is becoming apparent that, as with other flavivirus infections, severe neurological sequelae can occur. recent outbreaks have been associated with higher incidences of peripheral neuropathies such as guillian-barrë syndrome (gbs) cao-lormeau et al. ; cardoso et al. ; samarasekera and triunfol ) and other neurological diseases such as myelitis (anaya et al. ; dirlikov et al. ; mecharles et al. ) , encephalitis (carteaux et al. ; nicastri et al. ; soares et al. ) , seizures (asadi-pooya ), and various ophthalmological conditions smith et al. ). this is not surprising considering that related flaviviruses such as west nile virus (wnv) and japanese encephalitis virus (solomon et al. ) can cause myelitis and motor deficits (sejvar et al. ) . given the emerging nature of zikv, however, it is likely that we do not fully understand the acute and long-term consequences of zikv infection on the nervous system. therefore, investigating the pathobiology of zikv in animal models will help to understand the potential for zikv to cause neurological disease in human subjects. we focus herein on adult models because zikv-related motor deficits have been primarily associated with adult infection, as opposed to in utero infection (anaya et al. ; dirlikov et al. ; mecharles et al. ). prior to the zikv outbreak, a few animal models of zikv infection existed, but infection of rodents was largely non-productive unless a lab strain of the virus that had undergone several serial passages in mice was used (dick ) . after the recent outbreaks, efforts to develop animal models with more clinically relevant isolates of the virus have been renewed. a rapid series of publications found that adult mice that lack type (αβ; a and ifnar −/− strains) or types and (αβ/γ; ag strain) interferon receptors are susceptible to lethal infection (aliota et al. ; lazear et al. ; manangeeswaran et al. ; rossi et al. ; zmurko et al. ) . these models may have some relevance to human zikv infections in that, like many viruses, zikv gains advantages in human hosts by inhibiting interferon responses (best ; bowen et al. ; schulz and mossman ) . because viruses may not be able to inhibit mouse-specific interferon pathways (aguirre et al. ) , blocking them by other means, as in ag mice, may more closely mimic what happens in humans. although ag mice are deficient in innate immune responses, they do elicit acquired immune responses, such as vaccine-elicited protective immunity (sumathy et al. ; weger-lucarelli et al. ) . while complete knockout of the interferon responses produces a more severe disease in mice, it can provide insights into what happens when zikv gains access to the adult central nervous system (cns). besides a lethal infection, these mice manifest neurological symptoms such as btoe walking,^tremors, loss of balance, paralysis, and hunched posture (aliota et al. ; lazear et al. ; manangeeswaran et al. ; rossi et al. ) . to better understand the consequences of zikv infection of the adult nervous system and the pathobiology of the resulting neurological disease, we infected ag mice with a contemporary, low-passage zikv isolate and evaluated the neurological motor deficits occurring during the acute phase of the disease. we characterized the onset, course, and severity of behavioral hindlimb deficits and used electrophysiology and immunohistochemistry (ihc) to determine if such deficits are due primarily to myelitis, peripheral neuropathy, myositis, or encephalitis. this included histological analysis of motor cortex, spinal cord, peripheral nerve, and muscle. male and female ag mice (van den broek et al. ) were bred in-house in sterilized cages and maintained in a / light cycle. mice were randomly assigned to treatment groups based on weight, gender, and baseline measurements. a puerto rican isolate of zikv (prvabc , human/ / puerto rico, genbank ku ) was obtained from bei resources (cat no. nr- , lot no. ) . the certificate of analysis confirmed that the sequence of this stock (genbank kx ) is % identical to prvabc (genbank ku ). the bei stock was passaged two times in vero cells to make a stock with a titer of × pfu/ml for use in all experiments. infected cells were frozen once, thawed, centrifuged to remove cell debris, and aliquoted in frozen stocks. dilutions were made in minimal essential medium supplemented with μg/ml gentamicin to deliver pfu subcutaneously in the inguinal area on the right side in a volume of . ml. uninfected cells were prepared and diluted similarly for sham infections. the purpose of this experiment was to assess the time course and severity of motor deficits and collect tissues for histological analysis. seventy-two to -day-old ( . weeks) male and female ag mice were infected with zikv (n = females and males) or sham (n = females and males) inoculum and monitored for weight loss and survival (fig. ) . behavioral motor assessments were performed before infection (baseline); once after infection, but before symptom onset ( days post-infection (dpi)); and twice a day after symptom onset ( - dpi) (fig. ) . seizure-like activity observed during behavioral assessments was also noted (fig. ) . videos of motor deficits and seizure-like activity were obtained to provide examples of symptoms seen. observations were made by researchers who were blind to the infection status of each group. moribund mice were perfused for histological analysis of the brain, lumbosacral spinal cord, sciatic nerve, and gastrocnemius muscle. the purpose of this experiment was to collect electrophysiological data on mice with motor deficits. male and female ag mice aged - days old ( weeks) or days old ( weeks) were divided into groups. the zikv-infected group contained three males ( weeks old), four males ( weeks old), and four females ( weeks old). the shaminfected group contained two males ( weeks old), two males ( weeks old), and two females ( weeks old). mice were monitored for weight loss and survival. behavioral assessments were performed before infection (baseline) and after symptom onset so that mice with moderate to severe deficits (scores - , see below) could undergo electrophysiological assessment. compound muscle action potential (cmap) of the gastrocnemius muscle was recorded before infection (baseline) and after moderate to severe deficits had developed. after infection, one sham-infected mouse was recorded for every one to two zikv-infected mouse. measurements were obtained by a researcher who was blind to the infection status and deficit score of each mouse. cmap responses were recorded in response to stimulation at the sciatic notch and then the lumbosacral spinal cord. because stimulation of the lumbar spinal cord was an invasive procedure, we did not obtain baseline measurements for cmap in response to spinal cord stimulation. mice were analyzed for signs of tail and hindlimb paresis/ paralysis using a sensitive, open-field assay modified from the basso mouse scale used to assess paralysis in spinal cord-injured mice (basso et al. ) and a test used to track paralysis in amyotrophic lateral sclerosis mouse models (hatzipetros et al. ) . each mouse was placed on a tabletop and allowed to roam freely for min. hindlimb function was scored on a seven-point scale detailed in table by researchers who were blind to the infection status of each group. scoring was based on four main categories: tail position during walking, miss-step severity, weight bearing, and joint movement. miss-step severity was scored only on assessable walking passes, which was defined as a pass in which the animal moved three body lengths at a consistent speed and without turning (basso et al. ) . separate scores were given for the left and right hindlimbs to assess if symptoms were bilateral or unilateral. seizure-like activity noted during viral paresis scale (vps) assessments was recorded as mild if the animal had mild shakes or tremors or severe if the animal was violently seizing in the cage. mice were perfused transcardially with phosphate-buffered saline (pbs) followed by % paraformaldehyde. the brain, kidney, liver, pancreas, hindlimbs, and lumbosacral spinal column were removed and post-fixed in the same fixative overnight, rocking at °c. tissues were rinsed twice in pbs, and the lumbosacral spinal cord, sciatic nerves, and gastrocnemius muscles were isolated. all tissues except the gastrocnemius muscle on the right were cryoprotected in % sucrose in pbs for - days at °c, embedded in oct compound (ted pella), and frozen with a dry ice/ethanol bath. five sets of adjacent sections were cut on a cryostat at μm, mounted on slides, and stored at − °c until ready for further processing. for immunohistofluorescent staining, sections were encircled with a hydrophobic barrier pen (immedge, vector labs), rinsed with pbs, and blocked with % normal serum and % triton x- in pbs for h. primary antibodies were diluted in blocking solution as shown in table and applied to sections for incubation overnight at room temperature. secondary antibodies conjugated to alexa- , alexa- , or alexa- (from invitrogen or jackson immunoresearch) were diluted to μg/ml in blocking solution. sections were rinsed three times in pbs, incubated with secondary antibody solution for h at room temperature, rinsed three times in pbs, incubated with hoechst (invitrogen, / in pbs with . % triton x- ), and rinsed twice in pbs. coverslips were mounted with fluoromount g (southern biotech). for cd labeling, the amount of triton x- was decreased to . % in the blocking solution and . % in the antibody diluent. gastrocnemius muscles from the right side were sectioned longitudinally at μm on a vibratome ( plus, vibratome co.), collected in pbs, and stored at °c. ten to sections were obtained for each muscle, and the third or fourth and seventh or eighth sections were chosen for neuromuscular junction (nmj) and neurofilament (nf-h) staining as described in itoh et al. ( ) . briefly, sections were incubated free floating in . m glycine in pbs, ph . for min; blocked with % donkey serum (jackson immunoresearch laboratories), . % triton x- , and % bsa in pbs containing tetramethylrhodamine (tmr)-conjugated alphabungarotoxin (α-btx) for h; permeablized with % methanol at − °c for min; rinsed with pbs with . % triton x- (pbst); and incubated with primary antibody diluted in % bsa and . % triton x- in pbs at °c for h. after rinsing in pbst three times, sections were incubated at room temperature for h with secondary antibody (anti- correlation between highest vps score and seizure severity for zikvinfected animals. each dot represents one animal. the line is the best fit line if , , and are used for seizure categories of none, mild, and severe, respectively. slope is not significantly different from zero chicken conjugated to alexa fluor® , / , jackson immunoresearch laboratories) diluted in pbs. after rinsing in pbst three times, sections were dehydrated through a methanol series ( min each in , , , %), cleared with benzyl alcohol/benzyl benzoate ( : ) (zukor et al. ) , and stored at °c until ready to image. fluorescent images were obtained at × or × with a laser scanning confocal microscope (zeiss, lsm ) equipped with , , , and laser lines. for images taken for pixel-based quantification, identical settings were used for all images in a set. for images chosen for publication, distracting artifacts were removed in imagej (schneider et al. ) and levels were adjusted in photoshop to maximize the signal-to-noise ratio so that relevant features could be seen more clearly. for images chosen to highlight pixel-based quantification, sham and zikv group images were adjusted identically to enable equitable comparison. imagej or fiji was used for image quantification (schneider et al. ). the cell counter plugin was used for manual cell counts. for pixel-based quantification of a given antibody pab polyclonal antibody, mab monoclonal antibody a summary of primary antibodies and dilutions used signal, the area to be measured was defined by a region of interest (roi); then, thresholds of single-channel images were adjusted to select pixels with signal above noise (positive pixels). thresholds of all images in a set were adjusted identically for equitable comparison. then, positive pixels within the whole area roi were measured to obtain the area occupied by positive pixels. the area of positive pixels was divided by the whole area of the main roi to determine what proportion of the roi was positive for the antibody. for co-localization analysis, pixels that were positive for two antibody signals were measured. sagittal sections of the brain containing motor cortex were identified based on the morphology of the lateral ventricle about mm lateral of bregma (paxinos and franklin ) . a × μm region containing the pyramidal cell layer of the motor cortex, in a region of the motor cortex just dorsal to the lateral ventricle, was measured for pixel-based quantifications (see box in fig. (d) for location of the region measured). two sections per animal were measured for each parameter. for analysis of zikv in the hippocampus, one section per animal was measured, and the area measured corresponded to pyramidal cell layer (see fig. (b) for location of region measured). spinal cord levels were identified using the mouse spinal cord atlas (watson et al. ) and the location and morphology of clusters of choline acetyltransferase (chat) positive neurons. because motor neurons for the gastrocnemius muscle are located at the l -l level (mchanwell and biscoe ) , sections from this level were chosen for analysis when possible. for ventral motor neuron, astrocyte, and zikv infection level analyses, two sections per animal were analyzed. zikv−, chat+ and zikv+, chat+ neurons in the ventral horns were counted manually using the cell counter in imagej. because there were occasionally zikv+ cells in the ventral horns with neuronal morphology that had low or undetectable chat levels ( fig. (b -b )), these cells were also counted as ventral motor neurons. for analysis of ly g (neutrophils), cd (t cells), and iba (microglia/macrophages) signals, one section per animal was analyzed. for the nerve cross sections, a stereological counting grid containing × μm boxes where each box was spaced μm from the boxes above and below it was placed over the nerve to obtain counts from a sample of the nerve, representing approximately one fourth of the total area. an roi was placed around the whole nerve, and the counting grid was cut to eliminate all areas of the grid outside of the nerve (see fig. a ). stereological counting rules were used to count the number of myelinated axons, unmyelinated axons, and empty myelin sheaths within the boxes of the cut grid. images were converted to composite images, so the neurofilament and myelin basic protein (mbp) channels could be toggled on and off during counting. all counts were done by a researcher who was blind to the status of each animal. the area of the cut grid as well as the area of the whole roi were calculated and used to extrapolate the number of myelinated axons, unmyelinated axons, and empty myelin sheaths within the whole area. three sections per animal were analyzed. for nmj analysis, two sections per animal were analyzed. confocal z-stacks (~ -μm step size) of two optimal fields of view containing nmj endplates (α-btx+) and nerve fibers (nf-h+) were analyzed per section for a total of four fields of view per animal. the number of endplates and the number of innervated endplates (co-localized with some neurofilament signal) were counted manually with the cell counter in imagej. images were converted to composite images, so the nf-h and α-btx channels could be toggled on and off during counting. total numbers of innervated nmjs (nf-h+, α-btx+) and all nmjs (α-btx+) from all four fields analyzed were used to calculate the percentage of nmjs that were innervated per animal. cmap measurements were performed by a researcher who was blind to the infection status and vps score of each mouse. animals were anesthetized with isoflurane and maintained at °c body temperature with a rectal probe and heating pad, and their hindlimbs were shaved. monopolar needle electrodes (tai-chi brand, acupuncture needles) were inserted into the sciatic notch or epidural to the lumbosacral spinal cord (after surgical exposure) to stimulate the gastrocnemius muscle with . -ms pulses of current using a stimulus isolator (wpi isostim a ). for lumbosacral spinal cord stimulation, monopolar needle electrodes (biopac systems, el ) were used, and the anode was inserted between the t and l vertebrae, and the cathode was inserted between the l and l vertebrae (basoglu et al. ; harrison et al. ) . no laminectomy was necessary. muscle responses were recorded with -mm-diameter shielded ag/agcl surface recording electrodes (el s, biopac system) filled with electrode gel. electrodes were placed on the skin at the belly of the muscle and at the anterior aspect of the ankle and connected to a differential amplifier (dam , wpi) with a gain of × and filtered with a - k hz band pass filter. data were acquired at a k/s sampling rate with powerlab / and labchart software (adinstruments). responses to five pulses at hz were averaged, and the current was increased incrementally until a maximum amplitude was reached. maximal cmap amplitudes were measured from peak to peak of the m wave. for experiments with α-btx, uninfected, male ag mice, about . months of age, were used. after finding the stimulation current that gave the maximal cmap response, μl of . , . , or . μm α-btx (biotium, inc., # - ) was injected into the gastrocnemius muscle (with the gel pads kept in place) with a g needle (nakanishi et al. ) . cmap responses after maximal stimulation current were recorded at intervals for up to h after α-btx injection. dilutions of α-btx were made with saline, and thus, a μl saline injection was used in the control. data were graphed and analyzed with prism (graphpad software, inc.) for statistical significance using t tests or two-way anovas with post hoc t tests. linear regression was used for correlation analyses. experiment no. disease and behavioral motor deficits zikv-infected animals began losing weight by dpi and started succumbing to the disease by dpi (fig. ) . by dpi, all zikv-infected animals had died or been humanely euthanized. the onset of hindlimb motor deficits in zikv-infected mice ranged from to dpi (fig. a, b) . initial symptoms included tail weakness (tail not in the up position during walking) and subtle hindlimb lateral skidding and weakness (as indicated by a limp or wobble). disease progressed quickly after the onset of motor deficits and morbidity occurred within - days. the severity of motor deficits generally increased rapidly, with vps scores reaching as high as or before death (hindlimb dragging with little to no joint movement). mice were grouped into early ( - dpi) and late ( - dpi) onset groups in fig. c , d so that scores from later onset mice would not mask the progression of disease in the early onset mice. deficits were more severe on the right side compared with the left, corresponding to the side of virus inoculation. forelimbs were rarely affected. seizure-like activity was also observed in zikv-infected mice (fig. ) . six of zikv-infected mice had some seizurelike activity, and mice were scored as severe (fig. a) . seizure-like activity did not correlate with vps score (fig. b) . half of the mice in which severe seizure-like activity was noted had no to only subtle hindlimb deficits after seizure activity had subsided. additionally, there were mice with complete paralysis (vps score of ) that were never observed to have seizure-like activity. two other disease phenotypes, walking in circles and extreme mobile activity (not shown), were also observed (data not shown). interestingly, the severity of hindlimb paralysis and seizure-like activity did not correlate well with changes in body weight ( supplementary fig. s ), suggesting that mechanisms affecting neuroinvasion might operate independently from those affecting systemic infection and body weight changes. initial histopathologic analysis of our relatively thick, frozen sections with hematoxylin and eosin staining was considered to be preliminary. nevertheless, the following preliminary results were evidence of mild to moderate, multifocal encephalitis in the brain; moderate to severe, multifocal myelitis in the spinal cord; and no lesions in the gastrocnemius muscle or sciatic nerve (data not shown). to further identify anatomical features that might be associated with motor deficits, we performed immunohistofluorescent analyses of the motor cortex, lumbosacral spinal cord, sciatic nerve, and gastrocnemius muscle. the kidney, liver, and pancreas were collected to assess systemic infection level. using a polyclonal antibody against zikv envelope glycoprotein, zikv immunoreactive (ir) was seen in all parts of the adult ag mouse brain, including motor cortex, cerebellum, hippocampus, and brainstem ( fig. (a) ). in the motor cortex, zikv infects both neurons and astrocytes (arrows in fig. (e -e ) ), and many of the neurons have the morphology and location of layer v pyramidal neurons which are upper motor neurons that project to the spinal cord ( fig. (e and e -e ) ). quantification of a region of the motor cortex containing layer v (shown in box in fig. (d)) revealed that - % and up to - % of the area were zikv ir in zikv-infected mice (fig. (l) ). additionally, - % of astrocytes in this area were zikv ir ( fig. (m)), as measured by pixel-based quantification. there were also significant increases in astrogliosis ( fig. (e, e -e , and p)), t cell infiltration ( fig. (g, g , and q)), and neutrophil infiltration ( fig. (i, i , and r)) in the zikvinfected motor cortex compared to sham infected ( fig. (d, f, and h) ). interestingly, microglia and macrophages were virtually undetectable in the zikv-infected ag mouse brain (fig. (k and s) ), though resting microglia were apparent in sham-infected brains (fig. (j and j ) . the pyramidal cell layer of the hippocampus was also strikingly zikv ir in many zikv-infected mice ( fig. (c) ). quantification of this region (outlined in fig. (b) ) demonstrated that - % of this layer was zikv ir in three of eight mice (fig. (n) ). because of the association between the hippocampus and seizure activity, the severity of seizure-like activity was plotted against hippocampus pyramidal cell layer zikv infection levels, but no correlation was found (fig. (o) ). likewise, no correlation was found between motor cortex infection level and seizure-like activity (data not shown). zikv ir was also abundant in the lumbosacral spinal cord which contains lower motor neurons that innervate the hindlimbs (fig. (a-d) ). overall, - % of the crosssectional area was zikv ir (fig. (k) ). as with the brain, astrocytes (arrowheads in fig. (d -d and m) ) and neurons were infected. using a chat antibody to label ventral motor neurons, many were found to be zikv ir (arrows in fig. (b -b ) ) and a few ventral cells with clear neuronal morphology were strongly zikv ir, but only weakly chat ir (asterisks in fig. (b -b ) ). as many as - % of ventral motor neurons were zikv ir (fig. (l) ), which suggests that zikv can infect and replicate in lower motor neurons in the spinal cord. the number of motor neurons at the l -l and s levels was also diminished in many zikv-infected mice (fig. (n and o) ), but the difference from sham-infected mice did not reach statistical significance, likely because the disease progressed so rapidly. as in the brain, astrogliosis (fig. (c, d, and p) ), t cell infiltration (fig. (e, f, f , and q) , and neutrophil infiltration (fig. (g, h, h , and r) were markedly increased in zikvinfected mice compared to sham-injected mice. conversely, microglia and macrophages were markedly reduced or absent (fig. (j and s) ) in these zikv-infected ag mice, as observed in the brain, though resting microglia were evident in sham-infected spinal cords (fig. (i and i ) . zikv ir reactivity was detected in cross sections of the sciatic nerve ( fig. c-e) ; however, this did not appear to have a statistically significant effect on the number of myelinated axons (fig. a , b, f), unmyelinated axons (fig. g) , or empty myelin sheaths (fig. h) . gastrocnemius muscle, kidney, liver, and pancreas zikv ir and neutrophil infiltration were not detected in the gastrocnemius muscles of zikv-infected mice (data not shown). infection of lower motor neurons in the spinal cord and their axons in the sciatic nerve did not appear to lead to a statistically significant loss of nmj number or innervation (fig. ) . no zikv ir was detected in the kidney, liver, or pancreas of zikv-infected mice (data not shown). to determine which anatomical features might be correlated with hindlimb motor deficits, we used our data to make preliminary correlations between histological parameters of the spinal cord (fig. a-f ), motor cortex ( fig. g-k) , nmj (fig. l) , and sciatic nerve (fig. m) and vps scores. statistically significant correlations were only found with spinal cord parameters. increased numbers of zikv-infected ventral motor neurons in the lumbosacral spinal cord are associated with higher vps scores (fig. a , p < . ), and higher vps scores are associated with decreased survival of spinal motor neurons (fig. c, p < . ) . these data suggest that zikv infection of lower motor neurons and lower motor death adversely affects hindlimb function. interestingly, the level of neutrophil infiltration was inversely proportional to vps score (fig. f , p < . ), which suggests that the presence of neutrophils might mitigate motor deficits. to get a preliminary indication of how neutrophil invasion and spinal motor neuron viral infection are related to spinal motor neuron survival (as opposed to vps score), correlation graphs with these parameters were prepared as well and similar trends were observed (supplemental fig. s ). vps scores did not appear to correlate with virus levels in the motor cortex (fig. g , h) or with sciatic nerve infection levels (data not shown). overall, these results suggest that hindlimb motor deficits in these ag mice during the acute phase of zikv infection are likely caused primarily by spinal cord myelitis, though upper motor neuron disease and peripheral neuropathy may contribute. experiment no. reproduced the disease and motor phenotypes seen in experiment no. . zikv-infected animals began losing weight by dpi and started succumbing to the disease by dpi (supplemental fig. s ). all zikv-infected mice had died or been humanely euthanized by instead of dpi. vps assessments were done less frequently, but symptoms appeared at a similar timing and progressed at a similar rate (supplemental fig. s ). when mice in experiment no. had vps scores ≥ , gastrocnemius muscle cmaps were recorded in response to stimulation at the sciatic notch, followed by stimulation of the lumbosacral spinal cord to further delineate the cause of the hindlimb motor deficits. we reasoned that since motor deficits progress quickly after symptom onset and many motor neurons, though infected, have not died by the time severe deficits occur, axons and nmjs may still be healthy even if the neuronal cell body is sick. thus, cmap amplitudes may be normal in response to sciatic notch stimulation, but decreased upon lumbosacral spinal cord stimulation. responses to stimulation at these two sites, therefore, might distinguish between myositis/neuritis and myelitis. because cmap measurement in response to lumbar spinal cord stimulation was an invasive procedure, pre-infection baselines were only obtained in response to sciatic notch stimulation. figure a shows the cmap amplitudes for each animal pre-infection and post-infection in response to stimulation at the two sites. there are no statistically significant differences between groups when these babsolute^values are used. the use of relative values, however, are more compelling, and may be necessary because individual differences in the thickness and composition of tissue between the surface of the skin and the muscle can affect cmap amplitudes (nordander et al. ) . when post-infection cmap amplitude was expressed relative to pre-infection amplitude (both in response to sciatic notch stimulation), there was no statistically significant difference between the zikv-infected and sham-infected groups (fig. b , bpre minus post, at notch^), suggesting that the axons and nmjs are healthy after infection and motor deficits are not due to myositis or neuritis. when post-infection cmap amplitude in response to stimulation of the spinal cord was expressed relative to the amplitude in response to stimulation at the sciatic notch, however, there was a statistically significant relative decrease in amplitude in the zikv-infected group (fig. b , bpost, at s. cord minus notch,^p < . ). this suggests that, after infection and at the time motor deficits are observed, the axons and nmjs are healthy enough to generate a normal cmap response when axons are stimulated at the sciatic notch, but the neuronal cell bodies in the spinal cord are impaired and less able to generate normal cmap responses when stimulated at the lumbosacral spinal cord. this indicates that myelitis, rather than myositis or neuritis, contributes to the motor deficits. the fact that vps score did not correlate with the relative cmap amplitude in response to spinal cord stimulation (fig. c) , however, suggests that other factors also contribute to hindlimb motor deficits. to validate that the cmap indeed reflected muscle activity, and not just nerve activity, an inhibitor of the nmj nicotinic acetylcholine receptor, α-btx, was injected into the gastrocnemius muscles of uninfected adult ag mice, and cmaps were recorded for up to h after injection. cmap amplitudes decreased in a dose-dependent manner over the course of the h after α-btx injection, whereas cmap amplitude in the saline-injected animal was relatively stable (fig. ) . these data suggest that the cmap reflects mostly muscle rather than nerve activity, and thus, cmap measured the health status of all structures between the point of stimulation and the muscle (including the nerve and nmj). this study demonstrates that a contemporary strain of zikv (prvabc ) can widely infect astrocytes and neurons in the brain and spinal cord of adult ag mice and cause rapidly progressing hindlimb paralysis, as well as severe seizure activity, during the acute phase of disease. motor deficits in these circumstances appear to be primarily due to myelitis and possibly encephalitis as opposed to a peripheral neuropathy or gbs-like syndrome. this is an important new finding because the most severe histopathologies previously reported in ag mice were in the brain and muscle (aliota et al. ) . the finding that myelitis is likely the primary cause of zikv-induced paralysis is also in alignment with what has been seen with other flavivirus-induced motor deficits (sejvar et al. ; solomon et al. ). this conclusion is also supported by our data suggesting that muscle and peripheral nerve functions are normal. electrophysiological cmap amplitude measurements of sciatic nerve and sural muscles were normal in response to stimulation at the sciatic notch. additionally, the anatomy of the sciatic nerve and gastrocnemius muscle, as revealed by neurofilament/mbp and neurofilament/nmj staining, respectively, was minimally disrupted. in contrast, zikv ir was high, intense, and obvious in the brain and spinal cord. the severity of hindlimb motor deficits correlated with increased numbers of zikv-infected lumbosacral spinal motor neurons and decreased numbers of spinal motor neurons. relative cmap amplitudes upon stimulation at the lumbar spinal cord were also reduced when obvious motor deficits were present (vps score ≥ ). zikv infections in the brain and spinal cord were also associated with astrogliosis as well as t cell and neutrophil infiltration. thus, acute zikv infection of adult ag mice may be a useful model for studying the mechanisms of zikv-induced myelitis (anaya et al. ; dirlikov et al. ) , encephalitis, and seizure activity. the results of this study may be clinically relevant, because there have been several reports of myelitis during recent zikv outbreaks. in the february outbreak in guadeloupe, a -year-old girl developed hemiparalysis that was associated with detection of zikv in the cerebrospinal fluid. magnetic resonance imaging revealed extensive lesions in the cervical and thoracic spinal cord (mecharles et al. ) . investigators reporting on zikv-associated gbs in puerto rico noted cases of neurologic disorders other than gbs, of which were (dirlikov et al. ) . in a case-controlled study in colombia from a national population-based surveillance system (anaya et al. ) , patients with zikv-associated gbs were compared to matched controls. thirteen of these patients were reported to have other neurological syndromes: three with myelitis, three with peripheral facial palsy, six with transverse myelitis, and one with thoraco-lumbosacral myelopathy. the observation that zikv can infect and kill spinal motor neurons and that this correlates with hindlimb motor deficits is a unique finding of this report and may be clinically important. pathology reports of human zikv infection are limited, but do reveal neuronophagia in the brain , which suggests that zikv may be capable of eliciting neuron death in human neurological tissues as well as in the ag mouse. zikv infection has previously been reported in the spinal cords of ag mice (julander et al. ; zmurko et al. ) , and zikv-induced histopathology or myelitis has been observed in ifnar −/− (lazear et al. ) and swiss webster mice infected as newborns (fernandes et al. ) . the zikv-induced motor deficits in ag mice are reminiscent of motor deficits caused by other flaviviruses, such as wnv, in immunocompetent mice and hamsters (morrey et al. ; morrey et al. ; shrestha et al. ; siddharthan et al. ; xiao et al. ; zukor et al. ) . these flavivirus models also demonstrate virus infection and killing of spinal motor neurons together with mild to severe signs of motor impairment (zukor et al. ) . the vast majority of adults infected with zikv do not develop severe neurological disease such as encephalitis, yet the ag mice of this study are immunodeficient and develop encephalitis and myelitis. as such, the ag mouse model is probably more relevant to infection of immunocompromised patients. some clinical reports of infected immunocompromised patients describe severe neurological complications (henry et al. ; schwartzmann et al. ) . for example, an immunosuppressed heart transplant patient developed acute neurological impairment and mental deterioration after zikv infection, which culminated in death. autopsy revealed zikv infection of the brain and cerebral spinal fluid by rt-pcr, immunohistochemistry, and electron microscopy. the histopathology of the brain was consistent with meningoencephalitis (schwartzmann et al. ) . the fact that relative cmap amplitude after spinal cord stimulation did not statistically correlate with vps score suggests that damage upstream of spinal motor neurons may contribute to the hindlimb deficits. for example, cmap amplitude was not reduced in two infected mice with overt paralysis (vps score - , fig. c ). paralysis in these animals could be caused by motor cortex, cerebellum, or brainstem dysfunction, as extensive infection was observed in these areas of the brain. while upper motor neuron disease is associated with rigid, rather than flaccid paralysis, we occasionally saw symptoms that could be interpreted as rigidity, such as walking with high haunches and extended/stiff limbs, but it was difficult to reliably assess this with the vps test. another possible explanation for the paralysis in animals with normal relative cmap amplitudes is that spinal motor neuron infection damages dendrites and post-synaptic densities such that the neurons are not able to receive signals physiologically, but when stimulated externally with an electrode, the axon hillock and axon are healthy enough to propagate an action potential to the muscle. the observation of seizure-like activity in infected ag mice in this study and in immunocompetent mice infected as neonates in another study (manangeeswaran et al. ) may have clinical relevance since recent zikv infections have been associated with seizures in humans (asadi-pooya ; pastula et al. ) . notably, the seizure-like activity in this study was not correlated with the vps score, which suggests that the seizure activity was not simply a manifestation of motor deficits. to further investigate the relevance, seizures would need to be confirmed in zikv-infected mice with more detailed analyses. motor deficits seen in this study are not likely to be the result of peripheral neuropathy. while zikv ir was observed in the sciatic nerve, axon and myelin morphology did not appear to be altered. zikv also did not appear to alter the function of the sciatic nerve, which is supported by the observation that gastrocnemius cmap amplitudes in response to sciatic notch stimulation were not affected. the observation of viral antigen in axons is consistent with prior findings that four families of viruses, including flaviviruses, can undergo axonal transport and spread in the nervous system (samuel et al. ; taylor and enquist ; wang et al. ), as well as the finding that zikv appears to be able to travel in axons (van den pol et al. ). we were not able to determine what effect zikv might have on the development of peripheral neuropathies, such as gbs, in this study because the mice die during the acute infection and peripheral neuropathies tend to develop in chronic stages. in the current study, inflammatory cellular responses, such as astrogliosis, neutrophil infiltration, and t cell infiltration, were present in the brain and spinal cord. the possible influence of each of these responses to disease severity, whether causative or protective, is explored below. future work should comprehensively establish correlation with larger sample sizes and then determine which relationships are causative or protective, so that the cellular mechanisms underlying zikv-induced motor deficits can be understood. results from this study suggest that neutrophils play a role in mitigating or protecting zikv-infected ag mice from motor deficits, probably by enhancing immune responses that reduce viral load. increased numbers of neutrophils, as detected by the ly g antibody, were inversely correlated with vps score (p < . ). studies with wnv demonstrate that neutrophils can play complex roles during infection. they appear to be protective early in wnv infection, but detrimental later. when neutrophils were depleted before wnv challenge in c h/hej mice, mortality increased, but when they were depleted after wnv challenge, mortality decreased (bai et al. ) . another level of complexity regarding the role of neutrophils involves mosquito bites. when neutrophils are depleted and inflammasome activity is blocked, the ability of mosquito bites to promote infection and inflammation is abrogated (pingen et al. ) . therefore, the role of neutrophils in zikv-induced disease should be further investigated. the drastic reduction in iba ir (a marker for microglia and macrophages) in the brains and spinal cords of ag mice infected with zikv was a unique finding. it could mean that microglia and macrophages are present, but the iba marker is strongly downregulated, or it could mean that microglia are killed and macrophage infiltration is inhibited. after wnv infection in c bl mice, microglia and macrophages are strongly activated (zukor et al. ) . they are also strongly activated in wild-type c bl or s /svimj mice infected with zikv intracerebrally at e . (shao et al. ) . thus, the decrease in iba ir after zikv infection in our study might be unique to the ag mouse strain and could indicate a role for microglia and macrophages in protecting against lethal zikv infection. for example, m macrophages elicit proinflammatory responses and enable phagocytosis and killing of pathogens, and m macrophages function in protective and homeostasis of the cns (kabba et al. ) . a certain balance of these functions could be overall protective. to further elucidate immunological cellular responses, decreased iba ir cells and modulation of other immune cells should be validated with flow cytometric analysis in future studies. t cell infiltration was associated with zikv infection in the brain and spinal cord, though there was no correlation between the level of infiltration and the severity of motor deficits. this could indicate that t cells play both beneficial and detrimental roles (ousman and kubes ; prinz and priller ) . for example, cd (+) t cells contribute to resolution of wnv neurological infection by helping to clear wnv from neurons (mccandless et al. ; shrestha et al. ) . t cells also play beneficial roles in la crosse virus infection (winkler et al. ) . t cells, however, are the cause of fatal meningoencephalitis after tacaribe arenavirus infection (ireland et al. ) . future studies will delineate the role of t cells in the development of motor deficits in zikv-infected ag mice. astrogliosis in the spinal cord was strongly associated with zikv infection, and many astrocytes were infected by the virus. as astrocytes regulate synapse formation, function, and elimination (chung et al. ) and control glutamate excitotoxicity (murphy-royal et al. ) , this likely had a significant effect on motor neuron health and function. in a model of human coronavirus, expression of the primary glutamate transporter in the adult cns, glt- , was decreased in astrocytes. this was associated with flaccid hindlimb paralysis even in the absence of significant neuronal death. blockade of -amino- -( -methyl- -oxo- , -oxazol- -yl) propranoic acid (ampa) receptors with a non-competitive antagonist improved hindlimb function (brison et al. ) . in mouse models of experimental autoimmune encephalomyelitis, astrocytes are implicated in the temporary stripping of excitatory synapses from motor neurons in the spinal cord, leading to severe flaccid paralysis in the absence of neuronal cell death (blakely et al. ; marques et al. ). astrogliosis may not have correlated with vps scores in our studies because it may precede and be required for the onset of motor deficits (blakely et al. ) . in two independent experiments, female mice were found to die slightly earlier than male mice, though this was not statistically significant. other zikv studies with ag mice at our institute, however, suggest that males and females are equally susceptible (data not shown). additionally, experiment no. appeared to have two distinct groups of mice with an early versus late onset of symptoms, though this separation was not distinct in experiment no. . we interpret these trends (greater female susceptibility and two phases of disease onset) to be the result of stochastics and small sample sizes. the appearance of two onset groups in experiment no. occurred by chance because we did not sample mice with an intermediate onset of symptoms. the unique contributions of this study are that acute zikv infection of adult ag mice causes quantifiable, rapidly progressing hindlimb motor deficits that often culminates in full paralysis and that these deficits are likely due to myelitis and perhaps encephalitis rather than peripheral neuropathy or myositis. extensive histopathology and infection occur in the spinal cord and brain, but not in the sciatic nerve or muscle. the severity of motor deficits is only correlated with infection and survival levels of lumbosacral spinal motor neurons. moreover, gastrocnemius cmap amplitudes upon electrical stimulation of the lumbosacral spinal cord were impaired, but not upon stimulation of the sciatic nerve. these data should guide approaches for understanding zikv-induced acute myelitis or encephalitis in adults. denv inhibits type i ifn production in infected cells by cleaving human sting characterization of lethal zika virus infection in ag mice a comprehensive analysis and immunobiology of autoimmune neurological syndromes during the zika virus outbreak in cucuta, colombia zika virus-associated seizures a paradoxical role for neutrophils in the pathogenesis of west nile virus assessment of in vivo spinal cord conduction velocity in rats in an experimental model of ischemic spinal cord injury basso mouse scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains the many faces of the flavivirus ns protein in antagonism of type i interferon signaling astrocyte matricellular proteins that control excitatory synaptogenesis are regulated by inflammatory cytokines and correlate with paralysis severity during experimental autoimmune encephalomyelitis zika virus antagonizes type i interferon responses during infection of human dendritic cells guillain-barre syndrome associated with zika virus infection glutamate excitotoxicity is involved in the induction of paralysis in mice after infection by a human coronavirus with a single point mutation in its spike protein guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study outbreak of exanthematous illness associated with zika, chikungunya, and dengue viruses zika virus associated with meningoencephalitis do glia drive synaptic and cognitive impairment in disease zika virus. ii. pathogenicity and physical properties guillain-barre syndrome during ongoing zika virus transmission-puerto rico experimental zika virus infection induces spinal cord injury and encephalitis in newborn swiss mice vertebral landmarks for the identification of spinal cord segments in the mouse a quick phenotypic neurological scoring system for evaluating disease progression in the sod -g a mouse model of als chorioretinal lesions presumed secondary to zika virus infection in an immunocompromised adult cd and cd t cells mediate distinct lethal meningoencephalitis in mice challenged with tacaribe arenavirus zpk/dlk, a mitogen-activated protein kinase kinase kinase, is a critical mediator of programmed cell death of motoneurons efficacy of the broad-spectrum antiviral compound bcx against zika virus in cell culture and in a mouse model microglia: housekeeper of the central nervous system a mouse model of zika virus pathogenesis zika (prvabc ) infection is associated with t cell infiltration and neurodegeneration in cns of immunocompetent neonatal c bl/ mice spinal motoneuron synaptic plasticity during the course of an animal model of multiple sclerosis cxcr antagonism increases t cell trafficking in the central nervous system and improves survival from west nile virus encephalitis the localization of motoneurons supplying the hindlimb muscles of the mouse acute myelitis due to zika virus infection modeling hamsters for evaluating west nile virus therapies west nile virus-induced acute flaccid paralysis is prevented by monoclonal antibody treatment when administered after infection of spinal cord neurons astroglial glutamate transporters in the brain: regulating neurotransmitter homeostasis and synaptic transmission regulation of motoneuron excitability via motor endplate acetylcholine receptor activation guide for the care and use of laboratory animals. the national academies press influence of the subcutaneous fat layer, as measured by ultrasound, skinfold calipers and bmi, on the emg amplitude immune surveillance in the central nervous system zika virus disease for the neurointensivist host inflammatory response to mosquito bites enhances the severity of arbovirus infection the role of peripheral immune cells in the cns in steady state and disease characterization of a novel murine model to study zika virus concern over zika virus grips the world axonal transport mediates west nile virus entry into the central nervous system and induces acute flaccid paralysis nih image to imagej: years of image analysis possible association between zika virus infection and microcephaly-brazil viral evasion strategies in type i ifn signaling-a summary of recent developments zika virus meningoencephalitis in an immunocompromised patient acute flaccid paralysis and west nile virus infection zika virus infection disrupts neurovascular development and results in postnatal microcephaly with brain damage infection and injury of neurons by west nile encephalitis virus cd + t cells use trail to restrict west nile virus pathogenesis by controlling infection in neurons persistent west nile virus associated with a neurological sequela in hamsters identified by motor unit number estimation zika virus disease for neurologists fatal encephalitis associated with zika virus infection in an adult poliomyelitis-like illness due to japanese encephalitis virus neuropathology of zika virus infection protective efficacy of zika vaccine in ag mouse model axonal spread of neuroinvasive viral infections antiviral defense in mice lacking both alpha/beta and gamma interferon receptors zika virus targeting in the developing brain west nile virus preferentially transports along motor neuron axons after sciatic nerve injection of hamsters atlas of the mouse spinal cord development and characterization of recombinant virus generated from a new world zika virus infectious clone lymphocytes have a role in protection, but not in pathogenesis, during la crosse virus infection in mice west nile virus infection in the golden hamster (mesocricetus auratus): a model for west nile encephalitis the viral polymerase inhibitor -deaza- ′-cmethyladenosine is a potent inhibitor of in vitro zika virus replication and delays disease progression in a robust mouse infection model fluorescent whole-mount method for visualizing three-dimensional relationships in intact and regenerating adult newt spinal cords phrenic nerve deficits and neurological immunopathology associated with acute west nile virus infection in mice and hamsters acknowledgements we thank the following individuals from the institute for antiviral research at utah state university for their excellent technical assistance: mitchell stevens for help with vps scoring; brent hunter for help with immunohistofluorescent staining; christian morrill for help with image quantification; heidi julander for help with ag colony management; rich albrechtsen for help with setting up the cmap assays; skot neilson for overseeing animal studies; jared bennet, jason fairbourn, john mcclatchy, devin pfister, jean marie maxwell, taylor redding, rachelle stanton, and trevor truex for assisting with animal studies; and neil motter for maintenance and design of electrophysiological instruments. we also thank arnaud van wettere, dvm, ms, phd, dacvp at utah state university's veterinary diagnostic laboratory for histopathologic analysis of tissues. the authors declare that they have no conflict of interest.open access this article is distributed under the terms of the creative comm ons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- -hjx d rq authors: márquez-jurado, silvia; nogales, aitor; Ávila-pérez, ginés; iborra, francisco j.; martínez-sobrido, luis; almazán, fernando title: an alanine-to-valine substitution in the residue of zika virus ns a protein affects viral rna synthesis and attenuates the virus in vivo date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: hjx d rq the recent outbreaks of zika virus (zikv), its association with guillain–barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat zikv disease. in this respect, infectious clones constitute excellent tools to accomplish these goals. however, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. to bypass this problem, several alternative approaches have been applied for the generation of zikv clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. here, we report a simple and novel dna-launched approach based on the use of a bacterial artificial chromosome (bac) to generate a cdna clone of rio grande do norte natal zikv strain. the sequence was identified from the brain tissue of an aborted fetus with microcephaly. the bac clone was fully stable in bacteria and the infectious virus was efficiently recovered in vero cells through direct delivery of the cdna clone. the rescued virus yielded high titers in vero cells and was pathogenic in a validated mouse model (a mice) of zikv infection. furthermore, using this infectious clone we have generated a mutant zikv containing a single amino acid substitution (a v) in the ns a protein that presented reduced viral rna synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with zikv wild-type. this bac approach provides a stable and reliable reverse genetic system for zikv that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies. zika virus (zikv) is a recently emerged mosquito-borne member of the family flaviviridae, which was declared by the word health organization (who) as a global public health emergency on february [ , ] . like other flaviviruses, the viral particle is constituted by an inner nucleocapsid composed of the capsid (c) protein associated with the viral genomic rna (grna), surrounded by a lipid bilayer that contains the structural membrane (m) and envelope (e) proteins, which are arranged nucleus from the cmv promoter with a second amplification step in the cytoplasm driven by the viral polymerase. the recombinant virus rescued from the bac clone was fully infectious in vitro and in vivo. the zikv-rgn infectious clone was further used to evaluate the effect of a single amino acid change (alanine to valine) at residue of the ns a protein on viral rna synthesis and pathogenesis in vivo. we found that this unique single amino acid substitution impairs viral rna synthesis in cell culture and results in viral attenuation in a mice. remarkably, a single dose of the mutant virus was sufficient to induce protection against challenge with the parental wild-type (wt) zikv. these results demonstrate the reliability and potential of our bac approach to study zikv biology and to facilitate the development of vaccine and antiviral strategies. vero (a kidney epithelial cell line from an african green monkey) and a (an human adenocarcinomic alveolar epithelial cell line) cells were purchased from the american type culture collection (atcc, ccl- ) and were grown and maintained at • c and % co in growth medium, consisting in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) (hyclone, thermofisher scientific, madrid, spain), mm l-glutamine (sigma-aldrich, madrid, spain), % nonessential amino acids (sigma-aldrich), u/ml penicillin (sigma-aldrich) and µg/ml streptomycin (sigma-aldrich). the recombinant zikv-rgn wt (rzikv-rgn) or ns a mutant (rzikv-rgn-mns a) viruses were propagated in vero cells with virus growth medium (dmem supplemented with % fbs, mm l-glutamine, % nonessential amino acids, u/ml penicillin and µg/ml streptomycin) at • c and % co . for virus stocks preparation, to % confluent monolayers of vero cells were infected with a multiplicity of infection (moi) of . plaque forming units (pfu) per cell in virus growth medium and incubated at • c under % co . after - days of infection, the tissue culture supernatants were collected, clarified by centrifugation at × g for min, and stored in small aliquots at − • c. the bac plasmid pbelobac [ ] , kindly provided by h. shizuya (california institute of technology, pasadena, ca, usa), was used to assemble the zikv-rgn infectious cdna clone. this plasmid is a synthetic low-copy-number plasmid (one copy per cell) based on the escherichia coli (e. coli) f-factor [ ] that minimize the toxicity problems in the bacteria of exogenous sequences. e. coli dh b cells (invitrogen, thermofisher scientific) were used to amplify the bac plasmids. electrocompetent dh b cells (invitrogen, thermofisher scientific) were transformed by electroporation using a micropulser unit (bio-rad, madrid, spain), according to the manufacturer's instructions. bac-based plasmids were isolated and purified using the large-construct kit (qiagen, hilden, germany), following the manufacturer's specifications. we have assembled a zikv infectious cdna clone in the bac plasmid pbelobac , based on the data of the full-length sequence of the zikv clinical strain rgn [ ] deposited in the genbank (accession number ku ). this strain was selected because the full-length sequence was obtained directly from the virus-infected brain tissue of an aborted fetus with microcephaly in brazil in and therefore represents a good candidate to study zikv pathogenesis. the first step for the assembly of the full-length cdna clone was the selection of the restriction sites pmli, afei, and bstbi (genomic positions , and , respectively), which are unique in the viral genome ( figure a ). after that, four overlapping dna fragments covering the entire viral genome (zikv to zikv ) and flanked by the appropriate restriction sites, were generated by chemical synthesis (bio basic, inc., toronto, canada) ( figure b ). zikv fragment contained the cmv promoter precisely fused to the first nucleotides of the viral genome flanked at the '-end by apali and asci (absent in the viral genome) sites and at the '-end by a multiple-cloning site containing the selected restriction sites (pmli, afei and bstbi) followed by mlui (absent in the viral genome) and bamhi. fragments zikv (flanked by pmli and afei) and zikv (flanked by afei and bstbi) covered the genomic regions - and - , respectively. zikv fragment contained the restriction site bstbi, the last nucleotides of the viral genome, the hepatitis delta virus (hdv) ribozyme, the bovine growth hormone (bgh) termination and polyadenylation sequences, and the mlui restriction site. the infectious clone was assembled into pbelobac by sequential cloning of these four overlapping dna fragments. briefly, fragment zikv was digested with apali and bamhi and cloned into pbelobac −afei (a pbelobac without the afei restriction site) digested with the same enzymes, to generate the intermediate plasmid pbac-zikv . then, this plasmid was used as the backbone for the sequential cloning of the remaining overlapping dna fragments (zikv to zikv ) into the multicloning site of the intermediate plasmid (contains the restriction sites selected, pmli, afei, bstbi and mlui) to generate the full-length cdna clone pbac-zikv-rgn ( figure b) . the genetic integrity of the cdna clone was verified throughout the assembly process by extensive restriction analysis and sequencing. in all cases, the bacterial strain dh b (invitrogen, thermofisher scientific) was used as the e. coli host for all the cloning steps and the propagation of the bac cdna clone. to recover the infectious virus, vero cells on -well plates were grown to % confluence in growth medium without antibiotics, and transfected with µg of the bac cdna clone using µl of lipofectamine (invitrogen, thermofisher scientific), following the manufacturer's specifications. after h of incubation at • c, the transfection medium was replaced with fresh growth medium and the cells incubated at • c. aliquots of the culture supernatants were collected at h intervals for virus titer determination by plaque assay on vero cells. after five to seven days of transfection, when the cytopathic effect (cpe) was clear, cell culture supernatants were harvested and the recovered virus was cloned by three rounds of plaque purification. to determine the complete genome sequence of the rescued viruses, virions from supernatant of infected vero cells (moi of . pfu/cell) were purified through a % (w/v) sucrose cushion. viral rna was isolate from the purified virus with the qiaamp viral rna minikit (qiagen) following the manufacturer's instructions and deep-sequenced at the university of rochester genomics research center using illumina miseq (illumina, san diego, ca, usa). briefly, . µg of total viral rna was fragmented by controlled sonication and a dna library was generated using the nebnext mrna library prep master mix set for illumina (new england biolabs, ipswich, ma, usa), according to the manufacturer's instructions. after analyzing the library for size and quality (bio-analyzer; agilent technologies, inc., santa clara, ca, usa), deep-sequencing was performed using miseq (illumina) and the raw sequencing reads analyzed using swarm custom software. the genomic 'and '-terminal sequences were determined by the rapid amplification of cdna ends (race) using the '/ ' race second generation kit (roche, basilea, switzerland) with a polya-tail added to the cdna prior to the ' race reaction using polya polymerase (new england biolabs), following the manufacturer's instructions. to analyze the genetic stability of the recombinant zikv harboring the point mutation a v in the coding region of the ns a protein (rzikv-rgn-mns a), total rna was purified from vero cells infected with viruses from passage (p ) to passage (p ) using the rneasy minikit (qiagen), according to the manufacturer's specifications. purified rna ( ng) was reverse transcribed (rt) with random hexamer primers using the high-capacity cdna transcription kit (life technologies, thermofisher scientific), and the cdna was amplified by pcr with the forward primer zikv- vs viruses , , of ( '-gaggaatggtgctgcagg- '), spanning nucleotides to of the viral genome, and the reverse primer zikv- rs ( '-gcttgacatctccccag- ') , complementary to nucleotides to of the viral genome. finally, the amplicons generated covering the region encoding ns a and ns b proteins (genomic region - ) were sequenced by sanger sequencing (macrogen europe, amsterdam, netherlands) using specific oligonucleotides. vero cells seeded into -well plates at - % of confluence were infected with µl of serial -fold dilutions of the virus in virus growth medium without fbs for h at • c. after viral absorption, the viral inoculum was removed and the cells overlaid with ml of virus growth medium containing % deae-dextran (sigma-aldrich) and . % agar noble (difco, thermofisher scientific). after - days of incubation at • c under % co , the cells were fixed with % formaldehyde for h at room temperature, the overlaid removed, and the viral plaques visualized by staining with . % crystal violet in % methanol or by immunostaining with µg/ml of the pan-flavivirus e protein monoclonal antibody (mab) g (bei resources; nr- ) using the vectastain abc kit (vector laboratories inc., burlingame, ca, usa). visible plaques were counted and virus titers were calculated as pfu/ml. vero and a cells seeded into -well plates at % of confluence were infected with the indicated viruses diluted in virus growth medium without fbs at the specified mois. after h of absorption at • c in % co , the virus inoculum was removed, the cell monolayers washed twice with pbs, and . ml of fresh virus growth medium was added to each well. cells were incubated at • c under % co and at selected time points, aliquots of tissue culture supernatants were collected and virus titers determined by plaque assay in vero cells as described above. viral rna synthesis was evaluated by quantitative rt-pcr (rt-qpcr). total intracellular rna from uninfected or infected vero cells was purified using the rneasy minikit (qiagen) and total cdna was synthetized from ng of purified rna using random hexamer primers and the high-capacity cdna transcription kit (life technologies, thermofisher scientific), following the manufacturer's specifications. using this cdna, the level of viral rna was further quantified by qpcr using a custom taqman assay specific for zikv-rgn rna. this taqman assay is constituted by the forward primer '-gaagagcatccagccagagaa- ' (spanning nucleotides to of the viral genome), the reverse primer '-ctgggagccatgaactgaca- ' (complementary to nucleotides to of the viral genome), and the probe '-fam-tggagtaccggataatg- iabkfq- ' (covering nucleotides to of the viral genome). to normalize for differences in rna sampling, the expression of the histone h b (reference housekeeping gene) was analyzed using a specific taqman gene expression assay (rh _s ; life technologies, thermofisher scientific). data were acquired with a real-time pcr system (life technologies, thermofisher scientific) and analyzed with abi prism software v . . . the relative quantifications were performed using the cycle threshold ( −∆∆ct ) method [ ] . all experiments and data analysis were miqe (minimum information for publication of quantitative real-time pcr experiments) compliant [ ] . the expression of zikv e protein was analyzed by indirect immunofluorescence assay (ifa). vero cells grown on coverslips in -well plates at - % of confluence were infected with the rescued rzikvs at the indicated mois. at selected time points post-infection, cells were fixed with % paraformaldehyde in mm hepes ph . during min at room temperature and then permeabilized with . % triton x- in pbs for min. after that, cells were treated for h at room temperature with blocking solution ( % fbs in pbs) and incubated with µg/ml of the pan-flavivirus e protein mab g (bei resources; nr- ) in blocking solution for h at room temperature. after three washed with pbs, cells were incubated at room temperature for h with donkey anti-mouse antibody conjugated to alexa fluor (invitrogen, thermofisher scientific) diluted : in blocking solution, extensively washed with pbs, and incubated for min with dapi ( ', '-diamidino- -phenylindole) (sigma-aldrich) diluted : in pbs for nuclear staining. finally, coverslips were mounted in prolong gold antifade reagent (invitrogen, thermofisher scientific) and analyzed on a leica sp confocal microscope. immunofluorescence acquired images were processed and analyzed with imagej . b software [ ] . for the evaluation of the virus-specific antibodies levels present in the sera of vaccinated mice, enzyme-linked immunosorbent assays (elisas) were performed as previously described [ ] . briefly, -well plates were coated with cell lysates from mock-or zikv-infected vero cells and incubated overnight at • c. the coated wells were washed with pbs, blocked with % bsa in pbs, and then incubated with two-fold dilutions (starting dilution of : ) of mice sera for h at • c. after that, plates were washed with water and incubated with hrp-conjugated goat anti-mouse igg ( : ; southern biotech, birmingham, al, usa) for h at • c. reactions were developed with tetramethylbenzidine (tmb) substrate (biolegend, san diego, ca, usa) for min at room temperature, quenched with n h so , and read at nm in a vmax kinetic microplate reader (molecular devices, san jose, ca, usa). the in vivo studies were performed in type-i interferon (ifn) receptor deficient (ifnr-/-) a mice (the jackson laboratory, bar harbor, me, usa) maintained in the animal care facility at the university of rochester under specific pathogen-free conditions. in this animal model, subcutaneous (s.c.) or intraperitoneal (i.p.) infection with zikv induces neurological disease and the animals succumb to viral infection, with high viral load in blood, brain, spin cord, and testes, consistent with manifestations of zikv infection in humans. although deficient in innate ifn responses, a mice retain their adaptive immunity and have been successfully used as a suitable model for testing antivirals and vaccines [ ] [ ] [ ] . to evaluate virus pathogenicity, female -to- -week-old a mice (n = ) were first anesthetized i.p. with a mixture of ketamine ( µg per gram of body weight) and xylazine ( µg per gram of body weight), and then mock-infected (pbs) or infected s.c. in the footpad with the indicated doses of rzikv-rgn or rzikv-rgn-mns a diluted in pbs in a final volume of µl. after viral infection, animals were monitored daily for morbidity (body weight loss and disease signs, including hunching, ruffling and hind limb paralysis) and mortality (survival) over days. mice showing more than % of body weight loss or severe paralysis were considered to have reached the experimental endpoint and were humanely euthanized. to correlate development of clinical symptoms and death with virus replication, -to- -week-old mice (n = ) were infected as described above and the viral titers in serum were determined at days (n = ) and (n = ) by plaque assay and immunostaining using the pan-flavivirus e protein mab g as indicated before. to evaluate the protection efficacy of the rzikv-rgn-mns a, female -to- -week-old a mice (n = ) were first anesthetized i.p. as indicated above, and then mock-immunized (pbs) or immunized s.c. in the footpad with pfu of rzikv-rgn-mns a diluted in pbs in a final volume of µl. at days post-immunization, mouse sera were collected by submandibular bleeding and the presence of total antibodies against zikv-rgn was evaluated by elisa. twenty-four hours after bleeding, mice were challenged s.c. in the footpad with pfu of rzikv-rgn and their morbidity and mortality monitored over days as previously described. to determine viral replication, challenged -to- -week-old a mice (n = ) were bleeding at days (n = ) and (n = ) post-challenge and viruses , , of zikv viremia was determined by plaque assay and immunostaining using the pan-flavivirus e protein mab g as previously described. for quantitative analyses, a two-tailed, unpaired student t test was used to analyze differences in mean values between groups. all results were expressed as mean ± standard deviations of the means. p values of < . were considered significant. for mice experiments, the meier log-rank test was used to compare survival data and the reed and muench method to determine the mouse lethal dose (mld ). graphpad prism v . software was used for all statistical analysis. all animal protocols were approved by the university of rochester committee of animal resources (protocol number: ucar- - / ; approval date: / / ) and complied with the recommendations in the guide for the care and use of laboratory animals of the national research council [ ] . to overcome the toxicity problems associated to several flavivirus sequences during its propagation in bacteria, we used the bac plasmid pbelobac (a single-copy plasmid derived from the e. coli f-factor) [ ] to assemble a zikv infectious cdna clone, based on the genome sequence of the rgn strain of zikv (genbank accession number ku ) [ ] (figure ). this zikv-rgn strain was selected because it has no laboratory passage history and the full-length genome sequence was obtained from a zikv-infected fetus with microcephaly in [ ] , constituting a good candidate to further study zikv pathogenesis. after appropriate selection of unique restriction sites in the zikv-rgn genome ( figure a ), four overlapping dna fragments (zikv to zikv ), spanning the full-length viral genome and flanked for the selected restriction sites, were chemically synthesized, and sequentially cloned into pbelobac to generate the infectious cdna clone pbac-zikv-rgn ( figure b ). fragment zikv contained the cmv immediate-early promoter to allow the expression of the viral rna in the nucleus by the cellular rna polymerase ii [ ] and fragment zikv was flanked at the '-end by the hdv ribozyme followed by the bgh termination and polyadenylation sequences to produce synthetic rnas bearing authentic '-ends of the viral genome. this dna-lunched system ensures capping of the viral rna and allows the recovery of infectious virus from the transfected cdna clone without the need of an in vitro transcription step. once assembled, the full-length sequence of the zikv-rgn bac clone was determined and no changes were detected to that reported for the zikv-rgn strain (genbank accession number ku ). finally, to confirm the stability of this synthetic infectious cdna clone in bacteria, the bac clone was passaged in e. coli dh b cells for more than two hundred generations and the genetic integrity of the passaged infectious clone analyzed by restriction endonuclease analysis and sequencing. no differences were detected, demonstrating that the zikv-rgn bac clone was fully stable in bacteria and that the bac approach is a reliable and simple method to generate zikv infectious cdna clones. to recover the infectious virus ( figure ), vero cells were transiently transfected with the bac cdna clone using lipofectamine and virus production analyzed during seven days. in contrast to mock-transfected cells, increasing amounts of infectious virus were detected in the tissue culture supernatant of cells transfected with the infectious clone, with peak titers around pfu/ml on day five ( figure a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of . pfu/cell of the rescue virus and monitored for cpe induction and viral e protein expression by ifa using the pan-flavivirus e protein mab g ( figure b ). the rescued virus induced a clear cpe, characterized by the presence of rounded and birefringent cells, and high levels of e protein expression were detected in the perinuclear region of infected cells. these results demonstrated that the zikv-rgn infectious bac cdna clone produces high titers of rzikv-rgn directly after transfection of susceptible vero cells. to recover the infectious virus ( figure ), vero cells were transiently transfected with the bac cdna clone using lipofectamine and virus production analyzed during seven days. in contrast to mock-transfected cells, increasing amounts of infectious virus were detected in the tissue culture supernatant of cells transfected with the infectious clone, with peak titers around pfu/ml on day five (figure a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of . pfu/cell of the rescue virus and monitored for cpe induction and viral e protein expression by ifa using the pan-flavivirus e protein mab g ( figure b ). the rescued virus induced a clear cpe, characterized by the presence of rounded and birefringent cells, and high levels of e protein expression were detected in the perinuclear region of infected cells. these results demonstrated that the zikv-rgn infectious bac cdna clone produces high titers of rzikv-rgn directly after transfection of susceptible vero cells. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure ) , groups of five female -to- -week-old a mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn ( , and pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over days ( figure a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with pfu did not show disease symptoms, only a slight reduction in body weight was detected on days to , and all of them survived. in the case of mice infected with pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of % on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure a ). using the reed & muench method we determined that the mld of rzikv-rgn was approximately × pfu. to further analyze whether the virulence observed correlated with viral replication, viral titers in mouse sera were analyzed at days two and four post-infection ( figure b ). as expected, the viremia in the infected animals was dose dependent, reaching the highest titers at day two after inoculation. at day four post-inoculation a significant reduction of the viral titers was observed. in the case of animals infected with or pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high ( pfu) dose. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure ) , groups of five female -to- -week-old a mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn ( , and pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over days ( figure a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with pfu did not show disease symptoms, only a slight reduction in body weight was detected on days to , and all of them survived. in the case of mice infected with pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of % on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure a ). using the reed & muench method we determined that the mld of rzikv-rgn was approximately × pfu. to further analyze whether the virulence observed correlated with viral replication, viral titers in mouse sera were analyzed at days two and four post-infection ( figure b ). as expected, the viremia in the infected animals was dose dependent, reaching the highest titers at day two after inoculation. at day four post-inoculation a significant reduction of the viral titers was observed. in the case of animals infected with or pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high ( pfu) dose. week-old a mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with the indicated pfu of rzikv-rgn, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during days. mice that lost more than % of their initial body weight or presented hind limb paralysis were humanely euthanized. error bars represent standard deviations of the mean for each group of mice. asterisks indicate that the differences between viral doses of and are statistically significant when data are compared using the unpaired t test (*, p < . ; **, p < . ). (b) viral titers in mice sera. female -to- -week-old a mice (six mice per group) were infected with the indicated pfu of rzikv-rgn as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus e protein mab g . symbols represent data from individual mice and bars the geometric means of viral titers. asterisks indicate that the differences in viral titers between experimental samples are statistically significant when data are compared using the unpaired t test (**, p < . ; ***, p < . ). &: virus not detected in two mice; nd: virus not detected. the detection limit of the assay ( pfu/ml) is indicate as a dashed line. during the assembly of the pbac-zikv-rgn infectious clone, we detected the presence of a point mutation in the ns a protein, which was introduced during the chemical synthesis of fragment zikv . this mutation consists in a cytosine-to-thymidine substitution at genomic position , resulting in an alanine-to-valine change in the residue of the ns a protein (a v). because this mutation consists of a conservative amino acid change, we decided to explore the possibility of using this mutation as a genetic marker. to this end, the infectious clone pbac-zikv-rgn-mns a was generated by replacing the zikv wt fragment for that containing the ns a a v mutation. this infectious clone was fully stable in bacteria and no additional mutations were observed after sequencing the full-length clone. after that, vero cells were transfected with the mutant infectious clone and the recovery efficiency of the rzikv-rgn-mns a mutant virus was compared to that of the parental rzikv-rgn virus ( figure ). although the infectious virus was recovered in both cases, virus production was one logarithm lower in the case of the mutant rzikv-rgn-mns a, reaching maximum titers of pfu/ml at seven days post-transfection ( figure a ). when the plaque phenotype was analyzed, we found that the plaque size of the mutant rzikv-rgn-mns a was smaller (more than a -fold reduction) than that of the parental rzikv-rgn ( figure b ), indicating that the a v mutation, despite of being a conservative substitution, caused reduction in plaque size and virus production. in addition, an in silico analysis was performed to evaluate the frequency of amino acid residues of the ns a protein in more than zikv strains sequences deposited in the database [ ] (https://www.viprbrc.org/brc/home.spg?decorator=flavi). this analysis indicated that amino acid a is highly conserved, since the % of the analyzed zikv sequences contained an alanine residue at this position. to further confirm the effect of the ns a a v mutation on virus production, the growth kinetics at high ( pfu/cell) and low ( . pfu/cell) moi of the mutant virus were compared to those of the parental virus ( figure c ). again, a reduction of about one logarithmic unit in virus production was detected in vero cells infected with the mutant virus both at high and low moi ( figure c ). taken into consideration that flavivirus ns a protein is involved in regulation of rna replication and virus assembly [ ] , we further analyzed whether the reduction in plaque size and virus production of the mutant virus was associated with reduced viral rna synthesis. to this end, the production of viral rna in vero cells infected with either the parental or mutant viruses at an moi of . pfu/cell was analyzed at and hpi by rt-qpcr using a custom taqman assay specific for zikv-rgn genome ( figure d ). at both times, a -fold reduction in the levels of viral rna was observed in cells infected with the mutant virus ( figure d ), confirming that ns a a v mutation at least impairs viral rna synthesis. in agreement with these data, a reduction in the expression levels of zikv e protein was observed by ifa in vero cells infected with the mutant virus in comparison to cells infected with the parental virus ( figure e ). finally, to discard the presence of other undesired mutations, the full-length sequence of the mutant virus was determined by deep-sequencing, and no mutations other than ns a a v were detected. collectively, these results indicated that ns a a v mutation alone affected zikv growth in vero cells at least by impairing viral rna synthesis. to investigate whether the reduced rna synthesis of rzikv-rgn-mns a in vero cells could result in viral attenuation in vivo, the ability of the mutant virus to induce pathogenesis was analyzed in a mice and compared with that of the parental rzikv-rgn ( figure ). to that end, groups of five female -to- -week-old a mice were inoculated s.c. in the footpad with pfu of either rzikv-rgn or rzikv-rgn-mns a, or with pbs as a negative control, and the body weight loss and survival were monitored daily over days. in contrast to mice infected with rzikv-rgn that quickly lost weight and all of them died at day eight after infection, mice infected with the mutant rzikv-rgn-mns a did not presented any clinical signs of infection or weight loss and all of them survived to viral infection ( figure a ). to further analyze the correlation of the attenuation of the mutant virus with viral replication, presence of the virus in mice sera was analyzed at days two and four post-infection. in agreement with the pathogenicity data, mice infected with the mutant virus presented lower viremia than mice infected with the parental virus ( figure b ). the mutant virus was only detected at day two after infection and at lower titers (approximately . logarithms lower) than the parental virus. as an internal control of the experiment, the plaque phenotype of the viruses recovered from the blood of infected mice were analyzed. as expected, rzikv-rgn formed big plaques while the mutant rzikv-rgn-mns a formed small plaques ( figure c ). these results indicated that rzikv-rgn-mns a was highly attenuated in mice, as compared to rzikv-rgn, and that this attenuation may be due to a lower replication of the rzikv-rgn-mns a mutant virus. after elucidating that rzikv-rgn-mns a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure ) . to that end, groups of five female -to- -week-old a mice were vaccinated s.c. in the footpad with pfu of rzikv-rgn-mns a or mock-vaccinated with pbs. twenty days after vaccination, blood samples were collected to evaluate the humoral response. one day later, mice were challenged with a lethal dose ( pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over figure . pathogenesis of rzikv-rgn-mns a in a mice. (a) weight loss and mortality. female -to- -week-old a mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with pfu of rzikv-rgn or rzikv-rgn-mns a, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during days. mice that lost more than % of their initial body weight or presented hind limb paralysis were humanely euthanized. error bars represent standard deviations of the mean for each group of mice. (b) viral titers in mice sera. female -to- -week-old a mice (six mice per group) were infected with pfu of rzikv-rgn (wt) or rzikv-rgn-mns a (mut) as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus e protein mab g . symbols represent data from individual mice and bars the geometric means of viral titers. asterisks indicate that the differences between rzikv-rgn and rzikv-rgn-mns a are statistically significant when data are compared using the unpaired t test (***, p < . ). nd: virus not detected. the detection limit of the assay ( pfu/ml) is indicate as a dashed line. (c) plaque phenotype. vero cells at % confluence ( -well plate format) were infected with pfu of rzikv-rgn (left) or rzikv-rgn-mns a (right) recovered from infected mice at day two post-infection and the plaque size evaluated by plaque assay and immunostaining using the pan-flavivirus e protein mab g . after elucidating that rzikv-rgn-mns a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure ) . to that end, groups of five female -to- -week-old a mice were vaccinated s.c. in the footpad with pfu of rzikv-rgn-mns a or mock-vaccinated with pbs. twenty days after vaccination, blood samples were collected to evaluate the humoral response. one day later, mice were challenged with a lethal dose ( pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over days. as expected, mice vaccinated with pbs lost weight rapidly, showed clear symptoms of disease, and all of them succumbed to challenge with rzikv-rgn. in contrast, mice vaccinated with rzikv-rgn-mns a did not lose weight and all of them survived the challenge with rzikv-rgn ( figure a ), indicating that a single immunization dose with rzikv-rgn-mns a is enough to induce full protection against zikv-rgn. in agreement with these data, a strong humoral response against zikv-rgn was observed in mice vaccinated with rzikv-rgn-mns a ( figure b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure c ). figure a ), indicating that a single immunization dose with rzikv-rgn-mns a is enough to induce full protection against zikv-rgn. in agreement with these data, a strong humoral response against zikv-rgn was observed in mice vaccinated with rzikv-rgn-mns a ( figure b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure c ). once confirmed that rzikv-rgn-mns a was attenuated in vivo and induces protection against zikv-rgn in mice, the genetic stability of the mutant virus was analyzed in vero cells, in order to test the possible use of this mutant virus as a base for the development of a live-attenuated zikv vaccine (figure ). to this end, both mutant (rzikv-rgn-mns a) and parental (rzikv-rgn) viruses were passaged five times in vero cells (p to p ) and the virus plaque phenotype, growth kinetics and the sequence of ns a were analyzed for each passage. analysis of the plaque phenotype showed that the parental virus presented the expected plaque size throughout all passaging. in contrast, for the mutant virus a reversion to parental plaque phenotype was observed. this reversion started at p ( % of big plaques), clearly increased at p ( % of big plaques) and was complete at p ( figure a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of pfu/cell) of the mutant virus from p and p were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p , the virus from p replicated to the same levels as the parental virus ( figure b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns a coding region of mutant viruses from p to p was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a v mutation to the wt sequence. although the instability and reversion of the mutant virus to the wt sequence during its propagation in vero cells limits the use of this mutant for vaccine development, these data further support the importance of this ns a residue for virus replication. at hpi, cell culture supernatants were collected and used to infect fresh vero cells. this process was repeated four more times and virus stocks of passages to (p to p ) were generated. (a) plaque size. vero cells were infected with the different passages (p to p ) of rzikv-rgn or rzikv-rgn-mns a, and at four days post-infection the viral plaques were visualized by immunostaining using the pan-flavivirus e protein mab g . (b) growth kinetics. vero cells at % confluence ( -well plate format; triplicates) were infected (moi of pfu/cell) with p and p of rzikv-rgn or rzikv-rgn-mns a and at the indicated hpi, virus titers were determined by plaque assay. error bars represent standard deviations of the mean from three experiments. asterisks indicate that the differences between rzikv-rgn-mns a p and the experimental samples, rzikv-rgn p , rzikv-rgn p and rzikv-rgn-mns a p , are statistically significant when data are compared using the unpaired t test (***, p < . ). analysis of the plaque phenotype showed that the parental virus presented the expected plaque size throughout all passaging. in contrast, for the mutant virus a reversion to parental plaque phenotype was observed. this reversion started at p ( % of big plaques), clearly increased at p ( % of big plaques) and was complete at p ( figure a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of pfu/cell) of the mutant virus from p and p were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p , the virus from p replicated to the same levels as the parental virus ( figure b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns a coding region of mutant viruses from p to p was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a v mutation to the wt sequence. although the instability and reversion of the mutant virus to the wt sequence during its propagation in vero cells limits the use of this mutant for vaccine development, these data further support the importance of this ns a residue for virus replication. moreover, these data also suggest that zikv ns a protein represents a good target for the development of antivirals against zikv infection. the significance of zikv to public health due its association with guillain-barré syndrome and fetal abnormalities [ ] [ ] [ ] [ ] [ ] [ ] , together with the lack of approved antiviral agents or vaccines, have triggered a global effort to study this flavivirus in order to develop effective strategies to prevent and control zikv infection in humans. in this respect, the development and implementation of reverse genetic approaches for zikv provide investigators with a novel and powerful experimental tool to study both the biology and pathogenesis of zikv as well as the development of attenuated forms of zikv for their implementation as live-attenuated vaccines. however, as described for other flaviviruses, the generation of zikv infectious clones using traditional approaches are very difficult due to the toxicity and instability of some viral sequences when they were propagated as cloned cdna in bacteria [ ] [ ] [ ] . in the past two years, several approaches that overcomes this toxicity problem have been applied for the successfully generation of zikv infectious clones. these include the use of low-copy plasmids [ , ] , insertion of introns to disrupt toxic sequences [ ] [ ] [ ] , mutational silencing of cbps present in the viral genome [ ] , in vitro ligation of cdna fragments [ , , ] , the isa method [ , ] , and the cper approach [ ] . although very useful, some of these approaches are laborious, time consuming and present several disadvantages. for instance, most of them need in vitro ligation and transcription steps that complicate the assembly and reduce the recovery efficiency. moreover, these low recovery efficiencies increase the presence of undesired mutations that could result in in vitro and/or in vivo attenuation, limiting the use of these infectious clones for certain studies. others, such as the mutational silencing of cbps, in which a high number of silent mutations have to be introduced, could affect viral fitness. finally, the use of low-copy plasmids has been shown to be effective for several zikv strains but not for others, probably due the different degrees of toxicity of rna sequences of different strains [ , , , ] . here, we describe a powerful approach for the generation of an infectious cdna clone of the zikv-rgn strain in a single plasmid, based on the use of a combination of synthetic biology and bacs. the full-length cdna copy of the zikv-rgn strain was generated from four synthetic dna fragments and cloned in the bac plasmid pbelobac [ ] under the control of the cmv promoter, which allows the expression of the viral rna in the nucleus [ ] , and flanked at the '-end by the hdv ribozyme and the bgh polyadenylation and termination sequences to produce synthetic rnas bearing authentic '-ends of the viral genome. the bac cdna clone was fully stable during its propagation in bacteria and the functional infectious virus was rescued after direct transfection of susceptible vero cells that was pathogenic in a mice. a zikv-rgn infectious clone generated using the cper approach has been recently reported [ ] . however, in contrast to our results, the rescued virus was asymptomatic and nonlethal in female -to- -week-old a mice infected with doses of to ccid ( % cell culture infective doses) via the s.c. route. whether the differences in pathogenicity among this rzikv-rgn and ours are related to the experimental approach (cper versus bac), the age of the mice ( -to- -week-old versus -to- -week-old) or the moi used to infect the mice ( - ccid versus pfu) remain to be evaluated. although other zikv reverse genetic systems have been reported (discussed above), the bac approach constitutes an useful alternative that presents important advantages: (i) the bac plasmids present a strictly controlled replication leading to only one plasmid per cell and therefore minimize the toxicity associated with several flavivirus sequences when amplified in bacteria [ ] . this allows the easy and direct manipulation of the viral genome for molecular studies; (ii) similarly to other approaches using polii-driven promoters, the bac approach results in intracellular expression of the viral rna [ , , , , ] , allowing the capping of the viral rna and the recovery of infectious virus without the need of an in vitro transcription step. although some splicing events could occur during the nuclear expression of the viral genome, mainly due to the presence of donor and acceptor putative sequences in the viral genome, the efficiency of this phenomenon is very low and does not affect the recovery of infectious viruses [ ] ; (iii) like other systems based on transfection of dna constructs [ , , , , ] , bac cdna clones present a higher efficiency of transfection than rna transcripts in mammalian cells. this allows higher efficiencies of virus recovery, reducing the passages in cell culture to get a viral stock and therefore, the possibility of introducing undesired mutations by cell culture adaptation; (iv) the manipulation of bac cdna clones is relatively easy and similar to that of conventional plasmids with slight modifications due to the presence of only one plasmid copy per cell. in addition to standard protocols, the bac cdna clones could also be efficiently modified into e. coli by homologous recombination using a two-step approach that combine the red recombination system and counterselection with the homing endonuclease i-scei [ ] [ ] [ ] [ ] ; and (v) the bac approach has been successfully used to engineer infectious clones of other flaviviruses, including denv [ ] , and several coronaviruses that contain the largest viral rna genome known and similar toxicity problems to those described for flaviviruses [ , [ ] [ ] [ ] [ ] . these data highlight the potential of the bac approach for the rapid and reliable construction of stable infectious clones of emerging flavivirus and other similar rna viruses with unstable viral genomes when amplified as cdnas in bacteria. the zikv reverse genetic system described in this article was further used to study the effect of a single amino acid substitution (a v) in the viral ns a protein on virus growth in cultured cells and pathogenesis in vivo. our results suggested that this single amino acid change impaired viral rna synthesis and virus production in cell culture and highly attenuated the virus in mice. however, we cannot discard that this mutation in the ns a protein could affect other steps in the replication cycle of the virus. flavivirus ns a protein is a -kda hydrophobic protein associated with the endoplasmic reticulum that contains eight transmembrane domains. it is a multifunctional protein that has been involved in viral rna synthesis [ , ] , virus assembly [ , ] , membrane rearrangement [ ] , and immunomodulation of innate immune response [ ] [ ] [ ] . by homology with the denv ns a topology, the zikv ns a a v mutation maps in the last transmembrane domain, for which no specific function has been reported. therefore, our data constitute the first evidence of a role of this ns a domain in viral rna synthesis. on the other hand, it is important to note that the ns a a v mutation promoted a -fold reduction in viral rna synthesis and more than -fold reduction in virus production. this reduced virus production could be a consequence of the rna synthesis impairment. however, since the reduction in virus production is higher than that observed in rna synthesis and that flavivirus ns a protein is also involved in virus assembly, we cannot discard an additional effect of a v mutation in zikv assembly. in addition, we have found that the mutant virus was attenuated in a mice. this attenuation could be explained as a consequence of the lower rna synthesis of the mutant virus. however, an additional effect of the a v mutation on the putative immunomodulatory role of ns a protein [ ] [ ] [ ] , leading to virulence attenuation, cannot be discarded. future studies will be required to determine whether this mutation affects only viral rna synthesis or also virus assembly and immunomodulation of the host defenses. importantly, we have shown that immunization with a single dose of pfu of the mutant rzikv-rgn-mns a induced protection against a lethal challenge with the parental rzikv-rgn, suggesting the potential implementation of this ns a mutant as the base of a live-attenuated vaccine. unfortunately, the mutant rzikv-rgn-mns a was instable and reverted to the wt sequence during its propagation in vero cells, limiting the use of this mutation alone for vaccine development. however, this instability and the high conservation of the amino acid a of the ns a protein among zikv strains highlights the importance of this ns a residue for virus replication, and therefore the potential use of ns a protein as a good target for antiviral development against zikv infection. in summary, we have developed a powerful zikv reverse genetic system based on the use of bacs that has allowed us to identify a single point mutation in the ns a protein that attenuates the virus in vitro and in vivo. this infectious clone system provides a valuable tool to the research community to explore zikv molecular biology, viral determinants of zikv pathogenesis, virus-host interactions, and vaccine and antivirals developments. an update on zika virus infection who calls off global zika emergency the . å resolution cryo-em structure of zika virus probing molecular insights into zika virus(-)host interactions zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria simultaneous outbreaks of dengue, chikungunya and zika virus infections: diagnosis challenge in a returning traveller with nonspecific febrile illness the global threat of zika virus to pregnancy: epidemiology, clinical perspectives, mechanisms, and impact zika virus outbreak on yap island, federated states of micronesia zika virus, french polynesia, south pacific zika virus in the americas: early epidemiological and genetic findings four in florida are infected with zika from local mosquitoes zika virus was transmitted by sexual contact in texas, health officials report defining the syndrome associated with congenital zika virus infection the brazilian zika virus strain causes birth defects in experimental models guillain-barre syndrome after zika virus infection in brazil zika virus disrupts neural progenitor development and leads to microcephaly in mice zika virus infection during pregnancy in mice causes placental damage and fetal demise zika virus associated with microcephaly potential of selected senegalese aedes spp. mosquitoes (diptera: culicidae) to transmit zika virus viral determinants and vector competence of zika virus transmission transmission of zika virus through breast milk and other breastfeeding-related bodily-fluids: a systematic review the expanding spectrum of modes of transmission of zika virus: a global concern flavivirus reverse genetic systems, construction techniques and applications: a historical perspective successful propagation of flavivirus infectious cdnas by a novel method to reduce the cryptic bacterial promoter activity of virus genomes functional cdna clones of the flaviviridae: strategies and applications recovery of the zika virus through an in vitro ligation approach a reverse genetics platform that spans the zika virus family tree zika virus encoding nonglycosylated envelope protein is attenuated and defective in neuroinvasion an infectious cdna clone of zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors characterization of cis-acting rna elements of zika virus by using a self-splicing ribozyme-dependent infectious clone rescue of the zika virus prototype strain with a cytomegalovirus promoter-driven cdna clone a full-length infectious cdna clone of zika virus from the epidemic in brazil as a genetic platform for studies of virus-host interactions and vaccine development development and characterization of recombinant virus generated from a new world zika virus infectious clone simple reverse genetics systems for asian and african zika viruses a robust method for the rapid generation of recombinant zika virus expressing the gfp reporter gene a reverse genetics system for zika virus based on a simple molecular cloning strategy de novo generation and characterization of new zika virus isolate using sequence data from a microcephaly case development of a novel dna-launched dengue virus type infectious clone assembled in a bacterial artificial chromosome complete nucleotide sequence of two generations of a bacterial artificial chromosome cloning vector cloning and stable maintenance of -kilobase-pair fragments of human dna in escherichia coli using an f-factor-based vector analysis of relative gene expression data using real-time quantitative pcr and the (−∆∆ c(t)) method. methods the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments an open-source platform for biological-image analysis canine influenza viruses with modified ns proteins for the development of live-attenuated vaccines a susceptible mouse model for zika virus infection a mouse model of zika virus pathogenesis characterization of a novel murine model to study zika virus committee for the update of the guide for the care and use of laboratory animals sindbis virus dna-based expression vectors: utility for in vitro and in vivo gene transfer virus pathogen resource (vipr), faviviridae. available online engineering the largest rna virus genome as an infectious bacterial artificial chromosome targeted modification of a human β-globin locus bac clone using get recombination and an i-scei counterselection cassette a highly efficient escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of bac dna two-step red-mediated recombination for versatile high-efficiency markerless dna manipulation in escherichia coli a new logic for dna engineering using recombination in escherichia coli construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate molecular characterization of feline infectious peritonitis virus strain df- and studies of the role of orf abc in viral cell tropism recovery of a neurovirulent human coronavirus oc from an infectious cdna clone subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns a and ns a mutations in west nile virus nonstructural proteins that facilitate replicon persistence in vitro attenuate virus replication in vitro and in vivo mutations in the yellow fever virus nonstructural protein ns a selectively block production of infectious particles role of nonstructural protein ns a in flavivirus assembly analysis of adaptive mutations in kunjin virus replicon rna reveals a novel role for the flavivirus nonstructural protein ns a in inhibition of β interferon promoter-driven transcription a single amino acid substitution in the west nile virus nonstructural protein ns a disables its ability to inhibit α/β interferon induction and attenuates virus virulence in mice subversion of interferon by dengue virus we are grateful to carla gómez and snezhana dimitrova for technical assistance in the bac clone generation and mice experiments, respectively. we also thank sylvia gutiérrez and ana oña at the cnb advanced microscopy facility for their valuable support in immunofluorescence microscopy analysis. the authors declare no conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results. key: cord- -ypefqm authors: roberts, christine c.; maslow, joel n. title: assay challenges for emerging infectious diseases: the zika experience date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: ypefqm from the perspective of vaccine development, it is imperative to accurately diagnose target infections in order to exclude subjects with prior exposure from evaluations of vaccine effectiveness, to track incident infection during the course of a clinical trial and to differentiate immune reactions due to natural infections from responses that are vaccine related. when vaccine development is accelerated to a rapid pace in response to emerging infectious disease threats, the challenges to develop such diagnostic tools is even greater. this was observed through the recent expansion of zika virus infections into the western hemisphere in – . when initial zika vaccine clinical trials were being designed and launched in response to the outbreak, there were no standardized sets of viral and immunological assays, and no approved diagnostic tests for zika virus infection. the diagnosis of zika virus infection is still an area of active research and development on many fronts. here we review emerging infectious disease vaccine clinical assay development and trial execution with a special focus on the state of zika virus clinical assays and diagnostics. the declaration by the world health organization (who) that zika is a public health emergency of international concern in february led to a global effort to support vaccine development and control the spread of zika virus (zikv). our collaborative dna vaccine consortium focused and accelerated pre-clinical, manufacturing and early clinical development efforts to bring forward the first zika vaccine, gls- , into human clinical trials [ ] [ ] [ ] . at the outset, it was clear that gaps would need to be filled as the public health and science communities learned and shared new information on zika. one of the clear gaps affecting both public health efforts and vaccine development programs was a lack of standardized reagents and methods to test for evidence of current or prior zika infection. the need for fast and accurate diagnostic tests of infection in an outbreak situation is obvious: identify the source or epicenter so that appropriate healthcare measures can be quickly instituted. expanding that concept to the public health scale and attaining accurate infectious disease diagnoses allows for better understanding of the course and severity of an outbreak and aids decision-making for population-level countermeasure implementation. the clinical assays with which the immune response and pathogen presence are measured in vaccine trials become part of the basis for licensure for all vaccine products [ ] . because vaccines are tested in healthy populations through all phases of clinical development for immune response and/or pathogen presence whereas drugs/biologics (post-phase i) are most often tested in a population with specific disease to demonstrate improvement, the selected methods to measure vaccine responses and endpoints are of the utmost importance. the identification of an immune correlate of protection for each vaccine is highly desirable, though not always attainable [ ] . here we will use the recent experience with the zikv outbreak and ensuing public health countermeasures for containment and vaccine development as an example of challenges faced during emerging infectious disease emergencies. zika virus was discovered in during a survey to map the extent of yellow fever in the entebbe region of uganda. the virus was cultured from the serum of a sentinel macaque placed in the zika forest that developed fever but was otherwise well. initially restricted to equatorial regions of africa and asia, zikv started to spread eastward across the pacific ocean with an outbreak on yap island, micronesia in [ ] , in french polynesia in [ ] , and reaching brazil in late or early [ ] . zikv infection typically causes a self-limited illness that is minimally symptomatic for most individuals. zika infection presents similarly to dengue or chikungunya with fever in most, rash, malaise, myalgia, conjunctivitis and retro-orbital pain but may present with few, if any, discernable symptoms [ ] . however, documentation in brazil of severe neurologic complications associated with zikv infection beginning around october raised worldwide alert. the most publicized and dramatic complications are those that occur during fetal development. they include microcephaly, intra-uterine growth retardation, cerebral calcifications, ocular calcifications and other ocular abnormalities-with an attack rate estimated at - % of women who become infected during pregnancy [ ] . zikv can also directly infect the placenta and can result in spontaneous miscarriage with an unknown prevalence [ ] [ ] [ ] [ ] . in adults, the most common complication of zikv infection is guillain-barré syndrome occurring at an estimated rate of in cases [ ] . zika has also resulted in deaths among adults with and without other complicating factors [ , ] . there are no approved therapies or vaccines for zikv infection. there have been a number of vaccines developed for other flavivirus infections. live virus vaccines utilizing chimeric viral constructs have been approved for use to prevent yellow fever and dengue. dna vaccines have been developed and published for dengue [ , ] , west nile virus [ ] [ ] [ ] [ ] [ ] , and japanese encephalitis virus [ ] . notably, dna vaccines targeting west nile virus [ , ] and dengue virus [ ] have been tested as part of phase i clinical trials without vaccine associated toxicity. currently, as recently reviewed elsewhere, a number of vaccine modalities targeting zikv have reached early stage clinical trials and even more are in preclinical development [ ] [ ] [ ] . zikv is spread primarily through aedes species mosquitoes, mainly aedes aegypti, but can be carried by other mosquito species [ ] and does not appear to be transmission competent except for aedes species [ ] [ ] [ ] [ ] [ ] [ ] . aedes mosquitoes also transmit other arboviruses such as dengue and chikungunya [ , ] . in fact, these infections are co-endemic in most regions and may cause concurrent infections [ ] [ ] [ ] [ ] . alternative nonmosquito-borne routes of zikv spread include: blood transfusions, breast milk [ ] , sexual transmission [ ] [ ] [ ] , and may include urine and saliva [ ] [ ] [ ] [ ] . the additional potential routes of zikv transmission have increased the need for definitive monitoring and a variety of surveillance strategies to prevent disease spread [ ] [ ] [ ] [ ] [ ] . in an outbreak situation, such as with zika, it is important to have the ability to quickly develop both diagnostic kits for public health purposes and vaccine clinical assays to support pre-clinical studies and early stage clinical trials. both were largely unavailable on a commercial scale or for widespread use at the outset of the zika outbreak, though development ensued at a rapid pace upon the declaration of a worldwide public health emergency. because there is significant homology between zikv and other cocirculating flaviviruses, detection and diagnosis has had the extra challenge of avoiding cross-reactivity without sacrificing sensitivity. while the avoidance of cross-reactivity is more easily engineered into molecular tests of virus rna because primers or probes can be designed to be virus-, antigen-and serotype-specific [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it is not as easily achieved for immunoassays [ ] [ ] [ ] [ ] [ ] [ ] . diagnostic assays are often the same style tests as those used in vaccine development, but are for the intended purpose of identifying the source of a patient's illness to enable the initiation of appropriate treatments by healthcare professionals. the need for sensitivity and specificity as it relates to clinical disease identification for patient treatment is of the utmost concern. early in the zika outbreak, the us centers for disease control and some academic laboratories studying flaviviruses had assays developed for zikv that generally supported their own research interests [ ] [ ] [ ] [ ] [ ] [ ] [ ] . making sufficient quantities of these tests available for public health use while ensuring consistency and quality was a significant challenge. additionally, cross-reactivity in a number of immunological assays and the short time frame in which viremia can be detected in bodily fluids necessitated the institution of an algorithm to confirm zikv infection that was based on a combination of risk factors, clinical symptoms and diagnostic test results [ ] . the centers for disease control (cdc) issued testing guidance for healthcare providers using algorithms that included use of molecular testing for pregnant or non-pregnant and symptomatic or non-symptomatic individuals, the types of specimens and timing of collection of specimens that would provide the most reliable results [ ] . in addition, guidance algorithms were provided for the use and interpretation of zikv igm assays to indicate recent exposure with or without accompanying molecular test results [ ] . in july of , united states (us) health and human services (hhs) sponsored a hhs summit to accelerate zika diagnostics development, recognizing the important need. at that point in time, very few tests had gained emergency use authorization (eua) from the food and drug administration (fda). the five serological zika diagnostic kits currently authorized by the us fda are shown in table and fourteen viral diagnostic kits in table [ ] . at the fda medical countermeasures webpage, links can also be found for each eua approved molecular assay's key [ ] and performance [ ] characteristics including instruments approved for use and limits of detection. although no zika diagnostic kit, serological or viral, has been fully approved by the fda to date, the fda has worked collaboratively with developers to accelerate both eua approvals and the transition to formal full approvals of zika diagnostic kits using standard review timelines [ ] . a number of studies have evaluated the various eua tests both for independent sensitivity and specificity [ , , , , , , , ] assessments of assay performance and as part of comparative studies [ , [ ] [ ] [ ] [ ] to determine those best to use for the diagnostic or epidemiological surveillance need. a large number of companies have also developed tests that remain classified as "research use only" (ruo) for which emergency authorizations have not been granted. other authorizations for emergency use have been granted to different diagnostic kits by other agencies, such as who's emergency use assessment and listing procedures (euals) [ ] . while many diagnostic kits use methods similar or identical to those that may be used to support a vaccine clinical trial, often they are focused more toward the support of a clinical patient diagnosis and may not be sensitive enough for use to support vaccine development. often there is not enough data at the outset of a vaccine program, but especially in the case of emerging infectious diseases, to understand what assays will be most useful or informative or will perhaps even provide a correlate of protection for the vaccine into the future. a few general assumptions can be made at the outset about which types of assays will be needed to detect vaccine-induced humoral and cellular immune responses and these will evolve over time. the typical go-to methods used for vaccine clinical assays are: antibody binding-enzyme-linked immunosorbent assays (elisa); functional-virus neutralization or bactericidal; cellular-interferon gamma enzyme-linked immunospot assay (ifnγ-elispot) using the target antigen or antigen-derived peptide pools; and molecular or culture methods to detect the pathogen. the technologies used to develop and run these assays have improved over the years to allow for higher throughput, multiplexing, reduced sample volumes, and automation. often, more tests of a larger variety are done early in a program and are then whittled down based on the usefulness of the data generated so that just the relevant few remain to support large trials and licensure [ ] . when the first zikv vaccine trials commenced in late summer of , even though a few zika diagnostics had achieved eua status, none were commercially available and were initially restricted to public health use only. challenges to vaccine development centered around the determination of prior zikv exposure and immunity and determination of incident infection among study participants. each of the serological assays listed in table detect igm reflective of recent infection, however many, such as the mac-elisa and the inbios assay, will detect igm against the zikv envelope which is the target antigen for many vaccines. igg-based assays have not yet been validated primarily due to cross-reactivity between zikv and other flaviviruses, principally dengue. igm immunoassays targeted to the ns antigen are generally more specific than those directed to the viral envelope [ ] . in september , we initiated the first clinical trial of the gls- zikv dna vaccine in an endemic region, puerto rico, an area also known to have high rates of exposure to dengue. because no widely accepted standardized assays nor any international reference standards or reagents existed at the time of trial initiation, individual vaccine projects needed to rely on internally developed clinical assays to understand prior exposure and vaccine related immune responses. similar to patient diagnostic tests for zikv and as mentioned above, there was no accepted "gold standard" for any of the immunoassays one might choose to develop or use in a vaccine program. the extensive experience of our collaborative dna vaccine team allowed us to develop zikv-specific tests such as elisa, virus microneutralization and elispot around the development and pre-clinical testing of our zikv plasmid dna vaccine constructs [ , , , ] . these assays performed consistently on a pre-clinical scale and we were able to quickly expand their use to support our two phase gls- vaccine clinical trials. a concern always remains, however, that the lack of highly-characterized reagents and controls in early versions of vaccine clinical assays will result in difficulties in the maintenance of the assays through its full life cycle. sourcing, batch-to-batch variability and overall quality of critical reagents, standards and controls can become an issue over time. lack of standardization across the scientific field can be confounding in that interpretation of results from multiple "home brew" assays across labs are not directly comparable in the absence of an accepted international standard or a proficiency panel of samples [ ] . the main methodologies used to detect incident zikv infection are currently molecular-based, mainly reverse transcriptase polymerase chain reaction (rt-pcr) [ , , , , , , , , ] or varieties thereof [ , , ] , since culture methods can be both difficult and laborious [ ] [ ] [ ] . those viral detection systems with eua approval are shown in table . in clinical trials, identification of newly infected subjects over the treatment and follow-up periods are necessary to determine vaccine efficacy. as discussed earlier, for most individuals zikv infection is minimally symptomatic such that few present to clinical care. zikv is detectable in serum by rt-pcr for only a short interval following the onset of symptoms, typically for only seven days to a maximum of days [ , ] , though longer periods of rt-pcr detection (up to days) have been observed in serum of pregnant women [ ] , which may contribute to the incidence of zikv-related birth defects. zikv is excreted into the urine for approximately four weeks in most individuals, though this observation was not documented until over a year into the epidemic [ , , , ] . because of this, rt-pcr-based diagnosis of incident infections in a vaccine clinical trial would require very frequent sampling. other sample types have been evaluated for rt-pcr detection including saliva, whole blood, plasma, brain tissue, amniotic fluid and vaginal secretions [ , , , , , ] . a key concern from developers of both diagnostics and of vaccines or therapeutics for zikv highlighted at the hhs summit for diagnostics in july was the lack of well-characterized human zikv specimens which groups could use to fully understand the performance characteristics of the assays being developed and, eventually, work toward some standardization across the field. the who has initiated in, july , a collaborative study effort for the development of nucleic acid testing international standard for zika [ ] . additionally, in july , a plasma sample panel became available through the us fda for use in evaluating zikv immunoassay performance [ ] . reagents for newly emergent infectious diseases like zikv were not readily available from commercial vendors that had consistent production methods and quality controls in place, thus many reagents were not well characterized early on in the development of zikv clinical assays. because validated assays supporting vaccine efficacy endpoints need to support clinical and regulatory expectations over the life of the product, it is imperative that a line-of-sight is maintained such that reliable and qualified materials in appropriate quantity are available for resolving issues that arise. assays typically require some troubleshooting over time, changes to reagent lots or instruments, potential for multiplexing, platform changes to increase testing throughput, or a desire to bridge to a new technology [ ] . the development, qualification, validation and maintenance of vaccine clinical assays should be done in close consultation with biostatisticians and bench scientists to ensure the optimal assay design, control parameters and performance characteristics for the needs of the vaccine program from beginning to end. it should be noted that vaccine assay quality is highly dependent upon clinical study execution from collection through final data reporting. sample collection & handling are critical to the quality of the specimen and its ability to be used in an assay. implementing methods to assure the following are keys to successful vaccine clinical trials: proper sample storage and shipping conditions, processing and aliquotting with methods for contamination control, proper training of site and clinical research organization lab personnel, chain of custody verification of samples from collection to final valid test result, quality control checks and good data management. in the execution of vaccine clinical trials, there is the need for testing methodology to be reliable, reproducible (accuracy, specificity, robustness), and occasionally to provide rapid diagnosis (on-site or point-of-care testing, if needed), which contributes to patient care as well as to the understanding of vaccine efficacy or disease epidemiology. the diagnostic assay development response to zikv was quite rapid with the eua approval of different molecular detection assays and five serological assays in roughly months' time (tables and ) and the initiation of efforts to build international reference standards for both assay types. however, at the time of writing, there are still no fully approved diagnostics, no established "gold standard" detection methods nor any fully characterized and accepted international reference standards for zikv. while our understanding of the immune response to zikv has greatly expanded since the start of the most recent outbreak and leverages the years of vaccine research for other flaviviruses, such as dengue, there is still no established immune correlate of protection. other flavivirus vaccines have established immune correlates that are based on neutralization titers and it has been assumed that zikv will, as well [ ] . post-vaccination serum from participants enrolled in our group's gls- zika dna vaccine phase trial protected % of interferon α/β receptor knockout mice (ifnar) in a lethal-challenge model of zikv infection, however this protection was not dependent upon neutralizing antibody titers. other vaccines in clinical development have achieved neutralizing antibody titers in humans which were similar to the titers conferring protection in pre-clinical studies of the vaccines [ , ] . serum from participants in a phase clinical study receiving a purified inactivated zikv vaccine was also able to protect mice in a passive transfer mouse model [ ] . the development and validation of sensitive, specific and standardized zikv immunoassays to better characterize the immune response to vaccination are necessary to work toward the establishment of an immune correlate of protection in humans. efforts are being made through the work of the coalition for epidemic preparedness innovations (cepi) and others to ensure that rapid response mechanisms are in place to address emerging infectious diseases. well-seasoned development teams have accepted the challenges and funding to support building the vaccine design, manufacture and clinical assessment infrastructure needed to save lives in outbreak situations. scientific and quality principles must still apply although speed may be required when developing vaccine or diagnostic assays during an outbreak of a new emerging infectious disease. eid public health and countermeasure programs often have unique challenges for diagnostic and vaccine clinical assay development purposes including: . incomplete understanding of biology or epidemiology for appropriate target selection. lack of available reagent sources; inconsistency in quantity and/or quality of those available. difficulty in obtaining relevant human sample panels for the evaluation of test methods. challenges to produce clear line of sight sourcing and quality from early vaccine development through licensure. although the speed at which the diagnostic or vaccine development field needs to move will be dependent upon the urgency of the emerging infectious pathogen and its impact on human life, biological assay standardization is a critical component and requires three separate activities: • development of relevant, sensitive, specific and preferably quantitative biological assays. use of biostatistics to analyze assay performance data from development to validation to life cycle performance management. • ability to prepare stable and reproducible results over time to support diagnostic or vaccine program needs. if assay performance consistency and quality is not demonstrated, the validity of clinical study results may be questioned. assay standardization for any newly emergent infectious disease will be challenging and will: (i) take time to develop, collect and characterize quality reagents, (ii) to achieve sufficient sources of confirmed positive samples for establishment of serological standards, and (iii) to confirm new molecular standards, though this is more straightforward for molecular assays than serology. any efforts to prepare reagents, collect characterized samples, develop research materials and international standards in advance for eids for whom alerts have already been raised will leave the field better suited to respond in case of emergency. rapid response to an emerging infectious disease-lessons learned from development of a synthetic dna vaccine targeting zika virus safety and immunogenicity of an anti-zika virus dna vaccine-preliminary report vaccines for emerging infectious diseases: lessons from mers coronavirus and zika virus utilization of serologic assays to support efficacy of vaccines in nonclinical and clinical trials: meeting at the crossroads. vaccine nomenclature for immune correlates of protection after vaccination zika virus outbreak on yap island, federated states of micronesia zika virus, french polynesia, south pacific zika virus: history, epidemiology, transmission, and clinical presentation guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study teratogenic effects of the zika virus and the role of the placenta zika virus targets different primary human placental cells, suggesting two routes for vertical transmission zika virus on a spreading spree: what we now know that was unknown in the 's miscarriage associated with zika virus infection zika virus epidemic in brazil. i. fatal disease in adults: clinical and laboratorial aspects zika virus associated deaths in colombia evaluation of a prototype dengue- dna vaccine in a phase clinical trial development of a novel dna syncon tetravalent dengue vaccine that elicits immune responses against four serotypes a west nile virus dna vaccine utilizing a modified promoter induces neutralizing antibody in younger and older healthy adults in a phase i. clinical trial a west nile virus dna vaccine induces neutralizing antibody in healthy adults during a phase clinical trial coimmunization with an optimized il plasmid adjuvant enhances humoral immunity via stimulating b cells induced by genetically engineered dna vaccines expressing consensus jev and wnv e diii induction of potent th -type immune responses from a novel dna vaccine for west nile virus new york isolate (wnv-ny ) development of vaccines against zika virus zika virus structural biology and progress in vaccine development role of zika virus prm protein in viral pathogenicity and use in vaccine development zika virus emergence in mosquitos in southeastern senegal evolutionary enhancement of zika virus infectivity in aedes aegypti mosquitoes assessing seasonal risks for the introduction and mosquito-borne spread of zika virus in europe zika virus vector competency of mosquitoes aedes hensilli as a potential vector of chikungunya and zika viruses outbreak of zika virus infection, chiapas state, mexico, , and first confirmed transmission by aedes aegypti mosquitoes in the americas autophagy and viral diseases transmitted by aedes aegypti and aedes albopictus zika virus: following the path of dengue and chikungunya? lancet spatio-temporal coherence of dengue, chikungunya and zika outbreaks in merida zika might not be acting alone: using an ecological study approach to investigate potential co-acting risk factors for an unusual pattern of microcephaly in brazil viremia and clinical presentation in nicaraguan patients infected with zika virus, chikungunya virus, and dengue virus evidence of perinatal transmission of zika virus complete genome sequence of zika virus isolated from semen probable non-vector-borne transmission of zika virus potential sexual transmission of zika virus detection of zika virus in saliva persistence of zika virus in body fluids-preliminary report comparison of test results for zika virus rna in urine, serum, and saliva specimens from persons with travel-associated zika virus disease-florida potential use of saliva samples to diagnose zika virus infection use of blood donor screening data to estimate zika virus incidence, puerto rico zika virus outbreak and the case for building effective and sustainable rapid diagnostics laboratory capacity globally comparison of four serological methods and two reverse transcription-pcr assays for diagnosis and surveillance of zika virus infection seroprevalence of arboviruses among blood donors in french polynesia unexpected outbreaks of arbovirus infections: lessons learned from the pacific and tropical america evaluation of altona diagnostics realstar zika virus reverse transcription-pcr test kit for zika virus pcr testing molecular and serological techniques to detect co-circulation of denv, zikv and chikv in suspected dengue-like syndrome patients assay optimization for molecular detection of zika virus prolonged detection of zika virus rna in pregnant women simultaneous detection of zika, chikungunya and dengue viruses by a multiplex real-time rt-pcr assay attomolar zika virus oligonucleotide detection based on loop-mediated isothermal amplification and ac susceptometry single-reaction multiplex reverse transcription pcr for detection of zika, chikungunya, and dengue viruses detection of dengue virus genome in urine by real-time reverse transcriptase pcr: a laboratory diagnostic method useful after disappearance of the genome in serum cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites flavivirus-induced antibody cross-reactivity a multiplex microsphere immunoassay for zika virus diagnosis serodiagnosis of zika virus (zikv) infections by a novel ns -based elisa devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera a broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of e protein detection of virus-specific antigen in the nuclei or nucleoli of cells infected with zika or langat virus detection of anti-arboviral immunoglobulin g by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections evaluation of diagnostic tests: dengue approaches for the development of rapid serological assays for surveillance and diagnosis of infections caused by zoonotic flaviviruses of the japanese encephalitis virus serocomplex flaviviruses in europe: complex circulation patterns and their consequences for the diagnosis and control of west nile disease quantitative real-time pcr detection of zika virus and evaluation with field-caught mosquitoes interpretation of nucleic acid and immunoassay test results for suspected zika infection cdc zika testing guidance for healthcare providers fda zika emergency use authorizations table summary molecular assay characteristics fda zika eua table molecular assay performance characteristics fda zika virus diagnostic development high specificity of a novel zika virus elisa in european patients after exposure to different flaviviruses kinetics of anti-zikv antibodies after zika infection using two commercial enzyme-linked immunoassays diagnostic performance of commercial igm and igg enzyme-linked immunoassays (elisas) for diagnosis of zika virus infection evaluation of commercially available zika virus immunoassays advances in diagnosis, surveillance, and monitoring of zika virus: an update world health organization emergency use and listings procedures dna vaccination protects mice against zika virus-induced damage to the testes in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunisation with a prmenv dna vaccine genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia future developments in biosensors for field-ready zika virus diagnostics electrochemical biosensors for early stage zika diagnostics role of cell culture for virus detection in the age of technology isolation of infective zika virus from urine and saliva of patients in brazil prolonged detection of zika virus in vaginal secretions and whole blood detection of zika virus in urine prolonged detection of zika virus rna in urine samples during the ongoing zika virus epidemic in brazil congenital cerebral malformations and dysfunction in fetuses and newborns following the to zika virus epidemic in french polynesia detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study collaborative study to evaluate a candidate world health organization international standard for zika virus for nucleic acid amplification technique (nat)-based assays fda fda provides new tools for the development and proper evaluation of tests for detecting zika virus infection current status of zika vaccine development: zika vaccines advance into clinical evaluation preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated zika virus vaccine candidate: phase , randomised, double-blind, placebo-controlled clinical trials safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -p buyrcj authors: batista, mariana n.; braga, ana cláudia s.; campos, guilherme rodrigues fernandes; souza, marcos michel; de matos, renata prandini adum; lopes, tairine zara; candido, natalia maria; lima, maria leticia duarte; machado, francielly cristina; de andrade, stephane tereza queiroz; bittar, cíntia; nogueira, maurício l.; carneiro, bruno m.; mariutti, ricardo b.; arni, raghuvir krishnaswamy; calmon, marilia freitas; rahal, paula title: natural products isolated from oriental medicinal herbs inactivate zika virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: p buyrcj zika virus (zikv) has been associated with serious health conditions, and an intense search to discover different ways to prevent and treat zikv infection is underway. berberine and emodin possess several pharmacological properties and have been shown to be particularly effective against the entry and replication of several viruses. we show that emodin and berberine trigger a virucidal effect on zikv. when the virus was exposed to µm of berberine, a reduction of . % in the infectivity was observed; when emodin was used ( µm), this reduction was approximately . %. dynamic light scattering data showed that both compounds significantly reduce the hydrodynamic radius of virus particle in solution. we report here that berberine and emodin, two natural compounds, have strong virucidal effect in zika virus. zika virus (zikv), a member of the flaviviridae family and the flavivirus genus, is named after the forest where it was first identified in [ ] . for approximately years only a few minor epidemics have been identified, none of which had any major consequences. however, when this virus started to circulate in brazil in , a correlation was observed between zikv infection in pregnant women and poor fetal development, leading to severe conditions such as microcephaly and other neurological diseases [ ] . although the exact mechanism by which zikv triggers these diseases is still unclear, some relevant information is now available. for example, it is known that the virus can penetrate the placenta [ ] , infect progenitor neural cells, and disrupt development [ ] . in addition to fetal disease, zikv has also been implicated in the development of the guillain-barré syndrome in adults [ ] . strategies to combat zikv infection include the development of vaccines [ ] and the screening of molecules that inhibit the different phases of the viral lifecycle [ , ] . berberine is an isoquinoline alkaloid belonging to the structural class of protoberberines and is encountered in many plants including berberis vulgaris [ ] . it exhibits several pharmacological properties and is particularly effective against entry and replication of many viruses, including the human cytomegalovirus (hcmv) [ ] , herpes simplex virus (hsv) [ ] , influenza virus [ ] , chikungunya virus (chikv), and other alphaviruses [ ] . emodin, an anthraquinone derivative, is also a naturally occurring compound derived from the chinese herbs rheum palmatum [ ] , polygonum multiflorum [ ] , aloe vera [ ] , and cassia obtusifolia [ ] . emodin possesses a wide spectrum of pharmacological effects, including antiviral activity, against coxsakie b virus (cvb ), human respiratory syncytial virus (hrsv) [ ] , influenza a virus [ ] , epstein-barr virus (ebv) [ ] , herpes simplex virus (hsv) [ ] , hepatitis b virus (hbv) [ ] , and japanese encephalitis virus (jev), which is another flavivirus [ ] . in this study, we tested the activity of the natural compounds berberine and emodin for their ability to inhibit zikv infection. stock solutions of emodin and berberine were diluted in . % dimethyl sulfoxide (dmso, sigma-aldrich, st. louis, mo, usa). the working solutions were obtained by dilution, in different concentrations, of both drugs in dulbecco's modified eagle's medium (dmem) (sigma-aldrich, st. louis, mo, usa). vero e cells (atcc crl- tm ), donated by dr. maurício lacerda nogueira, were cultured in dmem (supplemented with % fetal bovine serum (cutlab, campinas, brazil), %, u/ml of penicillin, and µg/ml of streptomycin (invitrogen, new york, ny, usa) and maintained in a humidified % co incubator at • c. to test the inhibitory potential of berberine and emodin, a brazilian zika virus strain isolated from a febrile patient in northeast brazil [ ] was used. an aliquot of this virus was inoculated onto aedes albopictus mosquito cells, clone c / (atcc ® crl- ™), and incubated in leibovitz's l- medium (cutlab, campinas, brazil) supplemented with % fetal bovine serum, %, u/ml of penicillin, and µg/ml of streptomycin for - days until the first cytopathic effects were observed. the same viral passage was used in all experiments. the cytotoxicity of berberine and emodin was evaluated in order to select the optimal non-toxic concentration of each compound in vero e cells. for this assay, × vero e cells were seeded to each well of a -well plate and incubated for h at • c. media was removed and replaced with dmem containing different concentrations of berberine ( - µm) or emodin ( - µm). the effects of the compounds on the cells were analyzed at h, h and h after treatment. the supernatants were removed, and a solution of -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt; mg/ml) (sigma-aldrich, st. louis, mo, usa) was added to each well. the plates were then incubated for min at • c. subsequent to incubation, formazan crystals were solubilized with µl dmso (sigma-aldrich, st. louis, mo, usa), and the absorbance was measured at nm. the ability of berberine and emodin to inhibit zikv in a pre-entry step, acting directly on the viral particle, was tested by a virucidal assay. briefly, × cells per well were seeded in a -well plate. berberine or emodin were incubated at different concentrations (berberine: - µm and emodin: . - µm) with zikv ( plaque-forming unit/ ml (pfu/ml)) for h at • c. a control, zikv incubated with the drug diluent (dmem + dmso . %), was also performed. subsequently, the drug-containing supernatant was titrated in vero e cells and incubated for h. cells were fixed with % formaldehyde for min before staining with a crystal violet ethanolic solution. the titer of the virus treated with different concentration of each compound was determined and compared to the control (diluent only). we also tested the influence of berberine and emodin on cell receptors involved in virus entry. for these experiment, one day prior to the assay, × cells/well were seeded onto a -well. on the following day, the culture growth medium was replaced with dmem containing µm of berberine or µm of emodin and incubation proceeded for h at • c. after one hour, the medium containing the compounds was removed, and the cell monolayer was washed thrice with pbs. the control was incubated only with dmem + . % dmso (drug diluent). the treated cells were then infected with zikv and incubated at • c for h. the virus was then removed, culture media was added and cells were incubated for h at • c with % co . after fixation with % formaldehyde and staining with crystal violet, the number of foci was determined and compared to the non-treated control. the half maximal inhibitory concentration (ic ) and half maximal cytotoxic concentration (cc ) of berberine and emodin were calculated using non-linear regression analysis. the ic and cc values, were used to calculate the selectivity index (si) of each compound (si = cc /ic ). the si suggests potential effectiveness, where the higher the value, the more promising the drug. dynamic light scattering (dls) measurements of virions were carried out using freshly purified samples of zikv in polystyrene curettes with optical path lengths of cm and at a concentration of . × pfu/ml in filtered pbs buffer (ph . ) after incubation for h at • c, both in the absence and presence of µm emodin or µm berberine. the results presented are the average values of scans of s, each obtained with a zetasizer nano s (malvern, uk) equipped with a he-ne laser ( . nm). the scattered light was detected at • and the experimental data were analyzed using the zetasizer software. the results of viral inhibition were calculated as a percentage of the negative control (medium plus drug diluent). all statistical analyses were performed by one-way anova with tukey's post-test using the graphpad prism . software (graphpad software, san diego, ca, usa). we initially screened berberine and emodin for cellular cytotoxicity. the higher non-toxic concentrations were µm for berberine and µm for emodin (more than % cell viability) in all experimental times (figure ). by non-linear regression analysis, the cc values were calculated as µm for berberine and . µm for emodin. after determining the highest non-toxic concentration for each compound, a virucidal assay was performed to test the ability of these compounds to impair the virus particle infectivity. the supernatant containing infectious virus particles was incubated for h at different concentrations of each compound (berberine: - µm and emodin: . - µm) and subsequently used to infect vero e cells. after h, a considerable reduction of the foci numbers was observed for both compounds in a dose-dependent manner. when the viral particles were incubated with µm of berberine, plaques reduced from . × pfu/ml (control) to . × pfu/ml, a reduction of . % in zikv infectivity. when the particles were exposed to µm of emodin, this reduction was approximately . % (from . × pfu/ml to . × pfu/ml) ( figure ). the ic values for berberine and emodin were . µm and . µm respectively, leading to a si of . for berberine and . for emodin. although at reduced concentrations, berberine had a lower ability to inhibit the virus, all tested concentrations of berberine presented a significant reduction of foci number compared to the control (p < . ) (figure a) . emodin inhibited the virus with a significant reduction of foci number for all tested concentrations (p < . ), except for . µm ( figure b ). after determining the highest non-toxic concentration for each compound, a virucidal assay was performed to test the ability of these compounds to impair the virus particle infectivity. the supernatant containing infectious virus particles was incubated for h at different concentrations of each compound (berberine: - µm and emodin: . - µm) and subsequently used to infect vero e cells. after h, a considerable reduction of the foci numbers was observed for both compounds in a dose-dependent manner. when the viral particles were incubated with µm of berberine, plaques reduced from . × pfu/ml (control) to . × pfu/ml, a reduction of . % in zikv infectivity. when the particles were exposed to µm of emodin, this reduction was approximately . % (from . × pfu/ml to . × pfu/ml) (figure ). the ic values for berberine and emodin were . µm and . µm respectively, leading to a si of . for berberine and . for emodin. although at reduced concentrations, berberine had a lower ability to inhibit the virus, all tested concentrations of berberine presented a significant reduction of foci number compared to the control (p < . ) (figure a) . emodin inhibited the virus with a significant reduction of foci number for all tested concentrations (p < . ), except for . µm ( figure b ). finally, we tested the compounds as a pre-treatment, exposing the cells to each compound prior to infection. vero e cells were incubated with berberine or emodin for h at the highest non-toxic concentrations prior to zikv infection. pre-treatment with berberine had no effect on virus entry. on the other hand, emodin reduced entry by . % (plaques reduced from × pfu/ml (control) to . × pfu/ml) (figure ) . the hydrodynamic radius of zikv was determined both before and after incubation with berberine or emodin. the purified zikv particles are monodisperse (red line, figure ) with a correlation coefficient close to unity (supplementary figure s ) and the addition of µm of emodin finally, we tested the compounds as a pre-treatment, exposing the cells to each compound prior to infection. vero e cells were incubated with berberine or emodin for h at the highest non-toxic concentrations prior to zikv infection. pre-treatment with berberine had no effect on virus entry. on the other hand, emodin reduced entry by . % (plaques reduced from × pfu/ml (control) to . × pfu/ml) (figure ). finally, we tested the compounds as a pre-treatment, exposing the cells to each compound prior to infection. vero e cells were incubated with berberine or emodin for h at the highest non-toxic concentrations prior to zikv infection. pre-treatment with berberine had no effect on virus entry. on the other hand, emodin reduced entry by . % (plaques reduced from × pfu/ml (control) to . × pfu/ml) (figure ) . the hydrodynamic radius of zikv was determined both before and after incubation with berberine or emodin. the purified zikv particles are monodisperse (red line, figure ) with a correlation coefficient close to unity (supplementary figure s ) and the addition of µm of emodin the hydrodynamic radius of zikv was determined both before and after incubation with berberine or emodin. the purified zikv particles are monodisperse (red line, figure ) with a correlation coefficient close to unity (supplementary figure s ) and the addition of µm of emodin (green line, figure ) or µm berberine (blue, figure ) led to a significant reduction of the hydrodynamic radius of the samples. zikv incubated with emodin presented a sharper profile than when incubated with berberine. since the drugs were dissolved in dmso, dls measurements were also conducted with dmso at the same concentration as the diluent, and did not indicate any significant difference with the native virus in a pbs buffer. viruses , , x for peer review of (green line, figure ) or µm berberine (blue, figure ) led to a significant reduction of the hydrodynamic radius of the samples. zikv incubated with emodin presented a sharper profile than when incubated with berberine. since the drugs were dissolved in dmso, dls measurements were also conducted with dmso at the same concentration as the diluent, and did not indicate any significant difference with the native virus in a pbs buffer. berberine and emodin, when evaluated in vitro, have virucidal effects on zika virus. berberine reduced virus infectivity by almost % in vero e cells. this compound is present in two herbs widely used in natural therapies, coptis sp. and berberis sp. [ ] , and has been shown to be effective in controlling glycemia and lipid metabolism [ ] . from an antimicrobial point of view, berberine inhibited many viruses, in particular influenza virus [ ] , chikv [ ] , and hsvs [ ] . in these studies, berberine was effective at later stages of the viral cycle, such as during uncoating and replication. however, it should be noted that the viruses mentioned are not flavivirus. emodin is an anthraquinone derivative present in several types of herbs and used primarily in eastern medicine for the treatment of various health conditions [ ] . this compound has also been shown to inhibit several viruses using different mechanisms; for example, emodin can block the coronavirus channel protein and inhibit the process of viral particle release from cells [ ] . this compound also blocked the entry step of coxsackieviruses [ ] and hsv [ ] . we also observed a modest ( . %) effect of emodin as a pre-treatment, therefore interfering in virus entry. however, the mechanism of action is not clear. in previous studies, the effect of emodin on virus entry was berberine and emodin, when evaluated in vitro, have virucidal effects on zika virus. berberine reduced virus infectivity by almost % in vero e cells. this compound is present in two herbs widely used in natural therapies, coptis sp. and berberis sp. [ ] , and has been shown to be effective in controlling glycemia and lipid metabolism [ ] . from an antimicrobial point of view, berberine inhibited many viruses, in particular influenza virus [ ] , chikv [ ] , and hsvs [ ] . in these studies, berberine was effective at later stages of the viral cycle, such as during uncoating and replication. however, it should be noted that the viruses mentioned are not flavivirus. emodin is an anthraquinone derivative present in several types of herbs and used primarily in eastern medicine for the treatment of various health conditions [ ] . this compound has also been shown to inhibit several viruses using different mechanisms; for example, emodin can block the coronavirus channel protein and inhibit the process of viral particle release from cells [ ] . this compound also blocked the entry step of coxsackieviruses [ ] and hsv [ ] . we also observed a modest ( . %) effect of emodin as a pre-treatment, therefore interfering in virus entry. however, the mechanism of action is not clear. in previous studies, the effect of emodin on virus entry was determined through its high affinity for the envelope phospholipid bilayer, and this interaction can lead to the rupture and destruction of the hsv- and vhsv viral particles [ , ] . a wide spectrum of biological activities has been described for emodin through different mechanisms of action [ ] [ ] [ ] , ] and among these, some are directly related to induced changes of the viral envelopes, such as % disruption of the herpes simplex virus type (hsv- ) envelope [ ] . the interaction of emodin with phospholipids and its effects in the integrity of vesicles and lipid extracts from escherichia coli through the perturbation of the physical properties of the bilayer have been reported [ , ] , supporting the direct effect of this anthraquinone on biological membranes. berberine has also been described to act directly on biomembranes [ ] or by inducing the increase of membrane permeability [ ] . dls measurements indicated differences in the estimated hydrodynamic radii of the virion samples before and after incubation with emodin or berberine. the calculated hydrodynamic diameter is indicative of the apparent size of the dynamic hydrated/solvated particle including the hydration sphere which encapsulates the virus particle. thus, the observed change in the apparent hydrodynamic radius of the infectious viral particle upon addition of emodin and berberine could indicate disorder or disruption of the bilayer, or alterations in the solvation characteristics of the viral particles in solution. compounds that act inhibiting virus before the attachment to the host cell, such as berberine and emodin, can be used prophylactically at low concentrations since the number of infectious particles in a primary infection is relatively low. in addition, berberine can also spread to various organs of the body, such as the liver, kidneys, muscles, and brain, and remain there for a considerable period of time [ ] . this is a desired characteristic for an antiviral targeting zika virus when maintained at low concentrations, since many cell lines and tissues has been shown to be permissive to this virus [ ] [ ] [ ] . thus, infection via mosquito bites or through sexual contact could be controlled. another beneficial feature of berberine is that it has low toxicity and no serious adverse effects in humans [ ] [ ] [ ] . some applications of berberine and emodin have encountered a few obstacles in pharmaceutical development, such as poor aqueous solubility [ , ] and rapid metabolism [ , ] . recently, several nanoparticulate delivery systems for berberine and emodin have been reported, which have attempted to address the major pharmaceutical concerns associated with its systemic administration, with lipid-based nanocarriers being the most investigated [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . zika virus has drawn much attention within the scientific community since as a result of the outbreak in brazil. efforts have been made to identify effective drugs to treat this virus and to elucidate its fundamental characteristics. in this study, we showed that berberine and emodin, two natural compounds, have strong virucidal effect in vitro, directly impairing the zikv particles. after additional studies, given the high cellular viability in the active concentrations both compounds are good candidates for treatment of zika virus-infected patients and possibly for preventive treatment in low doses in endemic areas. zika virus. i. isolations and serological specificity zika virus infection during pregnancy and microcephaly occurrence: a review of literature and brazilian data zika virus infection during pregnancy in mice causes placental damage and fetal demise zika virus impairs growth in human neurospheres and brain organoids guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study a susceptible mouse model for zika virus infection nucleoside inhibitors of zika virus the viral polymerase inhibitor -deaza- -c-methyladenosine is a potent inhibitor of in vitro zika virus replication and delays disease progression in a robust mouse infection model berberis vulgaris and berberine: an update review antiviral activity of berberine and related compounds against human cytomegalovirus anti-herpes simplex virus effects of berberine from coptidis rhizoma, a major component of a chinese herbal medicine, ching-wei-san in vivo and in vitro antiviral effects of berberine on influenza virus discovery of berberine, abamectin and ivermectin as antivirals against chikungunya and other alphaviruses hepatotoxicity or hepatoprotection? pattern recognition for the paradoxical effect of the chinese herb rheum palmatum l. in treating rat liver injury comparison of the antioxidant and transmembrane permeative activities of the different polygonum cuspidatum extracts in phospholipid-based microemulsions dna degradation by aqueous extract of aloe vera in the presence of copper ions emodin isolated from cassia obtusifolia (leguminosae) seed shows larvicidal activity against three mosquito species antiviral effect of emodin from rheum palmatum against coxsakievirus b and human respiratory syncytial virus in vitro the antibacterial activity and action mechanism of emodin from polygonum cuspidatum against haemophilus parasuis in vitro inhibition of epstein-barr virus lytic cycle by an ethyl acetate subfraction separated from polygonum cuspidatum root and its major component the effect of emodin, an anthraquinone derivative extracted from the roots of rheum tanguticum, against herpes simplex virus in vitro and in vivo inhibitory effect of emodin and astragalus polysaccharide on the replication of hbv aloe-emodin is an interferon-inducing agent with antiviral activity against japanese encephalitis virus and enterovirus zika virus in the americas: early epidemiological and genetic findings traditional chinese medicine in treatment of metabolic syndrome emodin: a review of its pharmacology, toxicity and pharmacokinetics emodin inhibits current through sars-associated coronavirus a protein vitro and in vivo studies of the inhibitory effects of emodin isolated from polygonum cuspidatum on coxsakievirus b( ). molecules emodin is a novel alkaline nuclease inhibitor that suppresses herpes simplex virus type yields in cell cultures inactivation of enveloped viruses by anthraquinones extracted from plants membrane-related effects underlying the biological activity of the anthraquinones emodin and barbaloin anti-cytomegalovirus activity of the anthraquinone atanyl blue prl antiviral activity of aloe-emodin against influenza a virus via galectin- up-regulation experimental and theoretical studies of emodin interacting with a lipid bilayer of dmpc purification and characterization of s-adenosyl-l-methionine: norcoclaurine -o-methyltransferase from cultured coptis japonica cells triggering of erythrocyte cell membrane scrambling by emodin tissue distribution of berberine and its metabolites after oral administration in rats biology of zika virus infection in human skin cells jabrane-ferrat, n. zika virus reveals broad tissue and cell tropism during the first trimester of pregnancy differential cell line susceptibility to the emerging zika virus: implications for disease pathogenesis, non-vector-borne human transmission and animal reservoirs toxicology effects of berberis vulgaris (barberry) and its active constituent, berberine: a review efficacy of berberine in patients with type diabetes mellitus berberine and neurodegeneration: a review of literature solubility of berberine chloride in various solvents improving the dissolution and oral bioavailability of the poorly water-soluble drug aloe-emodin by solid dispersion with polyethylene glycol physicochemical characterization of berberine chloride: a perspective in the development of a solution dosage form for oral delivery differences in pharmacokinetics and ex vivo antioxidant activity following intravenous and oral administrations of emodin to rats berberine hydrochloride: anticancer activity and nanoparticulate delivery system improvement of anti-inflammatory and anti-angiogenic activity of berberine by novel rapid dissolving nanoemulsifying technique development of self-microemulsifying drug delivery system for oral bioavailability enhancement of berberine hydrochloride characterization, pharmacokinetics, and hypoglycemic effect of berberine loaded solid lipid nanoparticles stealth, biocompatible monoolein-based lyotropic liquid crystalline nanoparticles for enhanced aloe-emodin delivery to breast cancer cells: in vitro and in vivo studies formulation, antileukemia mechanism, pharmacokinetics, and biodistribution of a novel liposomal emodin a promising emodin-loaded poly (lactic-co-glycolic acid)-d-alpha-tocopheryl polyethylene glycol succinate nanoparticles for liver cancer therapy development of phenylboronic acid-functionalized nanoparticles for emodin delivery key: cord- -f uvohf authors: malmlov, ashley; bantle, collin; aboellail, tawfik; wagner, kaitlyn; campbell, corey l.; eckley, miles; chotiwan, nunya; gullberg, rebekah c.; perera, rushika; tjalkens, ronald; schountz, tony title: experimental zika virus infection of jamaican fruit bats (artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: f uvohf the emergence of zika virus (zikv) in the new world has led to more than , human infections. perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with guillain-barré syndrome (gbs). zikv is transmitted to humans by aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal aedes sp. mosquitos and non-human primates. in the s and ‘ s, several bat species were shown to be naturally and experimentally susceptible to zikv with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. because of zikv emergence in the americas, we sought to determine susceptibility of jamaican fruit bats (artibeus jamaicensis), one of the most common bats in the new world. bats were inoculated with zikv prvabc but did not show signs of disease. bats held to days post-inoculation (pi) had detectable antibody by elisa and viral rna was detected by qrt-pcr in the brain, saliva and urine in some of the bats. immunoreactivity using polyclonal anti-zikv antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular leydig cells. the virus likely localized to the brain via infection of iba (+) macrophage/microglial cells. jamaican fruit bats, therefore, may be a useful animal model for the study of zikv infection. this work also raises the possibility that bats may have a role in zika virus ecology in endemic regions, and that zikv may pose a wildlife disease threat to bat populations. introduction zika virus (zikv) was first isolated from a sentinel rhesus macaque in uganda in and subsequently from aedes africanus mosquitoes in the same location [ ] . the first human cases were identified in in nigeria and serosurveys found evidence of a broad geographic distribution for zikv throughout africa and asia with sporadic cases in humans [ , ] . the first recognized zikv epidemic occurred in yap state, federated state of micronesia in . an estimated % of residents were infected, and of those % presented with clinical disease [ ] . in , a second epidemic occurred in french polynesia with , cases reported. during the latter outbreak, the incidence rate of guillain-barré syndrome (gbs) increased -fold and first indication of a connection between zikv infection and gbs was established [ ] . the virus spread to brazil in [ , ] and has since disseminated throughout much of tropical south america, central america, the caribbean, and the southern united states, with more than , confirmed cases [ ] . zikv can also cause congenital zika syndrome (czs) in naïve populations and is therefore a virus of high concern [ ] . zika virus is maintained in an urban cycle, transmitted between an aedes mosquito vector and humans thereby maintaining endemicity [ ] . it is generally accepted that the virus transmits between non-human primates and vectors in a sylvatic cycle; however, the sylvatic cycle has not been well characterized in the old world and little is known about a new world sylvatic cycle [ , ] . molecular analysis of zikv to better understand viral phylogenetics suggests that animal hosts affected viral evolution and therefore may play an important role in viral ecology [ ] . in the s and ' s, the susceptibility of bats to zikv was investigated. shepherd and williams [ ] screened wild bats from different species in uganda for antibodies against zikv and found / little free-tail bats (tadarida pumila) and / angolan freetail bats (t. condylura) were seropositive by hemagglutination inhibition assay. additionally, two angolan free-tail bats were experimentally inoculated with zikv and serially bled to test for viremia. both animals were viremic on days , and as determined by paralysis in mice inoculated with the sera from those two bats [ ] . simpson and o'sullivan [ ] experimentally inoculated three straw-colored fruit bats (eidolon helvum), three egyptian fruit bats (rousettus aegyptiacusi), and five angolan free-tail bats. two of the straw-colored fruit bats were viremic and had seroconverted. one of the egyptian fruit bats was viremic and two had seroconverted. the angolan free-tail bats were euthanized on days , , , and days post inoculation and screened for viral tropism. at one day post infection, a kidney was trace positive [ ] . finally, reagan et al. [ ] inoculated new world little brown bats (myotis lucifigus) by different routes: intracranial, intraperitoneal, intradermal, intrarectal and intranasal. bats in all groups, with the exception of the intranasal group, developed fatal neurological disease - days post inoculation. brain tissue was virus-positive in all animals with clinical disease, determined by inoculation of mice with brain homogenate suspension [ ] . considering the evidence that african bats are naturally susceptible to zikv and that little brown bats develop disease, the question emerged: could bats serve as a natural reservoir host for zikv in the new world? to test this hypothesis, we inoculated jamaican fruit bats (artibeus jamaicensis), among the most abundant bats in the caribbean, central america and mexico, with zikv to examine virology, immunology and pathology of the infection. although virus was detected in several organs, including the testes and brains, no overt clinical signs were detected, and substantial viremia or viruria was not evident. these results suggest that jamaican fruit bats are unlikely to serve as amplification hosts but that zikv infection may constitute a wildlife disease threat to bats. bats for this project were obtained from the colorado state university breeding colony approved by the institutional animal care and use committee (protocol - a). two experimental infections were conducted; a pilot study and a time course study. in the pilotstudy, three male bats (aj-z , aj-z , aj-z ) were intradermally inoculated with . x plaque forming units (pfu) zikv, strain prvabc ; a high dose to assess susceptibility. no signs of disease were apparent during this day experiment; however, all three bats had antibody titers of on day (table ) . after demonstration of susceptibility in the pilot study, a time course study was conducted. six male bats (aj-z through aj-z ) were identically inoculated and two were euthanized at , and days post inoculation (dpi). no conspicuous signs of disease were observed in any of the inoculated bats. necropsies immediately followed euthanasia and no significant gross pathology was evident. quantitative probe-based reverse transcription pcr (qrt-pcr) was performed on seruminoculated vero cell supernatants, serum, brain, lung, liver, spleen, kidney, urinary bladder, prostate and testes from bats from both studies. in addition, urine collected during the time course study was similarly assayed. urine from bats aj-z at dpi and aj-z at dpi had low levels of vrna whereas bat aj-z , euthanized at dpi, had low levels of vrna in its brain ( fig ) . all other samples were negative. sera from aj-z at dpi, and aj-z and aj-z at dpi were negative by elisa. sera were blind passaged on vero e cells in an attempt to isolate zikv and all were negative for cpe and pcr. hematoxylin and eosin stain (h&e). heart, lung, liver, kidney, testes, prostate, urinary bladder, and brain were collected from all animals as well as salivary glands from / bats. all samples were blindly read by one pathologist. a summary of the consistent histopathology findings is listed in table . for the time course study, aj-z at dpi showed mild pulmonary congestion with multifocal areas of interstitial pneumonia, mild intra-alveolar hemorrhage and mild atelectasis. terminal airways had slightly increased amounts of mucus. kidneys had multifocal interstitial infiltrates of small numbers of lymphocytes. all other tissues were within normal limits. in aj- z at dpi, lungs showed milder pathology than aj-z with minimal interstitial to perivascular infiltrates predominately lymphocytes and macrophages with a band of collapsed air spaces subjacent to the pleural surface. there were focal lesions in the left ventricle of the heart where there was individual cell loss or else fragmentation of the sarcoplasm of scattered cardiomyocytes. degenerate/necrotic cardiomyocytes were accompanied by infiltrations of small numbers of macrophages, lymphocytes and satellite cells. all other tissues were within normal limits. lungs from aj-z at dpi had minimal focal interstitial histiocytic pneumonia with atelectasis. kidneys showed multifocal chronic lymphohistiocytic pyelitis with a few degenerate and detached epithelial cells accumulating in the renal pelvis and infiltration of pelvic stroma by small numbers of mixed inflammatory cells. mandibular salivary gland showed focal moderate cellular infiltrates of periductular lymphocytes and macrophages. affected salivary ducts contained detached and degenerate epithelial cells and leukocytes. occasional ducts were encircled by granulation tissue and a few heterophils. rare apoptosis was evident in the lining epithelium of such ducts. all other tissues were within normal limits. aj-z at dpi had lungs with minimal alveolar septal infiltrates scattered within collapsed lung parenchyma along with multifocal microscopic hemorrhages. kidneys had multifocal areas of mineralization. in the outer medulla and at the cortico-medullary junction were rare perivascular infiltrates of lymphoplasmacytes. esophagus and lymphoid tissue associated with palatine salivary gland showed focal mild lymphoplasmacytic inflammation. moderate numbers of lymphocytes and plasma cells were arranged in columns parallel to the respiratory mucosal epithelium of the nasophayrnx. the lumen contained increased amounts of mucus and a few inflammatory cells, mainly heterophils and lymphocytes. in the testicles, there was focal testicular degeneration manifested by presence of giant spermatids in the lumina of affected seminiferous tubules and accumulation of a small numbers of interstitial lymphocytes and macrophages. all other tissues were within normal limits. lungs from aj-z at dpi had minimal interstitial to perivascular infiltrates with multifocal atelectasis and microscopic hemorrhages. the left papillary muscle of the heart showed rare multifocal cardiomyocyte necrosis characterized by rounding up of individual cardiomyocytes. necrotic cardiomyocytes appeared with hypereosinophilic cytoplasm, devoid of cross striations or fragmented and rarely vacuolated. minimal interstitial hypercellularity due to increased activity of satellite cells and infiltration of small numbers of lymphocytes was observed in the vicinity of degenerate/necrotic cardiac muscle fibers. kidneys had an area of focal lymphoplasmactyic pyelitis. additionally, there was a focal area of mineralization and inflammation in the inner medulla. all other tissues were within normal limits. aj-z at dpi had occasional focal inflammation and cardiomyocyte degeneration in the left ventricle and interventricular septum. area ca of the hippocampus in the brain showed focal pyrimidal neuronal necrosis with a focal area of mineralization around a vessel in the cerebral cortex along with focal gliosis and individual neuronal necrosis (fig ) . all other tissues were within normal limits. testicular, neural and salivary glands' lesions are believed to be associated with zikv infection as they were not seen with other viral infections. in the pilot study bats, aj-z at dpi had more prominent interstitial pneumonia with congestion of the lungs compared to earlier time points. the heart had minimal cardiomyocyte degeneration and necrosis with hypercellular interstitium and increased amounts of mature fibrous connective tissue. the kidney had focal interstitial infiltrates of the cortical and outer medullary interstitium. the brain showed degenerate neurons in area a of the hippocampus. all other tissues were within normal limits. aj-z had minimal focal testicular degeneration (fig ) . all other tissues were normal. aj-z had perivascular lymphocyte pulmonary infiltrates and atelectasis. heart demonstrated locally extensive lymphocytic and histiocytic pericarditis. kidneys showed multifocal interstitial lymphocytic infiltrates. brain had focal, perivascular infiltrates of small numbers of lymphocytes at the subfornical commissure. the reticular formation showed multifocal neuronal degeneration/necrosis. immunohistochemistry and immunofluorescence. tissues were stained with a polyclonal antibody for zikv (cdc, fort collins). ajz- at dpi with inflammation of the mandibular salivary gland had moderate immunoreactivity in the lumen of affected ducts (fig ) . aj-z at dpi had immunoreactive cells in the brain and mononuclear cell immunoreactivity in the testes ( fig ) . additionally, aj-z demonstrated immunoreactivity in purkinje cells of the cerebellum (fig a) . aj-z at dpi had immunoreactive cells around the pulmonary arteries in the lungs ( fig a) . aj-z also had immunoreactivity perivascullarly in the tunica albuginea of the testes (fig a) . scrotal skin had focal lymphocytic dermatitis with immunoreactive mononuclear cells ( fig d) . cell morphology consistently identified mononuclear cells compatible with macrophages and fibroblasts as the primary cell types showing immunoreactivity against zikv antigen. brain and testicular tissues stained with both goat polyclonal goat anti-iba (green) and monoclonal g- flavivirus e specific antibodies (red) showed co-localization (yellow) of zikv antigen in cytoplasm of activated microglial cells with their characteristic morphology in the cerebral cortex of infected bats dpi in the time course study and day dpi in the pilot study (fig ) . increased microgliosis was noted in the vicinity of co-localization sites. the gliosis was also prominent in the cerebellum and hippocampus especially around dead neurons. in the testicles, occasional macrophages showed similar co-localization similar to that noted in the brain in the testicular interstitium, inner layer of tunica albuginea and scrotum. cells consistent in morphology with leydig cells were similarly highlighted by zika viral antigen only showing strong immunoreactivity using polyclonal anti-zikv antibody. two bat infection experiments were conducted in this investigation; ) a pilot study to determine susceptibility of jamaican fruit bats to zikv infection, and ) a time course study to better understand pathophysiology and chronology of events pertaining to the dynamics of viremia, viral tropism, replication and shedding of the virus in a new world bat species. the goal was to determine whether bats can be used as an animal model for zikv pathogenesis and to assess the possible role of bats in zikv ecology in the new world. in the pilot experiment, no signs of disease were apparent during the -day study. sera collected at euthanasia indicated modest antibody titers of for each bat by elisa (table ) , whereas the human -convalescent control serum titer was � , . bats typically have low to modest antibody titers, perhaps due to limited somatic hypermutation and affinity maturation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . concerning viremia, cell-serum supernatants, blind passage supernatants, and neat serum results were all negative. although serum is routinely used for zikv diagnostics in humans, it may not be the most suitable sample [ ] [ ] [ ] [ ] [ ] . in one investigation zikv patient had negative serum sample for the duration of the study, whereas whole blood yielded positive qrt-pcr results from days to [ ] . one possible explanation for the phenomenon of negative serum in human patients is that the virus during acute infection disseminates via a cell-associated viremia or as novel findings suggest that the virus gets phagocytized in neutrophils and therefore whole blood is a more sensitive diagnostic sample than serum. viruria is commonly detected in zikv-infected humans [ ] ; therefore, urine may be an equally important diagnostic sample with higher viral load in early infection when compared to blood in humans and other primates [ ] [ ] [ ] [ ] . although urine collection from bats was challenging, we collected urine from some of the inoculated bats in the time course study. aj-z exhibited viruria only at dpi, and aj-z was equivocal only at dpi, corroborating the findings in other mammals that urine may be a route of viral shedding early in infection. urine from one human patient was positive from the first time point ( dpi) through dpi and again on day . similarly, saliva from that same patient was positive from day nine through day and again on day [ ] . another investigation compared diagnostic samples of infected patients and showed that urine was positive in of them, whereas serum was only positive in patients by qrt-pcr. the study concluded that viral loads in urine were tenfold higher compared to serum and that uremia lasted longer [ ] . these data corroborated the first study that identified zikv shed in urine in which there was a higher viral load in urine for longer duration compared to serum [ ] . zikv rna in plasma was detected in the bats by qrt-pcr between and dpi, but between to dpi in urine [ ] . the lack of detectable viremia in the serum of bats is congruent with some of the human and nhp investigations in that viremia is low and short-lived. detached renal pelvic urothelial cells and degenerate salivary gland ductular epithelium as seen in the current study will make urine and saliva equally important fluids to collect in order to maximize detection of zikv in the acute and established stages of infections. for this experiment, all male bats were used because female bats are prioritized for colony expansion. zikv exhibited tropism for the testes with strong immunoreactivity in reproductive organs (figs & ) . histologically, minimal focal testicular degeneration in two bats ( fig ) suggests viral related pathology may be minimal. in humans it has yet to be completely elucidated what reproductive organs harbor zikv, it has been determined that semen contains zikv both in both vasectomized and unvasectomized men [ , ] . this suggests that zikv is sequestered in the testes and/or accessory sex glands. mouse models have demonstrated zikv infection and associated pathology in the testes [ ] [ ] [ ] of humanized blt mouse model with infection primarily targeting macrophages and leydig cells [ ] . limited investigation has been done relating to infection of accessory sex glands in mouse models, but one study that assessed the prostate found no virus, possibly due to differential expression of the receptor candidate in the testes but not in the prostate [ ] . for this experiment the finding of viral antigen and viral rna in the testes but not in the prostate is consistent with published animal models and may suggest the potential for bats to serve as another animal model. three bats had histopathological alterations in the hippocampus at later time points and one bat had viral nucleic acid present in the brain as determined by qrt-pcr demonstrating tropism for the cns, a tissue predilection also documented in humans and animal models. zikv has a predilection for nervous tissue in animal studies and disease manifestation in humans. as a neurological teratogen, zikv has been detected in the brain mononuclear cells in human newborns with fatal microcephaly and fetal miscarriages. histological lesions are varied but may include parenchymal calcification, microglial nodules, gliosis, cell degeneration, mononuclear infiltration and necrosis [ ] [ ] [ ] . in non-human animal models, evidence for viral tropism has been found in brain and/or peripheral nervous tissue [ ] [ ] [ ] [ ] . in immunocompromised mouse models, the virus has a predilection for the brain but with the mice engineered for specific immune traits it is difficult to know to what extent this recapitulates natural zikv pathophysiology [ ] . in the bats used in this experiment, evidence of zikvinduced pathology in the brain is consistent with what has been seen in human newborns and fetuses. the novel finding of co-localizing zikv antigen in bat iba + microglial/macrophage cells lends support to the earlier evidence of microglial cell infection via axl ligand bridging zikv particles to glial cells [ ] . iba (aka, allograft inflammatory factor , aif ) is a microglia/macrophage-specific calcium-binding protein, which has actin-bundling activity that participates in membrane ruffling and phagocytic activity of activated microglia. activated microglial cells appeared with increased ability of cell migration and phagocytosis, which is controlled by remodeling of membrane cytoskeleton [ ] . the morphology of cells with co-localization in the brain of infected bats is consistent with activated microglia depicting prominent branched processes. recent primate models in rhesus and cynomolgus macaques demonstrated similar viral distribution of zikv antigen to that in bats, described herein. high-level of zikv was evident in cerebellar neurons and the same studies documented involvement of iba positive microglial cells in cns infections. in primate models there is increasing evidence that zikv antigen was detected in individuals with the highest peak plasma viremia, which in part implies that zikv may initially seed the cns by a passive spillover from circulating monocytes to resident microglial cells. this is further substantiated in all of human and animal studies, which did not show any evidence of disruption to bbb or viral distribution reminiscent of circumventricular distribution seen in alphavirus animal models [ ] . in addition to brain and testes immunoreactivity, scrotal skin and mandibular salivary gland also harbored viral antigen. distribution of viral antigen in bat tissues suggests that infection in this species recapitulates human infection, which is thought to start with infection of epidermal and dermal cells with subsequent dissemination to multiple organs including salivary glands as viral rna can be detected in human saliva [ , ] . the histopathology for aj- zika virus infects new world bats z , dpi showed sialoadenitis and the presence zikv antigen by ihc (fig ) . this suggests zikv may be shed in the saliva, although additional animal experiments need to be performed to confirm such a route of shedding. the results presented here suggest that jamaican fruit bats may be a suitable animal model for examining zikv infection to elucidate its pathogenesis. jamaican fruit bats may also serve as a model to ascertain sexual transmission, in utero transmission, teratogenesis and neurological pathophysiology. it may be that zikv is a wildlife disease threat for bats that could lead to infertility in some males, which could impact bat populations. zikv is thought to be maintained in two different distinct cycles: sylvatic-cycling between non-human primates (nhp) and mosquito species, and urban-cycling between humans and mosquito species [ ] . while there are limited data on what mosquito species feed on jamaican fruit bats, evidence for natural flavivirus infection has been identified in wild new world bats. dengue virus (denv) rna and antibodies to denv were detected in multiple species of bats, including jamaican fruit bats, in mexico [ ] . additionally, antibodies to denv were detected in multiple bat species including those of the artibeus genus in costa rica and ecuador [ , ] . these data indirectly provide evidence for mosquito-bat interactions in the wild; either through consumption of bat-blood meals taken by mosquitoes or bat consumption of infected mosquitoes. as it pertains to a wildlife reservoir, wild nhps have antibody to zikv including several monkey species trapped near ziika forest [ ] , and wild and semi-captive orangutans in borneo [ ] . not only have nhp been found to be seropositive, but also many other mammals, including rodents, horses, cows, and goats [ , ] . furthermore, experimental inoculation of various north american species resulted in seroconversion (cottontail rabbits, boar goats, pigs, and leopard frogs) and demonstrated viremia (nine-banded armadillo and leopard frogs) [ ] . molecular epidemiology suggests animals play an important role in an enzootic cycle [ ] . much about the enzootic cycle of zikv has yet to be understood but it stands to reason that bats may be capable of maintaining the virus in nature. jamaican fruit bats are found in northern south america, central america, and the caribbean-areas that now have zikv potentially exposing bat populations to the virus [ , ] . however, the data presented here suggest it is unlikely that jamaican fruit bats can serve as amplification hosts of zikv, unless virus sequesters in some as-yet unidentified way that could lead to periodic shedding of virus. it may also be that some bats become persistently infected and can transmit sexually to maintain virus within populations of bats. further experimental and field studies will be necessary to fully understand the ecological role of bats in zikv maintenance. all animal procedures were approved by the colorado state university (csu) institutional animal care and use committee (protocol - a) and were in compliance with u.s. animal welfare act. bats csu has a captive colony of jamaican fruit bats (artibeus jamaicensis), a neotropical fruit bat indigenous to much of south america, central america and the caribbean [ ] . colony bats are kept in a free flight room measuring 'w x 'l x 'h. roosting baskets are hung from the ceiling throughout the room and drapes of different cloth material are positioned for hanging and roosting. ambient temperature is maintained between ˚c and ˚c, with humidity between % and %, and a hour light/ hour dark light cycle via a computer-controlled system. diets consist of a combination of fruits (shamrock foods, fort collins, co), tekald primate diet (envigo, huntington, uk), molasses, nonfat dry milk and cherry gelatin that are placed in multiple feeding trays around the room once a day. fresh water is provided. in addition, fruit is hung around the room to stimulate foraging behavior and serve as enrichment. for infection experiments, bats were trapped using a butterfly net and placed in an "d x "w x "h cage for hours prior to inoculations to allow for acclimation. hanging clothes were provided for roosting and coverage. food and water are placed in open trays in the bottom of the cage and changed daily. tray liners were changed every two days, and cages and hanging clothes are changed every two weeks. due to the social nature of these bats, minimums of two bats were kept in cages at all times to mitigate potential stress. two sets of experiments were performed; a pilot study and a time course study. zika virus strain prvabc . prvabc was isolated in by centers for disease control and prevention (fort collins, co) from an infected individual who traveled to puerto rico (genbank accession no. hq ). the virus stock titer is x plaque forming units (pfu) per ml of media, and the fourth passage was used for both studies. for the pilot study, three male bats were anesthetized with % to % isoflurane to effect with an oxygen flow rate of . l/min, administered with a gas mask. animals were placed on a heating pad to maintain body temperature and respirations continuously monitored. the dorsum of each animal was disinfected with % ethanol and ul containing . x p.f.u of virus was administered subcutaneously (sc) at the level of the scapula with a sterile hypodermic gauge needle in a biosafety cabinet. when procedures were finished, bats were removed from isoflurane and placed back in the cage in ventral recumbency. respirations were monitored until animal was fully awake and ambulated normally. bats were identified as aj-z , aj-z and aj-z . animals were euthanized at days post-inoculation (dpi). for the time course study, six male bats were anesthetized under the same protocol as the pilot study. animals were placed in ventral recumbency. after disinfecting the dorsum of each animal with % ethanol, . mls of % lidocaine was administered sc at the level of the last rib with a gauge sterile hypodermic needle as a local anesthetic. iptt transponders (biomedic data systems, inc., seaford, de) were inserted sc at the level of the caudal edge of the scapula. twenty-five microliters containing . x p.f.u of virus was administered sc at the level of the cranial edge of the scapula. recovery followed the same protocol as for the pilot study bats. animals were identified as aj-z through aj-z . aj-z and aj-z were euthanized at two dpi. aj-z and aj-z were euthanized at dpi. aj-z and aj-z were euthanized at dpi. female bats were excluded from the study because they are prioritized for breeding to sustain and expand upon the colony. for the pilot study, bats were visually monitored twice daily for fourteen days, and then monitored once a day for an additional fourteen days. for the time course study, bats were monitored twice a day throughout the experiment. for both studies, energy levels, behavior, ability to ambulate, respirations, presence of oral or nasal discharge, and fecal consistency were all assessed. during the time course study urine was collected at , , and dpi from as many bats as possible. urine was collected by allowing bats to grasp screen cloth with their feet and then the bat was placed in a clear solo cup (dart container, lake forest, il) with the screen covering the top of the cup as a lid, and kept in place with a rubber band. this allowed the bats to hang in a clear container. bats were monitored for minutes. if they urinated, bats were removed from the collection contraption and placed back in the cage without disrupting the urine. urine collection was attempted on all remaining bats at each time point, but not all bats would urinate at each collection attempt. urine was successfully collected as follows: two dpi from aj-z and aj-z ; three dpi from aj-z , aj-z and aj-z ; five dpi from aj-z , aj-z , aj-z and aj-z ; and ten dpi from aj-z and aj-z . urine was pipetted off the surface of the cup with a sterile pipette tip and put in a . ml microcentrifuge tube and stored at - ˚c for future use. urine volume ranged between ul and ul. bats were deeply anesthetized and maintained with % isoflurane and an oxygen flow rate of . l/min. deep pain was assessed by firmly pinching skin and toes with forceps and assessed for any response. a thoracotomy was then performed with sterile standard scissors to puncture through the skin, muscle and diaphragm just caudal to the sternum and cut through the wall of the chest cavity caudally to cranially-removing and preventing negative pressure from building in the thorax. cardiac blood was collected with a gauge sterile needle inserted into the apex of the heart. a maximum blood volume of between and . mls is collected in a syringe and transferred to a red top tube (rtt). rtts sat at room temperature for one hour to allow a clot to form and then centrifuged at x g for min at room temperature. serum was removed from the clot, placed in a new microcentrifuge tube and stored at - ˚c. serum from bats at and dpi were used to assess for viremia. serum from dpi and the dpi pilot study bats were used to determine antibody titers. because blood draws yield a small volume of blood ( μl whole blood for a non-terminal blood draw, μl whole blood for terminal blood draw) it was necessary to prioritize samples to optimize data retrieved. in order to assay the serum for viral rna and perform serology, earlier time points were used to assess for viremia and later time points for seroconversion. along with sample partitioning for data maximization, the small blood volume led to concerns that there would be an undetectably small viral load. to circumvent this issue, neat serum and : diluted serum were inoculated onto vero cells to amplify any virus that may have been present at low levels. one blind passage on vero cells was done and cell supernatants assayed by qrt-pcr. the remaining serum from three of the four bats was assayed directly for zikv rna. necropsies were performed immediately after euthanasia. bats were assessed for gross pathology. the following tissues were collected for both experiments: heart, lung, liver, spleen, kidney, urinary bladder, prostate, testes, and brain. a portion of tissues were collected and kept at - ˚c for rna extraction, and a portion placed in % buffered formalin for histology at a : weight to volume ratio for histology. for a negative control animal a male bat was trapped from the colony and euthanized under the same protocol as the experimental infection bats. vero e cells (atcc) were propagated to % confluency in a -well tissue culture plate and infected with zikv strain prvabc at an m.o.i. of . . after a one hour incubation period, unbound virus was removed and replaced with % fbs-dmem and incubated for a maximum of three days. media was then replaced with % acetone for minutes at - ˚c to fix virusinfected cells to plate and serve as an antigen for enzyme-linked-immunosorbent assay (elisa). plates were stored at ˚c until use and used within two weeks. plates were washed x with . % tween -pbs and blocked with superblock t (tbs) blocking buffer (thermo fisher scientific, waltham, ma) for one hour at room temperature. serum from an uninfected bat was used for a negative control. a convalescent human serum sample (kindly provided by b. foy, csu) was used as a positive control. a two-fold serial dilution was used starting at : to : . diluted serum was placed in wells and incubated for two hours at room temperature. serum was removed and plates washed. hrp-conjugated protein a/g (thermo fisher scientific, waltham, ma) was added at a concentration of μg/ml to each well, and incubated for minutes at room temperature. hrp-conjugated protein a/g was used in place of a secondary antibody as it targets the fc portion of an antibody, which is highly conserved and therefore can be used for multiple animal species [ ] . plates were washed and μl of abts peroxidase substrate ( component) (kpl, gaithersburg, md) added according to manufacturers' instructions, incubated at room temperature for minutes, and then μl of abts peroxidase stop solution (kpl, gaithersburg, md) added. plates were read on an emax plus microplate reader (cambridge scientific, watertown, ma). absorbance was measured at nm and the limit of detectable response was set at three standard deviation values above mean negative control serum. trizol reagent was used for rna extraction from serum-cell supernatants, serum, urine and tissues according to ambion, life technologies protocol. for tissues, approximately mg of tissue was homogenized with one ml of trizol reagent. a mm stainless steel bead (qiagen, valencia, ca) was used with a tissuelyser lt (qiagen, valencia, ca) at hz for minutes. one ml of trizol was added to urine to to μl of urine. one ml of trizol was added to μl of serum from aj-z , aj-z , and aj-z . two-hundred microliters of serum-cell supernatants were added to one ml of trizol. samples were then incubated at room temperature for minutes. chloroform (thermo fisher scientific, waltham, ma) was added, samples were mixed, incubated for minutes at room temperature and centrifuged at , x g for minutes at ˚c. the aqueous phase was removed, μg of glycogen (thermo fisher scientific, waltham, ma) and % molecular grade isopropanol added (thermo fisher scientific, waltham, ma). samples were incubated at room temperature for minutes and then centrifuged at , x g for minutes at ˚c. supernatant was removed and % molecular grade ethanol (thermo fisher scientific, waltham, ma) was added to rna pellet. samples were vortexed and centrifuged at x g for minutes at ˚c. wash was removed and air-dried. rna was resuspended in rnase-free water and stored at - ˚c for future use. vero cells were grown to to % confluency in a -well tissue culture plate with % fbs-dmem. media was removed and ul of bat serum from dpi bats and dpi bats was inoculated onto cells. additionally, serum from each bat was diluted -fold in % fbs (millipore sigma) pbs supplemented with % calcium and magnesium, and inoculated onto cells. samples were incubated for one hour at ˚c. inoculum was removed and cells washed twice in sterile pbs. two-percent fbs-dmem was added to wells and plates were incubated at ˚c, % co . cells were assessed daily for cytopathology (cpe) through day but none was observed. two-hundred microliters of the supernatant was removed on day and used for rna extractions. an additional μl of supernatant was blind passaged onto vero cells at to % confluency. cells were incubated for one hour at ˚c, washed twice with sterile pbs and % fbs-dmem added. on day seven, supernatant was removed and trizol extractions performed for rna recovery. serum was treated as such in an attempt to amplify viral load and increase assay sensitivity serum may not be the most sensitive diagnostic sample [ ] [ ] [ ] [ ] . if any serum was remaining it was directly used for trizol rna extractions. serum samples remained from aj-z at dpi, and aj-z and aj-z at dpi. no serum remained from aj-z . roche real time ready rna virus master kit (roche, indianapolis, in) was used on rna extracted from serum-cell supernatants, serum, urine and tissue to assay for zikv rna according to manufacturers' instructions. primers used were zikv (ccgctgcccaa cacaag) and zikv c (ccactaacgttcttttgcagacat). probe was zikv -fam (agcctaccttgacaagcagtcagacactcaa) [ ] . two-hundred nanograms of sample rna was added to each reaction. reactions were performed in duplicate. standards were a non-infectious clone of full length zikv strain prvabc by which concentration was determined through optical density. molecular weight of the genome sequence was used to calculate copy number [ ] . a log dilution series of the standard was made and linear regression used to determine copy number equivalents of positive samples. amplification was performed according to manufacturers' protocol for roche real time ready rna virus master kit (roche diagnostics corporation, indianapolis, in) with pcr conditions as follows: min at ˚c, s at ˚c, and cycles of s at ˚c, s at ˚c and s at ˚c. tissues fixed in %-buffered formalin were cut in and submitted to colorado state university veterinary diagnostic laboratory (csu vdl, fort collins, co) for paraffin embedding, sectioning and staining with hematoxylin and eosin, as well as immunohistochemistry (ihc). tissues cut in on bats to assess for histology included: heart, lung, liver, kidney, testes, prostate, urinary bladder and brain. additionally, for aj-z and aj-z mandibular salivary gland was cut in. aj-z had esophagus and lymphoid tissue that included palatine salivary gland cut in. antibody for ihc was a polyclonal rabbit antibody that targets prem and e proteins of zikv and was provided by csu vdl's pathology department. the bond-iii automated instrument (leica biosystems, wetzlar, germany) was used for ihc staining. all slides were blindly read by a diplomat of the american college of veterinary pathologists. brain tissues was prepared for immunohistochemical and immunofluorescence staining as previously reported [ ] . tissue was dehydrated by using a graded ethanol series of % ethanol for h, % overnight, % for h and % for h. brain tissues were then post-fixed in dimethylbenzene for min and embedded in dimethylbenzene-paraffin at ˚c for h, after which samples were embedded in a metal frame. sagittal sections were collected at um thick. all dewaxing, antigen retrieval and immunofluorescence staining was automated using a leica bond rxm. in short, sections were dewaxed using ethanol and then boiled in antigen retrieval solution for minutes. the cooled sections were incubated in % h o for min at room temperature and then blocked with % donkey and goat serum (millipore sigma) for hour. rabbit anti-iba (wako chemicals usa, irvine, ca) and g- flavivirus e specific monoclonal antibodies (cdc, fort collins) were diluted in tbs to final concentrations of : and : , respectively. sections were incubated in primary antibodies concurrently at room temperature for one hour. following removal of unbound primary antibodies by washing, goat anti-rabbit secondary (alexafluor- ) and donkey anti-mouse secondary (alexafluor- ) was added and incubated for hour at room temperature. finally, dapi counterstain (vector laboratories, burlingame, ca) was applied and sections were washed with tbs prior to cover slipping for imaging. stained sections were imaged on a ziess lsm with airyscan laser-scanning confocal microscope (ziess, oberkochen, germany) using a × oil immersion objective. each field of view was imaged as a z-stack ( - planes, . -μm step size) transformed into a single maximum projection image using the ziess zen (blue) imaging software. zika virus. i. isolations and serological specificity zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria zika virus: history, emergence, biology, and prospects for control zika virus outbreak on yap island, federated states of micronesia rapid spread of emerging zika virus in the pacific area zika virus outbreak, bahia, brazil. emerg infect dis first report of autochonous transmission of zika virus in brazil zika cases and congenital syndrome associated with zika virus reported by countries and territories in the americas potential for zika virus to establish a sylvatic transmission cycle in the americas yellow fever and zika virus epizootics and enzootics in uganda molecular evolution of zika virus during its emergence in the th century studies on viruses in east african bats (chiroptera). . haemagglutination inhibition and circulation of arboviruses studies on arboviruses and bats (chiroptera) in east africa. ii. isolation and haemagglutination-inhibition studies on bats collected in kenya and throughout uganda effect of zika virus and bwamba virus in the cave bat (myotis lucifugus) transmission studies of hendra virus (equine morbillivirus) in the fruit bats, horses and cats pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission antibody-mediated immune response in the bat, pteropus giganteus detection of specfic antibody responses to vaccinatin in variable flying foxes (pteropus hypomelanus) the little brown bat, m. lucifugus, displays a highly diverse vh, dh, jh repertoire but little evidence of somatic hypermutation tacaribe virus cases fatal infection of an ostensible reservoir host, the jamaican fruit bat replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) transcriptomic signatures of tacaribe virus-infected jamaican fruit bats assay optimization for molecular detection of zika virus a rhesus macaque model of asian-lineage zika virus infection zika virus testing considerations: lessons learned from the first eighty real-time rt-pcr-positive cases diagnosed in new york state detection of zika virus in urine long-term kinetics of zika virus rna and antibodies in body fluids of a vasectomized traveller returning from martinique: a case report persistence of zika virus in body fluids-preliminary report zika virus causes testis damage and leads to male infertility in mice zika virus infection damages the testes in mice a mouse model of zika virus pathogenesis zika viral infection and neutralizing human antibody response in a blt humanized mouse model notes from the field: evidence of zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses-brazil zika virus damages the human placental barrier and presents marked fetal neurotropism pathology of congenital zika syndrome in brazil: a case series zika virus infection of rhesus macaques leads to viral persistence in multiple tissues fetal brain lesions after subcutaneous inoculation of zika virus in a pregnant nonhuman primate nonhuman primate models of zika virus infection, immunity, and therapeutic development zika viral dynamics and shedding in rhesus and cynomolgus macaques overview of the current status of zika virus pathogenesis and animal related research axl mediates zika virus entry in human glial cells and modulates innate immune responses microglia/macrophage-specific protein iba binds to fimbrin and enhances its actin-bundling activity entry sites of venezuelan and western equine encephalitis viruses in the mouse central nervous system following peripheral infection detection of zika virus in saliva biology of zika virus infection in human skin cells denge virus in mexican bats neotropical bats that co-habit with humans function as dead-end hosts for dengue virus detection of dengue virus neutralizing antibodies in bats from costa rica and ecuador sylvatic transmission of arboviruses among bornean orangutans zika virus, vectors, reservoirs, amplifying hosts, and their potential to spread worldwide: what we know and what we should investigate urgently a sero-epidemiological survey for certain arboviruses (togaviridae) in pakistan investigating the potential role of north american animals as hosts for zika virus. vector borne zoonotic dis mammalian species: artibeus jamaicensis chimeric igg-binding receptors engineered from staphylococcal protein a and streptococcal protein g genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia rapid and specific detection of asian-and african-lineage zika viruses fluoxetine prevents lps-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress the authors would like to than brent davis, cdc, fort collins, co for supplying anti-zikv polyclonal and specific monoclonal antibodies. key: cord- - dljap authors: young, ginger; bohning, kelly j.; zahralban-steele, melissa; hather, greg; tadepalli, sambasivarao; mickey, kristen; godin, c. steven; sanisetty, srisowmya; sonnberg, stephanie; patel, hetal k.; dean, hansi j. title: complete protection in macaques conferred by purified inactivated zika vaccine: defining a correlate of protection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dljap a critical global health need exists for a zika vaccine capable of mitigating the effects of future zika epidemics. in this study we evaluated the antibody responses and efficacy of an aluminum hydroxide adjuvanted purified inactivated zika vaccine (pizv) against challenge with zika virus (zikv) strain prvabc . indian rhesus macaques received two doses of pizv at varying concentrations ranging from . µg − µg and were subsequently challenged with zikv six weeks or one year following the second immunization. pizv induced a dose-dependent immune response that was boosted by a second immunization. complete protection against zikv infection was achieved with the higher pizv doses of . µg, µg, and µg at weeks and with ug pizv at year following vaccination. partial protection was achieved with the lower pizv doses of . µg and . µg. based on these data, a neutralizing antibody response above . log( ) ec was determined as a correlate of protection in macaques. pizv elicited a dose-dependent neutralizing antibody response which is protective for at least year following vaccination. in and , large outbreaks of zika virus (zikv) occurred in the americas. these outbreaks were associated with clusters of congenital microencephaly and other severe neurological sequelae in infections in approximately of infants born to pregnant women with laboratory confirmed zika in the us and us territories . incidence of zikv infections subsequently declined in most of the americas throughout and . with the sporadic nature of zikv outbreaks and a very low incidence of symptomatic disease in both endemic and non-endemic areas, conducting phase clinical efficacy trials is not feasible. still, the risk of re-emergence and the severe consequences of infection in pregnant women demonstrate that the need for an effective zika vaccine remains. in such circumstances, alternative regulatory strategies such as animal rule approval or accelerated approval pathway may be relevant for licensure . non-human primate studies have contributed to the development of zikv vaccines by demonstrating protective efficacy and identifying biomarkers of protection against zikv. results to date have supported neutralizing antibodies as an immune marker that is reasonably likely to predict clinical benefit of several zikv vaccines , . indian rhesus macaques (macaca mulatta) are susceptible to zikv infection and have been used extensively as a model to study efficacy of zikv vaccines and pathogenesis of multiple zikv isolates [ ] [ ] [ ] [ ] [ ] . zikv infection can be performed by subcutaneous injection, which mimics infection via mosquito bite and causes consistent viremia [ ] [ ] [ ] [ ] [ ] . the kinetics of zikv infection are similar in rhesus macaques and humans where serum or plasma viremia typically peaks within the first six days of infection and resolves within - days , , . the purified inactivated zika vaccine (pizv) has previously been evaluated in mouse models and was immunogenic in ag and cd mice and protected ag mice against lethal zikv challenge . in those studies, baldwin et al. demonstrated that neutralizing antibodies correlate with protection in ag mice. pizv is currently being evaluated for safety and immunogenicity in phase trials (clinicaltrials.gov nct ). pizv elicits a dose dependent neutralizing antibody response. rhesus macaques were vaccinated with pizv on days and and challenged on day . serum neutralizing antibodies were tested using a zika reporter virus particle (rvp) assay. all macaques were seronegative on day and control macaques remained seronegative prior to zikv challenge (fig. ) . twenty-eight days following the first vaccine dose (study day ), little or no seroconversion was observed for the . and . µg pizv doses, while / macaques vaccinated with the . µg pizv dose seroconverted and / macaques receiving the and µg pizv dose seroconverted. immune responses were boosted in all groups after the second dose (all p-values ≤ . ). twenty-eight days after the second pizv dose (study day ), % of vaccinated macaques seroconverted. the magnitude of neutralizing antibody titers on day was similar after the second dose of . , , or µg, with no statistically significant difference detected among these groups. the titer on day was lower in the . (p = . ) and . µg groups www.nature.com/scientificreports www.nature.com/scientificreports/ (p = . ) compared to the . µg dose group. titers prior to zikv challenge on day were slightly decreased compared to day . following challenge, neutralizing antibody titers increased significantly in the placebo, . and . µg groups (all p-values ≤ . ), but not in the . , , and µg groups. neutralizing antibody levels after two pizv doses of . , , or µg were similar to post-challenge neutralizing antibody titers in the placebo group. in conclusion, we observed a dose-dependent neutralizing antibody response to one or two doses of pizv. after two pizv doses, the neutralizing antibody levels reached a plateau for vaccine doses above . µg, and the level of neutralizing antibody was comparable to infection with zikv challenge in the placebo group. the lack of anamnestic antibody responses after challenge in the . , , and µg pizv dose groups suggests that infection by zikv challenge virus may have been prevented by vaccination. in addition to evaluating neutralizing antibodies, we also quantified anti-zika-specific igg using a luminex-based assay. as with neutralizing antibodies, all pizv doses were immunogenic and no anti-zika igg was detected in the control group prior to zikv challenge. pizv elicited dose-dependent anti-zika igg responses (fig. ) in the vaccinated groups. anti-zika igg responses were significantly boosted after a second pizv dose for all vaccinated groups (all p-values ≤ . ). following zikv challenge, the anti-zika igg responses increased significantly in the placebo, . and . µg groups (all p-values ≤ . ), but not in the . , , and µg groups. similar to the neutralizing antibody response, the anti-zika igg titers following vaccination with the . , , or µg doses were similar to post-challenge anti-zika igg titers in the placebo group. pizv protects against zikv challenge. rhesus macaques were challenged subcutaneously on day with ffu prvabc . all macaques receiving the . , , or µg pizv dose were protected against zikv challenge, as no zika vrna could be quantified in any of the macaques in these groups. zika vrna was quantified in / macaques receiving the . µg dose, / macaques receiving the . µg dose, and in all macaques in the control group (fig. ) . the peak post-challenge zika vrna level decreased with increasing vaccine dose level. the peak zika vrna occurred on day ( days post-challenge), with a geometric mean level of . log copies/ ml in the control group (range . - . log copies/ml), . log copies/ml in the . µg dose group (range . - . log copies/ml) and . log copies/ml in the . µg dose group (range . - . log copies/ml). no clinical signs to the vaccine or challenge virus were seen throughout the studies. correlate of protection. pizv elicited a dose dependent neutralizing antibody immune response and an anti-zika igg response which correlated with a reduction in zikv vrna post-challenge (table ). an immune correlate analysis was subsequently performed to correlate zika vrna with neutralizing antibody titers (fig. ) and anti-zika igg (fig. ) , which demonstrated that an increase in both neutralization antibody and anti-zika igg titers correlates with a decrease in vrna concentration. correlation with protection was observed with both neutralizing antibodies ( . log ec ) and anti-zika igg ( . log u/ml). we chose the functional assay, neutralizing antibodies, to establish a correlate of protection in macaques as a neutralizing antibody titer of > . log ec , which conferred protection against zikv serum viremia in indian rhesus macaques. due to overlap among the distributions of neutralizing antibody titers between the protected and infected animals, titers in some protected animals were below the correlate of protection. to determine the kinetics of neutralizing antibodies over a year long period, a separate set of four male indian rhesus macaques were vaccinated with µg pizv on study days and and challenged with zikv prvabc on study day . zika neutralizing antibody levels were measured every days through day (except for days , , and ), as well as on day ( days post-zikv challenge). all macaques seroconverted following the first vaccine dose, with a significant boost in antibody titers following the second dose (p < . ; fig. a ). neutralizing antibody titers peaked on day , days following the second immunization, declined from day to day , and then remained stable from day www.nature.com/scientificreports www.nature.com/scientificreports/ to day . anti-zika igg levels also peaked on day and subsequently declined through study day (fig. b) . igg titers increased following zikv challenge. no zika vrna was detected in serum from any of the macaques following zikv challenge year following the second dose. in addition, no statistically significant increase in neutralizing antibodies was observed post-zikv challenge on day (log ec range of . - . on day compared to log ec range of . - . on day ). the combined results suggest that two doses of pizv prevented zikv infection year post-vaccination. in conclusion, two µg pizv vaccinations elicits persistent neutralizing antibodies and long-term protection in rhesus macaques. we demonstrated that pizv elicits a dose-dependent response of both zika neutralizing and anti-zika igg antibodies. pizv elicited neutralizing antibodies that persisted for at least year and protected against zikv challenge. vaccinating with a broad range of pizv dose levels enabled us to correlate both neutralizing and anti-zika igg antibody titers to protection against zikv infection. we determined a neutralizing antibody correlate of protection of . log ec , which we define as the maximum ec among the unprotected animals in the study. we chose the neutralizing antibody assay to establish a correlate of protection as the literature supports using functional neutralizing antibodies as a correlate of protection for zika and other flaviviruses. table . neutralizing antibody titers on day of zikv challenge, zika vrna levels post-challenge. summary of mean neutralizing antibody titers (log ec ), range of peak vrna post-zikv challenge (log copies/ml), and percent of macaques with quantifiable zika vrna post-zikv challenge. all reported and analyzed data is above the rt-qpcr lloq. < lloq = detected vrna was below the assay lower limit of quantitation. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ neutralizing antibodies directed against the envelope (e) protein have been identified as correlates of protection for vaccines japanese encephalitis virus (jev), yellow fever virus, and tickborne encephalitis viruses . other laboratories using different vaccine platforms (dna, rna, inactivated virus, protein subunit, adenovirus-vectored, vlp) have reported induction of zikv-specific neutralizing antibodies that conferred protection against live zikv challenge in animal models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these data support zikv e-specific neutralizing antibodies as a mechanism of protection against zikv infection. in flavivirus vaccine development, neutralizing antibody titers that can confer protection against viremia have be reported in animal models using several different assay methods: microneutralization (mnt), rvp, and plaque reduction neutralization (prnt). studies of other candidate zikv vaccines have demonstrated that a neutralizing antibody titer as low as , using a mnt assay, or a titer of , using an rvp assay can confer protection against viremia in animal models , , . these results are qualitatively similar to those of licensed vaccines against flaviviruses such as jev, where a neutralizing antibody titer of > (as measured by prnt) is considered to determine the correlate of protection, the neutralizing antibody titers (log ec ) of infected (positive for vrna at any given timepoint) and protected (negative for vrna) macaques were plotted. the correlate of protection was defined as the maximum neutralizing antibody titer across all unprotected macaques in this study. based on this definition, the correlate of protection was established as > . log ec . cop = correlate of protection. www.nature.com/scientificreports www.nature.com/scientificreports/ to correlate with protection [ ] [ ] [ ] . further supporting this correlate of protection, it has been reported that passive transfer of zika neutralizing monoclonal antibodies or antisera from vaccinated humans and animals to mice is sufficient to protect against zikv challenge , , , . adoptive transfer of serum from immunized mice fully protected against viremia, while splenocytes from the same donors provided marginal protection demonstrating that the mechanism of protection against zikv infection is antibody mediated. while zikv exists as three genotypes (west african, east african and asian), they all behave as a single serotype , . our previous studies demonstrated that pizv is capable of eliciting antibodies that neutralize both african and asian zikv isolates in vitro . others have shown that the magnitude or duration of viremia in unvaccinated macaques challenged with zikv isolates from brazil or puerto rico is similar , and that vaccinated macaques are protected against challenge with heterologous zikv strains , , . protection of animal models against heterologous challenge and cross-neutralization capabilities of antisera from multiple vaccine platforms , , , suggest that data from a single challenge strain may be sufficient to show cross protection against zikv strains from other lineages. several groups have developed rhesus macaque challenge models using zika strain prvabc , , , , which is a well-documented and characterized isolate from human serum and is representative of viruses that were circulating in the americas during the - outbreak . we therefore selected prvabc as the challenge strain for our studies. in this study, we extended our observation of pizv efficacy in mice to demonstrate efficacy in prevention of zikv vrna and to evaluate anamnestic antibody responses after zikv challenge in non-human primates. we did not assess presence of vrna in tissues. our working hypothesis is that prevention of serum zikv vrna may be a surrogate for prevention of the most serious sequelae of zikv infection in humans -fetal infection. by employing a pizv dose titration study design and a zikv rvp assay, we have established a minimum protective vaccine dose of . µg in rhesus macaques and established a neutralizing antibody correlate of protection against zikv challenge of . log ec . finally, we demonstrated that zikv neutralizing antibodies persist and are capable of preventing zikv infection for at least year post-vaccination. in the event that human phase efficacy studies are not feasible, identifying a correlate of protection in an appropriate non-human primate challenge model may be important to support licensure of a zika vaccine , , . altogether, these data support neutralizing antibodies as an immune marker that is associated with efficacy in a relevant animal model and that may predict a reasonable likelihood of clinical benefit in humans. vaccine. pizv is an aluminum hydroxide adjuvanted whole purified inactivated virus vaccine based on zikv strain prvabc which was originally isolated from serum from a human infected in puerto rico (genbank accession number ku ). pizv has been previously described and characterized by baldwin et al. . the same lot of pizv used in this study was also used in clinical trial zik (clinicaltrials.gov nct ). rhesus macaque challenge studies. thirty-five indian rhesus macaques were separated into groups. three male and three female macaques per group were vaccinated intramuscularly with either . , . . . , or µg pizv on study days and . to achieve statistical significance comparing vaccine doses, six animals/ group were selected to obtain > % power if the true infection rate among controls is ≥ % and the true infection rate among vaccinated animals is ≤ % based on a one-sided fisher's exact test with % type error rate . two male and three female macaques were vaccinated with placebo control (pbs) on study days and . one female from the control group was euthanized on the day of zikv challenge due to reasons unrelated to the study, resulting in macaques in the control group. macaques were challenged subcutaneously with ffu/ . ml zikv prvabc on study day . serum samples were collected and tested for antibody titers on study days , , , and , and , and for zikv rna on study days - , and study day . www.nature.com/scientificreports www.nature.com/scientificreports/ a second study was conducted to assess long-term pizv immunogenicity and efficacy. flavivirus seronegative male rhesus macaques (n = ) were vaccinated with the µg pizv dose on days and and challenged subcutaneously with ffu/ . ml zikv prvabc on day . serum samples were collected and tested for antibody titers monthly up to year post-vaccination and days following zikv challenge (study day ). to assess replication of zika vrna following zikv challenge, serum samples were collected on study days - , and . challenge virus. zika virus puerto rico strain prvabc was used for the challenge ( . ml containing a nominal dose of ffu). the seed stock was passaged three times on vero cells at cdc, fort collins, colorado. virus stocks were prepared by passaging two times on c / mosquito cells. stocks were from a master working virus bank and tested for sterility, mycoplasma and endotoxin prior to use. screening. to select flavivirus naïve macaques, baseline igg serostatus was determined using a multiplex luminex kit (ampersand biosciences flavivirus serological panel). briefly, macaque serum was diluted : , in sample diluent and added to multiplexed magnetic beads coupled with zika, dengue, yellow fever, japanese encephalitis, west nile, usutu, saint louis encephalitis, and chikungunya virus antigens. serum and beads were incubated on a plate shaker for hour at room temperature and then washed three times with assay buffer. detection antibody, anti-igg pe, was added to each sample and incubated on a plate shaker for minutes at room temperature. following another three wash cycles, beads were resuspended in assay buffer and read on the bio-plex magpix to measure median fluorescent intensity (mfi) for each bead set. macaques were considered flavivirus naïve and selected for the study if the mfi was below pre-defined assay cutoff criteria for seronegativity for all antigens. neutralizing antibodies. a zika rvp assay (sonnberg et al., manuscript in preparation) was used to determine neutralizing antibody titers in serum following the administration of pizv (study days , , , and ), and days post-zikv challenge (day ). briefly, macaque serum was heat inactivated at °c for minutes and then serially diluted -fold in assay media for an -point dilution series. diluted serum and rvp were plated in duplicate in a -well assay plate and incubated for hour at °c. vero cells were added to each well and incubated at °c for hours. renilla-glo substrate (promega, wi, usa) was then added to the plate and incubated for minutes at room temperature. finally, plates were analyzed by a luminometer. the effective concentration at % (ec ), was determined by a non-linear regression curve fit with the lower asymptote constrained to in graphpad prism. the lloq for the nhp assay is . log ec , below which the serum matrix interfered with the measurement. the upper limit of quantitation (uloq) of the standard assay is . log ec . any samples returning titers >uloq were retested with a higher initial pre-dilution. anti-zika igg binding antibodies. anti-zika igg antibody levels were measured for study days , , , and (prior to zikv challenge) using a luminex based anti-zika igg assay. heat-inactivated serum samples were diluted : in assay buffer and then serially diluted -fold for an -point dilution curve. magnetic beads covalently coupled with pizv antigen were added to each sample dilution in a -well plate and each sample dilution was tested in duplicate. plates were incubated on a plate shaker for minutes at room temperature and then washed two times with assay buffer. diluted anti-ig-pe detection antibody was then added to each sample and incubated on a plate shaker for hour at room temperature. after two wash cycles, beads were resuspended in assay buffer and read on the luminex flexmap d to measure the mfi. the igg antibody concentration was quantified using a reference standard serum with an assigned igg concentration in units/ml (u/ml). the lloq for the assay is . log u/ml. total rna from serum samples was extracted using the qiagen biorobot universal instrument and qiaamp virus biorobot mdx kit (qiagen; valencia, ca). extracted and purified rna was evaluated in two separate rt-pcr assays. primarily, zika vrna was detected and quantified using rt-qpcr with primers and probe specific to known zikv genotypes as described in lanciotti et al. and a standard curve generated from synthetic reference zika vrna (atcc). a qualitative extraction control rt-pcr was also performed. the extraction control rt-pcr utilized a primer/probe set, specific to macaca mulatta c galt c l mrna, confirmed adequate nucleic acid extraction from serum samples independent of zika vrna detection. the geometric mean, range of peak zika vrna detection, and the respective percentage of protected macaques (as defined by absence of vrna) was calculated using quantities greater than the assay lower limit of quantitation of . log copies/ml. samples with vrna concentration lower than the assay limit of detection of . log copies/ml were considered negative. correlate of protection. the mean neutralizing antibody titers (log ec ) determined by zika rvp assay was computed for each dose group at day (day of zikv challenge). zika vrna copies/ml determined by rt-qpcr assay were computed for each dose and timepoint post-challenge (study days - and ). peak vrna for each macaque was defined as the highest observed vrna concentration across all timepoints tested. macaques were considered protected if vrna was not detected or was below the assay lloq for all timepoints tested. the correlate of protection was defined as the maximum neutralizing antibody titer across all unprotected macaques in this study. this definition was conservative in that some protected macaques could have neutralizing antibody titer levels below the correlate of protection due to overlap between the distributions of protected and unprotected macaques. since the correlate of protection was not a statistical estimate, no confidence intervals were reported. statistical analysis. statistical tests were performed using the r software version . . .; log neutralization titers were compared for selected pairs of days for each dose group using a paired two-sided t-test. at day , tukey's test was used to compare all dose groups with each other. a two-sided spearman's test was applied to the unprotected macaques to check for an association between day neutralizing antibodies titers and peak vrna. comparisons between selected pairs of days were also performed with the log igg antibody levels in the main study and with the log neutralization titers from the long-term immunogenicity study using paired two-sided t-tests. a p-value threshold of . was used to determine statistical significance. the data that support the findings of this study are available from the corresponding author upon reasonable request and with permission of takeda vaccines, inc. vital signs: zika-associated birth defects and neurodevelopmental abnormalities possibly associated with congenital zika virus infection -u.s. territories and freely associated states who region of the americas/pan american health organization. plisa health information platform for the americas: cases of zika virus disease clinical development strategies and considerations for zika vaccine licensure correlates of protection induced by vaccination demonstrating vaccine effectiveness during a waning epidemic: a who/nih meeting report on approaches to development and licensure of zika vaccine candidates protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys rapid development of a dna vaccine for zika virus modified mrna vaccines protect against zika virus infection comparative pathogenesis of asian and african-lineage zika virus in indian rhesus macaque's and development of a non-human primate model suitable for the evaluation of new drugs and vaccines nonhuman primate models of zika virus infection, immunity, and therapeutic development macaque monkeys in zika virus research: -present characterization of a clinical isolate of zika virus in non-human heterologous protection against asian zika virus challenge in rhesus macaques a rhesus macaque model of asian-lineage zika virus infection zika virus infection of rhesus macaques leads to viral persistence in multiple tissues purified inactivated zika vaccine candidates afford protection against lethal challenge in mice protective efficacy of zika vaccine in ag mouse model zika virus protection by a single low-dose nucleoside-modified mrna vaccination in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunisation with a prmenv dna vaccine vaccine protection against zika virus from brazil durability and correlates of vaccine protection against zika virus in rhesus monkeys passive transfer of immune sera induced by a zika virus-like particle vaccine protects ag mice against lethal zika virus challenge report on a who consultation on immunological endpoints for evaluation of new japanese encephalitis vaccines yellow fever vaccine: direct challenge of monkeys given graded doses of d vaccine neutralizing antibodies protect against lethal flavivirus challenge but allow for the development of active humoral immunity to a nonstructural virus protein preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated zika virus vaccine candidate: phase , randomised, double-blind, placebo-controlled clinical trials a 'furry-tale' of zika virus infection: what have we learned from animal models? phylogeny of zika virus in western hemisphere a tale of two viruses: does heterologous flavivirus immunity enhance zika disease? zika viral dynamics and shedding in rhesus and cynomolgus macaques what is the predictive value of animal models for vaccine efficacy in humans? the importance of bridging studies and species-independent correlates of protection genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia correspondence and requests for materials should be addressed to g.y.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - zpkpdzu authors: sun, fang; xia, zhiqiang; han, yuewen; gao, minjun; wang, luyao; wu, yingliang; sabatier, jean-marc; miao, lixia; cao, zhijian title: topology, antiviral functional residues and mechanism of ifitm date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zpkpdzu interferon-inducible transmembrane proteins (ifitm / / ) have been reported to suppress the entry of a wide range of viruses. however, their antiviral functional residues and specific mechanisms are still unclear. here, we firstly resolved the topology of ifitm on the plasma membrane where n-terminus points into the cytoplasm and c-terminus resides extracellularly. further, krrk basic residues of ifitm locating at – of the conserved intracellular loop (cil) were found to play a key role in the restriction on the zika virus (zikv) and dengue virus (denv). similarly, krrk basic residues of ifitm / also contributed to suppressing zikv replication. finally, ifitm was revealed to be capable of restricting the release of zikv particles from endosome to cytosol so as to impede the entry of zikv into host cells, which was tightly related with the inhibition of ifitm on the acidification of organelles. overall, our study provided topology, antiviral functional residues and the mechanism of interferon-inducible transmembrane proteins. human interferon-induced transmembrane protein / / (ifitm / / ) is widely expressed in different tissues and organs, and the expression level of these proteins is regulated by interferon [ ] . they contain - amino acid residues and have two transmembrane domains. the topologies of ifitm , ifitm and ifitm are similar. they all consist of five parts, including short n-terminal domain (ntd) and c-terminal domain (ctd), intramembrane domain (im ) and intramembrane domain (im ), and short conserved intracellular loop (cil) [ ] . at least three possible topological models of ifitms have been reported [ ] . one model is that n-terminus and c-terminus reside extracellularly or in intracavity, im and im stretch across the phospholipid bilayer and cil resides in the cytoplasm. the second model is that n-terminus, c-terminus, and ctd are in the cytoplasm, and im and im are in the phospholipid bilayer. the third model is that n-terminus and cil reside in the cytoplasm, and ctd resides extracellularly or in intracavity [ ] . therefore, the exact topology of ifitms is still to be investigated. ifitm proteins are involved in the regulation of many activities, such as tumorigenesis [ ] and immune signal transduction [ ] . most importantly, it has been reported that ifitm proteins have a broad antiviral spectrum in recent decades. ifitm proteins can effectively block a variety of enveloped viruses from invading host cells, such as influenza a virus (iav) of orthomyxoviridae [ ] , respiratory syncytial virus (rsv) of pneumoviridae [ ] , zikv and denv of flaviviridae [ , ] , ebolavirus (ebov) and severe acute respiratory syndrome virus (sars-cov) of [ ] . on the other hand, ifitm proteins also restrict some nonenveloped viruses, such as reovirus (rov) [ ] . additionally, ifitms have almost no inhibitory effect on certain viral infections, such as adenovirus (adv) [ ] and sendai virus (sev) [ ] . although ifitms play critical antiviral roles in the innate immune system, their antiviral functional residues are mostly still unclear. there is a report showing im of ifitm is required for both the interaction of ifitm protein and the restriction of iav and denv [ ] ; therefore, vital antiviral functional residues of ifitm are to be determined. ifitm-mediated restriction on viruses mainly occurs at the stage of virus invasion into host cells [ ] . some studies suggest that ifitm proteins may suppress the entry of viruses by inhibiting the hemifusion of viral membrane and host cell membranes [ ] or restricting the formation of fusion pores following virus-endosome hemifusion [ ] . there is also a finding that ifitm disrupts intracellular cholesterol homeostasis to block viral release into the cytosol by interacting with vesicle-membrane-protein-associated protein a [ ] ; clearly, the antiviral mechanisms of ifitms are extremely diverse and complex, and still need to be studied. in our study, immunofluorescence and confocol microscopy analysis showed that ifitm was mainly distributed on the plasma membrane adopting the topological model that n-terminus of ifitm pointed into the cytoplasm and c-terminus extended extracellularly. alanine scanning and site-directed mutations found that krrk basic residues were key for the restriction of ifitm proteins on zikv and denv. by electron microscope and flow cytometry analysis, ifitm was revealed to restrict the release of zikv from endosome to cytosol, which was tightly related to its inhibition against the acidification of organelles. hek a, hek t, huh and vero cells were purchased from the china center for type culture collection (cctcc). all cells were cultured in dmem (gibco-invitrogen, new york, ny, usa) supplemented with % fbs (gibco-invitrogen) and % penicillin/streptomycin at • c in an incubator filled with % co . zika virus envelope (e) protein antibody (gtx ) was purchased from genetex (san antonio, usa). dengue virus type - antibody (ma - ) was purchased from invitrogen (shanghai, china). abs against flag (f ) and myc (a - ) were purchased from sigma-aldrich (shanghai, china) and genscript (nanjing, china), respectively. abs against ha ( - -lg) and gapdh ( - -lg) were purchased from proteintech group (wuhan, china). anti-sodium potassium atpase (na + /k + -atpase) antibody (ep y) was kindly offered by jing yao from the college of life sciences at wuhan university. alexa fluor ( es ) and alexa fluor affinipure donkey anti-mouse ( es ) were purchased from yeasen (shanghai, china). lysosensor™ green dnd- (l ) and thermo fisher scientific page ruler prestained protein ladder ( ) were purchased from invitrogen (shanghai, china). bestar ® sybr green qpcr master mix reagent (dbi- ) was purchased from dbi ® bioscience (shanghai, china). pcdna . -flag-ifitm /ifitm /ifitm , pcdna . -flag-ifitm-mutants, pcdna . -ha-ifitm -myc, pt-dimer-rab and pt-dimer-rab were constructed by our laboratory. pegfp-lamp was kindly offered by hongbing shu and zhiyin song from the college of life sciences, wuhan university. when covering about % of the culture flask, cells were transfected with recombinant plasmids using turbofect transfection reagent (invitrogen, r ) following the instructions; then, cells were cultured for h. zikv puerto rico strain (prvabc ) cdna plasmid was kindly provided by ren sun and danyang gong at ucla. denv serotype tsv strain (denv- tsv ) was kindly provided by bo zhang from wuhan institution of virology, chinese academy of sciences. sev and adv were kindly supplied by mingxiong guo and zan huang from the college of life sciences at wuhan university, respectively. after transfecting ifitm plasmid for h, cells were incubated with zikv (moi = . ), denv (moi = . ), sev (moi = . ) and adv (moi = ) for h at • c, respectively. then, the supernatant was removed and the cells were cultured with fresh dmem supplemented with % fbs and % penicillin/streptomycin for h. next, cells were collected to be detected. cells were fixed with precooled % paraformaldehyde for min at room temperature followed by washing them in pbs twice ( min each time). then, samples were treated without . % triton x- (intact cell membranes) or with . % triton x- (permeabilized cell membranes) for min followed by washing them in pbs twice ( min each time). then, nonspecific proteins were blocked for min with % bovine serum albumin (bsa) in pbs. next, cells were incubated with primary antibody at • c overnight and washed three times in pbs ( min each time). later, cells were incubated with alexa fluor-labeled secondary antibody for h at room temperature followed by washing them in pbs three times for min each time. cell nuclei were subsequently stained with dapi fluoromount-g ® (antgene, wuhan, china, ant ). the fluorescence was observed using a confocal laser-scanning microscope (leica tcs sp , wetzlar, germany). cells were lysed with % sds. samples were boiled in boiling water for min and centrifuged at , rpm for min at • c to remove cell debris. the total protein content of the supernatants was measured using a bca protein assay kit (thermofisher scientific, shanghai, china) and was mixed with × loading sample buffer (biosharp, bl a, hefei, china). then, samples were boiled in boiling water for min. equal amounts of proteins were separated by % sds-polyacrylamide gel electrophoresis (page) and transferred to nitrocellulose membranes (nc membrane, millipore, wuhan, china). nc membranes were incubated with % nonfat milk for h at room temperature to block nonspecific proteins. then, nc membranes were incubated with primary antibodies at • c overnight, followed by incubation with secondary antibodies for h at room temperature. antibodies were used following the instructions. later, nc membranes were detected by chemiluminescence using a westernbrighttm ecl western blotting detection kit (advansta, menlo park, usa). mrna of cells was extracted with trizol reagent (takara, beijing, china), and the first-strand cdna was reversed-transcribed by using the revert aid first-strand cdna synthesis kit (thermo scientific). the cdna was quantitated by qpcr with sev and gapdh primers using the bestar ® sybr green qpcr master mix reagent (dbi ® bioscience, shanghai, china). the primers of zikv, sev and gapdh were synthesized as our laboratory previously described [ ] . qpcr experiments were performed on an abi system (shanghai, china) according to the instructions. cells were incubated with µm lysosensor dnd- at • c for min. then, cells were digested with . % trypsin and passed through nylon net to obtain a single-cell suspension. flow cytometry measuring the intensity of fitc was performed on a cytoflex flow cytometer. if ifitm and -krrk alkalize acidic organelles, the peak of fitc would shift to the left relative to the negative control. vero cells were transfected with pcdna . , pcdna . -flag-ifitm and pcdna . -flag- -krrk in a cm dish, respectively. after h, cells were incubated with zikv (moi = ) at • c for h, followed by being cultured for h at • c in an incubator filled with % co . the supernatant was removed and then cells were fixed with % glutaraldehyde at • c for min. cells were collected and centrifuged at rpm for min. after that, cells were treated with % glutaraldehyde at • c for h to be detected. em samples were commissioned to wuhan institution of virology, chinese academy of sciences, to prepare and observe. samples were examined with a tecnai em with an accelerating voltage of kv and magnifications of × , and × . adobe photoshop cs and graphpad prism were used for statistical analysis. data were usually representative and presented as the mean ± standard deviation (sd) of the three independent experiments. p values were calculated by the student t-test. statistical significance was considered at p value less than . (* < . , ** < . and *** < . ). ifitm proteins widely expressed in different tissues and organs were found to play important antiviral roles in the innate immune system. although they share a high identity of amino acid sequences ( figure a ) and are able to inhibit a wide range of viruses, ifitm / / still have different antiviral activities and spectra. to determine if the intracellular distribution of ifitm proteins influences their antiviral activities, we analyzed their intracellular location. the cdna sequences of ifitm / / were cloned from hek t cells by rt-pcr and then were inserted into pcdna . plasmid with bamhi and xhoi, where a flag-tag was added to their n-terminus ( figure b ). the overexpression level of pcdna . -flag-ifitm / / was detected, and the result showed that they were greatly expressed compared with the control ( figure c ). na + /k + -atpase is a biomarker of the plasma membrane [ ] . we analyzed the colocalization of ifitm / / and na + -k + atpase by immunofluorescence using the anti-flag antibody and na + /k + -atpase antibody coupled with alexa fluor ® . the experimental data showed that ifitm distributed on the plasma membrane and in the cytoplasm. ifitm especially had strong colocalization with na + -k + atpase ( figure d ). in comparison, ifitm and ifitm appeared to reside only in cytoplasm ( figure d ). these data indicate ifitm distributes widely on the plasma membrane and cytoplasm. the colocalization of ifitm / / and na + -k + atpase in hek t cells; pcdna . and pcdna . -flag-ifitm / / were transfected in hek t cells. after h, cells were stained with anti-flag antibody, anti-na + -k + atpase antibody and dapi. then, cells were observed using confocal microscopy. flag-ifitm / / , red; na + -k + atpase, green; dapi, blue. previously, ifitms have been reported to have three topological models on the cell membranes ( figure a ) [ ] . to determine the detailed topological model that ifitm adopted on the cell membranes, we constructed pcdna . -ha-ifitm -myc plasmid to express ifitm protein fused n-terminal ha-tag and c-terminal myc-tag for the experiments. the sketch of the amino acid sequence of ha-ifitm -myc is shown in figure b . the location of ha-ifitm -myc was analyzed using anti-ha antibody or anti-myc antibody in huh cells when the plasma membranes were intact or permeabilized. we found that ha-ifitm -myc could not be dyed using anti-ha antibody when plasma membranes were intact, but it could be observed when cell membranes were permeabilized ( figure c,d) , which suggested that the n-terminus of ifitm points into the cytoplasm. differently, ha-ifitm -myc could be observed using anti-myc antibody when cells were treated with . % were transfected in hek t cells, respectively. after h, cells were collected, and the expression level of intracellular ifitm / / was analyzed by western blotting using anti-flag antibody. (d) the colocalization of ifitm / / and na + -k + atpase in hek t cells; pcdna . and pcdna . -flag-ifitm / / were transfected in hek t cells. after h, cells were stained with anti-flag antibody, anti-na + -k + atpase antibody and dapi. then, cells were observed using confocal microscopy. flag-ifitm / / , red; na + -k + atpase, green; dapi, blue. previously, ifitms have been reported to have three topological models on the cell membranes ( figure a ) [ ] . to determine the detailed topological model that ifitm adopted on the cell membranes, we constructed pcdna . -ha-ifitm -myc plasmid to express ifitm protein fused n-terminal ha-tag and c-terminal myc-tag for the experiments. the sketch of the amino acid sequence of ha-ifitm -myc is shown in figure b . the location of ha-ifitm -myc was analyzed using anti-ha antibody or anti-myc antibody in huh cells when the plasma membranes were intact or permeabilized. we found that ha-ifitm -myc could not be dyed using anti-ha antibody when plasma membranes were intact, but it could be observed when cell membranes were permeabilized ( figure c,d) , which suggested that the n-terminus of ifitm points into the cytoplasm. differently, ha-ifitm -myc could be observed using anti-myc antibody when cells were treated with . % triton x- or not ( figure c ,d), which indicated that the c-terminus of ifitm was located outside the plasma membranes. similarly, the location of ha-ifitm / -myc was analyzed using anti-ha antibody or anti-myc antibody in huh cells when plasma membranes were intact or permeabilized. we found that neither ha-ifitm -myc or ha-ifitm -myc could be dyed using anti-ha antibody or anti-myc antibody when plasma membranes were intact, but both of them could be observed when cell membranes were permeabilized ( figure s ). these results indicated that ifitm and ifitm are also located in the cytoplasm of huh cells. these data show that ifitm adopts the topological structure (figure a , right) on the plasma membranes, where the n-terminus points into the cytoplasm and the c-terminus resides extracellularly, consistent with the topology model that ntd and cil are in the cytoplasm and ctd is extracellular or intracavity. viruses , , x for peer review of triton x- or not ( figure c ,d), which indicated that the c-terminus of ifitm was located outside the plasma membranes. similarly, the location of ha-ifitm / -myc was analyzed using anti-ha antibody or anti-myc antibody in huh cells when plasma membranes were intact or permeabilized. we found that neither ha-ifitm -myc or ha-ifitm -myc could be dyed using anti-ha antibody or anti-myc antibody when plasma membranes were intact, but both of them could be observed when cell membranes were permeabilized ( figure s ). these results indicated that ifitm and ifitm are also located in the cytoplasm of huh cells. these data show that ifitm adopts the topological structure (figure a, right) on the plasma membranes, where the n-terminus points into the cytoplasm and the c-terminus resides extracellularly, consistent with the topology model that ntd and cil are in the cytoplasm and ctd is extracellular or intracavity. an amphipathic sequence of amino acids (residues - ) of ifitm has been reported to be required for the restriction on h n iav [ ] . however, the other antiviral functional residues of ifitm are still unclear. therefore, eight alanine scan (as) mutants of the ifitm cd domain were constructed and named ( figure a ). vero cells were kidney cells of the monkey chlorocebus sabaeus from africa, which was easily infected by zikv [ ] . we observed the location of ifitm / / fused with a flag-tag in vero cells; the result showed that over-expressed ifitm was located both on cell membranes and in the cytoplasm, while ifitm and ifitm appeared to reside only in the cytoplasm in vero cells ( figure s ). then, we compared the inhibitory effects of ifitm and eight as mutants against zikv (prvabc strain) in vero cells. the result showed that ifitm inhibited % zikv e protein compared with the control, while as, as, as, as, as, as, as and as mutants limited %, %, %, %, %, %, % and % intracellular zikv e protein, respectively ( figure b,c) . relative intracellular zikv e protein was analyzed using imagej. these results suggest that as, as, as, as and as mutants of ifitm have a weaker antiviral effect than ifitm does, and these mutated amino acid residues of ifitm may be potential antiviral functional determinants. to further analyze the key functional antiviral sites of ifitm , we constructed four single-point mutants of ifitm in the cil domain named k a, r a, r a and k a and a four-point mutant named -krrk ( figure d ). then, we analyzed the antiviral effects of these five mutants. the results showed that -krrk had the least inhibitory activity against zikv in vero cells, and r a and k a appeared to have less antiviral effect than the ifitm by qpcr ( figure e ) and western blotting ( figure f,g) . however, k a and r a still shared similar anti-zikv activity with the wild type ifitm ( figure e-g) . the amino acid sequences of ifitm and ifitm in the cd domain were almost the same as those of ifitm ( figure a ). to determine if the krrk basic residues of ifitm / are also key for their antiviral functions, flag-ifitm -krrk and flag-ifitm -krrk plasmids were constructed. k , r , r and k of ifitm , and k , r , r and k of ifitm were simultaneously mutated to alanine ( figure h) . similarly, ifitm / and / -krrk were used to analyze the antiviral function against zikv. we found that both ifitm and ifitm had a great limitation on the intracellular zikv e protein, while -krrk and -krrk had less restriction than their wild types ( figure i ,j). these data show the krrk basic residues of ifitm / are also significant to suppress zikv infection. denv, like zikv, is a member of the flavivirus genus. more than million people worldwide are infected each year by any of the four-denv serotypes [ ] . to further verify the importance of the krrk basic residues of ifitm against viruses, ifitm and krrk mutants were used to detect their restriction on the denv- tsv strain in vero cells. the result showed that ifitm could decrease the expression of intracellular denv e protein compared with the control, while -krrk had almost no influence on denv replication ( figure a,b) . therefore, the krrk basic residues of ifitm also played a key role in the restriction on denv. sev, a negative single-stranded rna virus, is a member of the paramyxoviridae family. adv is a nonenveloped double-strand dna virus. we asked whether the krrk basic residues of ifitm had an influence on the infection of sev and adv. the results showed that both ifitm and -krrk hardly had inhibition on the relative intracellular sev rna, compared to the control ( figure c ). similarly, they had almost no inhibitory effect on the infection of adv (figure d,e) . in short, the krrk basic residues of ifitm / / contributed specifically to their antiviral functions. importantly, we detected the intracellular distributions of five krrk residues mutants of ifitm . the results showed -krrk, k a, r a, r a and k a all had colocalization with na + -k + atpase in hek t cells, and five mutants all could be located in the cytoplasm ( figure ). in addition, we compared the location of ifitm and -krrk in vero cells. it was found that the localization of -krrk was almost the same as that of ifitm ( figure s ). these data indicate these basic amino acids hardly influence the intracellular distribution of ifitm and do not explain the reason that the antiviral effect of -krrk is weaker than that of ifitm . basic amino acids hardly influence the intracellular distribution of ifitm and do not explain the reason that the antiviral effect of -krrk is weaker than that of ifitm . after h, the fluorescence of cells was observed using fluorescence microscopy with the magnification of × (d). fluorescence intensity was counted (e). ns = no significance. viruses , , x for peer review of figure . the colocalization analysis of ifitm mutants and na + -k + atpase in hek t cells. pcdna . and pcdna . -flag- -krrk/k a/r a/r a/k a were transfected in hek t cells, respectively. after h, cells were stained with anti-flag antibody, anti-na + -k + atpase antibody and dapi. then, cells were observed using confocal microscopy. ifitm mutants, red; na + -k + atpase, green; dapi, blue. zikv is a single-stranded rna virus, and the diameter of its mature viral particles is - nm [ ] . the life cycle of zikv can be divided into four stages, including viral entry, genome replication, viral assembly and release [ ] . zikv enters host cells by clathrin-mediated endocytosis. in this process, the viral envelope and the endosome membrane of host cells are fused under the acidic figure . the colocalization analysis of ifitm mutants and na + -k + atpase in hek t cells. pcdna . and pcdna . -flag- -krrk/k a/r a/r a/k a were transfected in hek t cells, respectively. after h, cells were stained with anti-flag antibody, anti-na + -k + atpase antibody and dapi. then, cells were observed using confocal microscopy. ifitm mutants, red; na + -k + atpase, green; dapi, blue. zikv is a single-stranded rna virus, and the diameter of its mature viral particles is - nm [ ] . the life cycle of zikv can be divided into four stages, including viral entry, genome replication, viral assembly and release [ ] . zikv enters host cells by clathrin-mediated endocytosis. in this process, the viral envelope and the endosome membrane of host cells are fused under the acidic environment of the endosome; then, the viral genomic rna is released into the cytoplasm [ ] . previous studies have shown that ifitm proteins can inhibit the entry of zikv [ ] ; however, how ifitm inhibits this stage is still unknown. here, we detected the influence of ifitm and -krrk on the release of zikv from the endosome to the cytosol. by electron microscopy, we observed ifitm prevented more virus particles from being released into the cytoplasm, compared with the negative control, while -krrk virtually unlimited the release of virus particles ( figure a ). specific results were counted and shown in figure b , suggesting that ifitm did restrict the release of zikv from the endosome to the cytosol. in addition, the colocalization of ifitm and acidic organelles was detected. rab and rab , members of the ras gtpase superfamily, are mainly located in early endosomes and late endosomes, respectively [ ] . lamp is the main constituent protein of the endosome/lysosome pathway [ ] . therefore, we detected the colocalization of ifitm and rab /rab /lamp . indeed, we found that ifitm was partially co-located with early endosomes, late endosomes and lysosomes ( figure c ). this was consistent with previous reports that exogenous ifitm can localize on cell membranes and in highly acidified late endosomes and lysosomes [ , ] . we then detected the organelles acidity of huh cells after being over-expressed by ifitm and -krrk by a flow cytometer using a nm laser for excitation. compared with the control group, the curve of ifitm shifted to the left, and the curve of -krrk was between ifitm and the control ( figure d ). these data suggested that ifitm could inhibit the acidification of organelles, and -krrk had weaker suppressive power than ifitm did. therefore, ifitm can restrict the release of zikv from endosome to cytosol, which is related with its inhibition on organelles acidification. more importantly, the krrk basic residues of ifitm are vital in these processes. viruses , , x for peer review of environment of the endosome; then, the viral genomic rna is released into the cytoplasm [ ] . previous studies have shown that ifitm proteins can inhibit the entry of zikv [ ] ; however, how ifitm inhibits this stage is still unknown. here, we detected the influence of ifitm and -krrk on the release of zikv from the endosome to the cytosol. by electron microscopy, we observed ifitm prevented more virus particles from being released into the cytoplasm, compared with the negative control, while -krrk virtually unlimited the release of virus particles ( figure a ). specific results were counted and shown in figure b , suggesting that ifitm did restrict the release of zikv from the endosome to the cytosol. in addition, the colocalization of ifitm and acidic organelles was detected. rab and rab , members of the ras gtpase superfamily, are mainly located in early endosomes and late endosomes, respectively [ ] . lamp is the main constituent protein of the endosome/lysosome pathway [ ] . therefore, we detected the colocalization of ifitm and rab /rab /lamp . indeed, we found that ifitm was partially co-located with early endosomes, late endosomes and lysosomes ( figure c ). this was consistent with previous reports that exogenous ifitm can localize on cell membranes and in highly acidified late endosomes and lysosomes [ , ] . we then detected the organelles acidity of huh cells after being over-expressed by ifitm and -krrk by a flow cytometer using a nm laser for excitation. compared with the control group, the curve of ifitm shifted to the left, and the curve of -krrk was between ifitm and the control ( figure d ). these data suggested that ifitm could inhibit the acidification of organelles, and -krrk had weaker suppressive power than ifitm did. therefore, ifitm can restrict the release of zikv from endosome to cytosol, which is related with its inhibition on organelles acidification. more importantly, the krrk basic residues of ifitm are vital in these processes. previous reports have revealed that ifitm adopts the topological model on organelle membranes where its ntd and cil are in the cytoplasm, and ctd is intracavity by using the combined electron paramagnetic resonance (epr) and solution nuclear magnetic resonance (nmr) [ ] . ifitm is high homologous with ifitm and has been reported with at least three topological models [ , ] . here, we support that ifitm adopts a topological model on cell membranes where the n-terminus points into the cytoplasm and the c-terminus reside extracellularly. this is consistent with previous reports [ ] . the anti-viral function of ifitm proteins was found initially against iav, west nile virus (wnv) and denv in [ ] . over the next years, ifitm proteins have been reported to restrict many other viruses. zikv and denv are an arthropod-borne virus (arbovirus) in the flavivirus genus of the flaviviridae family; both zikv and denv have shown a threat to global health, and the effective vaccines for them still need to be developed [ , ] . ifitm is an important downstream gene of interferon; there are reports that show the inhibitory effect of ifitm on zikv and denv replication [ , ] . in our study, we conducted a series of experiments to screen the antiviral functional residues of ifitm against zikv. we found that the krrk basic residues of ifitm were important for the restriction of ifitm on zikv and denv. it has been reported that the as and as mutants of ifitm have weaker restrictions on iav and denv replication than wild type [ ] . to further illustrate the importance of these four residues, we also compared the antiviral effects of ifitm /ifitm and their corresponding krrk mutant. indeed, the krrk basic residues of ifitm /ifitm also played a key role in their restriction on zikv. significantly, we found that ifitm can restrict the release of zikv from endosome to cytosol to prevent the entry of viral particles into host cells, which was associated with its inhibition on organelles acidification. attractively, -krrk had almost no restriction in this process. significantly, we found krrk residues of ifitm were very conservative in many species by aligning their amino acid sequences ( figure s ). we hypothesize that krrk residues of ifitm proteins of other species may play important roles in their antiviral effects. most of the previous reports have studied the effect of ifitm on viral replication, while our work mainly studied the restriction of ifitm on viruses, including zikv, denv, adv and sev. importantly, we found that the krrk basic residues of ifitm proteins played a vital role in their antiviral processes. in addition, our work provided new insights into the antiviral mechanism of ifitm related to inhibiting organelles acidification. the previous research also showed that the endosomes in hulec cells with a high basal level of ifitm were less acidic than in mdcks [ ] . the mechanism may be applicable to explain how ifitm restricts other viruses entering cells by clathrin-mediated endocytosis. however, our work could not explain why the krrk basic residues of ifitm are vital for ifitm to inhibit viral entry and suppress organelles acidification. the isoelectric point (pi) of ifitm with a flag-tag is . , predicted by the protparam website, and the ph values of the late endosome and lysosome are . - . and . - . , respectively [ ] . therefore, we hypothesize that ifitm may alkalize the acidic organelles by its own properties; besides, the pi of -krrk with a flag-tag is . predicted by the protparam website, which may explain that -krrk hardly affects the acidification of organelles. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s . the location of ha-ifitm / -myc in huh cells. figure s . the localization analysis of ifitm / / in vero cells. figure s . the localization analysis of ifitm / -krrk in vero cells. figure s . the amino acid sequence alignment of ifitm across nine species. the small interferon-induced transmembrane genes and proteins a membrane topology model for human interferon inducible transmembrane protein ifitm-family proteins: the cell's first line of antiviral defense the anti-inflammatory ifitm genes ameliorate colitis and partially protect from tumorigenesis by changing immunity and microbiota the ifitm protein family in adaptive immunity palmitoylation on conserved and nonconserved cysteines of murine ifitm regulates its stability and anti-influenza a virus activity defining the range of pathogens susceptible to ifitm restriction using a knockout mouse model the ifitms inhibit zika virus replication the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replicatio interferon-induced transmembrane protein-mediated inhibition of host cell entry of ebolaviruses interferon-inducible transmembrane protein (ifitm ) restricts reovirus cell entry the antiviral restriction factors ifitm , and do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus ifitms restrict the replication of multiple pathogenic viruses ifitm proteins-cellular inhibitors of viral entry ifitm proteins restrict viral membrane hemifusion ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry a scorpion venom peptide ev restricts viral late entry by alkalizing acidic organelles physical mapping and characterization of the human na,k-atpase isoform, atp a a sorting signal suppresses ifitm restriction of viral entry ifitm requires an amphipathic helix for antiviral activity identification of various cell culture models for the study of zika virus dengue virus infection of blood-brain barrier cells: consequences of severe disease zika virus replication in the mosquito culex quinquefasciatus in brazil development of small-molecule inhibitors against zika virus infection probing molecular insights into zika virus host interactions the autophagic roles of rab small gtpases and their upstream regulators: a review disruption of endolysosomal rab / efficiently eliminates colorectal cancer stem cells ifitm inhibits influenza a virus infection by preventing cytosolic entry the interferon-induced transmembrane proteins, ifitm , ifitm , and ifitm inhibit hepatitis c virus entry combined approaches of epr and nmr illustrate only one transmembrane helix in the human ifitm ifitm proteins mediate the innate immune response to influenza a h n virus, west nile virus and dengue virus zika virus: new clinical syndromes and its emergence in the western hemisphere dengue vaccine: hypotheses to understand cyd-tdv-induced protection ha-dependent tropism of h n and h n influenza viruses to human endothelial cells is determined by reduced stability of the ha, which allows the virus to cope with inefficient endosomal acidification and constitutively expressed ifitm ion flux and the function of endosomes and lysosomes: ph is just the start: the flux of ions across endosomal membranes influences endosome function not only through regulation of the luminal ph we thank ren sun and danyang gong of ucla for providing zikv puerto rico strain (prvabc ) cdna plasmid and bo zhang from wuhan institution of virology, chinese academy of sciences for giving denv serotype tsv strain (denv- tsv ). the authors declare no conflict of interest. key: cord- -nmt tu authors: goh, gerard kian-meng; dunker, a. keith; foster, james a.; uversky, vladimir n. title: zika and flavivirus shell disorder: virulence and fetal morbidity date: - - journal: biomolecules doi: . /biom sha: doc_id: cord_uid: nmt tu zika virus (zikv) was first discovered in in africa. since then, sporadic zikv infections of humans have been reported in africa and asia. for a long time, this virus was mostly unnoticed due to its mild symptoms and low fatality rates. however, during the – epidemic in central and south america, when millions of people were infected, it was discovered that zikv causes microcephaly in the babies of mothers infected during pregnancy. an examination of the m and c proteins of the zikv shell using the disorder predictor pondr vlxt revealed that the m protein contains relatively high disorder levels comparable only to those of the yellow fever virus (yfv). on the other hand, the disorder levels in the c protein are relatively low, which can account for the low case fatality rate (cfr) of this virus in contrast to the more virulent yfv, which is characterized by high disorder in its c protein. a larger variation was found in the percentage of intrinsic disorder (pid) in the c protein of various zikv strains. strains of african lineage are characterized by higher pids. using both in vivo and in vitro experiments, laboratories have also previously shown that strains of african origin have a greater potential to inflict higher fetal morbidity than do strains of asian lineage, with dengue- virus (denv- ) having the least potential. strong correlations were found between the potential to inflict fetal morbidity and shell disorder in zikv (r( ) = . ) and denv- (denv- + zikv, r( ) = . ). a strong correlation between cfr and pid was also observed when zikv was included in an analysis of sets of shell proteins from a variety of flaviviruses (r( ) = . ). these observations have potential implications for antiviral vaccine development and for the design of cancer therapeutics in terms of developing therapeutic viruses that penetrate hard-to-reach organs. the zika virus (zikv) was first isolated in when three owl monkeys from the zika forest, uganda, were found to have a fever [ , ] . even though the first human case was reported in and although this virus has been circulating in asia and africa, very few zikv cases were noticed and recorded before the / epidemic, since the symptoms were generally mild [ , ] and fatality was the appropriate proteins and their sequences were carefully searched and checked upon retrieval from uniprot (http://www.uniprot.org) or ncbi (http://www.ncbi.nlm.nih.gov/structure/). after downloading the sequences and respective protein data bank (pdb) files, the necessary information was imported into the database using tools implemented in java [ ] . the corresponding protein sequences in fasta format were fed into the pondr vlxt neural network (http://www.pondr.com). the results were given as scores from to . residues with scores of . and over represent disordered residues [ , ] . an important ratio used in this and previous studies is the percentage of intrinsic disorder (pid), which is defined as the number of residues predicted to be disordered in a protein chain divided by the total number of residues in the chain. the pid can range from % (perfectly ordered) to % (totally disordered). the predicted intrinsic disorder-related information was retrieved and fed into a mysql database that has been mentioned in previous papers [ ] . another java-dbi program then automatically created codes that were readable in jmol (www.jmol.com), which generated a d representation of protein crystal structures with disorder annotations that could be visualized from the colors provided by the codes. the jmol software also required the respective pdb (protein data bank) files, which could be obtained from ncbi, as mentioned above. a pondr vlxt predictor was used in this study, since it is one of the most informative tools for structural proteins, namely for viral shells, where protein-protein interactions play vital roles in order-disorder transitions [ ] [ ] [ ] [ ] . it has successfully been utilized to study the shell disorder of a large variety of viruses, including hiv, smallpox, and polio viruses [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pondr represents a suite of computational tools for sequence-only predictions of the intrinsic disorder predisposition of query in proteins. pondr vlxt was the earliest predictor created at the end of the last century [ , ] . it involves the use of neural networks that are trained to recognize protein sequences that are ordered and those that are not [ ] . statistical methods, including regression analysis and analysis of variance (anova), were used to analyze the pondr vlxt results along with data on virulence and morbidity. the statistical package r version . . . [ ] was used to obtain the results. before obtaining the necessary results, the respective codes had to be written in r. while zikv is not known for its virulence, fetal morbidity, as already mentioned, is a threat from zikv infection [ ] [ ] [ ] ] . zikv strains are generally categorized as of african or asian lineage, and the epidemic that occurred in - involved asian strains that had crossed the pacific ocean to reach the americas. at least two laboratories have found that zikv strains of african lineage are more dangerous to fetuses than those of asian lineage [ , ] . the classical isolate for the strains of african lineage is mr- , which was discovered in uganda in [ , ] . a pid-based wide selection of zikv strains and isolates allowed us to see that the strains can be divided into four groups that are consistent with their lineages but that are independent of the dates of the isolates ( figure a and table ). interestingly, our results show that there are at least two mr- isolates from uganda- with distinct shell disorder features (table ) . furthermore, denv- has also been shown to cause microcephaly in fetuses and newborns, but to a lesser extent when compared to zikv [ , , ] . this has been observed in several laboratories using in vivo and in vitro methods. chicken liver cells, human nerve cells, and mouse models were used, and the levels of destruction by zikv and denv- were observed according to the amount of cells destroyed [ , , ] . the pids of denv- and zikv are compared in figure and table . various statistical methods were used to reaffirm not just the statistical significance of the grouping by shell pid (especially the c protein), but also the statistical significance of a strong correlation between the pid of the c protein and fetal morbidity, given the evidence of greater fetal morbidity in infections by mr- . regression analyses and an anova conducted on the data shown in table provided evidence that the shell pid differences between strains of the african and asian lineages are statistically significant (table ; regression: r = . , f = , p < . ; one-way anova with c pid as the independent variable: f = , p < . ). when data related to denv- were added to the analysis, a strong correlation that was statistically significant was found (r = . , f = , p < . ). table . percentages of intrinsic disorder (pids) of m and c shell proteins from zika virus (zikv) strains with uniprot accession. differences between the shell disorder in asian and african strains are statistically significant (regression: r = . , f = , p < . ; one-way analysis of variance (anova) with c percentage of intrinsic disorder (pid) as the sole independent variable: f = , p < . ). higher pids in c proteins were found in the african strains even as the pid levels could differ for c proteins in strains from the same lineage. the regression was performed using the assumption that the african strains have greater fetal morbidity than do the asian strains [ , , ] . (a) correlation between fetal morbidity and zikv-dengue virus (denv- ) shell disorder (regression: r = . , f = , p < . ; independent variables: c and m pids). see figure s for more details on the data points; (b) correlation between flavivirus case fatality ratio (cfr) and shell disorder (regression: r = . , f = , p < . ; independent variables: c and m pids). with the exception of the zikv data, much of the data can be found in a previous publication [ ] . while figure a and table involve the determination of the correlation between the fetal morbidity associated with zikv infection and zikv shell disorder (pid), figure b and table represent the results of the analysis of the correlation between the case fatality ratio ((cfr), i.e., virulence) and the shell disorder of various flaviviruses, including zikv. information on the cfrs of various flaviviruses is publicly available [ , [ ] [ ] [ ] [ ] . because the symptoms of zikv are usually very mild and fatalities are extremely rare, for a long time, the virus was shrouded in mystery [ , ] . it is therefore likely that the cfr is very close to zero, even if most of the currently available data are those from infections arising in the americas, which involved strains of asian lineage, not african lineage [ , [ ] [ ] [ ] . a strong correlation was found between flavivirus cfr and shell disorder, as seen in figure b and table . while a correlation between flavivirus cfr and shell disorder has already been reported [ , ] , the data presented here include results for zikv, which was not previously considered. a a regression model using cfr as the dependent variable with m and c pids as independent variables yielded r = . , f = , p < . . interaction between the two independent variables was seen as statistically significant. the pid are in percentages (%). b a regression model using cfr as the dependent variable with c pid as the independent variable yielded r = . , f = , p < . . c a regression model using cfr as the dependent variable with m pid as the independent variable yielded r = . , f = , p < . . tick-borne encepthalitis virus(tbev). d the standard error is denoted by the prefix "±". yfv: yellow fever virus; wnv: west nile virus. in a previous study, we established that both c and m proteins play roles in flavivirus virulence, as represented by cfr [ ] . according to our research on other viruses, the capsid association with virulence involves a mechanism of protein promiscuity, whereby intrinsic disorder allows the capsid protein (the c protein, in the case of flaviviruses) to bind to a greater number of proteins, thereby allowing the virus to multiply more rapidly before the host immune system can take action [ , , ] . the matrix protein (the m protein in flaviviruses), on the other hand, is likely to play a somewhat different role. a more disordered matrix allows the virus to penetrate organs, namely vital organs, as in the case of hiv- , but still be able to evade the host immune systems via matrix and surface glycoprotein motions [ , , ] . a closer look at tables and and figure reveals other specific trends. as in previous studies, the pid of the c protein, as the sole independent variable, provided a weaker correlation with flavivirus cfr that was of statistical significance (r = . , f = , p < . ). similarly, the pid of the m protein, as the sole independent variable, also did not provide a very strong correlation with flavivirus cfr, even if this correlation was statistically significant (r = . , f = , p < . ). the use of pids of both m and c proteins as independent variables, on the other hand, gave a strong correlation with flavivirus cfr that was definitely stronger than the pids of c or m proteins alone (r = . , f = , p < . , figure b ). obviously, both m and c proteins complement each other in their combined contributions to virulence, which can be seen in both figure b and table . there was also a statistically significant interaction between the two factors (p < . ). figure and tables and demonstrate that zikv is a peculiar virus, since it has a relatively disordered m protein but also a relatively ordered c protein. this accounts for the zikv characteristics of low mortality rates and yet high fetal morbidity rates. however, the use of pid values is inadequate in providing a closer look at the differences in the disordered regions among the various viruses. figures and provide us with the opportunity for a detailed look at the disorder differences by region of the proteins among several flaviviruses. figure a shows that disordered regions of the m proteins of zikv and yfv are relatively large and overlap. with the disordered regions being found mostly around the n-termini of the m proteins of the three flaviviruses shown, a much shorter disordered region is found in the denv- m protein. these observations could be used as a template for designing a zikv vaccine, especially when looking at the pondr vlxt plot of the zikv m protein. the asterisk (*) in figure shows areas that could be potential targets in the development of a zikv vaccine. table ), wnv (uniprot: q q ), and denv- (uniprot: p ). regions with scores of . or above represent disorder. disorder differences between c proteins can be traced mainly to mutations and disorder near the n-and c-termini. the regions with an asterisk (*) denote potential targets for the development of a zikv vaccine. with the exception of msia (malaysia ) zikv, the strains were randomly chosen. msia was deliberately chosen as a representative asian strain that has a low c pid. the yfv m protein was chosen for (a), since it has one of the highest pids among flaviviruses. wnv was not necessary for (a), as specific strains of denv- have an even lower m pid (pid: %) than does the zikv (pid: %). on the other hand, wnv was chosen for (b), as the low wnv c pid (among flaviviruses) allows us to suggest strategies for zikv vaccine development. the pondr vlxt plot for the c proteins of three flaviviruses, zikv, denv- , and wnv, can be found in figure b . as mentioned, studies have shown that disorder in the inner shells of viruses in general tend to be associated with greater virulence because of the ability of proteins to bind more promiscuously, thus providing the viruses with greater ability to replicate more rapidly. similarly, figure b and table suggest that the reason that the zikv's cfr is extremely low has to do with the relatively low mean pid of its c protein ( ± %, cfr~ ). while these results tell us about the differences with regard to the mean c pids among the various flaviviruses, they do pinpoint the disorder differences among the various flaviviruses along the entire stretch of the various flavivirus c proteins, especially with respect to zikv. figure b attempts to address this problem by showing that the disorder differences between c proteins from zikv and other flaviviruses lie in two areas near the n-and c-termini, respectively. while figure b , figure b , and table provide evidence that disorder in the c protein contributes to the virulence of flaviviruses, figure a , figure a , figure , and table point to the contribution of zikv c protein disorder to fetal morbidity. more specifically, a rather large disordered region is missing near the n-terminus of the strains of asian lineage, which, as already mentioned, inflict morbidity to fetuses at a lower rate than do their african counterparts. there are also two distinct patterns of disorder for the two separate groups of mr- -like strains (zikv/macaca mulatta/uga/mr- -veroe -ac -p _ / vs zikv/macaca mulatta/uga/mr- / /east_africa) shown in table . while figure a reports that the residues that are disordered only in the mr- -related strains can be found near the n-terminus, figure b identifies the residues responsible for the disorder differences. the residues marked with "x" represent mutations. in general, the more polar and charged residues tend to induce more disorder, and such can be observed in this case. disorder differences between c proteins from the asian and african lineages can be traced mainly to mutations and disorder near the n-termini. table for further details) strains. disorder differences between c proteins from the asian and african lineages can be traced mainly to mutations and disorder near the n-termini. the structure of immature flaviviruses is somewhat different from mature ones. an immature flavivirus virion includes a precursor membrane protein, prm. during maturity, the prm is cleaved such that the m remains as the membrane protein. this characteristic raises important questions about the roles of prm and m proteins, especially with regard to protecting the virion. while such roles have seldom been explored in the past, hints of the potential roles of prm and m proteins can be found in the pid values obtained in this study. we have seen that with the exception of yfv and zikv, most other flaviviruses have highly ordered m proteins to protect their respective mature virions ( figure b and table ). figure a shows that the m protein is relatively more disordered (in red) than in denv- ( figure b ). as with the pondr vlxt plots in figure , the disordered regions of both zikv and denv- are restricted to segments near the n-termini. while we have seen that the m proteins of zikv and yfv are relatively disordered, the same cannot be said about pr. the pr proteins for yfv and zikv are quite ordered, as seen in figure c -e. the pr disorder levels are relatively low for most flaviviruses [ , ] , as seen in the cases of zikv, denv, and wnv. with pr being mostly ordered for most flaviviruses and m being the same for most flaviviruses (with the exception of yfv and zikv), one can conclude that both pr and m contribute to the order of the prm proteins for most flaviviruses, and this is especially important with respect to protecting the immature virion. the following question then arose: how do zikv and yfv protect their immature virion given their abnormally high m disorder? a hint to the answer to this question lies in the pid of their pr proteins. as we can see in figure c -e, the pr proteins of both zikv and yfv are quite ordered, well within the range of most flaviviruses. as a result, fairly ordered prm proteins are seen in both zikv and yfv. the study therefore shows that the immature virions of zikv and yfv are more protected against environmental insults than are their corresponding mature virions, which could suggest a slightly different life cycle for yfv and zikv. while mature viruses are of course infectious, experimental evidence has suggested that fully immature flaviviruses are generally not infectious, even if they may become infectious upon interactions with antibodies [ ] . partially mature viruses are likely more infectious than fully immature ones [ ] . zikv and yfv could therefore rely more on lesser mature viruses for their spread, given our evidence showing that their virions become more delicate upon maturation. the data in figures - and tables and show correlations between the intrinsic disorder predisposition of proteins from the inner and outer shells and mortality and fetal morbidity rates. this flavivirus-wide statistical study shows that both m and c proteins play roles in pathogenesis. similarly, the statistical analyses reveal that disorder in both the c and m proteins also contributes to fetal morbidity. the regression analysis reported here suggests the presence of interaction effects between c and m pids. given these observations and the fact that similar proteins play similar roles even among nonrelated viruses, what do other viruses have to offer with respect to clues about trends in the correlation between virulence and shell disorder? while very few viruses have shown positive correlations with statistical significance between their virulence and membrane/matrix disorder (as the sole independent variable), hiv offers a unique insight into the role of the matrix (membrane) protein. of the hiv and simian immunodeficiency virus (siv) family, the hiv- matrix has the greatest known pid [ , ] . hiv- and its closest sibling, sivcpz, are the most pathogenic when compared to their other cousins and siblings, including hiv- . hiv- patients are known to have much greater viral loads in their bloodstream than patients with hiv- . without the help of retroviral drugs, over % of hiv- patients would die within years of the infection, whereas those infected with hiv- take a much longer time to succumb to the disease, if at all [ , ] . the roles of the matrix protein in virulence have to do with viral ability to evade the host immune system and also the ability to bind promiscuously to more host proteins [ , , , ] . an important characteristic of hiv is its ability to penetrate vital organs, including the brain, with ease [ ] . apparently, the promiscuous binding potential arising from the disordered matrix shell allows hiv to have easier access to specialized cells. we shall use this argument again in the cases of zikv and yfv, which have high pids for their m proteins ( . ± . % and + %, respectively). while disorder in the matrix shell is associated with virulence and the immune evasion found in hiv, disorder in the inner shell (capsid or nucleocapsid) of other viruses, such as ebola virus and nipah virus), is often also strongly associated with virulence [ , ] . virulence arising from disorder in the inner shell involves a different strategy of immune evasion, which can be referred to as trojan horse, since the virus attempts to replicate rapidly before the host immune system is able to recognize its presence [ , ] . it does so by using the disordered inner shell proteins that play important roles in the replication of the virus, where disorder assists the process by allowing for greater promiscuous (and therefore more efficient) binding of host proteins. curiously, a conflicting trend has also been observed in hiv: when the outer shell is disordered, then the inner shells tend to be more ordered [ , ] . conversely, if the outer shell is more ordered, the inner shells tend to be more disordered. these conflicting trends can be seen in a variety of viruses, including nipah, corona, and ebola viruses, not just hiv [ , , [ ] [ ] [ ] . the reason for this has to do with the fact that both shell layers play their roles in protecting the virion from environmental insults. if the outer layer is more disordered, the inner layer compensates by being more ordered. if, on the other hand, the outer layer is more ordered, the inner layer, depending on the type of environment the virus is exposed to, has the luxury of being less ordered. the two aforementioned seemingly conflicting trends that are seen in other viruses can also be observed in flaviviruses and zikv. it is for this reason that we observed statistical significance in the interaction effects between the pids of c and m proteins as independent variables. furthermore, strong flavivirus cfr correlations were not attainable until both c and m pids were used as independent variables (r = . , f = , p < . ). a closer look at the data in figure b allows us to conclude that some flaviviruses do indeed have more ordered m proteins when their c proteins are disordered and vice versa. for example, tick-bone encephalitis virus (tbev) viruses have higher pids in their c proteins than do denv viruses, but they have lower pids for m proteins. similarly, zikv has a low pid value for c but a high pid for m. there is also evidence of this trend within zikv strains themselves: we can notice a slightly lower m disorder in mr- strains (pid: vs %, table ) despite their higher c pid values even as m pids remain relatively high for all zikv strains. this trend is, however, not the case with every flavivirus. a striking exception is yfv, which provides some evidence for the other trend that involves strategies of immune evasion using disorder in either or both m and c shells. more specifically, disorder in m and c proteins is likely to play different but complimentary roles in maintaining higher viral load, especially in vital organs necessary to evade the immune system, and yfv is somewhat unique in using both c and m disorder to a much greater extent than other flaviviruses do. this will be further described in the next section. relative to other flaviviruses, yfv is characterized by highly disordered c and m proteins. two modes of action are at work here. a highly disordered yfv has an overwhelmingly high pid in its c protein (~ %). interestingly, the mean pid of its m protein (~ %) was also far higher than the corresponding values for all other flavivirus species found in our sample. it is likely that the highly disordered c protein allows for more rapid replication via its promiscuity in protein binding. as a result of the rapid replication, greater viral loads are more attainable, leading to greater virulence. a higher disorder in the m protein, however, allows the virus to more easily penetrate different organs, including vital organs such as the liver [ , ] . apparently, yfv's extremely high pathogenicity, which reaches above a % cfr, arises from its unique ability to utilize disorder from both its m and c proteins to maintain a viral load as a means of evading the immune system. while yfv has greater disorder in both its m and c proteins, zikv offers us a unique opportunity to study a flavivirus with a high pid in its m protein ( ± %) but a low pid in its c protein ( . ± %). if we use the aforementioned model based on our knowledge of shell disorder, the model will predict zikv as a virus that is of low pathogenicity but has the ability to penetrate vital organs. this prediction is extremely accurate in the case of zikv. we are able to see that zikv infection is usually nonfatal, with cfr being close to %, but zikv also has an uncanny ability to penetrate the brain and placenta, as seen in its ability to cause microcephaly [ ] . we have seen that flaviviruses use disorder in m and c proteins to evade host immune systems. yfv uses both m and c to penetrate vital organs and proliferate rapidly. the two modes become a deadly mix. the zikv uses its high m disorder to penetrate the placenta and brain, but we have also noticed that, while zikv c pid values are generally low, there is noticeable variation in the c pids seen in the different lineages and strains. this pid variation in the c protein correlates with fetal morbidity that is dependent on the lineage of zikv. the results fit perfectly with what has been observed in other viruses. the "trojan horse" theory tells us that the higher c disorder allows for more efficient proliferation of the virus in the larger variety of organs that it is able to penetrate [ , ] . while variation in zikv m pid is only %, zikv c pid can be as low as % and as high as %. inspection of the pondr vlxt plot shows that the disordered regions lie in both the n-and c-termini even if the zikv c mutations arise entirely within the n-terminus (figure ). this provides clues to the evolution of zikv with respect to its ability to cause fetal morbidity in its hosts. it is likely that zikv had to evolve with the primate species that it most commonly infects. for certain primate hosts, it finds greater fitness when a greater viral load is necessary for it to infect a larger number of hosts because of the peculiarities found in the hosts' immune system: having a more disordered c protein does the trick. it is therefore likely that the different pids of c proteins from the various zikv strains and lineages are the result of the types of primates that the peculiar zikv strain primarily infects and its ability to fine-tune its c disorder to meet the optimal viral load necessary for more efficient transmission of the virus in a given host, as seen in the case of other viruses and other flaviviruses [ , , , , , ] . experiments do provide evidence pointing to the important roles of c and m proteins in replication and immune evasion. flavivirus protein c, along with other viral proteins, assists in the assembly and packaging of viral particles during replication, in addition to assisting the protein m to move to the endoplasmic reticulum (er). additionally, the c protein is trafficked to the nucleus through binding with importin [ ] . upon doing so, it interacts with histones to force the cell to stay longer in the g stage of the cell cycle so as to buy more time for viral replication [ ] . in the cytoplasm, protein c binds to the sec protein in order to inhibit sec proteasomal antiviral activity [ ] . as for the m protein, there is still much to be uncovered about its functions. it is, however, known that prm binds to the er for two reasons. it protects the e proteins from damage by host enzymes via the formation of a beta barrel [ ] . the other reason is that prm prevents e from being so embedded in the host membrane that it cannot be removed in the later stages of the virion replication process. while the pr segment is ordered to protect e from the proteases, the greater flexibility of m in many flaviviruses is likely needed to ensure greater efficiency in its binding to the er. similarly, greater disorder in the c protein offers plenty of opportunity for greater binding efficiency, given its aforementioned experimentally proven functions. for instance, the greater disorder in the c protein could help its binding to histones with greater efficiency. protein disorder not only provides greater efficiency in protein-protein binding but also promotes the binding of proteins to nucleic and fatty acids as well. figure reiterates the minimum requirements necessary in the development of a zikv vaccine: the pid of the c protein has to be as low as possible. figure b , on the other hand, suggests ways that a zikv can be used for a zikv vaccine developed by comparing the pondr vlxt plots of the zikv c protein to those of other flaviviruses. more specifically, it shows a region near the c-terminus that is ordered in wnv but disordered in zikv. this region could be a good target for the development of an attenuated zikv, as it indicates a region that is not necessarily disordered but is still tolerated by a cousin, wnv. just as relatively high disorder levels in the m protein (pid: %, %) are a hallmark of the zikv, an effective vaccine needs to have an m protein with a lower pid so that an attenuated virus will not penetrate the placenta of pregnant women. figures a and show that the disordered regions of the m protein are near the n-terminus, and mutations to attenuate the virus must be in that region. since the m protein of denv has a much shorter disordered region ( figures b and a ,b), figure a identifies an area that is ordered in denv and would be a good target of mutations during the development of an attenuated zikv. again, since that region is ordered in denv- , order there could be tolerated by zikv. there were two main zikv oddities discovered in this research. the first has to do with the relatively high disorder in the zikv m protein, which is at a level that is seldom found among other flaviviruses, with the exception of yfv. the other is the somewhat large variation in the disorder of zikv depending on the strain and lineage of the virus. these oddities are consistent with what we now know as characteristics of zikv. just as other viruses (such as wnv and hiv- ) that have high outer shell (matrix/membrane) pids are able to penetrate vital organs, zikv can also penetrate the placenta and brain [ ] . furthermore, scientists have discovered that strains of the african lineage possess a greater potential to cause fetal morbidity. this fact correlates with the variations in the disorder levels of the zikv c protein. disorder in the inner shell has already been associated with virulence and higher viral loads in other flaviviruses and other unrelated viruses, such as in the ebola and nipah viruses. there are important implications for these findings. the results presented here could lead to the development of a new vaccine through the creation of attenuated strains resulting from more ordered c and m proteins. the regions for potential vaccine targets are disordered regions of the zikv m and c proteins that are ordered in other flavivirus species. exploration of a zikv vaccine could include mutations in these areas. other applications could include building a more comprehensive model that could anticipate the behaviors of new or little-known viruses. lastly, the ability of zikv and yfv to penetrate vital organs as a result of shell disorder adds to a growing body of knowledge that encompasses a variety of viruses, such as hiv, that use such strategies to enter or to hide in hard-to-reach organs. an important potential application of this is in the field of cancer oncolysis, which involves the use of viruses to "attack" tumors, since both share the same pathways [ , ] . viruses could be engineered to evade host immune systems that impede the effectiveness of therapeutic viruses. moreover, since cancers in hard-to-reach organs such as the brain are often problematic for virotherapies and chemotherapies [ , ] , a better understanding of the roles of shell disorder in the viral penetration of such organs will lead to improved therapies. zika virus: history, epidemiology, transmission, and clinical presentation viral shapeshifters: strange behaviors of hiv and other viruses zika virus outbreak in brazil why zika virus infection has become a public health concern? concern over zika virus outbreak: another alarming global threat the african strain of zika virus causes more severe in utero infection than asian strain in a porcine fetal transmission model zika virus infection at mid-gestation results in fetal cerebral cortical injury and fetal death in the olive baboon de freitas, a.c. in vitro and in vivo models for studying zika virus biology an immunocompetent mouse model of zika virus infection microencephaly in fetal piglets following in utero inoculation of zika virus zika virus infection in pregnant rhesus macaques causes placental dysfunction and immunopathology using immunocompromised mice to identify mechanisms of zika virus transmission and pathogenesis hiv vaccine mystery and viral shell disorder. biomolecules fundamentals of molecular virology zika virus structure, maturation, and receptors structure of the immature zika virus at Å resolution intrinsically unstructured proteins: re-assessing the protein structure-function paradigm why are "natively unfolded" proteins unstructured under physiologic conditions? intrinsically unstructured proteins intrinsically disordered side of the zika virus proteome molecular recognition features in zika virus proteome correlating flavivirus virulence and levels of intrinsic disorder in shell proteins: protective roles vs. immune evasion structural disorder in viral proteins orderly order in protein intrinsic disorder distribution: disorder in proteomes from viruses and the three domains of life disordered interactome of human papillomavirus protein intrinsic disorder and human papillomaviruses: increased amount of disorder in e and e oncoproteins from high risk hpvs protein intrinsic disorder and influenza virulence: the h n and h n viruses protein intrinsic disorder as a flexible armor and a weapon of hiv- intrinsic disorder mediates hepatitis c virus core-host cell protein interactions the intrinsic disorder status of the human hepatitis c virus proteome unstructural biology of the dengue virus proteins functional correlations of respiratory syncytial virus proteins to intrinsic disorder deciphering the dark proteome of chikungunya virus structural disorder in the proteome and interactome of alkhurma virus (alkv) the african zika virus mr- is more virulent and causes more severe brain damage than current asian lineage and dengue virus evolution of two major zika virus lineages: implications for pathology, immune response, and vaccine development protein intrinsic disorder toolbox for comparative analysis of viral proteins predicting binding regions within disordered proteins identifying disordered regions in proteins from amino acid sequence mining α-helix-forming molecular recognition features with cross species sequence alignments † coupled folding and binding with α-helix-forming molecular recognition elements † nipah virulence and v shell disorder a comparative analysis of viral matrix proteins using disorder predictors a comparative analysis of envelope and tegument proteins of suid herpesvirus , bovine herpesvirus and bovine herpesvirus viral disorder or disordered viruses: do viral proteins possess unique features? understanding viral transmission behavior via protein intrinsic disorder prediction: coronaviruses prediction of intrinsic disorder in mers-cov/hcov-emc supports a high oral-fecal transmission detection of links between ebola nucleocapsid and virulence using disorder analysis shell disorder, immune evasion and transmission behaviors among human and animal retroviruses sequence complexity of disordered protein r: a language and environment for statistical computing; r foundation for statistical computing zika virus induced more severe inflammatory response than dengue virus in chicken embryonic livers the diversification of zika virus: are there two distinct lineages? genome boil factors associated with dengue mortality in latin america and the caribbean flavivirus vaccine tick-borne encephalitis-clinical course and outcome epidemiology: the study of disease, injury and death in the community immature dengue virus: a veiled pathogen? functional importance of dengue virus maturation: infectious properties of immature virions viral sex: the nature of aids specific interaction of capsid protein and importin-α/β influences west nile virus production dengue virus capsid protein binds core histones and inhibits nucleosome formation in human liver cells west nile virus and dengue virus capsid protein negates the antiviral activity of human sec protein through the proteasome pathway the flavivirus precursor membrane-envelope protein complex: structure and maturation fighting cancer with viruses: oncolytic virus therapy in china. hum fighting cancer with oncolytic viruses oncolytic virotherapy versus cancer stem cells: a review of approaches and mechanisms this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -qtwcbn m authors: gao, yaning; tai, wanbo; wang, ning; li, xiang; jiang, shibo; debnath, asim k.; du, lanying; chen, shizhong title: identification of novel natural products as effective and broad-spectrum anti-zika virus inhibitors date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qtwcbn m zika virus (zikv) infection during pregnancy leads to severe congenital zika syndrome, which includes microcephaly and other neurological malformations. no therapeutic agents have, so far, been approved for the treatment of zikv infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat zikv-caused diseases. after screening a natural product library, we have herein identified four natural products with anti-zikv activity in vero e cells, including gossypol, curcumin, digitonin, and conessine. except for curcumin, the other three natural products have not been reported before to have anti-zikv activity. among them, gossypol exhibited the strongest inhibitory activity against almost all zikv strains tested, including six recent epidemic human strains. the mechanistic study indicated that gossypol could neutralize zikv infection by targeting the envelope protein domain iii (ediii) of zikv. in contrast, the other natural products inhibited zikv infection by targeting the host cell or cell-associated entry and replication stages of zikv. a combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-zikv compound, exhibited significant combinatorial inhibitory effects against three zikv human strains tested. importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (denv) human strains in vitro. taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against zikv and other flaviviruses, such as denv. zika virus (zikv) is a mosquito-borne flavivirus in the same genus as other important human pathogens, including dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and tick-borne encephalitis virus (tbev) [ ] . zikv was originally isolated in a rhesus macaque in [ ] , but this virus has only recently claimed worldwide attention owing to its close association with congenital zika syndrome (czs), as represented by microcephaly, fetal demise, central nervous system abnormalities, and other neurological complications [ ] [ ] [ ] [ ] [ ] . no antiviral therapeutics for the treatment of zikv-associated human diseases, particularly congenital syndrome a plaque reduction inhibition assay was carried out to measure the inhibitory activity of natural products in a natural product collection library from microsource discovery systems (gaylordsville, ct, usa) against infections of zikv and denv, as previously described [ ] [ ] [ ] [ ] . briefly, zikv (strain pan , ~ plaque-forming unit (pfu); multiplicity of infection (moi) ~ . ) was incubated with -fold serial dilutions of natural products (including curcumin, digitonin and conessine, as well as anti-zikv compound control, bortezomib) or dmso ( . % vol/vol) control at °c for h. the compound-virus mixtures were then transferred to vero e cells ( /well) and incubated at °c for h. for gossypol, zikv (strain pan , ~ . × pfu; moi ~ . ) was incubated with serial dilutions of this natural product at °c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was then incubated with vero e cells at °c for h. the cells were washed thoroughly with pbs and overlaid with dmem containing % carboxymethyl cellulose and % fbs, followed by in vitro culture at °c for - days, and then stained with . % crystal violet. the inhibitory activity of all natural products tested against denv- - , as described above, was performed following the above procedures, except that llc-mk cells were used for infection, and cells were cultured at °c for - days before staining with . % crystal violet. the % inhibitory concentration (ic ) and ic of natural products were calculated based on the concentrations at % and % plaque reduction, respectively, using the calcusyn computer program, as described before [ ] . the cytotoxicity of natural products in vero e for zikv or llc-mk cells for denv- - was evaluated using the cell counting kit- (cck , sigma, st. louis, mo, usa), according to the manufacturer's instructions. briefly, -fold serial dilutions of each of the natural products ( l/well) were added to equal volumes of cells ( . × /well) in -well plates and cultured at °c for days. the cells were then incubated with cck solution and absorbance was measured at nm (a value) using a microplate reader (infinite f pro, tecan, morrisville, nc, usa). the % cytotoxic concentration (cc ) of natural products was calculated based on the percent cytotoxicity a plaque reduction inhibition assay was carried out to measure the inhibitory activity of natural products in a natural product collection library from microsource discovery systems (gaylordsville, ct, usa) against infections of zikv and denv, as previously described [ ] [ ] [ ] [ ] . briefly, zikv (strain pan , ~ plaque-forming unit (pfu); multiplicity of infection (moi) . ) was incubated with -fold serial dilutions of natural products (including curcumin, digitonin and conessine, as well as anti-zikv compound control, bortezomib) or dmso ( . % vol/vol) control at • c for h. the compound-virus mixtures were then transferred to vero e cells ( /well) and incubated at • c for h. for gossypol, zikv (strain pan ,~ . × pfu; moi~ . ) was incubated with serial dilutions of this natural product at • c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was then incubated with vero e cells at • c for h. the cells were washed thoroughly with pbs and overlaid with dmem containing % carboxymethyl cellulose and % fbs, followed by in vitro culture at • c for - days, and then stained with . % crystal violet. the inhibitory activity of all natural products tested against denv- - , as described above, was performed following the above procedures, except that llc-mk cells were used for infection, and cells were cultured at • c for - days before staining with . % crystal violet. the % inhibitory concentration (ic ) and ic of natural products were calculated based on the concentrations at % and % plaque reduction, respectively, using the calcusyn computer program, as described before [ ] . the cytotoxicity of natural products in vero e for zikv or llc-mk cells for denv- - was evaluated using the cell counting kit- (cck , sigma, st. louis, mo, usa), according to the manufacturer's instructions. briefly, -fold serial dilutions of each of the natural products ( µl/well) were added to equal volumes of cells ( . × /well) in -well plates and cultured at • c for days. the cells were then incubated with cck solution and absorbance was measured at nm (a value) using a microplate reader (infinite f pro, tecan, morrisville, nc, usa). the % cytotoxic concentration (cc ) of natural products was calculated based on the percent cytotoxicity using the calcusyn computer program [ , ] . the combinatorial cytotoxicity of gossypol with other natural products to vero e cells was detected using a similar approach as described above, except for mixing gossypol with one of the natural products (curcumin, digitonin, conessine, or bortezomib) throughly before adding them to the cells. this experiment was carried out as previously described, with some modifications [ ] [ ] [ ] [ ] [ ] [ ] [ ] . briefly, vero e cells ( /well) or zikv were incubated at different infection steps as described below, with or without the tested natural products at the specified concentrations of µm for gossypol, µm for curcumin, . µm for digitonin, or µm for conessine for h before zikv infection, h after infection, or the same time during infection. anti-zikv compounds, such as temoporfin [ ] , -hydroxycholesterol [ ] , bortezomib [ ] , and nitd [ ] , were included as controls for these steps. after the culture of the zikv-or compound-treated cells at • c for - days, plaques were visualized with crystal violet staining, as described above, and the percent inhibition of natural products was calculated. specifically, the following six stages of zikv infection were tested: (a) pretreatment of zikv (pan ,~ . × pfu; moi~ . ) with natural products at • c for h before incubation with cells; (b) pretreatment of cells with natural products at • c for h before incubation with zikv (pan ,~ pfu; moi~ . ); (c) cotreatment of cells with zikv (pan ,~ pfu; moĨ . ) and natural products at • c for h; (d) cotreatment of cells, zikv (pan ,~ pfu; moĨ . ), and natural products at • c for h; (e) preincubation of cells with zikv (pan ,~ pfu; moi~ . ) at • c for h and then incubation with natural products at • c for h; and (f) preincubation of zikv (pan ,~ pfu; moi~ . ) and cells at • c for h, followed by incubation with natural products at • c for h. the binding between natural products and zikv full-length e protein (aviva systems biology, san diego, ca, usa) or envelope protein domain iii (ediii) protein (e residues - fused with a c-terminal human fc) [ ] was carried out by elisa, as previously described [ , , , ] . briefly, elisa plates were precoated with the proteins described above at • c overnight and blocked with % fat-free milk at • c for h. serial dilutions of natural products or dmso (negative control) were then added to the plates and incubated at • c for h. the plates were washed with pbs containing tween- (pbst) and incubated at • c for h with zikv ediii-specific human monoclonal antibody (mab) zka -lala ( . µg/ml) for binding to zikv full-length e and ediii proteins. the plates were washed with pbst and incubated with horseradish peroxidase (hrp)-conjugated anti-human igg-fab ( : , abcam, cambridge, ms, usa) antibody at • c for h. the , , , -tetramethylbenzidine (tmb) substrate (sigma) was added to the plates, and the reaction was stopped by n h so . absorbance at nm (a value) was measured by elisa microplate reader (tecan). ec ( % effective concentration) was calculated based on the calcusyn computer program, as described above [ ] . to determine the ability of gossypol to inhibit binding between zikv ediii protein and ediii-specific human mabs (smzab , zka -lala, zv- , or z ) or edi/ii-specific human mab control (zka ) [ ] , elisa was carried out, as described above, except that serially diluted gossypol or dmso (negative control) was added in the presence of mabs ( . µg/ml), followed by sequential incubation with hrp-conjugated anti-human igg-fab antibody and tmb substrate and detection for a value. the percent inhibition of natural products was calculated, and ic (concentration causing % reduction in ediii-mab binding) was obtained using the calcusyn computer program [ ] , as described above. the interactions between natural products and zikv full-length e were analyzed at • c using the biacore t system (ge healthcare, port washington, ny, usa), as previously described [ , ] . briefly, zikv e was immobilized on a sensor chip (cm ) using the amine coupling kit (ge healthcare). natural products at various concentrations were injected as analytes, and pbs-p ( mm phosphate buffer containing . mm kcl, mm nacl, and . % surfactant p , ph . ) was used as the running buffer. the data were analyzed using biacore evaluation software (t version . ), and the curve was fitted with a : binding model. the potential combinatorial effect of gossypol with other natural products was carried out as previously described [ , ] . briefly, zikv (strain pan , flr, or prvabc , . × pfu; moĨ . ) was incubated with serially diluted gossypol at • c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was incubated with vero e cells at • c for h in the presence of dmem containing serial dilutions of each of the other three natural products identified, such as curcumin, digitonin, and conessine, or anti-zikv compound control (bortezomib). the unbound viruses and natural products were removed, the cells were cultured at • c for - days, and stained with . % crystal violet. the natural products without combinations were used as controls. the ic values of natural products were calculated using the calcusyn computer program [ ] , as described above. the natural products were then analyzed for combinatorial effects based on the combination index (ci) [ ] and ic values using the calcusyn computer program, as previously described [ , , ] . specifically, ci values < and > indicate synergy and antagonism, respectively. synergy was further identified as five different categories: ci values < . , . - . , . - . , . - . , and . - . indicate very strong synergism, strong synergism, synergism, moderate synergism, and slight synergism, respectively. fold enhancement of anti-zikv potency is expressed as the ratio of molar concentrations of natural products tested alone and in the mixture using the formula ((ic alone)/(ic in the mixture)- ). using a plaque-based assay, we initially screened natural products (at µm of concentration) from a natural product library for their inhibitory activity against infection of a recent zikv human strain (pan ). based on table , four "hit" natural products, including gossypol, curcumin, digitonin, and conessine ( figure a -d), were selected, since they demonstrated inhibitory activity against zikv infection with no obvious cytotoxicity in vero e cells when observed under a microscope. these natural products were subsequently ordered from sigma with ≥ % purity, and tested for their cytotoxicity using a cell-based cytotoxicity assay (cck kit), with cc values ranging from . to . µm (table , figure s ). among these natural products, curcumin has been previously reported to inhibit zikv infection [ ] , whereas the other three natural products have not been previously reported to have anti-zikv activity. gossypol, curcumin, digitonin, and conessine were retested to confirm their anti-zikv (strain pan ) activity, with ic values of . , . , . , and . µm, respectively ( table ). the ic values of these natural products against zikv pan strain were also calculated, equal to about . , . , . , and . µm, respectively, for gossypol, curcumin, digitonin, and conessine ( figure s ), which were slightly higher than the respective ic values, but much lower than the corresponding cc values. table . initial screening of a natural product library for identification of potential anti-zikv inhibitors. natural products from microsource discovery systems for screening primary hits (with inhibition against zikv strain pan ) a ( . ) primary hits (with high cytotoxicity in vero e cells) b ( . ) specific hits (primary hits with low cytotoxicity) ( . ) specific hits (available to purchase) ( . ) specific hits (ordered from sigma and retested to confirm anti-zikv activity) ( . ) specific hits displaying ic < cc ( . ) a a total of primary hits inhibited zikv (strain pan ) infection by more than % at µm, whereas the negative control (dmso) had inhibitory activity less than %. b observed cytotoxicity of natural products (at µm) under a microscope. the cytotoxicity was recorded as grades (−, ±, +, ++, +++, ++++), and the natural products with cytotoxicity greater than or equal to grade ++ were referred to as "high cytotoxicity in vero e cells", and discarded for further testing. viruses , , of table . initial screening of a natural product library for identification of potential anti-zikv inhibitors. no. (%) natural products from microsource discovery systems for screening primary hits (with inhibition against zikv strain pan ) a ( . ) primary hits (with high cytotoxicity in vero e cells) b ( . ) specific hits (primary hits with low cytotoxicity) ( . ) specific hits (available to purchase) ( . ) specific hits (ordered from sigma and retested to confirm anti-zikv activity) ( . ) specific hits displaying ic < cc ( . ) a a total of primary hits inhibited zikv (strain pan ) infection by more than % at μm, whereas the negative control (dmso) had inhibitory activity less than %. b observed cytotoxicity of natural products (at μm) under a microscope. the cytotoxicity was recorded as grades (−, ±, +, ++, +++, ++++), and the natural products with cytotoxicity greater than or equal to grade ++ were referred to as "high cytotoxicity in vero e cells", and discarded for further testing. the identified natural products were further studied for their broad-spectrum activity against nine additional zikv strains, including those isolated from different hosts, namely humans, mosquitos, and rhesus macaques, at different time periods in different countries, including mexico, panama, columbia, honduras, puerto rico, thailand, nigeria, and uganda ( figure ). the results showed that these natural products could inhibit infections of all nine zikv strains tested with various ic values ( table ). gossypol exhibited the most potent inhibitory activity for ic values, ranging from . to . m, against all nine zikv strains tested (table ) . it was also more potent than bortezomib, the anti-zikv previously reported compound as an active compound control, against all zikv strains tested ( table ). the ic values of these natural products against the selected zikv strain, prvabc , were calculated, among which gossypol had the lowest ic value (about . m, slightly higher than its ic value but lower than its cc value) ( figure s ), confirming its strong and broad-spectrum anti-zikv activity. the identified natural products were further studied for their broad-spectrum activity against nine additional zikv strains, including those isolated from different hosts, namely humans, mosquitos, and rhesus macaques, at different time periods in different countries, including mexico, panama, columbia, honduras, puerto rico, thailand, nigeria, and uganda ( figure ). the results showed that these natural products could inhibit infections of all nine zikv strains tested with various ic values (table ). gossypol exhibited the most potent inhibitory activity for ic values, ranging from . to . µm, against all nine zikv strains tested (table ) . it was also more potent than bortezomib, the anti-zikv previously reported compound as an active compound control, against all zikv strains tested ( table ). the ic values of these natural products against the selected zikv strain, prvabc , were calculated, among which gossypol had the lowest ic value (about . µm, slightly higher than its ic value but lower than its cc value) ( figure s ), confirming its strong and broad-spectrum anti-zikv activity. comparison of zikv e protein sequences revealed that most of the amino acid sequences were highly conserved, but that some variations occurred among the zikv strains used for evaluation of the inhibitory activity of natural products, including the pan strain tested earlier ( figure s ). the above data demonstrate that the identified natural products, particularly gossypol, were able to block infections of divergent human, mosquito, and monkey zikv strains isolated from different time periods and countries, including six recent zikv human strains, confirming their broad-spectrum anti-zikv activity. to identify which steps of zikv infection in its life cycle were blocked by these natural products, we carried out a time-of-addition experiment by incubating natural products with zikv or cells at different time points during zikv and cell interaction, and then calculated the percent inhibition based on the number of plaques formed [ , , , ] . to test whether a natural product can neutralize zikv infection or inhibit viral entry by targeting the viral proteins, zikv was pretreated with the natural product at • c before incubation with the host cells ( figure a(a) ). to evaluate whether a natural product can bind to the cellular receptors or cofactors to block virus-receptor binding, cells were pretreated with the natural product at • c before incubation with zikv ( figure a(b) ). to determine whether a natural product can inhibit attachment of zikv to target cells, but cannot block the virus-cell membrane fusion, cells were cotreated with zikv at • c in the presence of the natural product ( figure a (c)). to assess whether a natural product can inhibit attachment of zikv to target cells and subsequent virus-cell membrane fusion, the cells were cotreated with zikv and the natural product at • c ( figure a(d) ). to investigate whether a natural product can inhibit zikv fusion with the cell membrane and then entry into the cell, cells were pretreated with zikv at • c first and then incubated with the natural product at • c ( figure a (e)). to study whether a natural product can inhibit zikv infection at postentry stages (i.e., viral replication, virion assembly, or release), cells were pretreated with zikv and then incubated with the natural product at • c ( figure a (f)). after completing the above approaches, we gained insight into the potential mechanisms of the natural products responsible for inhibiting zikv (pan ) infection. after pretreatment of zikv with gossypol at • c before incubation with the target cells, zikv completely lost its infectivity, whereas it maintained its infectivity after other treatments described above ( figure b ), suggesting that gossypol can effectively neutralize zikv infection by targeting the virus, rather than the cell or cell-associated entry or replication stages. the results from curcumin revealed that about - % of zikv infection was blocked when curcumin was incubated with zikv only at • c, or coincubated with zikv and cells at • c or • c, whereas there was low to no impact on zikv infection when curcumin was pretreated with cells or postincubated with zikv-treated cells at • c and • c, respectively ( figure c ). these results suggest that curcumin inhibits zikv infection at the early stages of viral entry, particularly the viral attachment stage. pretreatment of vero e cells with digitonin and then with zikv or cotreatment of cells with zikv and digitonin at • c significantly (≥ %) blocked zikv infection, whereas preincubation of cells with zikv and then with digitonin at • c, pretreatment of cells with zikv at • c and then with digitonin at • c, or cotreatment of cells with digitonin and zikv at • c inhibited about - % of zikv infection ( figure d ). in contrast, preincubation of digitonin and zikv had no effect on zikv infection ( figure d ). these results suggest that digitonin could not directly neutralize zikv infection, but inhibited zikv infection by binding to the viral receptors or inhibiting viral entry (i.e., attachment and membrane fusion or postentry steps). the data from conessine indicated that coincubation of cells with conessine and zikv at • c or postincubation of conessine with zikv-treated cells at • c or • c resulted in - % inhibition of zikv infection, whereas pretreatment of cells with conessine before zikv incubation blocked about % of zikv infection. nevertheless, preincubation of conessine and zikv at • c or cotreatment of cells with conessine and zikv at • c had very low to no effect on zikv infection ( figure e ). these data suggest that conessine does not block zikv attachment to the host cell, but inhibits zikv infection by targeting virus-cell fusion or postentry step. the above steps of inhibition of zikv infection were further proven inhibition of zikv infection were further proven by the respective anti-zikv compound controls ( figure f-i) . therefore, the above data confirm the potent inhibitory activity of the identified natural products, particularly gossypol, in blocking zikv infection at various stages of the viral life cycle. anti-zikv inhibitor, temoporfin [ ] , was used as a control for step a. (g) an anti-zikv entry (especially in inhibition of the internalization/fusion step) inhibitor, -hydroxycholesterol [ ] , was used as a control for steps b and e. anti-zikv compound bortezomib was used as a control for step d (h), and a replication inhibitor, nitd [ ] , for step f (i). the natural product curcumin (c), which has been previously reported to inhibit the attachment of zikv to host cells [ ] , was used as a control for stage c. the percent inhibition was calculated in the presence or absence of serially diluted natural products. the data are expressed as mean ± s.e.m. (n = ). the experiments were performed in vero e cells and repeated three times, with similar results. (f) a potent anti-zikv inhibitor, temoporfin [ ] , was used as a control for step a. (g) an anti-zikv entry (especially in inhibition of the internalization/fusion step) inhibitor, -hydroxycholesterol [ ] , was used as a control for steps b and e. anti-zikv compound bortezomib was used as a control for step d (h), and a replication inhibitor, nitd [ ] , for step f (i). the natural product curcumin (c), which has been previously reported to inhibit the attachment of zikv to host cells [ ] , was used as a control for stage c. the percent inhibition was calculated in the presence or absence of serially diluted natural products. the data are expressed as mean ± s.e.m. (n = ). the experiments were performed in vero e cells and repeated three times, with similar results. to identify the potential binding regions of the natural products in zikv proteins, we first carried out an elisa-based approach by coating the plates with zikv full-length e or ediii proteins. we then tested for binding affinity using a zikv ediii-specific mab, zka -lala, for e or ediii binding. results revealed that gossypol bound potently to the full-length e and ediii proteins, with ec values of . and . µm, respectively, whereas curcumin had much lower binding affinity to zikv full-length e and ediii proteins ( figure a,b) . otherwise, digitonin, conessine, bortezomib (anti-zikv compound control), and dmso (negative control) did not bind to any zikv proteins tested ( figure a,b) . we then evaluated the binding of gossypol using an spr assay, and the result showed that it had binding affinity values of . µm to zikv e protein ( figure c ). zikv full-length e and ediii proteins ( figure a,b) . otherwise, digitonin, conessine, bortezomib (anti-zikv compound control), and dmso (negative control) did not bind to any zikv proteins tested ( figure a,b) . we then evaluated the binding of gossypol using an spr assay, and the result showed that it had binding affinity values of . m to zikv e protein ( figure c ). since gossypol bound to zikv e protein, potentially in the ediii region, we further carried out an elisa completion assay to identify its possible binding site(s) in the ediii. accordingly, zikv ediii protein was coated on the plates, and binding between zikv ediii and ediii-specific mabs (smzab , zka -lala, zv- , or z ), or a zikv edi/dii-specific mab control (zka ) was evaluated in the presence of serially diluted gossypol. the results showed that gossypol potently blocked binding of ediii-specific mabs smzab , zka -lala, zv- , or z to ediii protein in a dose-dependent manner, with ic values of . , . , . , and . m, respectively, whereas the dmso control showed no blockage of this binding ( figure d ). in the meantime, there was no binding between the control mab zka and ediii protein, so no obvious inhibition of gossypol was detected ( figure d ). the above zikv ediii-specific mabs have been previously shown to potently neutralize zikv infection [ ] and recognize epitopes, including the lateral ridge, such as residues - , - , , , , and - of zikv ediii protein [ ] [ ] [ ] . therefore, the above data suggest that gossypol most likely binds to the lateral ridge of the zikv ediii protein to block the ediii-mab binding. the ability of gossypol to inhibit the binding between zikv ediii and ediii-specific neutralizing mabs. the concentrations of zikv ediii and mabs were . and . µg/ml, respectively. the percent inhibition in the ediii-mab binding was measured in the presence or absence of serially diluted gossypol using the formula (( -(ediii-mab-gossypol)/(ediii-mab) × ), which, in turn, formed the basis for calculating % inhibitory concentration (ic ) values. zikv edi/dii-specific mab (zka ) and dmso were used as controls. the data are expressed as mean ± s.e.m. (n = ) . the experiments were repeated twice, with similar results. since gossypol bound to zikv e protein, potentially in the ediii region, we further carried out an elisa completion assay to identify its possible binding site(s) in the ediii. accordingly, zikv ediii protein was coated on the plates, and binding between zikv ediii and ediii-specific mabs (smzab , zka -lala, zv- , or z ), or a zikv edi/dii-specific mab control (zka ) was evaluated in the presence of serially diluted gossypol. the results showed that gossypol potently blocked binding of ediii-specific mabs smzab , zka -lala, zv- , or z to ediii protein in a dose-dependent manner, with ic values of . , . , . , and . µm, respectively, whereas the dmso control showed no blockage of this binding ( figure d ). in the meantime, there was no binding between the control mab zka and ediii protein, so no obvious inhibition of gossypol was detected ( figure d ). the above zikv ediii-specific mabs have been previously shown to potently neutralize zikv infection [ ] and recognize epitopes, including the lateral ridge, such as residues - , - , , , , and - of zikv ediii protein [ ] [ ] [ ] . therefore, the above data suggest that gossypol most likely binds to the lateral ridge of the zikv ediii protein to block the ediii-mab binding. the data described here demonstrate that the identified natural product gossypol bound strongly to zikv e protein, potentially the conserved ediii, thus blocking ediii-mab binding at important neutralizing epitopes and inhibiting viral entry into the target cell. these data reasonably explain the potent broad-spectrum antiviral activity of gossypol against infections of multiple zikv strains. since gossypol demonstrated the highest antiviral activity individually against all zikv strains tested, we next investigated the potential combinatorial effects of the combination of gossypol with three other natural products identified, namely curcumin, digitonin, and conessine, as well as anti-zikv compound control (bortezomib). results demonstrated significant combinatorial inhibitory effects against three zikv strains (pan , flr, and prvabc ) tested when combining gossypol with any of these natural products, and the ci values ranged from . to . µm, . to . µm, and . to . µm for zikv pan , flr, and prvabc strains, respectively (tables - ). the combinations of gossypol with each of these natural products also resulted in the highest enhancement of anti-prvabc activity among the three zikv strains tested (table ). these data show that gossypol can be combined with other inhibitors described above to further increase overall inhibitory activity against current and future emergent zikv strains. the above results on the combinatorial effects against zikv infection could not exclude the possibility that the observed decrease in ic in the presence of another natural product might result from the enhanced cell death caused by the natural product in combination. therefore, we also assessed the change of cc of the natural products alone and in combination, and compared the enhancement of cc with that of ic against zikv strain prvabc . as shown in table , the cytotoxicity of gossypol in combination with curcumin, digitonin, or conessine was not enhanced. the cytotoxicity of gossypol in combination with bortezomib was slightly increased ( . -fold), but it was much lower than the enhancement in anti-prvabc activity of gossypol ( . -fold) in combination with bortezomib. in addition, the cytotoxicity of curcumin and bortezomib, respectively, in combination with gossypol was not enhanced. the cytotoxicity of digitonin or conessine in combination with gossypol was slightly increased by about . -and . -fold, respectively, while they were still lower than the enhancement in anti-prvabc activity of digitonin ( . -fold) or conessine ( . -fold) in combination with gossypol. moreover, the ci values of gossypol with any of the four natural products tested were greater than ( table ), indicating that there was no combinatorial effect on the cytotoxicity to the test cells. these results suggest that the observed decrease in the ic values of the natural products in combination was not due to the enhancement in cytotoxicity of these natural products. note: gossypol with one of the natural products, including curcumin, digitonin, conessine (three lead natural products) or bortezomib (anti-zikv compound control), were first mixed according to the molar ratio identified in the above combinational experiment against zikv strain prvabc , and then added to vero e cells to determine cytotoxicity. the combinatorial cytotoxicity of natural products to vero e cells is expressed as cc identification of broad-spectrum anti-flavivirus inhibitors is crucial to treat infections caused by zikv and other flaviviruses, such as denv. hence, using a plaque assay similar to zikv, we evaluated the antiviral activity of natural products against infections of four serotypes of denv human strains in llc-mk cells. even though all four natural products could inhibit denv- - infections, results showed that gossypol had the highest potency against denv- , denv- , and denv- infections, with ic values of . , . , and . µm, respectively (table ) . also, the anti-denv- activity of gossypol (ic value: . µm) was only slightly higher than that of curcumin (ic value: . µm) ( table ) . the cytotoxicity of these natural products on llc-mk cells was investigated by a cytotoxicity assay, with the cc values ranging from . to . µm ( table ) . the above data indicate the potent anti-denv activity of the natural products, particularly gossypol, against infections of four denv human strains with low to no cytotoxicity. the experiments were performed on llc-mk cells, and the cytotoxicity of natural products in this cell line is expressed as cc . the inhibitory activity of natural products against infections of denv- - is expressed as ic . the data are expressed as mean ± s.e.m. (n = ). the experiments were repeated twice, with similar results. as described earlier, gossypol targeted zikv e protein, potentially ediii. although a number of variations have been identified in the amino acid sequences of e proteins of zikv and denv strains tested in this study ( figure s ), gossypol could still inhibit all zikv and denv strains tested, suggesting that it potentially targeted the conserved sequences in zikv and denv ediii proteins. our data further explain the potent, broad-spectrum activity of gossypol against infections of at least two flaviviruses, including zikv and denv. development of safe and effective antiviral therapeutics is urgently needed to treat zikv infections and zikv-caused diseases, particularly zikv-associated microcephaly, fetal death, or neurological diseases [ , [ ] [ ] [ ] [ ] . here, we have identified four small-molecule-based natural products, namely gossypol, curcumin, digitonin, and conessine, with robust anti-zikv activities in vero e cells. among them, gossypol had the greatest potency to block infections of zikv for almost all strains isolated from to from nine countries and three hosts, including six recent human strains associated with congenital zika syndrome and other neurological malformations. time-of-addition-based mechanistic studies indicated that gossypol could neutralize zikv infection by targeting the virus rather than the cell or cell-associated zikv entry or replication steps. inhibition assays revealed that gossypol strongly bound to zikv e protein, particularly the conserved ediii, and blocked the binding between zikv ediii and ediii-specific neutralizing mabs, rationalizing its efficacious and broad-spectrum anti-zikv activity. it appears that the binding between gossypol and zikv e/ediii protein might be nonspecific, since gossypol is also active against other enveloped viruses, including herpes simplex virus type , parainfluenza virus type , influenza virus, and hiv- [ ] [ ] [ ] [ ] [ ] . because of the potential toxicity and side effects of gossypol to humans [ ] [ ] [ ] , it might not be a good idea to use this natural product as a drug to treat human diseases, including zikv-associated microcephaly and other flavivirus-caused diseases. nevertheless, the goal of this study is to identify the best active, natural product, then modify it to improve its antiviral activity and drug-like properties and reduce its toxicity. due to the symmetric nature of the gossypol molecule, there will be better opportunity to defragment its structure to smaller drug-like molecules with higher activity and lower toxicity. therefore, it is suggested not to use gossypol as the lead molecule, but rather the final drug. unlike gossypol, digitonin and conessine did not neutralize zikv directly, but instead inhibited zikv infection at various stages of the life cycle, including viral attachment, membrane fusion, or postentry steps. notably, digitonin is a glycoalkaloid saponin detergent. it is widely used as a cell membrane permeabilizing agent [ , ] and a detergent for selective solubilization of membrane proteins [ ] [ ] [ ] ; thus, the antiviral activity of this natural product is likely to be very nonspecific. by comparison, conessine appears to be a good candidate with a decent anti-zikv activity and very low cytotoxicity (higher selectivity). however, it is a steroidal alkaloid [ ] , which might not be ideal for further optimization. similar to gossypol, curcumin may directly neutralize zikv infection, but it seems to mainly inhibit the early stage (attachment) of virus infection, the mechanism of which is similar to that seen in a previous report using different approaches [ ] . our data have also demonstrated strong in vitro ability of these natural products-gossypol in particular-against four serotypes of denv human strains with low to no cytotoxicity, and the ic values against denv were similar to those against zikv. we have found that gossypol bound to zikv e protein, potentially ediii, suggesting that it may recognize highly conserved regions and sites in the e proteins of zikv and denv. previously, curcumin was shown to potentially inhibit denv- infection through direct effects on viral particle production or various cellular systems [ ] . we have not found studies in the literature reporting on the antiviral activity of curcumin on other serotypes of denv or the inhibitory effects of gossypol, digitonin, and conessine on zikv and other flaviviruses. thus, the present study provides a rationale for further understanding of anti-denv and anti-zikv mechanisms of these natural products and identifying their potential binding sites in the viral proteins. except for demonstrating the individual in vitro anti-zikv activity of natural products identified in this study, particularly gossypol, we have confirmed important combinatorial anti-zikv effects of gossypol in combination with other natural products. it is interesting to note that combining gossypol with curcumin, digitonin, or conessine resulted in increased combinatorial effect against prvabc strain compared to the flr strain of zikv, potentially because gossypol had more potent anti-flr activity than anti-prvabc activity when tested individually. also, such combinations enhanced the individual antiviral activity of curcumin, digitonin, and conessine against the test viruses. we have shown that the combination of gossypol and bortezomib, a licensed proteasome inhibitor previously reported inhibiting zikv infection [ , ] , led to significant combinatorial activity against the three epidemic zikv human strains tested. therefore, our study demonstrates the possibility of combining gossypol with other natural products or compounds to enhance antiviral activity. overall, this study has evaluated the antiviral activity of four natural products in vitro, among which three are newly identified natural products with strong anti-zikv ability. future observations will be needed to evaluate the protective efficacy of these natural products individually or in combination, or using their defragmented, small drug-like molecules (in the case of gossypol) against zikv-caused congenital infection and fetal demise in available animal models. modification of the structure of gossypol and identification of its derivatives with better antiviral activity, but without cytotoxicity, would be essential for developing a safe and effective anti-zikv agent for human use. also, future studies to evaluate the antiviral activity of modified gossypol or its derivatives against other flaviviruses, both in vitro and in vivo, will be helpful for identification of an effective and safe pan-flavivirus inhibitor. taken together, the broad-spectrum ability of the identified natural products, especially gossypol, against zikv and denv infections indicates the potential for further development of these small molecules or their derivatives as the lead compounds or effective anti-flavivirus inhibitors. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s : association between inhibitory activity of natural products against zikv strain pan and their cytotoxicity. figure s : association between inhibitory activity of natural products against zikv strain prvabc and their cytotoxicity. figure s : multiple sequence alignment of amino acid (aa) sequences of e protein of zikv strains and denv- - human strains used in this study. author contributions: s.j., a.k.d., and l.d. designed the study. y.g., w.t., n.w., and x.l. performed the experiments and analyzed the data. y.g. and n.w. carried out sequence alignment analyses. y.g., s.j., a.k.d., l.d., and s.c. wrote and revised the manuscript. zika virus: new clinical syndromes and its emergence in the western hemisphere zika virus (i). isolations and serological specificity zika virus infection in pregnancy: maternal, fetal, and neonatal considerations implications of zika virus and congenital zika syndrome for the number of live births in brazil congenital zika syndrome in zika virus infection with prolonged maternal viremia and fetal brain abnormalities zika virus infection in pregnant women in rio de janeiro full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika virus entry mechanisms in human cells therapeutic approaches for zika virus infection of the nervous system discovery of immunologically inspired small molecules that target the viral envelope protein the immunopathology of dengue and zika virus infections dengue: a continuing global threat critical neutralizing fragment of zika virus ediii elicits cross-neutralization and protection against divergent zika viruses a screen of fda-approved drugs for inhibitors of zika virus infection rational design of zika virus subunit vaccine with enhanced efficacy transfusion-transmitted zika virus infection in pregnant mice leads to broad tissue tropism with severe placental damage and fetal demise n-substituted pyrrole derivatives as novel human immunodeficiency virus type entry inhibitors that interfere with the gp six-helix bundle formation and block virus fusion theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies triterpenoids manipulate a broad range of virus-host fusion via wrapping the hr domain prevalent in viral envelopes broad-spectrum antiviral agents: secreted phospholipase a targets viral envelope lipid bilayers derived from the endoplasmic reticulum membrane identification of a small-molecule entry inhibitor for filoviruses discovery of pentacyclic triterpenoids as potential entry inhibitors of influenza viruses a bivalent recombinant protein inactivates hiv- by targeting the gp prehairpin fusion intermediate induced by cd d d domains a peptide-based viral inactivator inhibits zika virus infection in pregnant mice and fetuses time-of-addition and temperature-shift assays to determine particular step(s) in the viral life cycle that is blocked by antiviral substance(s) existing drugs as broad-spectrum and potent inhibitors for zika virus by targeting ns b-ns interaction -hydroxycholesterol protects host against zika virus infection and its associated microcephaly in a mouse model adenosine analog nitd is a potent inhibitor of zika virus. open forum introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines enhanced ability of oligomeric nanobodies targeting mers coronavirus receptor-binding domain cross-reactivity, and function of antibodies elicited by zika virus infection a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov anti-hiv antibody and drug combinations exhibit synergistic activity against drug-resistant hiv- strains combining a fusion inhibitory peptide targeting the mers-cov s protein hr domain and a neutralizing antibody specific for the s protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd curcumin inhibits zika and chikungunya virus infection by inhibiting cell binding inhibitory effects of curcumin on dengue virus type -infected cells in vitro differential human antibody repertoires following zika infection and the implications for serodiagnostics and disease outcome structural basis of zika virus-specific antibody protection recurrent potent human neutralizing antibodies to zika virus in brazil and mexico association between zika virus and microcephaly in french polynesia infection-related microcephaly after the and zika virus outbreaks in brazil: a surveillance-based analysis who zika causality working group. zika virus infection as a cause of congenital brain abnormalities and guillain-barré syndrome: systematic review zika virus disease-associated guillain-barré syndrome-barranquilla antiviral activity of gossypol and apogossypol synthesis and anti-hiv activity of , -dideoxygossypol and related compounds study of antiviral effect of gossypol on chick embryo model selective inhibition of human immunodeficiency virus type replication by the (-) but not the (+) enantiomer of gossypol investigations on gossypol: past and present developments mechanism of drug and toxic actions of gossypol: focus on reactive oxygen species and electron transfer gossypol-induced suicidal erythrocyte death permeabilization of mdck cells with cholesterol binding agents: dependence on substratum and confluency glycoalkaloids selectively permeabilize cholesterol containing biomembranes streptolysin-o induces release of glycosylphosphatidylinositol-anchored alkaline phosphatase from ros cells by vesiculation independently of phospholipase action differential solubilization of lipids along with membrane proteins by different classes of detergents specific release of membrane-bound annexin ii and cortical cytoskeletal elements by sequestration of membrane cholesterol anti-malarial property of steroidal alkaloid conessine isolated from the bark of holarrhena antidysenterica clinical use of proteasome inhibitors in the treatment of multiple myeloma this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no competing interests. key: cord- -cr zskcw authors: lu, chien-yi; lin, chen-sheng; lai, hsueh-chou; yu, ya-wen; liao, chih-yi; su, wen-chi; ko, bo-han; chang, young-sheng; huang, su-hua; lin, cheng-wen title: the rescue and characterization of recombinant, microcephaly-associated zika viruses as single-round infectious particles date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cr zskcw zika virus (zikv) is transmitted by aedes mosquitoes and exhibits genetic variation with african and asian lineages. zikv natal rgn strain, an asian-lineage virus, has been identified in brain tissues from fetal autopsy cases with microcephaly and is suggested to be a neurotropic variant. however, zikv natal rgn strain has not been isolated; its biological features are not yet illustrated. this study rescued and characterized recombinant, single-round infectious particles (srips) of the zikv natal rgn strain using reverse genetic and synthetic biology techniques. the dna-launched replicon of zikv natal rgn was constructed and contains the egfp reporter, lacks prm-e genes, and replicates under cmv promoter control. the peak in the zikv natal rgn srip titer reached . × ( ) tcid /ml in the supernatant of prm-e-expressing packaging cells h post-transfection with a zikv natal rgn replicon. the infectivity of zikv natal rgn srips has been demonstrated to correlate with the green florescence intensity of the egfp reporter, the srip-induced cytopathic effect, and zikv’s non-structural protein expression. moreover, zikv natal rgn srips effectively self-replicated in rhabdomyosarcoma/muscle, glioblastoma/astrocytoma, and retinal pigmented epithelial cells, displaying unique cell susceptibility with differential attachment activity. therefore, the recombinant zikv natal rgn strain was rescued as srips that could be used to elucidate the biological features of a neurotropic strain regarding cell tropism and pathogenic components, apply for antiviral agent screening, and develop vaccine candidates. zika virus (zikv) was first isolated in monkeys in uganda in , and then detected in aedes africanus mosquitoes in . zikv belongs to a mosquito-borne flavivirus of the family flavivirus, which spread from africa to south-eastern asia through transmission by aedes mosquitoes, such as a. to produce single-round infectious particles (srips). the packaging cells carrying the recombinant plasmid encoding the viral structural proteins are transfected with dna-launched replicons and then self-replicate viral subgenomes and express all viral proteins, allowing viral assembly and release as srips. several flavivirus, replicon-based srips, including jev, dengue virus, west nile virus, and tick-borne encephalitis virus have been generated and demonstrated as safer vaccine candidates [ ] [ ] [ ] . subsequently, replicon-based srips are suitable systems for investigating the mechanism of the zikv life cycle, zikv-host interaction, and virulence. the zikv natal rgn strain that is associated with microcephaly was recognized as the representative, epidemic, zikv asian strain in - [ ] . the zikv natal rgn strain is detected in brain tissues from fetal autopsy cases with microcephaly in the natal region of brazil; its genome has been sequenced using next-generation sequencing (genbank accession number ku ) [ ] . the zikv natal rgn strain contains a unique genotype and phenotype; therefore, this study aimed to generate srips of the zikv natal rgn strain using synthetic and reverse genetic technologies. this paper characterizes the infectivity of zikv natal rgn srips in different cell types. moreover, the paper analyzes the expression pattern of type i interferons and apoptosis-related genes induced by zikv natal rgn srips. te (human, rhabdomyosarcoma/muscle), sf (human brain, glioblastoma/astrocytoma), and arpe- (human, retinal pigmented epithelium) cells were cultured in minimum essential medium containing % fetal bovine serum, mm glutamine, mm pyruvate, and × penicillin-streptomycin at • c with % co . components of two dna segments we synthesized, i and ii, consisted of the cmv immediate-early promoter (cmvp), cdna fragments of the zikv natal rgn genome (genbank accession number ku ), enhanced green fluorescent protein (egfp), fmdv- a (f- a), hepatitis delta virus ribozyme (hdvr), and bovine growth hormone polya signal (bgh-pa), which were purchased from bio basic inc. (ontario, canada). the construction of synthesized dna fragments, plasmid information, and restriction enzyme digestion analysis of the dna fragments we purchased are shown in supplementary figures s and s and figure a . to construct the zikv natal rgn replicon, the plasmid pbr -linker, described in our prior report [ ] , was chosen as the vector to assemble fragments a, b, c, and d of the cmvp-driven zikv natal rgn replicon with the egfp reporter via the cloning sites of ecori, noti, clai, rsrii, and xhoi ( figure b ). fragments a, c, and d were generated using the platinum ® pcr supermix high fidelity reaction with the dna segments we synthesized, i and ii, as the templates (life technology, carlsbad, ca, usa). fragment b was produced through two-round pcr with the gibson assembly reaction of two first-round pcr products (b and b ) as the template (new england biolabs, ipswich, ma, usa). the pairs containing the restriction enzyme site(s) indicated, are listed in supplementary table s . the resultant plasmid carrying the cmvp-driven zikv natal rgn replicon with the egfp reporter was propagated in e. coli dh α and then sequenced by sanger sequencing assays with sequencing primers (supplementary table s ). the nucleotide and deduced amino acid sequence alignment analysis of the cmvp-driven zikv natal rgn replicon and its parent strain (genbank accession number ku ) was performed using the lasergene dnastar megalign software. figure . construction of the pbr -based zikv natal rgn replicon and pcdna . -zikv prme. two synthetic dna segments in the puc plasmid contained the entire zikv natal rgn strain genome: cmvp, egfp, fmdv- a, and bgh-pa sequences (a). four pcr products (fragments a-d) were cloned into the indicated restriction sites (ecori, noti, clai, rsrii, and xhoi) of the pbr plasmid and then assembled as the in-frame fusion of the zikv natal rgn replicon with the egfp reporter under cmv-promoter control (b). those four pcr products were analyzed using agarose gel electrophoresis (c). the pcr product of zkiv prm and e genes was cloned into restriction sites (ecori and xhoi) of the pcdna . -his-c plasmid (d). initially, te cells were transfected with pcdna . -zikv prm-e, selected with g for weeks, analyzed to examine zikv prm and e protein expression, and then recognized as the packaging cell line ( figures d, a middle, a middle). subsequently, the packaging cells were transfected with the zikv natal rgn replicon, and then cpe and the egfp reporter expressions were examined ( figure ). in the packaging cells, egfp reporter expression and cpe showed slight levels h post-transfection, but exhibited significant changes h after transfection with the zikv natal rgn replicon (figure bottom) . moreover, in vitro translation and transcription of the zikv natal rgn replicon in packaging cells were further characterized ( figure ). immunofluorescent staining indicated that the zikv e c-terminus, ns , and ns proteins were massively expressed in replicon-transfected packaging cells ( figure a bottom), but not in mock and packaging cells ( figure a top and middle). the real-time, reverse transcription pcr assay showed large amounts of positive and negative-sense zikv subgenomes in replicon-transfected packaging cells h post-transfection, but not in mock cells ( figure b top and bottom) . the results demonstrated efficient translation and replication of the zikv subgenomes in the packaging cells using the dna-launched zikv natal rgn replicon. figure . construction of the pbr -based zikv natal rgn replicon and pcdna . -zikv prme. two synthetic dna segments in the puc plasmid contained the entire zikv natal rgn strain genome: cmvp, egfp, fmdv- a, and bgh-pa sequences (a). four pcr products (fragments a-d) were cloned into the indicated restriction sites (ecori, noti, clai, rsrii, and xhoi) of the pbr plasmid and then assembled as the in-frame fusion of the zikv natal rgn replicon with the egfp reporter under cmv-promoter control (b). those four pcr products were analyzed using agarose gel electrophoresis (c). the pcr product of zkiv prm and e genes was cloned into restriction sites (ecori and xhoi) of the pcdna . -his-c plasmid (d). to detect self-amplifying rna genomes of the cmvp-driven zikv natal rgn replicon, the synthesis of positive and negative-sense rna subgenomes in vitro was examined using sybr green-based real-time pcr. total rnas of te- cells transfected with the zikv natal rgn replicon were extracted using the purelink mini total rna purification kit (thermo fisher scientific, waltham, ma, usa), reverse transcribed into cdna with specific-capture primers, and measured using real-time pcr with zikv ns -specific primer pairs (supplementary table s ), according to our prior report [ ] . relative levels of self-amplifying zikv sense and antisense genomes were normalized to glyceraldehyde -phosphate dehydrogenase (gapdh). the absolute copy number of the zikv genome was determined according to the standard curve of real-time pcr for serial dilutions of the plasmid containing the zikv ns gene at a known concentration. to explore the expression of replicon-based egfp and zikv reporter proteins, replicon-transfected cells were initially photographed using immunofluorescence microscopy; then, an immunofluorescence assay (ifa) was performed with primary antibodies against zikv ns and ns (genetex, inc., taiwan) and secondary af goat anti-rabbit igg (thermo fisher scientific). the immunofluorescent staining assay was carried out as described in our prior report [ ] . the fluorescence intensity of replicon-based egfp and zikv proteins in zikv natal rgn replicon-transfected cells was counted by image j. to establish the packaging cells expressing zikv structural proteins, the prm/m-e gene was amplified using pcr with specific primers (supplementary table s ) and synthesized dna segment i as the template. the pcr product of the zikv prm-e gene was digested with ecori and xhoi, and then cloned into the ecori and xhoi sites of the expression plasmid pcdna . -hisc. the resultant plasmid pcdna-zikv prme was transfected with te- cells at % confluence in a -well plate with lipofectamine ltx (invitrogen, carlsbad, ca, usa) according to the manufacturer's guidelines. the transfected cells were selected in the culture media with µg/ml g for weeks; the expression of zikv prm and e proteins in a stably transfected cell line (packaging cell line) was validated by real-time rt-pcr with zikv e-specific primers (supplementary table s ) and immunofluorescence staining with primary antibodies against zikv e protein (genetex, inc.), as described above. the packaging cells grown at % confluence in -well plates were transfected with or without the zikv natal rgn replicon using lipofectamine ltx. the cytopathic effect (cpe), replicon-based egfp and zikv proteins, and viral sense and antisense genomes in mock and transfected cells, were measured and h post-transfection using immunofluorescent staining assays, as described above. moreover, real-time rt-pcr assays with ns -specific primer pairs, listed in supplementary table s , were performed to examine the positive and negative-sense viral genomes in replicon-transfected packaging cells, as well as the viral genome in srips. zikv natal rgn srips in the cultured media of transfected packaging cells were concentrated using the peg virus precipitation kit (abcam, cambridge, ma, usa). the cultured media were centrifuged at × g for min at • c; then, the supernatant ( ml) was collected from each and incubated with . ml peg solution overnight at • c. the pellets of zikv natal rgn srips were harvested after centrifugation at × g for min at • c, re-suspended in µl virus re-suspension solution, and then stored at − • c. to analyze the antigenicity of zikv natal rgn srips, µl each of one-fold and -fold srip stock dilution was spotted onto a nitrocellulose membrane for the dot-blot assay with anti-zikv e antibodies. the membrane was subsequently blocked with % skim milk in tbst (tris-buffered saline, . % tween- ) buffer at • c for h, incubated overnight with anti-zkiv e antibodies (genetex, inc., taiwan), and then reacted with hrp-conjugated anti-mouse igg antibodies (invitrogen, carlsbad, ca, usa) after washing with tbst. immunoreactive signals for the e proteins of zikv natal rgn srips were amplified with ecltm western blotting detection reagents (ge healthcare, chicago, il, usa), and then imaged by the multi-function gel image system (multigel- ) (gentaur, san jose, ca, usa). the infectious titer of the zikv natal rgn srip stock was determined by a median tissue culture infectious dose (tcid ) assay. serial dilutions of the zikv natal rgn srip stock were added and incubated on the % confluent monolayer of packaging cells in -well plates. after incubating for h at • c, the cpe in each well was observed and recorded to determine the tcid titer of the zikv natal rgn srip stock. in the infectivity assay, packaging cells were cultured in -well plates overnight and infected with different doses of zikv natal rgn srips at multiplicity of infections (mois) of , . , . , and . . cpe, replicon-driven egfp, and zikv ns expression in the srip-infected cells were examined h post-infection, and fluorescence microscopy and immunofluorescent staining were performed as described above. in cell line susceptibility assays, te- , sf- , and arpe- cells were infected with zikv natal rgn srips at a moi of . tcid /cell. after incubating for h, the cells were photographed to examine cpe and explore replicon-driven egfp reporter expression using immunofluorescence microscopy. moreover, the cells were fixed with paraformaldehyde at • c for h, and then immunofluorescence staining was performed with anti-zikv ns antibodies, as mentioned above. in the attachment assay, zikv natal rgn srips at a dose of tcid /well (moi = . tcid /cell) were added onto the monolayers of arpe- , te , and sf- cells and incubated at • c for h. after washing with cold pbs, zikv natal rgn srips attached on the cell surface were harvested, and the relative levels of extracted viral genomes were quantitated using two-step, sybr, green i, real-time rt-pcr with zikv ns -specific primer pairs, as described above. all data from independent experiments were analyzed using student's t-tests or χ tests. statistical significance for each assay was judged at p < . . the zikv asian natal rgn prototype strain, a microcephaly-associated zikv, was not isolated from the brain tissues from fetal autopsy cases; therefore, this study intended to recover the recombinant zikv natal rgn strain using reverse genetics and synthetic biology techniques based on the published genome sequence (genbank accession number ku ). two synthetic dna segments in the puc plasmids comprised the entire zikv natal rgn strain genome, cmvp, egfp, fmdv- a, and bgh-pa sequences (supplementary figures s and s and figure a ,b). the synthetic dna segments were used as templates via pcr to amplify the gene fragments (a-d) that were cloned into the low-copy-number plasmid pbr to construct a dna-launched zikv natal rgn replicon under the control of the cmv promoter ( figure b,c) . fragment a consisted of cmvp, zikv '-utr, zikv c protein, the reporter egfp, and fmdv- a. fragment b containing the -residue c-terminal region of zikv e protein, ns , ns a, ns b, and the n-terminal region of ns , was produced using gibson assembly cloning ( figure c ). in order to join fragments b and c, the reverse primer for fragment b and the forward primer for fragment c contained the restriction enzyme site clai (supplementary table s ), which is recognized as the marker nt atcgat of the replicon in which thymidine at nucleotide of the zikv natal rgn genome was exchanged with adenosine, but it caused a silent mutation ( figure b) . interestingly, the rsrii restriction site within the ns b gene ( nt cggaccg ) of the zikv natal rgn genome was a unique restriction site for ligation between fragments c and d. in addition, hdvr was fused with the '-utr of zikv in the replicon to verify the accuracy of the '-end of the transcribed zikv natal rgn rna genomes ( figure b,c) . furthermore, sequencing analysis of the zikv natal rgn replicon indicated that only two amino acid substitutions appeared within the ns protein (l r and e n) (supplementary figure s ) . therefore, the dna-launched zikv natal rgn replicon was effectively constructed to generate the in-frame fusion of zikv c protein, reporter egfp, fmdv- a, the -residue c-terminal region of zikv e protein, and all non-structural zikv proteins under the control of the cmv promoter. initially, te cells were transfected with pcdna . -zikv prm-e, selected with g for weeks, analyzed to examine zikv prm and e protein expression, and then recognized as the packaging cell line ( figure d to examine the production of zikv natal rgn srips, the supernatant of replicon-transfected packaging cells was harvested h post-transfection and analyzed using dot-blotting, real-time rt-pcr, and tcid assays (figure ) . the dot-blotting assay with anti-zikv e protein demonstrated the antigenic structure of the e protein ( figure a) , and real-time rt-pcr assay elucidated the viral subgenomes of zikv natal rgn srips from the supernatant of the replicon-transfected packaging cells ( figure b) . moreover, the titer of zikv natal rgn srips in the supernatant was determined by tcid assay with the packaging cells. the titer of the zikv natal rgn srip stock was . × tcid /ml ( figure c ). to examine the production of zikv natal rgn srips, the supernatant of replicon-transfected packaging cells was harvested h post-transfection and analyzed using dot-blotting, real-time rt-pcr, and tcid assays (figure ) . the dot-blotting assay with anti-zikv e protein demonstrated the antigenic structure of the e protein ( figure a) , and real-time rt-pcr assay elucidated the viral subgenomes of zikv natal rgn srips from the supernatant of the replicon-transfected packaging cells ( figure b) . moreover, the titer of zikv natal rgn srips in the supernatant was determined by tcid assay with the packaging cells. the titer of the zikv natal rgn srip stock was . × tcid /ml ( figure c ). to examine the infectivity of zikv natal rgn srips in vitro, the packaging cells were infected with -fold dilutions (mois of , . , . , and . ) of zikv natal rgn srips to determine sripinduced cpe and srip-driven expression of egfp reporter and zikv ns ( to examine the cell line susceptibility to zikv natal rgn srips in vitro, the three cell lines te- , sf- , and arpe- were used ( figure ). zikv natal rgn srips at a moi of . tcid /cell induced more observable and higher cpe levels in te- cells than those in arpe- and sf cells (figure a top) . the cpe level induced by zikv natal rgn srips correlated with the fluorescence intensity of the egfp reporter encoded within the srip genome in the following order: te- > arpe- > sf cells infected with zikv natal rgn srips ( figure a middle) . in addition, immunofluorescent staining with anti-zikv ns antibodies indicated that zikv ns protein was expressed in these three zikv natal rgn srip-infected cell lines, in which the fluorescence intensity of the ns protein was consistent with the cpe level induced by zikv natal rgn srips ( figure a bottom). meanwhile, tcid of the srips per well was added onto the monolayer of these three cell lines to examine the attachment activity of zikv natal rgn srips ( figure b) . interestingly, the srips showed the highest level of attachment activity to te cells, in which the highest viral genome number was discovered h after attachment at °c. however, the srips showed the lowest level of attachment activity to sf cells. compared to arpe- and sf cells, it was found that te cells exhibited the highest susceptibility to zikv natal rgn srips and allowed the highest number of srips to attach to the surface. to examine the cell line susceptibility to zikv natal rgn srips in vitro, the three cell lines te- , sf- , and arpe- were used ( figure ). zikv natal rgn srips at a moi of . tcid /cell induced more observable and higher cpe levels in te- cells than those in arpe- and sf cells (figure a top) . the cpe level induced by zikv natal rgn srips correlated with the fluorescence intensity of the egfp reporter encoded within the srip genome in the following order: te- > arpe- > sf cells infected with zikv natal rgn srips ( figure a middle) . in addition, immunofluorescent staining with anti-zikv ns antibodies indicated that zikv ns protein was expressed in these three zikv natal rgn srip-infected cell lines, in which the fluorescence intensity of the ns protein was consistent with the cpe level induced by zikv natal rgn srips ( figure a bottom). meanwhile, tcid of the srips per well was added onto the monolayer of these three cell lines to examine the attachment activity of zikv natal rgn srips ( figure b) . interestingly, the srips showed the highest level of attachment activity to te cells, in which the highest viral genome number was discovered h after attachment at • c. however, the srips showed the lowest level of attachment activity to sf cells. compared to arpe- and sf cells, it was found that te cells exhibited the highest susceptibility to zikv natal rgn srips and allowed the highest number of srips to attach to the surface. the zikv natal rgn strain found in brain tissues from fetal autopsy cases with microcephaly in the natal region of brazil has not been isolated [ ] . this study, firstly, generated srips of the zikv natal rgn strain using synthetic and reverse genetic technologies (figures - and supplementary figures s -s ) . the viral yield of zikv natal rgn srips in prm-e-expressing packaging cells transfected with the zikv replicon was . × tcid /ml (figure ) , which indicated the replication capacity of the cmv promoter-launched zikv natal rgn replicon. in addition, a green fluorescence reporter, egfp, encoded within the zikv natal rgn replicon, represented an observable marker to examine replication of the zikv natal rgn replicon and srips (figures , , and ). in our laboratory, the pbr plasmid has been used to construct a jev replicon that is genetically stable in e. coli [ ] . the jev genome sequence encoding for structural proteins (c, prm/m, and e) was toxic in e. coli, which resulted in genetic instability of the jev infectious clones and replicons [ ] . similarly, construction of zikv natal rgn infectious clones was not accomplished due to the genetic instability of zikv natal rgn infectious clones, particularly within the ns gene region (supplementary figures s and s ) . however, the recombinant pcdna . plasmid containing prm and e genes and the pbr plasmid containing ' and '-utrs, c, and all ns genes, showed genetic stability. two zikv infectious clones obtained by cloning the entire fulllength native cdna have been reported [ , ] , and other zikv infectious clones were reported using strategies to avoid the toxicity of the zikv genome sequence, including in vitro assembly of viral cdna fragments and insertion of eukaryotic introns [ ] [ ] [ ] [ ] [ ] [ ] . particularly, the zikv natal rgn infectious clone was produced using bacterial artificial chromosomes (bacs) [ ] , in which a singlecopy pbelobac plasmid containing the full-length genome of the zikv natal rgn strain under the cmv promoter control was used to maintain zikv genome stability and reduce toxicity in bacteria. in addition, our prior report demonstrated that jev srips carrying the egfp reporter were a more sensitive, effective, and efficient platform for quantitating antiviral efficacy using flow cytometry compared to the traditional plaque assay [ ] . like jev srips, the zikv natal rgn srips the zikv natal rgn strain found in brain tissues from fetal autopsy cases with microcephaly in the natal region of brazil has not been isolated [ ] . this study, firstly, generated srips of the zikv natal rgn strain using synthetic and reverse genetic technologies (figures - and supplementary figures s -s ) . the viral yield of zikv natal rgn srips in prm-e-expressing packaging cells transfected with the zikv replicon was . × tcid /ml (figure ) , which indicated the replication capacity of the cmv promoter-launched zikv natal rgn replicon. in addition, a green fluorescence reporter, egfp, encoded within the zikv natal rgn replicon, represented an observable marker to examine replication of the zikv natal rgn replicon and srips ( figure , figure , and figure ). in our laboratory, the pbr plasmid has been used to construct a jev replicon that is genetically stable in e. coli [ ] . the jev genome sequence encoding for structural proteins (c, prm/m, and e) was toxic in e. coli, which resulted in genetic instability of the jev infectious clones and replicons [ ] . similarly, construction of zikv natal rgn infectious clones was not accomplished due to the genetic instability of zikv natal rgn infectious clones, particularly within the ns gene region ( supplementary figures s and s ) . however, the recombinant pcdna . plasmid containing prm and e genes and the pbr plasmid containing ' and '-utrs, c, and all ns genes, showed genetic stability. two zikv infectious clones obtained by cloning the entire full-length native cdna have been reported [ , ] , and other zikv infectious clones were reported using strategies to avoid the toxicity of the zikv genome sequence, including in vitro assembly of viral cdna fragments and insertion of eukaryotic introns [ ] [ ] [ ] [ ] [ ] [ ] . particularly, the zikv natal rgn infectious clone was produced using bacterial artificial chromosomes (bacs) [ ] , in which a single-copy pbelobac plasmid containing the full-length genome of the zikv natal rgn strain under the cmv promoter control was used to maintain zikv genome stability and reduce toxicity in bacteria. in addition, our prior report demonstrated that jev srips carrying the egfp reporter were a more sensitive, effective, and efficient platform for quantitating antiviral efficacy using flow cytometry compared to the traditional plaque assay [ ] . like jev srips, the zikv natal rgn srips carrying the egfp reporter could be applicable as a drug screening platform. therefore, the reverse genetics system for the production of zikv natal rgn srips provided an alternative, applicable, and rapid approach to studying the biological features of the unique zikv strains. the human rhabdomyosarcoma/muscle te cells were used as packaging cells expressing zikv prm and e proteins (figure ), in which transfection with the zikv natal rgn replicon significantly generated recombinant srips with the high yield of . × tcid /ml (figure ) . interestingly, te cells expressing jev c, prm, and e proteins were also used as a packaging cell line that was efficient at producing jev srips post-transfection with the jev replicon [ ] . the recombinant zikv natal rgn srips showed infectivity and self-replication in the packaging cells ( figure ). two amino acid substitutions within ns (l r and e n) (supplementary figure s ) seemed to have no influence on the activities of ns methyltransferase and rna-dependent rna polymerase. a recent study indicated that human embryonic kidney t cells had been used to establish different flavivirus prm-e-expressing packaging cells, including denv - , zikv, jev, west nile virus, yellow fever virus, and tick-borne encephalitis virus, and the cells were transfected with the denv reporter replicon to generate a chimeric flavivirus srip-based neutralization assay [ ] . therefore, recombinant zikv natal rgn srips generated in this study could be used to analyze the zikv life cycle, elucidate viral pathogenesis, screen antiviral agents, design faster and safer diagnostic tools, and even develop attenuated vaccines against zikv infection. the full-length zikv natal rgn strain genome was directly sequenced using next-generation sequencing with the total rna extracted from brain tissues from fetal autopsy cases with microcephaly (genbank accession number ku ) [ ] . in this study, recombinant zikv natal rgn srips were rescued using reverse genetic and synthetic biology techniques (figures - and supplementary figures s -s ) . three cell lines, te , sf , and arpe- , were further tested for cell susceptibility to zikv natal rgn srips ( figure ). the order of cell susceptibility to zikv natal rgn srips was te > arpe- > sf cells. the cpe level, egfp reporter intensity, and zikv ns protein expression were most obvious (> % involvement) in te cells, but were mild in arpe- cells and limited in sf cells h post-infection with srips at an moi of . tcid /cell. the results indicated that zikv natal rgn srips had a broad range of human tissue tropism with differential cell susceptibilities ( figure a ). in addition, zikv natal rgn srips had the highest attachment activity to te cells among those three cell lines ( figure b ). srip attachment to te cells was suggested to be involved in the infectivity of zikv natal rgn in te cells. this finding implied that zikv natal rgn srips had tropism for rhabdomyosarcoma/muscle and retinal pigmented epithelial cells, which was consistent with myalgia, arthralgia, retinitis, chorioretinal atrophy, and pigmentary maculopathy in symptomatic zikv infection [ , ] . therefore, the cell line susceptibility results revealed the unique features of zikv natal rgn srips and suggested the possible pathogenesis of zikv infection. in conclusion, the approach for generating recombinant zikv srips with an egfp reporter showed the genetic stability of zikv prm-e genes in the mammalian expression vector and zikv replicon sequence in the low-copy plasmid pbr , providing a faster and safer platform to study the biological aspects of zikv infection. zikv natal rgn srips were rescued by the combination of prm-e-expressing packaging cells and the zikv natal rgn replicon under cmv promoter control, which mimicked self-replicating, positive-sense, single strand rna viruses. the biological features of zikv natal rgn srips were revealed, including cpe, infectivity, cell susceptibility, and attachment activity. therefore, recombinant zikv natal rgn srips could be further used to investigate cell tropism, persistent infection, and vaccine candidates, and to establish reliable, effective, and efficient assays for screening antiviral agents and diagnosing viral infections. table s . primer pairs for real-time quantitative rt-pcr of zikv e and ns rna copies; supplemental figure s . the construction of in-frame gene fusion (a), plasmid information and restriction enzyme digestion analysis (b) of the synthesized dna segment i; supplemental figure s . the construction of in-frame gene fusion (a), plasmid information and restriction enzyme digestion analysis (b) of the synthesized dna segment ii; supplemental figure s . sequencing analysis of zikv asian natal rgn replicon with the primers listed in supplemental table s a . the amino acid substitutions were also shown in the figure. the diversification of zika virus: are there two distinct lineages? molecular evolution of zika virus during its emergence in the (th) century evolution of two major zika virus lineages: implications for pathology, immune response, and vaccine development virus pathogenesis and tissue tropism the origin and spread of a mosquito-borne virus zika virus-associated neurological disease in the adult: guillain-barré syndrome, encephalitis, and myelitis an update on zika virus infection comparative genomic analysis of pre-epidemic and epidemic zika virus strains for virological factors potentially associated with the rapidly expanding epidemic an evolutionary insight into zika virus strains isolated in the latin american region a single mutation in the prm protein of zika virus contributes to fetal microcephaly an evolutionary ns mutation enhances zika virus evasion of host interferon induction development of dengue type- virus replicons expressing gfp reporter gene in study of viral rna replication development and characterization of the replicon system of japanese encephalitis live vaccine virus sa - - noncytopathic flavivirus replicon rna-based system for expression and delivery of heterologous genes single-round infectious particle antiviral screening assays for the japanese encephalitis virus production of single-round infectious chimeric flaviviruses with dna-based japanese encephalitis virus replicon increased expression of capsid protein in trans enhances production of single-round infectious particles by west nile virus dna vaccine candidate sars coronavirus papain-like protease induces egr- -dependent up-regulation of tgf-β via ros/p mapk/stat pathway an infectious cdna clone of zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors novel genetically stable infectious clone for a zika virus clinical isolate and identification of rna elements essential for virus production a robust method for the rapid generation of recombinant zika virus expressing the gfp reporter gene development and characterization of recombinant virus generated from a new world zika virus infectious clone a reverse genetics platform that spans the zika virus family tree characterization of cis-acting rna elements of zika virus by using a self-splicing ribozyme-dependent infectious clone a full-length infectious cdna clone of zika virus from the epidemic in brazil as a genetic platform for studies of virus-host interactions and vaccine development rescue of recombinant zika virus from a bacterial artificial chromosome cdna clone high-throughput neutralization assay for multiple flaviviruses based on single-round infectious particles using dengue virus type reporter replicon rescue of the zika virus prototype strain with a cytomegalovirus promoter-driven cdna clone risk factors associated with the ophthalmoscopic findings identified in infants with presumed zika virus congenital infection all authors declare no potential conflict of interest. key: cord- -xx l r e authors: kodani, andrew; knopp, kristeene a.; di lullo, elizabeth; retallack, hanna; kriegstein, arnold r.; derisi, joseph l.; reiter, jeremy f. title: zika virus alters centrosome organization to suppress the innate immune response date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: xx l r e zika virus (zikv) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. inherited forms of microcephaly (mcph) are associated with disrupted centrosome organization. similarly, we found that zikv infection disrupted centrosome organization. zikv infection disrupted the organization of centrosomal proteins including cep , a mcph-associated protein. the zikv nonstructural protein ns bound cep , and expression of ns was sufficient to alter centrosome architecture and cep localization. loss of cep suppressed zikv-induced centrosome disorganization, indicating that zikv requires cep to disrupt centrosome organization. zikv infection or loss of cep decreased the centrosomal localization and stability of tank-binding kinase (tbk ), a regulator of the innate immune response. zikv infection or loss of cep also increased the centrosomal accumulation of the cep interactors, mindbomb (mib ) and dtx , ubiquitin ligases that respectively activate and degrade tbk . therefore, we propose that zikv ns binds cep to increase centrosomal dtx localization and destabilization of tbk , thereby tempering the innate immune response. in addition to identifying a mechanism by which cep controls the innate immune responses in zikv infection, we propose that the altered centrosomal organization caused by altered cep function may contribute to zikv-associated microcephaly. in the summer of , cerebral malformations were linked to mosquito-transmitted zika virus (zikv). inherited forms of microcephaly (mcph) are characterized by reduced head and brain size, resulting in severe intellectual disability and motor movement defects. many forms of mcph are caused by autosomally recessive mutations in genes encoding centrosomal proteins required for centrosome biogenesis and mitotic progression . given the similar pathologies between zikv-associated and inherited mcph, we hypothesized that both disorders were due to centrosomal defects leading to disrupted brain development. in mammalian cells, centrosomes serve as the microtubule organizing center of the cell to facilitate neuronal migration and cell division - . the centrosome is composed of centrioles surrounded by a pericentrosomal matrix that nucleates microtubules , . during s phase, the centrosome duplicates by recruiting specialized proteins to the centriole base , - . many mcph-associated proteins are recruited to the centrosome in a hierarchical manner to promote centrosome duplication , [ ] [ ] [ ] [ ] . defects in centrosome organization and biogenesis leading to cell death or premature differentiation in neural progenitors may underlie the pathology of many forms of mcph , , . cells infected with zikv have disrupted centrosome organization and mitotic abnormalities, leading to altered neural progenitor differentiation [ ] [ ] [ ] [ ] [ ] . however, the mechanism by which zikv disrupts centrosome architecture remains unclear. we found that zikv alters the function of the mcph-associated protein, cep . more specifically, the zikv nonstructural protein, ns , localizes to the centrosome and binds cep . zikv ns overaccumulates cep at centrosomes of zikv-infected cells and recruits the ubiquitin ligases, mib and dtx . mib and dtx promote the degradation of tbk , a regulator of the innate immune response [ ] [ ] [ ] [ ] [ ] . consequently, zikv-infected cells express lower levels of interferon b (ifnb), a key anti-viral signal. we propose that zikv recruits cep to the centrosome to degrade tbk , dampening anti-viral responses, and disrupted centrosomal architecture in zikv-infected cells may also perturb neural development. to explore whether microcephaly associated with zikv infection involves the centrosome, we infected human induced neural precursor cells (inpcs) derived from induced pluripotent stem cells with zikv and examined the organization of their centrosomes. sixteen hours post infection (hpi) with zikv, inpcs displayed supernumerary foci of centrin, a component of the centriolar distal lumen (figure a, c) . similarly, zikvinfected u and h cells exhibited supernumerary centrin foci (figure b , c, and supplemental figure a ). like zikv-infected cultured cells, npcs isolated from human fetal neocortical tissue infected by zikv exhibited supernumerary centrin foci ( figure d ). in zikv-infected inpcs and u cells, the supernumerary centrin foci co-localized with the distal centriole protein cp ( figure e and supplemental figure b ), indicating that zikv-associated supernumerary centrin foci can recruit centriolar proteins. however, the supernumerary centrin foci do not accumulate the distal appendage protein cep or pericentrosomal protein g-tubulin ( figure f , supplementary figure b , c). zikv infection did not alter the levels of cp or g-tubulin (supplemental figure d ). electron microscopy revealed that zikv infection did not alter centriole number or distal appendages hpi ( figure g) . instead, zikv-infected cells accumulated electron dense particles in the vicinity of the centrosome. thus, acute zikv infection does not cause centrosome overproduction, but rather disorganizes the centrosome. many mcph-associated proteins dynamically localize to the centrosome during its biogenesis [ ] [ ] [ ] . given that zikv infection alters centrosome organization, we investigated whether the localization of mcph-associated proteins is also altered. zikv infection in u cells did not affect either the localization or levels of the mcph-associated proteins plk , stil, sas , cdk rap , cep , or wdr (supplemental figure a-d) . in striking contrast, zikv infection caused cep , another mcph-associated protein , to overaccumulate at supernumerary centrin foci (figure a , and quantified in figure b ). as in u cells, zikv infection of inpcs and npcs isolated from dissociated fetal brain tissue relocalized cep to supernumerary centrin foci ( figure c) . although zikv infection did not dramatically affect cep protein levels, it did increase the levels of a higher molecular weight species of cep ( figure d ). because mcph-associated proteins function in centrosome biogenesis, we hypothesized that the disruption of cep localization participates in zikv-associated centrosomal disorganization. to test this, we infected control and cep gt/gt mouse embryonic fibroblasts (mefs) with zikv and assessed centrin organization. in contrast to control cells, zikv did not induce supenumerary centrin foci in the absence of cep (figure e-f) . together, these findings indicate that cep is required for zikv-associated reorganization of the centrosome. in zikv-infected inpcs, the non-structural viral protein ns localized to one or two foci among the supernumerary centrin foci at hpi ( figure a) . a yeast two-hybrid analysis suggested that cep binds the non-structural protein ns of flaviviruses related to zikv . as zikv infection causes cep to overaccumulate at centrosomes, we determined whether zikv ns interacts with cep . immunoprecipitation of endogenous cep in zikv-infected cells revealed that cep interacts with zikv ns (figure b ). to confirm the interaction of zikv ns with cep , we transfected cells with myc-tagged brazilian zikv ns and found that myc-ns co-precipitated with endogenous cep , but not cp , another centrosomal protein that misaccumulates at the centrosome upon zikv infection ( figure c ). as zikv ns interacts with cep , we investigated whether cep was required to localize ns to the centrosome. to test this, we infected control and cep gt/gt mefs with zikv and examined ns localization hpi. similar to control cells, zikv ns localized to centrosomes in cep gt/gt mefs ( figure d ) suggesting that ns affects cep localization, but not vice versa. ns acts together with another nonstructural protein, ns b, to form a proteolytic complex ( figure e ) . to gain insight into how zikv disrupts centrosomes, we examined whether the ns b/ns complex could induce supernumerary centrin foci or if ns alone is sufficient. expression of either brazilian zikv myc-ns b/ns or myc-ns induced supernumerary centrin foci ( figure f-h) , suggesting ns alone is sufficient to perturb centrosome organization. additionally, myc-ns increased the centrosomal accumulation of cep ( figure i and quantified in supplementary figure d ) and had no effect on the localization or stability of cep , cdk rap and wdr ( figure i and supplemental figure b -d). therefore, brazilian zikv ns interacts with cep , localizes to the centrosome, and is sufficient to reorganize centrosomal architecture akin to zikv infection. microcephaly is associated with south american strains of zikv, but not uganda zikv [ ] [ ] [ ] . given the ns proteins of the brazilian and ugandan strains of zikv differ by amino acids, we examined whether ugandan ns was sufficient to induce supernumerary centrin foci and misrecruit cep . like brazilian ns , ugandan zikv ns localized to centrosomes ( figure j ). in striking contrast to brazilian zikv ns , the ugandan ns did not induce supernumerary centrin foci or increase centrosomal cep localization ( figure j , and quantified in supplementary figure e , f). together, these findings indicate that brazilian zikv ns has acquired the ability to increase centrosomal cep localization and induce supernumerary centrin foci. to assess how centrosomal disorganization participates in zikv biology, we examined whether centrosomal disorganization contributes to viral production. we found that loss of cep did not alter zikv production ( figure a ). as cep is not required for zikv production, we hypothesized that it may alter the cellular response to zikv infection. zikv inhibits type i interferon effector signaling by degrading stat , and by an unknown mechanism, inhibits interferon induction , . during viral infections, the kinase tbk phosphorylates and activates the transcription factor irf , and is then degraded, to regulate interferon induction , , . tbk localizes to centrosomes [ ] [ ] [ ] [ ] [ ] [ ] and proximity interaction studies have suggested that tbk and cep interact . through co-immunoprecipitation, we confirmed that endogenous cep and tbk interact ( figure b ). an activated form of tbk , phospho-tbk (p-tbk ), is removed from centrosomes upon zikv infection during mitosis , we confirmed p-tbk is removed during interphase ( figure c , and quantified in supplemental figure a ). as p-tbk is absent from centrosomes of zikv-infected cells, we assessed whether zikv infection induces interferon beta (ifnβ) using reverse-transcription digital droplet pcr (rt-ddpcr). following zikv infection, ifnβ expression gradually peaked to hpi, ( figure d ). ifnβ expression was abruptly curtailed at to hpi in zikv-infected cells, corresponding to the timing of when centrosome begin to disorganize. zikv infection decreased levels of tbk and phosphorylated tbk (p-tbk ) at hpi ( figure e ). therefore, we propose that zikv may suppress the innate immune response by suppressing tbk stability and centrosome organization. to test whether zikv disrupts tbk function in a cep -dependent manner, we examined whether centrosomal p-tbk localization depends upon cep . indeed, p-tbk was absent from centrosomes in cep gt/gt mefs ( figure f , and quantified in supplemental figure b ), indicating that cep is required for the centrosomal localization of p-tbk . similarly, tbk and p-tbk levels were decreased in cep gt/gt mefs ( figure g ). these data are consistent with zikv interfering with cep function to disrupt tbk stability. how might zikv reorganize the centrosome in a cep -dependent manner to affect tbk stability? in response to sendai virus infections, mindbomb (mib ) binds and activates tbk through k -linked ubiquitylation , , and proximity interaction studies of cep identified mib as an interactor . we confirmed the interaction of mib to tbk and cep using reciprocal co-immunoprecipitation of endogenous mib with tbk and cep ( figure a and supplemental figure a ). furthermore, we assessed whether zikv infection induced a change in the centrosomal localization of mib . zikv infection increased centrosomal accumulation of mib in inpcs, fetal npcs, and u cells ( figure b and supplemental figure b , c). zikv infection mildly altered the stability of mib , but significantly increased the levels of a higher molecular weight species ( figure c ). we assessed whether cep restricts mib localization to the centrosome. like zikv infection, depletion of cep increased mib accumulation at the centrosome and resulted in the appearance of a higher molecular weight species of mib (supplemental figure d -f), indicating that mib localization to centrosomes is restricted by cep . we then tested whether cep regulates mib activity by examining the levels of mib dependent k -ubiquitylation of tbk in cep gt/gt mefs. k -ubiquitylation of tbk was significantly increased in cep mutant cells, suggesting that increased centrosomal mib may facilitate its ability to ubiquitylate tbk at the centrosome (supplemental figure g -h). similar to the loss of cep , k -ubiquitylation of tbk was dramatically increased in zikv infected cells ( figure d and supplemental figure i ). together, these data suggest that zikv disrupts cep to affect the levels and function of mib at the centrosome. once activated the innate immune response is attenuated by k ubiquitin-mediated degradation of tbk by the ubiquitin ligase dtx . as tbk localizes to the centrosome, we examined whether dtx is, like mib , present at centrosomes. interestingly, dtx partially co-localized with g-tubulin (supplemental figure j ). the specificity of the antibody was confirmed by sirna (supplemental figure j ). in accordance with previously published data, depletion of dtx led to the stabilization of tbk (supplemental figure k ). as tbk stability is disrupted in the absence of cep , we hypothesized that dtx interacts with and is localized to the centrosome by cep . reciprocal immunoprecipitation of cep and dtx confirmed that these two proteins interact we have found that zikv-produced ns binds cep to alter centrosome organization, but not centrosome amplification , increases centrosomal mib and dtx , decreases tbk levels, and disrupts the innate immune response. as cep mutations cause microcephaly in humans , disrupted cep function may underlie the pathogenesis of zikv-associated microcephaly. moreover, the centrosomal cep interactors mib and dtx , regulators of neurogenic notch and innate immune signaling, respectively are altered upon either loss of cep or zikv infection. based on these results, we propose that zikv ns disrupts cep function to both dampen the innate immune response and to disrupt developmental signaling during brain development. previous studies have reported conflicting effects on centrosome biogenesis in zikv infected cells , , , , . our results using human neural progenitor cells from fetal tissue, induced pluripotent stem cells, and established neural cell lines indicate that acute zikv infection disrupts centrosome organization but does not lead to centrosome amplification. as studies by others on centrosome biogenesis examined later time points following zikv infection, centrosomal overabundance in zikv-infected cells may be secondary to multiple rounds of abnormal cell division. we found that zikv-produced ns localizes to the centrosome to induce the formation of supernumerary centrin foci by interacting with and localizing increased amounts of cep to the centrosome. the failure of zikv infection in cep mutant mefs to induce supernumerary centrin, supports our assertion that zikv disrupts centrosome organization in a cep -dependent manner. we and others have shown that cep promotes centriole duplication , , , . however, its function in other cellular processes has not been explored. here, we have provided evidence that cep controls tbk stability, a central component of innate immune signaling. given that cep interacts with and limits the localization of the centrosomal ubiquitin ligases, mib and dtx , to the centrosome, a direct role for cep in promoting cellular signaling at the centrosome is implicit. mib , a k -linked ubiquitin ligase is a key regulator of notch signaling, a pathway that controls neural progenitor maintenance and cell division . a less studied role for mib , is in the innate immune response . we have found that mib over accumulates at the centrosome and k -ubiquitylates tbk in response to zikv infection or cep loss. the increased activity of mib in zikv-infected neural progenitors or cep -depleted cells suggests that mib activity in the notch signaling pathway could also be affected. in agreement with this, a recent publication has demonstrated that mib levels are affected in response to zikv infection , , raising the possibility that zikv-associated microcephaly is a side effect of zikv altering the centrosome to evade host immunity. we found that dtx localizes to the centrosome and promotes the k -ubiquitylation of tbk . similar to mib , dtx accumulates at the centrosome in zikv infected cells in a cep -dependent manner. as tbk is ubiquitylated by dtx to promote its degradation, we propose that zikv mediated recruitment of dtx to the centrosome may limit tbk activity and stability, and thus the innate immune response, to zikv infection. it will be interesting to determine whether other viruses with ns homologues such as sars-cov- m pro and nsp which interact with centrosome proteins can similarly suppress innate immunity by altering centrosome organization. in summary, we have found that zikv ns localizes to the centrosome, recruits cep and its binding partners mib and dtx to ubiquitylate and degrade tbk , a key regulator of the innate immune response. these findings provide mechanistic insight into how zikv specifically targets the centrosome with implications for how it may both evade viral detection and alter brain development. hela and t/ cells (ucsf tissue culture facility) were cultured in advanced dulbecco's modified eagle's medium (dmem, thermo fisher scientific) supplemented with % fetal bovine serum (fbs, thermo fisher scientific) and glutamax-i (thermo fisher scientific). u and h cells were cultured in dmem (thermo fisher scientific) supplemented with % fbs and l-glutamine (thermo fisher scientific). cep gt/gt mefs and control cep +/-mefs were cultured in amniomax c- (thermo fisher scientific). t/ and hela cells were transfected with plasmids using fugenehd (promega) or lipofectamine (thermo fisher scientific), respectively, according to manufacturer's instructions and analyzed h later. npcs were derived from pluripotent stem cells (nih human embryonic stem cell registry line wa (h ) at passages - ) according to a recently published protocol and maintained in neural media composed of dmem/f with sodium pyruvate and glutamax, n , b , heparin and antibiotics. medium was either supplemented with growth factors epidermal growth factor ( ng/ml) and fibroblast growth factor ( ng/ml). de-identified fetal brain tissue samples were collected with previous patient consent in strict observance of the legal and institutional ethical regulations from elective pregnancy termination specimens at san francisco general hospital. protocols were approved by the human gamete, embryo and stem cell research committee (an institutional review board) at the university of california, san francisco. blocks of cortical tissue spanning the ventricle to the cortical plate were dissected away from meninges and germinal zone using a stereomicroscope, and then minced using a razor blade. cells were dissociated by incubation with papain (worthington biochemical corporation) at °c for - min, followed by addition of dnase i and trituration. the cells were collected by centrifugation for min at g, the supernatant was removed, and the cells were resuspended in sterile dmem containing n- , b- supplement, penicillin, streptomycin and glutamine and sodium pyruvate ( . mg/ml) (all invitrogen). the suspension was passed through a μm strainer (bd falcon) to yield a uniform suspension of single cells. cells were plated at a density of . x cells/well on mm coverslips (neuvitro -gg-pdl) precoated with high concentration growth factor-reduced matrigel (bdbiosciences, ) and cultured at °c, % co , % o . we acquired cep tm . (komp)vlcg (also called pibf tm . (komp)vlcg ) heterozygous mice (jackson laboratory). cep tm . (komp)vlcg is a deletion of chr : - (grcm .p ), covering all coding exons of cep . we isolated mefs from littermate e . embryos produced from a heterozygous intercross. mefs were genotyped by quantitative pcr using express sybr greener qpcr supermix, with premixed rox (invitrogen) on a real-time pcr machine (applied biosystems) and cep genotyping primers listed in the reagents table. zikv strain prvabc was propagated in vero cells. viral titers were determined by focus assay . briefly, serial dilutions of viral stock were added to vero cells in -well plates. h post-infection, inoculum was removed and cells were fixed with . % pfa for minutes. foci were visualized by immunofluorescent staining for flavivirus envelope. zikv infections of npcs, u and h cell lines, at mois of , were carried out by incubating cells with inoculum for h and then replacing the inoculum with fresh media. zikv was added to dissociated fetal brain cells at mois of and incubated for h unless stated otherwise. mock and zikv-infected cells were fixed - h post infection in chilled methanol for min at - °c and processed for immunofluorescence. wild type human codon optimized zikv ns b and ns open reading frame flanked by attb sites were synthesized by gibson assembly (sgi). gateway cloning into pdonr generated pentr-ns zikv. subsequent gateway-mediated subcloning into pdest-cmv-myc (gift of keith yamamoto) generated pcmv-myc-ns brazilian and ugandan zikv, encoding n-terminally myc-tagged ns and pcmv-myc-ns b brazilian zikv encoding an n-terminally myc-tagged fusion of ns b and ns . the s a mutant form of ns was generated using site-directed mutagenesis to create pcmv-myc-ns -s a brazilian zikv (quikchange ii, agilent). immunoprecipitations were performed as previously described . in brief, mock or zikv infected u cells or t/ cells were collected in dulbecco's phosphate-buffered saline (dpbs), lysed in lysis buffer ( % igepal ca- , mm tris-hcl ph . , mm nacl, . mm kcl, . mm kh po , . mm na hpo - h o) supplemented with protease and phosphatase inhibitors (emd millipore). myc-tagged proteins were immunoprecipitated with ag myc monoclonal agarose beads (emd millipore), washed three times in lysis buffer and boiled in x laemmli reducing buffer (bio-rad). samples were separated on - % tgx precast gels (bio-rad), transferred onto protran ba nitrocellulose membrane (ge healthcare) and subsequently analyzed by immunoblot using ecl lightening plus (perkin-elmers) or supersignal west dura (thermo fisher scientific). cells were fixed in - °c methanol for minutes followed by permeabilization in blocking buffer ( . % bsa, . % triton-x , . % nan in dpbs) for min. primary and secondary antibodies were diluted in blocking buffer and incubated with cells for at least h. to detect cells in s-phase, cells were co-stained with antibodies to centrin and cyclin a to determine centriole number and s-phase/g cells, respectively. to detect phospho-tbk , u and mefs were fixed in % paraformaldehyde for minutes, blocked in % fbs and . % tritonx diluted in dpbs. permeabilized cells were stained overnight at room temperature using the p-tbk antibody at a dilution of : in % fbs and . % tritonx diluted in dpbs. samples were mounted in gelvatol and imaged with an axio observer d or lsm (zeiss). images were processed using adobe photoshop and analyzed using fiji. (d) immunoblot of mock or zikv-infected u cell lysates probed for ns , cp , or gtubulin. actin served as a loading control. name sequence cep wt l '-ggaaaccattttattgcgacag- ' cep wt r '-ctcaaagtctcgcagatttcg- ' cep gt l '-ctcatcaatgtatcttatcatgtctgg- ' cep gt r '-tcgactactaggaaagcaacgag- ' cep -wt l '-gtaggaccaggccttagcgttag- ' cep -wt r '- bridging centrioles and pcm in proper space and time a primary microcephaly protein complex forms a ring around parental centrioles cep and cep cooperate to ensure centriole duplication molecular architecture of a cylindrical self-assembly at human centrosomes microcephaly proteins wdr and aspm define a mother centriole complex regulating centriole biogenesis, apical complex, and cell fate centriolar satellites assemble centrosomal microcephaly proteins to recruit cdk and promote centriole duplication human cep and cep cooperate in plk recruitment and centriole duplication subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material katanin p regulates human cortical development by limiting centriole and cilia number acentriolar mitosis activates a p -dependent apoptosis pathway in the mouse embryo zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells zika virus differentially infects human neural progenitor cells according to their state of differentiation and dysregulates neurogenesis through the notch pathway. emerging microbes & infections recent zika virus isolates induce premature differentiation of neural progenitors in human brain organoids zika virus ns localizes at centrosomes during cell division mapping a dynamic innate immunity protein interaction network regulating type i interferon production ikkepsilon and tbk are essential components of the irf signaling pathway socs drives proteasomal degradation of tbk and negatively regulates antiviral innate immunity differential requirement for tank-binding kinase- in type i interferon responses to toll-like receptor activation and viral infection nemo binds ubiquitinated tank-binding kinase (tbk ) to regulate innate immune responses to rna viruses most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles cep and cp suppress a cilia assembly program cep , a novel centriole appendage protein required for primary cilium formation gamma-tubulin is a highly conserved component of the centrosome a missense mutation in the pisa domain of hssas- causes autosomal recessive primary microcephaly in a large consanguineous pakistani family mutations in stil, encoding a pericentriolar and centrosomal protein, cause primary microcephaly mutations in plk , encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy flavivirus ns and ns proteins interaction network: a highthroughput yeast two-hybrid screen functional characterization of cis and trans activity of the flavivirus ns b-ns protease detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study zika virus associated with microcephaly comparative genomic analysis of pre-epidemic and epidemic zika virus strains for virological factors potentially associated with the rapidly expanding epidemic. emerging microbes & infections , e zika virus targets human stat to inhibit type i interferon signaling zika virus inhibits type-i interferon production and downstream signaling triggering the interferon antiviral response through an ikkrelated pathway nlrp negatively regulates type i interferon signaling by targeting the kinase tbk for degradation via the ubiquitin ligase dtx contribution of a tank-binding kinase -interferon (ifn) regulatory factor pathway to ifn-gamma-induced gene expression recent insights into the complexity of tank-binding kinase signaling networks: the emerging role of cellular localization in the activation and substrate specificity of tbk zika virus disrupts phospho-tbk localization and mitosis in human neuroepithelial stem cells and radial glia tank binding kinase is a centrosome-associated kinase necessary for microtubule dynamics and mitosis a dynamic protein interaction landscape of the human centrosome-cilium interface zika virus disrupts phospho-tbk localization and mitosis in human neuroepithelial stem cells and radial glia cep recruits cdk to the centrosome: implications for regulation of mitotic entry, centrosome amplification, and genome maintenance mind bomb is essential for generating functional notch ligands to activate notch zika virus increases mind bomb levels, causing degradation of pericentriolar material (pcm ) and dispersion of pcm -containing granules from the centrosome dysregulation of astrocyte extracellular signaling in costello syndrome quantification of lymphocytic choriomeningitis virus with an immunological focus assay in -or -well plates kif a interacts with dynactin subunit p glued to organize centriole subdistal appendages cdk rap (green), cep (green) or wdr (green). (b-c) the fluorescence intensities ± s.d. of plk , stil, sas , cdk rap , cep and wdr were quantified in mock and zikv infected u cells scale bars indicate μm for all images. (c) immunoblot of hela cells expressing myc-tagged ns probed for c-myc, cdk rap , cep , wdr , and cep . actin served as a loading control. asterisk denotes specific band for cep . (d) quantification of mean fluorescence intensities ± s.d. of cdk rap , cep , wdr , and cep in hela cells transfected with an empty c-myc vector (control) or brazilian myc-ns . for fluorescence quantifications cells were analyzed per experiment (n= ). asterisk denotes p< . (student's t test). (e) quantification of hela cells transfected with ugandan myc-ns with greater than four centrioles. (f) quantification of mean fluorescence intensities ± s.d. of cep in control and ugandan myc-ns expressing hela cells. supplemental figure : (a) quantification of mean fluorescence intensities ± s.d. of centrosomal p-tbk in mock and zikv-infected u cells. for fluorescence quantifications cells were analyzed per experiment (n= ) asterisk denotes p< . (student's t test). (d) s phase sc and cep sirna transfected hela cells co-stained for centrin (red) and mib (green). (e) mean fluorescence intensity quantifications of mib in sc and cep sirna-treated hela cells. (f) lysates from control and cep gt/gt mefs immunoblotted for mib . asterisk denotes a higher molecular weight species of mib actin served as a loading control. (i) mock and zikv-infected u cell lysates used to immunoprecipitate tbk in figure d were analyzed by western blot using antibodies to ns and tbk . actin served as a loading control. (j) sc or dtx -depleted hela cells were co-stained for g-tubulin and dtx . (k) immunoblot of hela cells transfected with sc or dtx sirna probed for dtx and tbk . actin served as a loading control. (l) quantification of mean fluorescence intensities ± s.d. of dtx in mock or zikv-infected u cells. (m) quantification of centrosomal dtx in sc and cep depleted hela cells expressed as a mean fluorescence intensities ± s.d. of the control. (n) total cell lysate from mock and zikv-transfected u cells hpi were analyzed by western blot using antibodies to zikv ns and dtx . actin served as a loading control. (o) immunoblot analysis of lysate from sc and cep sirna-treated hela cells using antibodies to cep and dtx . actin served as a loading control. (p) control and cep gt/gt mef lysates used to immunoprecipitate tbk in figure m were analyzed by western blot using antibodies to tbk . actin served as a loading control. (q) cell lysates from mock and zikv-infected u cells used to immunoprecipitate key: cord- -g kqqpy authors: bramhachari, pallaval veera; mohana sheela, ganugula; prathyusha, a. m. v. n.; madhavi, m.; satish kumar, k.; reddy, neelapu nageswara rao; berde, chanda parulekar title: advanced immunotechnological methods for detection and diagnosis of viral infections: current applications and future challenges date: - - journal: dynamics of immune activation in viral diseases doi: . / - - - - _ sha: doc_id: cord_uid: g kqqpy diagnosis and identification of viruses is an important component of diagnostic virology laboratory. although various modes of diagnostic methods are now available at disposal, a vast majority of the diseases across the globe remain undiagnosed. this is largely due to the overlapping undifferentiated set of symptoms across myriad set of rna and dna viral diseases. as such, it becomes critical to take into consideration several factors for viral diagnosis ranging from the type and quality of specimen collected, time of specimen collection, mode of transport, accuracy, specificity, sensitivity, and the type of diagnostic method used. this chapter broadly emphasizes various methods on diagnostic virology ranging from the classical methods of diagnosis to the most recently developed molecular methods of detection of virus. viruses are obligate intracellular parasites and their detection and identification is an imperative component clinically. viral infectious signify a major portion in public health perspectives with thousands of deaths annually. notwithstanding from highly contagious infections and serious pandemics to common influenza episodes, clinical diagnosis of viral infection often relies on early detection. therefore, effectual detection of viruses is indispensable aid to avert transmission, initiate befitting therapy, and scrutinize response to treatment which leads to effective disease control and management (souf ) . viral diagnosis is a dynamic process. although prophylaxis is better than cure, an accurate diagnosis of any virus fundamental to the infection is equally vital. generally, diagnostic tests are categorized into three groups: direct detection, isolation of virus, and serology tests. direct examination methods are merely faster and examined directly for presence of virus particles, virus antigen, or viral nucleic acids. immunofluorescence assay is extensively used for rapid identification of virus infections through detection of virus antigen or virus-specific antibodies in clinical specimens. viral diagnosis is a crucial revolution and renders more accessible and makes it potential to standardize the recently developed diagnostic methods. serology essentially comprises bulk of work of any virology laboratory. a serological diagnosis can be identified by increasing titers of antibody among acute and convalescent stages of infection, or by the detection of immunoglobulin m (igm). the diagnosis of viral infections were enhanced noticeably all through s, with the onset of highly sensitive methods, viz. enzyme-linked immunosorbent assay (elisa) and pcr are superseded for this reason. nevertheless, during the last years this technique is being utilized, due to its unique nature, making diagnosis probable through visualization of virus. then after, introduction of elisa then revolutionized viral diagnosis by simplifying detection and shortening the time (torrance and jones ) . molecular diagnostic tools for viral diagnosis has trialed speedy advancements in last few years (hayden and persing ) , and revolutionized diagnosis of infectious diseases, particularly viral diseases. the use of amplification techniques, viz. pcr, rt-pcr, or nasba (nucleic acid sequence-based amplification) (van belkum and niesters ) for detection of virus, genotyping, and quantification have several advantages such as high reproducibility and sensitivity, including broad dynamic range (ebner et al. (ebner et al. , . several molecular diagnostic techniques were recently swapped by fully automated devices that use less time, maneuver smaller volumes of liquids, and provide quantified results with improved accuracy. the current review emphasizes the advanced immunotechniques, how the specific characteristics of diagnostic methods revolutionized field of viral diagnosis clinically from a decade. transmission electron microscopy (tem) is a merely imaging technique that permits direct image of viruses attributable to its nanoscale resolution. this is a classical technique owing to its advantage of direct visualization of virus. amid the s and s, tem contributed a breakthrough and doled out as a diagnostic tool for recognizing numerous virus unswervingly in any biological samples. for that reason, in modern years, role of tem in diagnosis of anonymous infectious agents in specific viral outbreaks or transmission clusters shifted from regular use to an initial screening test. tem is also considered an important method for assessment of viral safety in biopharmaceutical products. application of tem understands the unknown viruses for which there is an imminent risk of contamination, and tem will undeniably be functional for documenting the subsistence of viruses or virus-like particles in these cells and the products derivative from them (roingeard et al. ). the immunoassays are primarily antibody/antigen dependent assays. this principle works on immunization with the antigens and activating, or infection. therefore, measuring of igg immunoglobulin and quantifying the antibodies are preferentially used as diagnostic markers. the antigens or antibodies are coated with conjugated labels like metals, radioactive isotopes, fluorescent tags, respectively. as a part of modern research on immunotechniques, a diagnostic approach for chronic hepatitis c infection (chc), detects specific antibody to hcv (anti-hcv) (indirect tests) and assays that can detect, quantify, or characterize components of hcv viral particles, viz. hcv rna and core antigen (direct tests). quantification of hcv core antigen (cag) as a one-step procedure has been indispensable. hence, in order to evaluate the performance of cag quantification in diagnosing chc and how it is predisposed by concomitant hiv or hbv infections, cross-sectional validation assays, i.e., hcv ag quantification as a one-step procedure in diagnosing chc in cameroon was designed to abridge the diagnostic process (duchesne et al. ) . it is pertinent to note that a multiplex microsphere immunoassay (mia) recently developed possess diagnostic power to incarcerate viral envelope protein, which evokes to be strong cross-reactive antibodies to other flaviviruses and differential power of viral nonstructural proteins ns and ns was. interestingly this serologic assay needs to be employed in rapid clinical diagnosis of zikv and/or dengue virus infections for screening immune responses in vaccine trials (wong et al. ) . it is noteworthy that oral fluid is a noninvasive biospecimen that can harbor pathogen-specific antibodies and reach potential to replace blood-based testing protocols. therefore, a saliva-based oral fluid immunoassay was developed to assess past and recent hepatitis e virus (hev) infections from noninvasive sampling methods. the sensitivity and specificity of this assay was comparable to serum-based elisas. this salivary assay could improve our understanding of the ecology and natural history of hev (pisanic et al. ) . diagnosing zikv remains a great challenge, as detection of viral rna is only possible merely few days after onset of symptoms. conversely, novel highthroughput image-based fluorescent neutralization method for identification of zikv was thoroughly evaluated and developed which reported higher sensitivity than plaque reduction neutralization test (prnt) and mac-elisa, respectively. this test might employ for clinical diagnosis, clinical trials, and confirmation and seroprevalence studies of zikv infection (koishi et al. ) . in one of the recent studies, detection of serum hev antigen (ag) is deemed to be sensitive and promising biomarker for hev antigen diagnosis with hev rna in both acute and chronic genotypes. strikingly an antigen assay was recently evaluated for diagnosing hev genotypes with higher sensitivity than commercial anti-hev igm and hev rna elisa tests (zhang et al. ) . nonetheless, recent studies on respiratory syncytial virus (rsv) developed luciferase immunoprecipitation systems (lips) assay to detect igg antibodies against human rsv g-glycoprotein. moreover, human rsv g-glycoprotein also acts as biomarker for natural exposure or immunization. rsv genes encoding native and mutated g (mg) proteins from subgroups a and b strains were cloned, expressed as luciferase-tagged proteins, and experimented separately to spot anti-rsv-g specific igg antibodies employing a high-throughput luciferase immunoprecipitation system (lips-g). it was pertinent to note that rsv monoclonal antibodies and polyclonal antisera explicitly bound in lips-g a and/or -g b assays (crim et al. ) . the diagnosis of (zikv) and dengue virus (denv) infections against viral envelope protein and nonstructural proteins (ns) was developed using flavivirus multiplex microsphere immunoassay (mia). however mia could not differentiate more recent from past infections, which represents a key diagnostic challenge; therefore, in a most recent report an immunoglobulin g (igg)-based avidity assay was developed for its diagnostic performance to accurately differentiate between recent zika and past dengue virus infections. this assay was found useful in patients with high risk of zika complications, viz. pregnant women and monitoring immune responses in vaccine trials (furuya et al. ). consecutively to develop serological diagnosis of zikv-iga and zikv-igg, avidity assays were evaluated to characterize zika infections in want of viremia. these assay facilitated construed low avidity of igg and iga results, enhanced the serological diagnosis of zikv (amaro et al. ) . in another study, homologous proteins of diverse flaviviruses exhibited high degrees of sequence uniqueness, mainly within subgroups. this led to prevalent immunological cross-reactivity. therefore, a proportional deconvolution of complex b cell responses against zikv and other flavivirus were deliberated by screening with a microarray chip-based high-resolution serological analysis primed from overlapping peptides covering the whole amino acid sequence of zikv genomic polyprotein was developed. additionally with advent of this assay several infections, viz. dengue, yellow fever, tick-borne encephalitis, and west nile viruses shall be diagnosed (hansen et al. ). enzymes are extensive tool for diagnosing virus which have various applications like enzyme immune assay, elisa. enzyme immune assay has different applications like fluorescence polarization immune assay (fpia), micro-particle immune assay (meia), chemiluminescent (clia). enzyme immune assays work with antigen-antibody interaction with the conjugated tags like fluorescent tags, chemiluminescent tags which are complemented with substrates like polarized light and fluorescent substrates. as a part of most advanced immunotechniques, an ultrasensitive colorimetric assay called magnetic nano(e)zyme-linked immunosorbent assay (maglisa) was developed, wherein silica-shelled magnetic nanobeads (magnbs) and gold nanoparticles were pooled to monitor influenza a virus up to femtogram per milliliter concentration (oh et al. ) . sensitive and specific detection of crimean-congo hemorrhagic fever virus (cchfv) was developed employing specific igm and igg antibodies in human sera using recombinant cchfv nucleoprotein as antigen in μ-capture and igg immune complex (ic) elisa tests (emmerich et al. ) . recently, truncated forms of hev and niv (hendra and nipah virus) g proteins as well as full-length niv nucleocapsid (n) protein were used for detection of hendra and nipah virus specific antibodies in pigs. these recombinant proteins were expressed through diverse expression systems and an indirect elisa was developed for detection (fischer et al. ) . a rapid diagnostic platform for colorimetric differential detection of denv and chikv viral infections was recently developed with a possibility to alter clinical diagnosis of acute febrile illnesses in resource-limited settings. this platform principally facilitates consistent and accurate multiplexed detection of chikungunya and dengue igm/igg antibodies in human clinical samples within short stint ). immunofluorescence (if) is extensively used for speedy detection of viral infections clinically started in early s. if is used for diagnosis of virus antigen and virusspecific igg/iga/igm antibody in clinical specimens. in this technique, fluoresceinlabeled antibody to stain specimens containing specific virus antigens, were used for uv illumination. as a part of modern research on immunotechniques, new recombinant rabies virus expressing green fluorescent protein (rrv-gfp) is more rapid, simpler, and less expensive detection and for quantification of virus neutralizing antibodies in blood sera. this technique simplified multistep rapid fluorescent focus inhibition test (rffit) procedure by purging immunostaining step (qin et al. a, b) . nucleic acid (dna and rna) amplification assays are conventionally known as polymerase chain reactions (pcrs). distinct pcrs exists based on type of nucleic acid and information known a propos the sequences of genomes for immunodetection. rous sarcoma virus (rsv) is one of the most significant causative agents of respiratory tract infection in children and related with high morbidity and mortality. however, very little is known with reference to effects of respiratory viral infections. a reverse transcription recombinase polymerase amplification assay (rt-rpa) is nucleic acid probe based on novel isothermal amplification technique which has been widely employed to detect human rsv. the results exemplified that concurrence rates between rt-rpa assay and qrt-pcr assay for clinical samples was %, demonstrating that rt-rpa assay holds better diagnostic presentation on clinical samples in remote rural areas in developing countries (xi et al. ). using quantitative pcr (qpcr) for common respiratory viruses and for two genes (ccl /cxcl ) is recognized to be extremely upregulated in viral infections. notably, rna-seq virus detection achieved % sensitivity compared to qpcr-based screening in asthmatic children which consequently drives immune cell airway infiltration, cellular remodeling, and alteration of asthmogenic gene expression (wesolowska-andersen et al. ). nevertheless as a part of latest advancements on immunotechniques, isothermal reverse transcription and recombinase polymerase amplification (rt-rpa) of synthetic rna (ebola virus) employing paper microfluidics devices was developed initially. later on based on rna detection and multiplexed analysis for ebola virus diagnostics were optimized and demonstrated with high sensitivity. additionally, nine-spot multilayered device achieving parallel detection of three distinct rna sequences opens a route in the direction for detection of multiple viral pathogens (magro et al. ) . rabies virus (rabv) is one of the most significant global zoonotic pathogens. two sensitive real-time quantitative rt-pcr assays were developed and validated for large-spectrum detection of rabv, with a focus on african isolates. the primer and probe sets were targeted for highly conserved regions of nucleoprotein (n) and polymerase (l) genes. effective detection and high sensitivity of these assays can be effectively functional in general research and used in diagnostic process and epizootic surveillance (faye et al. ) . however, in recent times a fluorescent reverse transcription loop-mediated isothermal amplification (rt-lamp) employing quenching probes for detection of middle east respiratory syndrome coronavirus was developed. additionally, detection efficacy of qprobe rt-lamp was comparable to that of rt-pcr assay (azhar ) . furthermore this assay can as well be used as authoritative diagnostic tool for rapid detection and surveillance of mers-cov infections (shirato et al. ). consecutively to assist detection of zikv infections, and distinguish these infections from denv and chikv, trioplex real-time rt-pcr assay was recently developed. however the performance of trioplex real-time rt-pcr assay was particularly employed for the detection of zikv, denv and chikv viruses. simultaneous testing of more than one specimen type from each patient affords a superior diagnostic sensitivity of this technique (santiago et al. ). an in situ hybridization (rna-ish) assay was recently developed to detect viral hemorrhagic septicemia virus (vhsv), an oie listed piscine rhabdovirus, in infected fish cells with fathead minnow (fhm) as model cell line. two antisense rna probes targeting fragments of n and g genes were amplified by rt-pcr employing vhsv-specific primers trailed by transcription reaction in presence of digoxigenin dutp has competently localized vhsv mrnas in infected cells. the diagnostic sensitivity of rna-ish assay was better than immunocytochemistry, qrt-pcr and tcid (qadiri et al. ) . development and evaluation of a new one-step, real-time rt-pcr assay was developed for detecting latest h n influenza viruses competent of causing human infection. the sensitivity of one-step, real-time rt-pcr assay was generally determined to be used in vitro transcribed rna, devoid of any cross-reactivity against rna from h - subtypes of influenza viruses and other viral respiratory pathogens with no nonspecific reactions (saito et al. ). in the current trends mass spectrometry (ms) is a benchmark for qualitative and quantitative diagnosis of viruses clinically. in clinical laboratories, maldi (matrixassisted laser desorption ionization) and es (electrospray) ionization methods are most frequently used as they allow ionization of analyte in considerable amounts. the combination (rt-pcr/esi-ms) was able to detect viral pathogens (acute viral respiratory infections and influenza a viruses) usually for those viruses which are undetected by regular testing methods as well as provides rapid and detailed data (about types and subtypes of virus) in short period (deyde et al. ; chen et al. ). yet another study reported that near-infrared spectroscopy (nirs) is a rapid, reagent-free, and cost-effective tool used to detect zikv noninvasively in heads and thoraces of intact aedes aegypti mosquitoes with high prediction accuracies relative to quantitative rt-qpcr reaction. perhaps this technique could be extended upon for identifying probable arbovirus hotspots to guide spatial prioritization of vector control (fernandes et al. ) . recently developed surface plasma resonance (spr) spectroscopy was developed to be a valuable optical biosensor and potential method for diagnosis of dengue virus e-protein and also for identification of antibodies to denv antigen. the diagnosis limit, sensitivity, and selectivity of spr sensing in denv antigen was amazingly high. this technique was introduced as a novel d-pamam-sam-au multilayer thin film for future research on spr sensing applications (omar and fen ) . mirna are conserved small noncoding rna with - nucleotides which regulates post-transcriptional modification. mirnas are transcribed by rna polymerase ii into pre-mirna which is processed by dorsa/dgcr- and dicer and transported to cytoplasm. risc in cytoplasm processes pre-mirna into mature mirna and regulates post-transcriptional activities. as a part of the most advanced immunotechniques, exosomal micrornas were recently studied as potential diagnostic markers for various malignancies, including hepatocellular carcinoma (hcc). serum exosomal micrornas combined with alpha-fetoprotein as diagnostic markers of hepatocellular carcinoma . in another study, synthetic mirna-based approach was developed to express neutralizing antibodies directly in lung via aerosol, to prevent from human rsv and influenza infections. engineered mrnaexpressed antibodies prevented rsv infection. it is noteworthy that an expressing membrane-anchored broadly neutralizing antibody in lungs could potentially be promising pulmonary prophylaxis approach (tiwari et al. ) . in a most recent study, endogenous micrornas (mirna) are evolutionarily conserved and their presence in biological fluids signifies regulatory role of circulating mirnas in pathogenesis, immune responses, and viral infections. on the other hand, noninvasive diagnostic approach, using biomarkers, currently plays a central role in early diagnosis of viral infections. given the fact, a recent report depicted numerous circulating microrna biomarkers viz. mir and mir in influenza; mir , mirvp p, and mir in arboviruses; and mir b and mir in hepatitis infection for diagnostic function, respectively (ojha et al. ) (table . ). next-generation sequencing (ngs) is one of the noteworthy achievements recorded in the current era. ahead from genome sequencing of known organisms, permitted breakthrough of new viruses dependable for unknown human diseases, for tracking viral outbreaks and pandemics as influenza to comprehend their emergence and transmission profiles. viral diversity from next-generation sequencing of hiv- samples provides more exact and precise estimates of time since infection, consequently, the infection regencies are also crucial for hiv- surveillance and understanding of viral pathogenesis. ngs-derived average pair-wise diversity exhibited higher sensitivity and specificity compared to fraction of ambiguous nucleotides (carlisle et al. ). it is noteworthy that metagenomic next-generation sequencing assay (mngs) for pan-pathogen detection has been effectively tested in patients with acute illness of several viral etiologies. in this connection a customized bioinformatics pipeline, surpi+, was recently designed to quickly analyze mngs data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results (miller et al. ) . panpathogen metagenomic sequencing assay for slev (st. louis encephalitis virus) infection in csf (cerebrospinal fluid) is an unbiased approach to infectious disease testing, although several challenges still remain relating to test availability, interpretation, and validation that were reported. however, recently metagenomic next-generation sequencing was employed to diagnose fatal case of meningoencephalitis caused by slev (chiu et al. ) . in another study, metagenomic viral sequencing is the potential diagnostic test for influenza which also provides insights on transmission, drug resistance, evolution, and simultaneously detects other viruses. it is pertinent that oxford nanopore technology was employed to metagenomic sequencing of respiratory samples. this technology operated with very high sensitivity compared to current diagnostic standard techniques and certainly this approach may show a great promise for nanopore platform to be used in diagnosis and genetic analysis of influenza and other clinical respiratory viruses (lewandowski et al. ) (table . ). in the recent past designing diagnostic and therapeutic platforms based on aptamer technology is undoubtedly a potential approach in viral infections. nevertheless the oligonucleotide aptamers which are potential alternatives for monoclonal antibodies based detection could be aimed against any protein in infected cells and any components of viral particles are deemed as probable novel diagnostic molecules against viral hepatitis. it is noteworthy that these aptamer molecules could be a favorable substitute for monoclonal antibody in near future (mirian et al. ). a sensitive and selective electrochemical immunosensor for label-free zikv protein detection was recently developed, employing functionalized interdigitated microelectrode of gold (ide-au) array. this zikv immune-sensing chip can be integrated with miniaturized potentiostat (mp)-interfaced with smart phone for rapid zikv-infection detection is obligatory for early stage diagnostics at point-ofcare application (kaushik et al. ). however, a cost-effective and portable graphene-enabled biosensor to detect zikv with a highly specific immobilized monoclonal antibody was recently developed. field effect biosensing (feb) with monoclonal antibodies covalently linked to graphene enables the real-time, quantitative detection of native zikv antigens. this assay is first-of-its-kind grapheneenabled zika biosensor which makes it an ideal candidate for the development as a medical diagnostic test (afsahi et al. ). an accurate and timely diagnosis of any new reassortment of avian influenza is very crucial for controlling disease outbreaks. in view of this, a simple strategy for rapid and sensitive detection of h n virus was achieved by employing an intensitymodulated surface plasmon resonance (im-spr) biosensor technique integrated with newly generated monoclonal antibody. this novel antibody demonstrates noteworthy specificity to identify h n virus compared to homemade target-captured elisa, qrt-pcr, and rapid influenza diagnostic test (ridt) with high sensitivity (chang et al. ) . in a recent study, sandwich-type electrochemiluminescence (ecl) immunosensor was developed for ultrasensitive determination of hbv surface antigen. the primary antibody of hbs (ab ) was immobilized on surface of the carboxylmodified magnetic nanoparticles (mnps). then, the pamam dendrimer with many amine functional groups was employed as carrier for immobilizing cdte@cds quantum dots (qds) and the secondary antibody (ab ) amplified ecl signal of qds considerably improved sensitivity. strikingly, this ecl sensor was designed based on signal amplification with dendrimer-quantum dots structures (babamiri et al. ) . in another study, hemagglutinin (ha), a glycoprotein present on the surface of influenza a subtype virus h n , virus binds to human cells with sialic acid on membrane of upper respiratory tract. for early detection of swine flu (h n ) infection in human, an impedimetric hemagglutinin gene-based biosensor was developed by immobilizing amino-labeled single-stranded dna probe onto cysteine modified gold surface of the screen printed electrode for early and rapid detection of h n (swine flu) in human (mohan et al. ). microfluidic technique permitted research of viral measurement of fusion kinetics, viral infectivity, and screening of viral responses to neutralizing molecules. besides that, microfluidic platforms also signify promising and innovative clinical tools with applications in clinical diagnostics including drug screening. microfluidics specifically emphasizes an array of techniques concerned with the precise control and manipulation of fluids, within microscopic channels (whitesides ) . this moderately clear-cut perception underpins a range of biological research techniques, from flow cytometric and dna analyses to enzyme and immunoassays (duncombe et al. ) . a recent study investigating the use of an integrated microfluidic system for diagnostics utilized aptamers against iav (h n ) for viral detection (tsang et al. ) was studied. remarkably, two recent studies into the infectivity of murine norovirus (mnv) also utilized droplet-based microfluidic platforms to test the convenience of neutralizing antibodies and explored their impact on virus-cell interactions (fischer et al. ) . since viral infections are complex and highly dynamic process, appreciably affected by the physical and chemical environment, studies into infection biology should preferably occur in microfluidics system the use of crispr/cas system for rna-based gene therapy is currently raising numerous potential therapeutic applications. crispr/cas is the latest and unique rna targeted gene therapy successfully applied in and observed in staphylococcus pneumonia. the crispr is rna sequence repeats which targets the foreign dna cleavage by binding to the pam flanking sequences which mediates the endonuclease called cas through guide rna (g-rna) and responsible for the double strand breaks in the host or foreign dna and silences the gene expression by nonhomologous end joining (nhej). there are different kinds which are mediated by different cas proteins. notably like class , type ii has cas where g-rna complements cas protein and other system, eventually class ii and type v complements with cas a and the crispr codes crrna acts as guide rna by complementing the type v cas protein complex. there are other crisper/cas systems that were identified (makarova et al. ) . the application of crispr/cas in viral diagnostics makes a breakthrough and it increased specificity. the type v crispr-cas a is used for the detection of the viral dna. the cas a is designed specific to the viral dna. the ssdna is tagged with a reporter which has fluorophore and quencher on both ends. the cleavage activity of viral dna by cas a confers the cleavage of the ssdna. as a result flourophore reporter, the cleavage of ssdna emits the fluorescence which is detected by quenchers and further amplified by recombinase polymerase pcr. the technique was characterized by detectr. there are other methods like sherlock (gootenberg et al. ) which works consiently based on the cas system. this tool is specially applied in the zika virus diagnosis (table . ). a novel arrayed crispr screen is aptly based on the plasmid library expressing single-guide rna (sgrna) and disrupted genes, encoding kinases, proteins related to endocytosis, and golgi-localized proteins, individually using sgrnas. this crispr screen uncovered host factors indispensable for infection by coxsackievirus b (cvb ) which comprises human pathogens causing diverse diseases. this technique is more sensitive as compared to arrayed screens based on sirna-mediated knockdown ).the lenti viral-based crispr screen-based sg-rna plasmid pool can be employed as potential diagnostic tool for hpv and can target other viral diseases. an automated poc system for ebv detection with rna-guided rna endonuclease cas a, employing its collateral rna degradation subsequent to its activation was recently developed by qin and colleagues. followed by an automated microfluidic mixing and hybridization, nonspecific cleavage products of cas a were allowed to quantify by a custom-integrated fluorometer. this crispr-cas a based diagnostic method is rapid, amplificationfree, simple, and sensitive, thus establishing a key technology toward a useful poc diagnostic platform (qin et al. a, b) . the prologue of a new class of nanoscale materials with manifold exceptional properties and functions has sparked series of breakthrough applications in biomedical and diagnostic applications. it is pertinent to note that some recent advances in nanoparticle-based lateral flow immunoassay as point-of-care diagnostic tool for infectious agents and diseases has been recently developed for the detection of infectious viral agents. lateral flow immunoassay (lfia) technology is a paperbased, point-of-care strip biosensor designed to detect a specific analyte in virusinfected samples (banerjee and jaiswal ) . aunp-based detection techniques were reported by various groups of clinically relevant viruses with a unique focus on applied types of bio-aunp hybrid structures, virus detection targets, and assay modalities and formats were recently developed (draz and shafiee ). the most recently developed diagnostic viral techniques are redesigning the field of clinical virology, which could contribute to reduction in incidence of serious infectious viral diseases. the foremost advantages of molecular techniques are its higher sensitivity and specificity matched up with other diagnostic methods, viz. serological assays and culture methods, as well as its rapidity and possibility of automation. nevertheless, the technological capabilities alone are inadequate if not sustained by health promotion policies to boost the consciousness a propos the significance of early detection of infectious viral diseases outbreak and its spread. in conclusion, good quality diagnosis has a cost that only developed countries can afford in regular practice so far, and this is delaying the execution of new-fangled methods in developing world and in disease endemic areas. conversely, it is anticipated that asserted efforts may persist toward developing new high-quality tests inexpensive in low-income countries, which would considerably reinforce disease control strategies. novel graphene-based biosensor for early detection of zika virus infection the application and interpretation of igg avidity and iga elisa tests to characterize zika virus infections development of fluorescent reverse transcription loop-mediated isothermal amplification (rt-lamp) using quenching probes for the detection of the middle east respiratory syndrome coronavirus ultrasensitive electrochemiluminescence immunosensor for determination of hepatitis b virus surface antigen using cdte@ cds-pamam dendrimer as luminescent labels and fe o nanoparticles as magnetic beads recent advances in nanoparticle-based lateral flow immunoassay as a point-of-care diagnostic tool for infectious agents and diseases viral diversity from next-generation sequencing of hiv- samples provides precise estimates of infection recency and time since infection simple strategy for rapid and sensitive detection of avian influenza a h n virus based on intensitymodulated spr biosensor and new generated antibody rapid identification viruses from nasal pharyngeal aspirates in acute viral respiratory infections by rt-pcr and electrospray ionization mass spectrometry diagnosis of fatal human case of st. louis encephalitis virus infection by metagenomic sequencing development of luciferase immunoprecipitation systems (lips) assay to detect igg antibodies against human respiratory syncytial virus g-glycoprotein genomic signature-based identification of influenza a viruses using rt-pcr/electro-spray ionization mass spectrometry (esi-ms) technology applications of gold nanoparticles in virus detection hcv ag quantification as a one-step procedure in diagnosing chronic hepatitis c infection in cameroon: the anrs study microfluidics: reframing biological enquiry molecular detection and quantitative analysis of the entire spectrum of human adenoviruses by a two-reaction real-time pcr assay typing of human adenoviruses in specimens from immunosupressed patients by pcr-fragment length analysis and real-time quantitative pcr sensitive and specific detection of crimean-congo hemorrhagic fever virus (cchfv)-specific igm and igg antibodies in human sera using recombinant cchfv nucleoprotein as antigen in μ-capture and igg immune complex (ic) elisa tests development and validation of sensitive real-time rt-pcr assay for broad detection of rabies virus rapid, noninvasive detection of zika virus in aedes aegypti mosquitoes by near-infrared spectroscopy indirect elisa based on hendra and nipah virus proteins for the detection of henipavirus specific antibodies in pigs use of the immunoglobulin g avidity assay to differentiate between recent zika and past dengue virus infections multiplexed and portable nucleic acid detection platform with cas , cas a, and csm diagnosing zika virus infection against a background of other flaviviruses: studies in high resolution serological analysis diagnostic molecular microbiology review a sensitive electrochemical immunosensor for label-free detection of zika-virus protein arrayed crispr screen with image-based assay reliably uncovers host genes required for coxsackievirus infection development and evaluation of a novel high-throughput image-based fluorescent neutralization test for detection of zika virus infection metagenomic nanopore sequencing of influenza virus direct from clinical respiratory samples. biorxiv an updated evolutionary classification of crispr-cas systems paper-based rna detection and multiplexed analysis for ebola virus diagnostics laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid oligonucleotide aptamers: potential novel molecules against viral hepatitis hemagglutinin gene based biosensor for early detection of swine flu (h n ) infection in human magnetic nanozyme-linked immunosorbent assay for ultrasensitive influenza a virus detection emerging role of circulating microrna in the diagnosis of human infectious diseases recent development of spr spectroscopy as potential method for diagnosis of dengue virus e-protein development of an oral fluid immunoassay to assess past and recent hepatitis e virus (hev) infection development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (vhsv) mrnas in a cell culture model rapid and fully microfluidic ebola virus detection with crispr-cas a a new recombinant rabies virus expressing a green fluorescent protein: a novel and fast approach to quantify virus neutralizing antibodies virus detection by transmission electron microscopy: still useful for diagnosis and a plus for biosafety development and evaluation of a new real-time rt-pcr assay for detecting the latest h n influenza viruses capable of causing human infection performance of the trioplex real-time rt-pcr assay for detection of zika, dengue, and chikungunya viruses development of fluorescent reverse transcription loop-mediated isothermal amplification (rt-lamp) using quenching probes for the detection of the middle east respiratory syndrome coronavirus recent advances in diagnostic testing for viral infections engineered mrna-expressed antibodies prevent respiratory syncytial virus infection recent developments in serological methods suited for use in routine testing for plant viruses ultrasensitive detection of ebola virus oligonucleotide based on upconversion nanoprobe/nanoporous membrane system nucleic acid amplification and related techniques in microbiological diagnosis and epidemiology serum exosomal micro rna s combined with alpha-fetoprotein as diagnostic markers of hepatocellular carcinoma a rapid diagnostic platform for colorimetric differential detection of dengue and chikungunya viral infections dual rna-seq reveals viral infections in asthmatic children without respiratory illness which are associated with changes in the airway transcriptome february) immune suppression in cancer: effects on immune cells, mechanisms and future therapeutic intervention a multiplex microsphere immunoassay for zika virus diagnosis development of a reverse transcription recombinase polymerase amplification assay for rapid detection of human respiratory syncytial virus evaluation of an antigen assay for diagnosing acute and chronic hepatitis e genotype infection the authors are grateful to their respective academic institutions for the support extended. the authors declare that they have no competing interests. key: cord- -rr qet c authors: counotte, michel j; meili, kaspar w; low, nicola title: emergence of evidence during disease outbreaks: lessons learnt from the zika virus outbreak date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: rr qet c introduction: outbreaks of infectious diseases trigger an increase in scientific research and output. early in outbreaks, evidence is scarce, but it accumulates rapidly. we are continuously facing new disease outbreaks, including the new coronavirus (sars-ncov- ) in december .the objective of this study was to describe the accumulation of evidence during the - zika virus (zikv) outbreak in the pacific and the americas related to aetiological causal questions about congenital abnormalities and guillain-barre syndrome. methods: we hypothesised that the temporal sequence would follow a pre-specified order, according to study design. we assessed ) how long it takes before findings from a specific study design appear, ) how publication of preprints could reduce the time to publication and ) how time to publication evolves over time. results: we included publications published between march , and january , . in the - zikv outbreak, case reports, case series and basic research studies were published first. case-control and cohort studies appeared between - days after zikv was first detected in the region of the study origin. delay due to the publication process were lowest at the beginning of the outbreak. only . % of the publications was available as preprints. discussion: the accumulation of evidence over time in new causal problems generally followed a hierarchy. preprints reduced the delay to initial publication. our methods can be applied to new emerging infectious diseases. introduction outbreaks of infectious diseases trigger an increase in scientific research and output. early in outbreaks, evidence is scarce, however, it accumulates rapidly as time progresses. as we are continuously facing new disease outbreaks, as we do at the moment with the emergence of the new coronavirus (sars-ncov- ), understanding the accumulation of evidence is vital. here, we describe the accumulation of evidence during the zika virus outbreak and summarize the lessons learnt. causality is a principal theme in epidemiological research. establishing that an exposure causes a specific health outcome is based on evidence and may inform guidance about public health measures. the concepts and types of evidence required to conclude that an association is causal are the subject of ongoing debate. vandenbroucke proposed a hierarchy of evidence based on the best chance for discovery and explanation of phenomena [ ] . observations published in case reports and case series, or findings in data and literature drive discovery. verification of these discoveries happens in observational studies and in randomized controlled trials, given that exposures can be randomized. the value of evidence used for public health guidance is thought to follow an inverse pattern of the hierarchy for discovery; here, case reports and other anecdotal evidence is considered to be evidence that provides the least certainty on an effect or association. cohort studies, randomized controlled trials and case-control studies are considered to provide evidence with a higher certainty (figure ). the zika virus (zikv) outbreak in the pacific and the americas between -- presented several aetiological causal questions. in - , zikv caused an outbreak in french polynesia [ , ] . during this period, investigators documented some severe neurological conditions, including people with guillain-barré syndrome (gbs). gbs is usually a rare sporadic condition. often triggered by infection, an autoimmune response affects the peripheral nerves, leading ascending paralysis, which can be fatal if it involves the respiratory nerves [ ] . at the time, the reports did not attract much attention and the investigators refrained from making a causal connection because dengue was also circulating at the time [ ] . in november , the ministry of health in brazil reported a cluster of births affected by microcephaly in the north east. microcephaly is a birth defect, indicative of impaired brain development, which can be caused by congenital infection. at around the same time, zikv had been identified for the first time in brazil [ ] . in december , the pan american health organization announced heightened surveillance owing to an "increase of congenital anomalies, guillain-barré syndrome, and other zikv circulation [ ] . retrospective assessment of the french polynesia outbreak identified an increase in adverse congenital outcomes as well [ ] . the pheic and the extensive outbreak catalysed the research on zikv. early public health guidance about the prevention of zikv infection and its potential consequences was based on limited evidence, however [ ] . systematic reviews were developed to address the pheic recommendation for research about the causal relationships between zikv infection and adverse congenital outcomes, including microcephaly and between zikv and autoimmune outcomes, including gbs [ ]. the reviews organized the findings around a 'causality framework' with ten dimensions derived from those proposed by bradford hill [ , ] . an expert committee reviewed the evidence collected by these systematic reviews up to may and reached the conclusion that "the most likely explanation of the available evidence" was that zikv is a cause of adverse congenital outcomes and a trigger of gbs [ ] . this review has been kept up to date as a living systematic review, by periodically incorporating new results [ , ] . the additional evidence has reinforced the conclusions of causality. a temporal sequence for the emergence of evidence was already hypothesised during the planning of the systematic reviews in early ( figure ). acknowledging that 'astute observations' of new causes of disease often start an aetiological investigation [ ] , case reports and case series were eligible for inclusion in the systematic reviews. these study designs are often excluded from systematic reviews because they are the lowest level of the "hierarchy of evidence" that applies to evaluation research. vandenbroucke proposed a reverse hierarchy for discovery in which 'anecdotal' forms of evidence are at the top [ ] . cross-sectional, case-control and retrospective follow-up studies follow because they are quickest study designs that include a control group. prospective cohort studies take longer to set up and rcts only provide additional information if a treatment or vaccine is available. in addition to epidemiological studies, basic and clinical laboratory science start early in the search for causes. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted march , . figure . hypothetical accumulation of evidence over time, by study design. the objective of this study was to examine the body of evidence that was used to establish the causal relation between zikv infection and adverse outcomes. we hypothesised that the temporal sequence would follow figure . for all included studies, we retrieved the received and published date, the location of the study and the study design (table ) . for epidemiological studies, we extracted the study location and the number of patients with both exposure and the outcome according to the case definition provided in the publication. we excluded modelling studies or surveillance and outbreak reports. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted march , . . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . we defined the publication date as the earliest date the publication was available. if the publisher's website did not state an exact date, we assigned the 'epub' date from medline via pubmed or 'page created' date for specific online journals (eid and mmwr). we also recorded the date the manuscript was received by the publisher (received date) and the date of acceptance for publication (accepted date). we defined the time to publication as the time between introduction of zikv virus in the region and the publication date. for basic research studies, many of which were done in countries unaffected by zikv, we assigned the time to publication as the time between february (the pheic declaration) and the first available publication date. the delay resulting from the publication process (publication delay) was defined as the time between the 'received date' and the first available publication date. we assessed ) how long it takes before findings from a specific study design appear, ) how publication of preprints could reduce the time to publication and ) how time to publication evolves over time. we provide a descriptive analysis of the total time to publication and the publication delay by / . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint publication. of these durations, we provide the median and interquartile range (iqr) by study design and over time. we compare the publication delay by three month period (quarter). results during the period of the first review [ ] and subsequent update [ ], we screened , publications. during the remaining period, between january , and january , , we screened an additional , publications. figure shows the evolution of the volume of the published zikv research over time is provided. year number/month is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . figure a shows the comparison of publications published between the pheic and the end of the was a result of a retrospective study looking back at the french polynesia outbreak [ ] . the median total time to publication was longer for more robust study designs (cohort studies, case-control studies). we see a similar pattern for epidemiological studies if we consider the data up to january , and consider the time to publication between the regional introduction of zikv and the publication date ( figure b ). . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . a strength of this study is the pre-specified hypothesis about the time to publication of aetiological research and the use of data from systematic reviews that had screened and selected studies that addressed the causal relationship between zikv infection and its adverse outcomes. we calculated additional measures related to the time to publication of research, including delays due to the publication, and thus the time that could have been gained by publishing preprints. the limited information extracted about each study was a limitation. the time between introduction of zikv and the actual publication of a research study is dependent on factors both within and between study designs. there is substantial variation in the time to publication within the study designs. we did not quantify several factors that likely influence this duration such as the size of the outbreak, the research capacity or outbreak preparedness. small outbreaks or small population sizes limit the opportunity to enrol sufficient patients with adverse outcomes, and unless involved in multi-centre/multi-region studies, these regions are less likely to produce high quality epidemiological studies. the same holds true for regions with limited research capacity, such as appropriate diagnostic facilities and expertise. outbreak preparedness likely increased over time, with funding increasing after the pheic, meaning that initiation of studies started relatively late for regions that were affected earliest by the outbreak. countries that were affected later in time by zikv, might have already had surveillance and diagnostic methodology in place. the publication delay is a proxy measure, which could not be calculated for all studies; for only % / . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint ( / ) studies the "received date" was provided. it is unclear whether these data are missing at random. furthermore, the recorded publication delay could only be calculated for the journal in which a study was published. the true publication delay includes the time taken up by rejection and resubmission. the publication date also ignores dissemination of the findings at conferences or within collaborations. however, here the information is only available to a limited audience. the timing of zikv introduction is also a proxy measure, which does not capture the first actual case, but signals the moment at which the health authorities and the research community noted the introduction in that region and thus serves its purpose as a proxy for when research start intensifying. phylogenetic data suggest that zikv was often introduced months before formal detection and notification [ ] . the sequence of emergence of evidence about causality was not exactly as hypothesised (figure ). while case reports and case series were the first types of study to be published, findings from animal research were also published quickly. this finding might have been influenced by the more frequent use of preprints to disseminate laboratory research than clinical science [ ] . in our study, the time taken to publication of case-control studies and cohort studies was similar, particularly for studies of congenital outcomes. case-control studies are widely assumed to be quicker to organise and conduct than cohort studies [ , ] . in the zikv outbreak, one case-control study about gbs was published soon after the pheic declaration because it used data already collected from the earlier zikv outbreak in french polynesia. an important consideration is the short duration of pregnancy. the cohorts that were fastest to produce results, were cohorts that were already in place for other disease (dengue, influenza) [ ] . in a disease outbreak with adverse outcomes that are new or incompletely understood, the full spectrum of evidence needs to be assessed to establish causality. early in an outbreak, we need anecdotal evidence to drive discovery and explanation [ ] . studies across the different scientific disciplines are . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . problems based on study design and timing of evidence will provide more insight in how evidence accumulates. the zikv outbreak in the americas was unique by its size; making rare as the sars-ncov- outbreak continues to unfold, we will apply the same methodology as discussed here to keep track of the accumulation of evidence, the delay in publication and the use of pre-print publishing. the accumulation of evidence over time in new causal problems seems to follow a hierarchy where case reports and case series were rapidly followed by basic research. during the zikv outbreak, robust epidemiological studies, such as case-control studies and cohort studies, took - days to appear. causal inference based on a wide spectrum of evidence is therefore essential for early public health guidance in emerging causal problems. publishing preprint does reduce the delay, and especially in epidemiological research this is an underused tool. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted march , . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted march , . observational research, randomised trials, and two views of medical science zika virus infection complicated by guillain-barré current zika virus epidemiology and recent epidemics guillain-barré syndrome: an update world health organization. who statement on the first meeting of the international health ihr ) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations syndrome outbreak associated with zika virus infection in french polynesia: a case-control study world health organization. publications, technical guidance on zika virus infection as a cause of congenital brain abnormalities and guillain-barré syndrome: systematic review the environment and disease: association or causation? world health organization. who -zika causality statement zika virus infection as a cause of congenital brain abnormalities and guillain-barré syndrome: from systematic review to living systematic review zika virus infection as a cause of congenital brain abnormalities and guillain-barré syndrome: a living systematic review in defense of case reports and case series timeline of emergence of zika virus in the americas polynesia - : anatomy of a completed outbreak spread of zika virus in the americas preprints: an underutilized mechanism to accelerate outbreak science seroprevalence, risk factor, and spatial analyses of zika virus infection after the world health organization. who zika virus research agenda basic: animal experiment basic: in vitro experiment/other epi: case−control study epi: case report epi: case series epi: cohort study epi: cross−sectional study key: cord- -kwzo puo authors: si, lulu; meng, yu; tian, fang; li, weihua; zou, peng; wang, qian; xu, wei; wang, yuzhu; xia, minjie; hu, jingying; jiang, shibo; lu, lu title: a peptide-based virus inactivator protects male mice against zika virus-induced damage of testicular tissue date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: kwzo puo zika virus (zikv) was a re-emerging arbovirus associated with guillain–barré syndrome in adult and congenital zika syndrome in fetus and infant. although zikv was mainly transmitted by mosquito bites, many sexual transmission cases have been reported since the outbreak in . zikv can persist in testis and semen for a long time, causing testicular tissue damage and reducing sperm quality. however, no drug has been approved for prevention or treatment of zikv infection, especially infection in male testicular tissue. previously reported peptide z could inactivate zikv, inhibiting zikv infection in vitro and in vivo. importantly, z could inhibit vertical transmission of zikv in pregnant mice, reducing zikv infection in fetus. here we showed that intraperitoneally administered z could also be distributed to testis and epididymis, resulting in the reduction of zikv rna copies in testicular tissue and protection of testis and epididymis against zikv-induced pathological damage and poor sperm quality in type i interferon receptor-deficient a mice. thus, z , a zikv inactivator, could serve as an antiviral agent for treatment of zikv infection and attenuation of zikv-induced testicular tissue damage. zika virus was a re-emerging arbovirus (gao, ; pettersson and bohlin, ) , like dengue virus (denv) and japanese encephalitis virus (jev), belonging to flavivirus genus in the flaviviridae family. since the outbreak in brazil in , zikv has rapidly spread to countries and territories (baud et al., a; gorshkov et al., ) , attracting global attention. about % of those infected with zikv presented with asymptomatic, or only mild illness (petersen e. et al., ; pierson and diamond, ) . however, zikv infection has been associated with more severe complications: guillain-barré syndrome in adult (brasil et al., ; peixoto et al., ) and congenital zika syndrome in fetus and infant (baud et al., b; gurung et al., ) . zikv is mainly transmitted through mosquito bites (petersen l.r. et al., ) , while it can also be transmitted via in utero from mother to fetus (besnard et al., ) , blood transfusion (tai et al., ) and intercourse (duggal et al., ; mead et al., ; sakkas et al., ) . it is reported that zikv was transmitted through sexual contact, perhaps up to days after the onset of symptoms (turmel et al., ) , and infective virions were still isolated from semen days after infection (arsuaga et al., ; garcia-bujalance et al., ) . the viral load in semen was , times that in the blood at weeks postinfection (mansuy et al., ) , and viral rna was still detected up to days after illness onset (barzon et al., ) . it is also reported that zikv infection caused patients to have a decreasing total sperm count in the acute phase of infection (joguet et al., ) and abnormal spermogram results year after infection (avelino-silva et al., ) , suggesting zikv was harmful to human spermatozoa production. testis explants from uninfected donors were also proven to be susceptible to zikv infection (matusali et al., ) . as determined from an in vitro human testicular organoid culture system, zikv-infected testicular organoids may lead to multiple kinds of cell death (strange et al., ) . although little was known about zikv infection in human testis and epididymis, except for semen, many murine models were used to study damage to testicular tissue. govero et al. ( ) performed a study in wild-type c bl/ mice in the presence of the anti-ifnar antibody and revealed that zikv preferentially infected spermatogonia and sertoli cells in the testis. this led to cell death and destruction of the seminiferous tubules in association with testis damage and poor sperm quality (govero et al., ) . ma et al. ( ) also established a mouse model using ifnα/β receptor-deficient mice (ifnar −/− knockout mice), and demonstrated that zikv infection induced inflammation in the testis and epididymis, leading to severe damage to testes at days post-infection. taken together, these findings suggested that zikv could persist in testicular tissue for a long time, causing severe damage to testis and epididymis and reducing sperm quality. currently, no approved drug is available to inhibit zikv infection (da silva et al., ) , especially infection in testicular tissue. ebselen (ebs), an antioxidant in clinical trials, was reported to alleviate testicular pathology in zikv-infected mice by reducing the level of oxidative stress and proinflammatory cytokines. however, it only had a weak effect on zikv directly, and its safety for pregnant women was unknown (simanjuntak et al., ) . this calls for the development of safe and effective drugs to prevent zikv-induced testicular damage. the testis is a male reproductive organ, mainly producing spermatozoa and androgen. specifically, spermatogenesis is a complex cellular event taking place in the seminiferous epithelium of seminiferous tubules and protected by sertoli cells that form the blood-testes barrier (btb) by tight junction protein (su et al., ) . the btb provides a specialized microenvironment for spermatogenesis by preventing harmful agents from entering the seminiferous tubule, but this was found to pose a major obstacle to the delivery of therapeutic drugs to the seminiferous epithelium (cheng and mruk, ) . therefore, any drug able to prevent zikvinduced damage in testicular tissue should be able to cross the btb into seminiferous tubules, or reach the testicular tissue, to inhibit zikv from entering into seminiferous tubules. the most promising anti-zikv drugs so far include small-molecule compounds (deng et al., a; chan et al., ; li et al., ) , antibodies (zhang et al., ; wang et al., wang et al., , , and peptides (yu et al., ; jackman et al., ) . compared with small-molecule compounds, peptides were safer, especially for pregnant women. as a macromolecular substance, passing through the btb was challenging for antibodies, and it is reported that the concentration of specific igg entering into the rete testis was . % of that in blood serum (knee et al., ) . therefore, the safer and cheaper peptide drugs, which consisted of dozens of amino acids, began to gain gradual acceptance. we previously demonstrated that an amphipathic peptide z , derived from the stem region of zikv e protein (figure a) , inhibited zikv infection in vivo and in vitro, suggesting its promise as an anti-zikv candidate drug. what's more, z had a protective effect in pregnant mice and their fetuses, suggesting it was able to cross the placental barrier (yu et al., ) . however, whether z could enter the seminiferous tubules and protect testicular tissue against zikv infection remained unknown. in this work, we showed that intraperitoneally injected z could be distributed in the testicular tissue. it then inhibited zikv infection, resulting in significant reduction of viral loads and protection of testis, epididymis, and sperm from zikvinduced pathological damage. these results suggest that z has the potential to be further developed as an anti-zikv agent for treatment and prevention of zikv-induced damage in testicular tissue. (sirohi et al., ) ]; di, dii, diii, stem and transmembrane domain are shown in red, yellow, blue, orange, and wheat, respectively; the fusion peptide is shown in cyan, and z ( - ) is shown in green. tm cells were infected with zikv strain sz (b), mr (c) and flr (d), treated with z at different concentration, and then viral copies in the supernatant at h were measured by qrt-pcr. data were presented as means ± sd. (e) z -treated zikv of different strain lost infectivity on tm cells irreversibly. after incubation with z at • c for h, zikv particles were separated from the unbound z by peg to measure their infectivity on tm cells. each sample was tested in triplicate and the experiments were repeated at least once. the data from two independent experiments were presented as mean ± sd. mixture f- (dmem/f , thermo fisher scientific, waltham, ma, united states) supplemented with % horse serum and . % fbs at • c and % co . zika virus (zikv) strain sz / (genbank no. ku ) was kindly provided by dr. cheng-feng qin (deng et al., b) and preserved in our laboratory. zikv strains mr (#vr ) and flr (#vr ) were obtained from atcc. zikv was propagated in vero-e cells. briefly, vero-e cells were infected with the virus at multiplicity of infection (moi) of . . the supernatants were harvested at days post-infection, centrifuged at , rpm for min to remove cellular debris, and stored at − • c as stock. peptides z (mavlgdtawdfgsvggalnslgkgihqifg aaf), z -cy and scrambled peptide (ldiiaglsagfq ggatfvdahgmvkasflggnw) were synthesized at kangbei bio, co., ltd. (ningbo, china) with % purity. peptides were solubilized in dimethyl sulfoxide (dmso) at mm and stored at − • c. virus titer was detected by plaque forming assay as shown below. bhk- cells were seeded onto a -well plate with × cells per well and cultured overnight at • c and % co . serially fold diluted virus were added to each well and incubated for h at • c. then the supernatant was replaced with ml dmem containing % low melting agarose and % fbs. after agarose solidification, the cells were cultured at • c and % co for days. then cells were fixed with % formalin and stained with % crystal violet overnight. finally, the plaque forming units were counted, and virus titer was calculated. six male a mice ( - weeks old) were randomly divided into two groups. mice in each group (n = ) were injected intraperitoneally with z -cy ( µg in µl pbs) or pbs (vehicle in µl pbs) as control. after anesthetization with pentobarbital sodium, mice were imaged by the ivis lumina k series iii in vivo imaging system from perkinelmer (waltham, ma, united states) for h. to determine the distribution of z -cy in the testicular tissue, mice were sacrificed using pentobarbital sodium, and all testes and epididymides were removed for imaging. the radiant efficiency (ps − cm − sr − ) (µw − cm − ) in mouse body and testis and epididymis was calculated by living image . . software. to determine the protection of z against zikv-induced testis damage, male a mice ( - weeks old) were randomly divided into three groups (n = ): z -treated group, vehicletreated group, and mock-infected group. mice in the z -or vehicle-treated group were intraperitoneally injected (i.p.) with pfu zikv with z ( mg/kg) or vehicle on day , followed by i.p. administration of z ( mg/kg) or vehicle once daily for six consecutive days, respectively. mice in the mock-infected group only received pbs as normal control. the body weight was monitored daily for days, and blood was collected at , , , , and days post-infection (d.p.i.) for detection of zikv copies in sera. at d.p.i., mice were euthanized by co inhalation, and the testes and epididymides were removed. the weight and size of testes were measured as previously described (de la vega et al., ) . after imaging, the left testes and epididymides were immersed in bouin's for hematoxylin-eosin (h&e) staining. the right testes and epididymides were soaked in rnaiso plus reagent at − • c for further detection of zikv copies. for safety analysis of z in testicular tissue, male balb/c mice ( - weeks old) were randomly divided into two groups. five mice in each group were i.v. administered with z at mg/kg of body weight or pbs control for days. the body weight of mice was monitored every other day for days. blood was collected at h, as well as , , and days post-injection and sera were separated from the blood samples for use in elisa to detect the concentration of testosterone and inhibin b as well as the titer of z -specific antibody. at days, mice were sacrificed, and the testes and epididymides were removed for histological examination. mature sperm in the cauda epididymis of the three groups of male a mice were collected and placed in ml pbs (preincubated at • c) immediately after euthanasia. the sperm suspension was analyzed for total sperm count and motility by computer-assisted sperm analysis (casa), as previously described (goodson et al., ) , using hamilton thorne ivos ii (beverly, ma, united states). then, after smear, desiccation and fixation, remaining sperm were stained by the papanicolaou staining method for manual morphological analysis. sperm morphology was observed in each mouse. zikv-infected mice were euthanized at days post-infection. testes and epididymides were homogenized with beads in ml rnaiso plus reagent (takara, japan) using tissuelyser- (jingxin, shanghai, china) after weighing. homogenized tissue were centrifuged for min at , rpm at • c, then total rna in tissues was extracted according to the operating manual and stored at − • c for the next step. sperm collected in pbs were placed in rnaiso to extract total rna under the same procedure. viral rna in sera samples on specific days was extracted using the easypure r viral dna/rna kit (transgen, china) and stored at − • c for the next step. zikv rna was examined by one-step real-time quantitative reverse transcription pcr (qrt-pcr) using the mastercycler r ep realplex real-time pcr system (eppendorf, germany). zikv rna copies were calculated based on the standard curve which was determined by plasmid containing specific sequence. the following primers were used: zikv-f: -ttggtcatgatactgctgattgc- ; zikv-r: -ccttccacaaagtccctattgc- ; zikv-probe: -fam-cggcatacagcatcaggtgcataggag-bhq - . the concentration of testosterone and inhibin b in the sera of balb/c mice was detected by mouse testosterone (ml , mlbio, shanghai, china) and inhibin b elisa kit (ml , mlbio, shanghai, china), respectively. according to the manual, µl standard or testing samples were added to a -well plate, which was coated with purified mouse testosterone or inhibin b antibody combined with hrp labeling. hrp-conjugate reagent was added to each well, except blank well (no sample; hrpconjugate reagent added as background). the plate was closed with closure plate membrane and incubated at • c for min. after washing, chromogen solution was added and incubated for min at • c. stop solution was added to each well, and absorbance was read at nm. peptide z was dissolved in dmso and diluted to different concentration by dmem/f . then µl of different zikv strains were incubated with z for h at • c. the mixture was added to × cells seeded into a -well plate and incubated at • c for h. after the culture supernatant was replaced by dmem/f- with % horse serum, cells were cultured for h at • c. then the culture supernatant was collected to detect zikv rna copies by qrt-pcr, as described above. the ability of z to inactivate different zikv strains was determined as follows. briefly, µl z or z -scr, at graded concentration were added to µl zikv ( × pfu/ml), followed by incubation at • c for h. then, peg- and nacl were added to the treated virus at final concentration of % and . m, respectively. after incubation on ice for h, the mixture was centrifuged at , rpm for h. the supernatants were removed, and the pellet was resuspended in µl dmem with % fbs. the infectivity of the zikv particles in the pellet was determined by cck- on bhk- cells or qrt-pcr on tm cells. the testis and epididymidis of z -or vehicle-treated zikvinfected mice and mock-infected mice were all collected post mortem. tissues were fixed in bouin's overnight, dehydrated, embedded in paraffin and sectioned. then the sections ( µm thick) were stained by h&e. subsequently, observation was made via panoramic scanner ( d histech pannoramic midi, hungary). student's unpaired two-tailed t-test was used to monitor the distribution of z in male a mouse body and testicular tissue and to analyze the difference of viral rna level in sera or tissues between z -and vehicle-treated a mice. one-way anova was used to examine the effect of z on the weight, length and width of testes, as well as sperm count, sperm motility and progressive sperm motility among the three groups. p-value was calculated by graphpad prism software, v. . , and significant difference was achieved with p-value less than . . * p < . ; * * p < . ; * * * p < . ; * * * * p< . . to determine the protective effect of z on zikv infection of testicular tissue, we tested if z could inhibit infection by different zikv strains of asian and african lineages in mouse sertoli tm cells, which are nurse-like cells that support spermatogenesis (wei et al., ) and important target cells for zikv testicular infection. zikv sz (asian lineage), flr (asian lineage), or mr (african lineage) was pretreated with z at different concentration before addition of tm cells and incubation at • c for h. after replacement of culture medium and further incubation for h, the viral copies in the supernatant were examined by qrt-pcr. as shown in figures b-d, z treatment resulted in a decrease of zikv copies in a dosedependent manner. considering that z -mediated inhibition of zikv infection is possibly attributed to its viral inactivation activity (yu et al., ) , different strains of zikv were incubated with z at different concentration for h at • c, followed by separating virions from the unbound free peptide by peg and detecting the infectivity of z -treated zikv in tm cells ( figure e ) and bhk- cells (supplementary figure s ) . we found that z -treated zikv strains lost their infectivity in a dose-dependent manner with % effective concentration (ec ) of . ± . µm (for sz ), . ± . µm (for mr ) and . ± . µm (for flr), respectively, suggesting that z inhibits infection of zikv strains of both asian and african lineages in tm and bhk- cells via inactivation of virions. to determine whether z entered testicular tissue of male mice, we employed cy -conjugated z peptide (z -cy ) to detect the distribution of z in the organs of male mice. as shown in figure a , the bodies of the z -cy -treated mice showed a strong fluorescence signal with average radiant efficiency of about . × (ps − cm − sr − ) (µw − cm − ), which is significantly higher than that in pbs-treated mice (p = . , student's two-tailed t-test; figure b) . then, the testes and epididymides were collected for examination of the fluorescence signal in the testicular tissue. as expected, both testes and epididymides of mice treated with z -cy showed a strong fluorescence signal, while those in the pbs-treated mice displayed no significant fluorescence signal (figure c) . the average radiant efficiency in testes and epididymides of z -cy -treated mice was significantly figure | distribution of z in the testicular tissue of male a mice. (a) imaging of male a mice treated with z -cy or pbs by the ivis lumina k series iii from perkinelmer. male a mice were injected intraperitoneally with µg z -cy (n = ) or pbs (n = ) as control, followed by imaging analysis. (b) the statistical analysis of fluorescence signal intensity in mouse body. data were presented as means ± sd. (c) imaging of the testes and epididymides from male a mice. (d) statistical analysis of fluorescence signal intensity in testis. each sample was tested in triplicate and the data were presented as mean ± sd. (e) statistical analysis of fluorescence signal intensity in epididymis. data were presented as means ± sd. * p < . ; * * * * p < . , student's two-tailed t-test. higher than that of pbs-treated mice (p < . , student's twotailed t-test; figures d,e) . these results suggest that z peptide can be distributed in the testis and epididymis of male mice. to determine the protective effect of z against zikv infection of male mice, pfu zikv were intraperitoneally injected into male a mice (type i interferon receptor-deficient). the infected mice were i.p. administered with z at mg/kg of body weight or vehicle, respectively, daily for days (figure a) . mice in the mock-infected group received pbs as normal control. results showed that the z -treated mice had neither weight loss ( figure b ) nor obvious clinical symptoms, consistent with mice in the mock-infected group (data not shown). however, in the vehicle-treated group, mouse body weight began to decline from the fifth day post-infection (d.p.i.) (figure b) , and some symptoms, like hunched posture and ruffled fur, appeared. we then examined viral copies in sera of z -or vehicle-treated mice at different time points by qrt-pcr. a high level of viral load was detected in sera of vehicle-treated mice, e.g., about copies/ml at d.p.i. (figure c) . however, viral load in sera of z -treated mice (figure c ) was as low as copies/ml at all time points tested, significantly lower than that of vehicle-treated mice. these results suggest that z can exert protection against zikv infection of male mice. to further evaluate the protective effect of z against zikvinduced damage of testicular tissue in male mice, all mice were sacrificed at d.p.i. and their testes were collected for analysis of weight and size. we found that testis weight in vehicle-treated mice was around mg, which was significantly lower than that in z -treated mice (∼ mg) (p < . , one-way anova, figure a ). the length and width of testes in vehicle-treated mice were both significantly decreased compared with those of testes in z -treated mice the change of mouse body weight was monitored daily for days. (c) the change of zikv rna level in mouse sera were detected by qrt-pcr at days , , , , and after zikv infection. male a mice were intraperitoneally injected with pfu zikv with z ( mg/kg) or vehicle on day , followed by daily injection of z ( mg/kg) or vehicle for six consecutive days. each sample was tested in triplicate and the data were presented as mean ± sem, * * * p < . ; * * * * p < . , student's two-tailed t-test. (p = . and p < . , one-way anova, figures b,c) . no significant difference in weight (p > . , one-way anova, figure a ), as well as length (p > . , one-way anova, figure b ), and width (p > . , one-way anova, figure c ) of testes were noted between z -treated zikvinfected mice and mock-infected mice. the representative image of testes from the three groups of mice were shown in figure d . subsequently, we examined the testes and epididymides for histopathological changes. the results of h&e staining of testes in vehicle-treated mice revealed that the normal architecture of the seminiferous tubule was seriously destroyed and replaced with an infiltrate of mixed inflammatory cells and necrotic debris, accompanied by degeneration of the spermatogenic lineage ( figure e, upper panel) . the connective tissue areas surrounding the seminiferous tubule had also been infiltrated by a large number of inflammatory cells (figure e, upper panel) . however, the architecture of the seminiferous tubule in the testes of both z -treated and mock-infected mice was intact and clear. spermatogenic cells at different stages were organized tightly and identified clearly (figure e, upper panel) . histological analysis of epididymis showed that epididymides from mice in the vehicle-treated group were also damaged. sperm in the lumen of caput epididymides decreased precipitously, only to be replaced by secretions and numerous necrotic epithelial cells (figure e, middle panel) . the lumens of cauda epididymis contained degenerating spermatozoa and a small number of normal spermatozoa, accompanied by scattered necrotic epithelial cells and inflammatory cells (figure e , lower panel). however, histological analysis of the caput epididymis and cauda epididymis showed no apparent microscopic differences between z -treated and mock-infected mice (figure e middle, lower panel). the architecture of the caput epididymis and cauda epididymis in these two groups was normal with no obvious morphological damage, suggesting that z protected testicular tissue against zikv-induced pathological damage. we used casa to evaluate the protective effect of z on the count and motility of mouse sperm. as shown in figure , the figure | z effectively attenuated damage to testes and epididymides of zikv-infected male a mice. (a) the weight, (b) length, and (c) width of testes from z -or vehicle-treated zikv-infected and mock-infected male a mice at day . each symbol represents one testis; all horizontal bars indicate mean, and error bars reflect sem. (d) the representative image of testes from z -or vehicle-treated zikv-infected and mock-infected male a mice at day . scale bar, mm. (e) histopathological analyses of testes and epididymides collected from z -or vehicle-treated zikv-infected male a mice and mock-infected mice used as a control. scale bar: µm. upper panel, testes; middle panel, caput epididymides; lower panel, cauda epididymides. * * p < . ; * * * * p < . ; one-way analysis of variance with tukey's multiple comparison post hoc tests. sperm count of z -treated mice was significantly higher than that of the vehicle-treated group (p = . , one-way anova; figure a ). meanwhile, the percentages of total ( figure b ) and progressively ( figure c ) motile sperm in z -treated mice were dramatically higher than those in the vehicle-treated group (p = . and p = . , one-way anova), but similar to that in the mock-infected mouse group (p > . , oneway anova). papanicolaou staining of morphological spermatic features revealed more noticeable teratogenesis of sperm in vehicle-treated mice compared to the other groups ( figure d) . to investigate whether the protective effect of z on testicular tissue results from the reduction of local viral load, we examined the viral copies in different testicular tissues. results showed a high level of viral rna ( - equivalents per g) detected in the testis and epididymis of vehicle-treated mice at d.p.i., much higher than that ( - equivalents per g) in z -treated mice (figures a,b) . notably, zikv rna was also detected (up to equivalents per ml) in the mature sperm collected from cauda epididymis in vehicle-treated mice, which was significantly higher than that in z -treated mice (p = . , student's twotailed t-test; figure c ). finally, to examine the safety of z for male mice, male balb/c mice were injected intravenously with z at mg/kg of body weight (n = ) or pbs (n = ). results showed that body weight change of mice was nearly consistent in the two groups ( figure a) , indicating that z peptide did not cause significant harm to the male mice. since the levels of testosterone and inhibin b reflect testicular function and sperm count, the concentration of these hormones in mouse sera at the indicated time points was measured. we found no significant difference between the z -and pbs-treated groups at all time points tested (figures b,c) , suggesting z may not affect the function of testis or sperm. we then compared the potential histopathological changes between the two groups. as shown in figure d , h&e analysis of testis and epididymis revealed no obvious pathological abnormality in mice treated with z compared with the pbs group. besides, the titer of z -specific antibody in sera of mice at and days post-injection was detected. as shown in figures e,f , no significant level of z -specific antibody was detected in the sera of mice that were intravenously injected with high doses of z peptide, consistent with the finding from our previous report for studying anti-mers-cov peptides (xia et al., ) . this result suggests that z peptide consisting of amino acids is unable to elicit a significant z -specific antibody response after it is intravenously administered in the absence of figure | z inhibited zikv replication in testes, epididymides and sperm. zikv rna copies in (a) testes, (b) epididymides, and (c) sperm of z -or vehicle-treated zikv-infected male a mice at day were detected by qrt-pcr. each symbol represents data from individual mice; all horizontal bars indicate mean, and error bars reflect sem. experiment was repeated at least twice. * * * p < . or * * * * p < . respectively, student's two-tailed t-test. figure | safety analysis of z for male balb/c mice. balb/c mice were injected with z ( mg/kg/day, i.v.) for days (n = ), and another group of mice (n = ) received pbs as a control. (a) body weight change of balb/c mice at different time points. data were presented as means ± sem. concentration of (b) testosterone and (c) inhibin b in sera before and after z injection. data were presented as means ± sem of triplicate experiments. (d) histological analysis of the testis and epididymis collected from z -or pbs-treated male balb/c mice. scale bar: µm. (e) z -specific antibody response in mice days after i.v. administration of z or pbs. (f) z -apecific antibody response in mice days after i.v. administration of z or pbs. each sample was tested in triplicate and the data were presented as mean ± sd. adjuvant. therefore, z is safe for male mice, especially for their testicular tissue. currently, many studies have reported the deleterious effects of zikv on male testicular tissue, causing severe damage of testis and epididymis, even leading to infertility uraki et al., ) . two dna vaccines were reported to reduce zikv persistence in the testicular tissue and zikv-associated pathological lesion (griffin et al., ) , or partially prevent infertility of male mice (de la vega et al., ) . however, no effective and safe antiviral agent has ever been reported to prevent or treat zikv infection in testicular tissue. our previous study has demonstrated that z peptide is highly effective in inhibiting zikv infection in vivo and in vitro (yu et al., ) . noticeably, it can penetrate the placental barrier to inhibit vertical transmission of zikv in pregnant mice. however, whether z could cross the btb and protect testicular tissue against zikv infection remained unknown. several studies have reported that sertoli cells play an important role in the entry of zikv into the seminiferous tubules and support long-term replication of zikv in the testicular tissue (siemann et al., ; kumar et al., ) . we found that z peptide possesses potent antiviral activity against zikv infection in bhk- and vero cells (yu et al., ) . in this study, we found that z was also highly effective in inhibiting infection of divergent zikv strains with asian and african lineages in tm cells, the mouse sertoli cell line. particularly, z treatment via intraperitoneal injection resulted in dramatically decreased zikv rna level in the testis of a mice, suggesting that z can protect testis against zikv infection in sertoli cells. meanwhile, we employed z -cy to examine whether z could enter seminiferous tubule, and we found that intraperitoneally injected z could be distributed in the testicular tissue of male a mice, consistent with the observation in mice intravenously administered with z (yu et al., ) . however, because of the intricate structure of capillary vessel and seminiferous tubule in mouse testis, we could not obtain sufficient evidence to prove that z crossed btb into seminiferous tubule. h&e analysis showed no obvious pathological damage in the testicular tissue of z -treated mice, but it did reveal severe pathological damage in the testis and epididymis of vehicle-treated mice, consistent with the findings of other studies (govero et al., ; ma et al., ) . when combined with evidence that viral load in mature sperm of z -treated a mice was significantly decreased, we speculate that z may, indeed, cross the btb and enter seminiferous tubule to inhibit zikv infection in the sperm. zika virus infection in the testicular tissue not only damages male testicular tissue, resulting in pathological lesion of testes and epididymides, but also produces zikv-infected semen, causing infertility. in addition, zikv in semen of an infected male can be sexually transmitted to his pregnant partner (russell et al., ; nelson et al., ) , who can further pass the virus to her fetus, causing congenital zika syndrome in the newborn (yarrington et al., ) . sexual transmission may also contribute to the spread of zikv in regions where the aedes mosquito is not endemic (rowland et al., ) . here we found that z treatment could significantly reduce viral load in sperm of zikv-infected a mice and improve the number and motility of sperm, implying that application of z can limit the damage to testicular tissue and sperm caused by zikv infection and reduce the risk of sexual transmission of zikv. conclusion z administered via intraperitoneal or intravenous injection could be distributed in mouse testicular tissue, protect the tissue against zikv infection and zikv-induced pathological damage and poor sperm quality, suggesting that z peptide has the potential to be further developed as an anti-zikv therapeutic for treatment of zikv infection and attenuation of zikv-induced damage in the testicular tissue. all datasets generated for this study are included in the manuscript/supplementary files. the animal study was reviewed and approved by shanghai public health clinical center animal welfare and ethics committee institutional laboratory animal care and use committee at fudan university. probable sexual transmission of zika virus from a vasectomised man potential effect of zika virus infection on human male fertility? virus and antibody dynamics in travelers with acute zika virus infection an update on zika virus infection zika virus: a new threat to human reproduction evidence of perinatal transmission of zika virus guillain-barre syndrome associated with zika virus infection novel antiviral activity and mechanism of bromocriptine as a zika virus ns b-ns protease inhibitor the blood-testis barrier and its implications for male contraception a review of the ongoing research on zika virus treatment zika-induced male infertility in mice is potentially reversible and preventable by deoxyribonucleic acid immunization adenosine analog nitd is a potent inhibitor of zika virus isolation, identification and genomic characterization of the asian lineage zika virus imported to china frequent zika virus sexual transmission and prolonged viral rna shedding in an immunodeficient mouse model from "a"iv to "z"ikv: attacks from emerging and re-emerging pathogens persistence and infectivity of zika virus in semen after returning from endemic areas: report of cases classification of mouse sperm motility patterns using an automated multiclass support vector machines model zika virus: origins, pathological action, and treatment strategies zika virus infection damages the testes in mice dna vaccination protects mice against zika virus-induced damage to the testes zika virus infection at mid-gestation results in fetal cerebral cortical injury and fetal death in the olive baboon therapeutic treatment of zika virus infection using a brain-penetrating antiviral peptide effect of acute zika virus infection on sperm and virus clearance in body fluids: a prospective observational study transport of igg across the blood-luminal barrier of the male reproductive tract of the rat and the effect of estradiol administration on reabsorption of fluid and igg by the epididymal ducts human sertoli cells support high levels of zika virus replication and persistence -hydroxycholesterol protects host against zika virus infection and its associated microcephaly in a mouse model zika virus causes testis damage and leads to male infertility in mice zika virus: high infectious viral load in semen, a new sexually transmitted pathogen? zika virus infects human testicular tissue and germ cells zika virus as a sexually transmitted pathogen knowledge of the sexual transmission of zika virus and preventive practices against zika virus among u.s. travelers guillain-barre syndrome associated with zika virus infection in brazil: a cost-of-illness study rapid spread of zika virus in the americas-implications for public health preparedness for mass gatherings at the brazil olympic games zika virus re-visiting the evolution, dispersal and epidemiology of zika virus in asia the emergence of zika virus and its new clinical syndromes zika virus infection in semen: a call to action and research male-to-female sexual transmission of zika virus-united states an update on sexual transmission of zika virus zika virus infects human sertoli cells and modulates the integrity of the in vitro blood-testis barrier model ebselen alleviates testicular pathology in mice with zika virus infection and prevents its sexual transmission the . Å resolution cryo-em structure of zika virus human testicular organoid system as a novel tool to study zika virus pathogenesis drug transporters, the blood-testis barrier, and spermatogenesis transfusion-transmitted zika virus infection in pregnant mice leads to broad tissue tropism with severe placental damage and fetal demise late sexual transmission of zika virus related to persistence in the semen zika virus causes testicular atrophy a human bi-specific antibody against zika virus with high therapeutic potential structural basis for neutralization and protection by a zika virus-specific human antibody b -h promoted proliferation of mouse spermatogonial stem cells via the pi k signaling pathway a pancoronavirus fusion inhibitor targeting the hr domain of human coronavirus spike congenital zika syndrome arising from sexual transmission of zika virus, a case report a peptide-based viral inactivator inhibits zika virus infection in pregnant mice and fetuses neutralization mechanism of a highly potent antibody against zika virus we are very grateful to the staff at the animal experiment department of shanghai public health clinical center for their contribution to this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -ena usqv authors: long, rory k. m.; moriarty, kathleen p.; cardoen, ben; gao, guang; vogl, a. wayne; jean, françois; hamarneh, ghassan; nabi, ivan r. title: super resolution microscopy and deep learning identify zika virus reorganization of the endoplasmic reticulum date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ena usqv the endoplasmic reticulum (er) is a complex subcellular organelle composed of diverse structures such as tubules, sheets and tubular matrices. flaviviruses such as zika virus (zikv) induce reorganization of endoplasmic reticulum (er) membranes to facilitate viral replication. here, using d super resolution microscopy, zikv infection is shown to induce the formation of dense tubular matrices associated with viral replication in the central er. viral non-structural proteins ns b and ns b associate with replication complexes within the zikv-induced tubular matrix and exhibit distinct er distributions outside this central er region. deep neural networks trained to identify zikv-infected versus mock-infected cells successfully identified zikv-induced central er tubular matrices as a determinant of viral infection. super resolution microscopy and deep learning are therefore able to identify and localize morphological features of the er and may be of use to screen for inhibitors of infection by er-reorganizing viruses. the endoplasmic reticulum (er) is a highly dynamic network composed of - nm ribosome- studded rough er sheets and convoluted networks of smooth er tubules ( , ) . er shaping proteins such as the lumenal sheet spacer protein cytoskeleton-linking membrane protein (climp- ), in order to study er morphology following zikv infection, we first generated stable u glioblas- for hours. cells were fixed with % paraformaldehyde/ . % glutaraldehyde to preserve er archi- tecture ( , , , ) and labeled for dsrna, a marker for zikv replication factories ( ). maximum figure . d sted microscopy reveals zikv-induced er reorganization in human u glioblastoma cells. a) ermoxgfp or sec β-gfp stably transfected u cells were mock-infected or infected for hours with the prvabc zikv strain (moi= ). er reporter gfp and immunostained dsrna-labeled zikv replication factories were imaged by d sted microscopy. b) fluorescence intensity of ermoxgfp of infected cells using a spectrum heat map and a segmentation mask of the er that colocalizes with dsrna (grey), both generated on imaris x . . (imaris), are depicted. yellow squares in the panels indicate the magnified rois shown in the adjacent panels. quantification of the mean normalized er density ((intensity sum of mask/total cell intensity sum)/ (volume sum of mask/ total cell volume sum)) was performed for both dsrna-positive and dsrna-negative ermoxgfp and sec β-gfp in zikv-infected cells by imaris segmentation. scale bar= microns. cells per biological replicate (n= ). statistics were done using unpaired student's t tests: **= p< . , error bars represent sem. were imaged by d sted microscopy. magnified rois (yellow rois identified by red roman numerals) show that the per extends over - sections ( nm step size) and cer > sections. graph shows average per and cer z-height for each ermoxgfp or sec β-gfp labeled cell. a z-height cutoff of . microns (red line) was used to identify per and cer objects. b) segmented er labeling from -hour zikv-or mock-infected ermoxgfp or sec β-gfp stably transfected u cells (moi= ) was visualized using a z-height spectrum heat map and cer (green; > . µm) and per (red; < . µm) masks are shown. c) volume percentage (left) and mean normalized density (right) of cer and per masks between mock-and zikv-infected cells. cells per biological replicate were analyzed for a total of n= . anova with post-hoc tukey hsd: *= p< . , **= p< . , and ***= p< . . error bars represent sem. scale bar= microns. (green) and per (red) masks overlaid with the dsrna-positive er mask (white) for zikv-infected ermoxgfp and sec β-gfp transfected u cells. enlarged images of ermoxgfp transfected cells show x µm rois of the mock-infected cer (green box) and of the zikv-infected dsrna-positive (red box) and dsrna-negative (yellow box) cer shown in b. graphs show the volume percent of the cer or per region that contains dsrna-positive er (left) and the volume percent of the dsrna-positive er that resides within the cer mask. b) d images of er (white) and dsrna (red) labeling in x µm rois of the zikv-infected dsrna-positive (red) and dsrna-negative (yellow) cer and mock-infected (green) cer are shown above imaris d surface rendering of x µm regions of the above rois. graph shows mean normalized er density for each of the three cer zones by imaris segmentation and masking. cells per biological replicate were analyzed for a total of n= . anova with post-hoc tukey hsd: *= p< . , **= p< . , and ***= p< . . error bars represent sem. scale bar: microns ( nm for zoomed rois). projections of d sted image stacks show high intensity ermoxgfp and sec β-gfp labeling in a cer region and low intensity labeling in per tubules in mock-infected cells ( figure a ), as reported previously by diffraction limited confocal microscopy ( ). upon zikv infection, the cer reorganizes to form an intensely labeled crescent-shaped region surrounding a lower intensity perinuclear re- gion ( figure a) . interestingly, the crescent-shaped zikv-induced perinuclear er overlapped exten- sively with dsrna ( figure a ). imaris bitplane software fragments the er into distinct segments that can then be analyzed for different features, including reporter density, segment z-height and segment overlap with other labels, such as dsrna. density-based segmentation of the ermoxgfp- and sec β-gfp-labelled er of zikv-infected cells showed that the higher density crescent-shaped cer region exhibited significant overlap with dsrna-positive er structures relative to the rest of the er ( figure b ). this suggests that zikv dsrna associates with an er region of high density for both lumenal and membrane er reporters. figure . ultrastructural analysis of zikv-infected cerebral brain organoids. transmission em images of nm thin sections of -hour mock-and zikv-infected cerebral brain organoids (moi= ). yellow boxes show rois shown of adjacent higher magnification images that highlight rough er sheets in mock-infected and tubular matrices (convoluted membranes) in zikv-infected cells. scale bars: nm and nm for zoomed image rois. we then investigated the relation- figure d ). overlaying the cer and per masks with the dsrna-positive er mask showed that the dsrna- positive er (> % volume/volume) is predominantly included within the cer mask ( figure a ). in- deed, only % of per volume contains dsrna while % of cer volume contains dsrna for both er reporters ( figure a ). morphological comparison of the dsrna-positive and -negative cer of zikv-infected cells with the cer of mock-infected cells showed that the cer was composed of a convoluted network of tubules for both the ermoxgfp-and sec β-labeled er ( figure b ). d re- constructions confirmed that these regions were predominantly tubular with a few small sheet-like structures, similar to the tubular matrix morphology of peripheral sheets ( ). d voxel-based visu- alization and quantification showed that the density of er tubular structures in the dsrna-positive er is higher, for both the ermoxgfp or sec β-gfp er reporters, than in the dsrna-negative cer regions of zikv-infected cells or the cer of mock-infected cells ( figure b ). the lower er reporter density reflects reduced spacing between tubules in the dsrna-positive er, suggesting that zikv infection induces tubular matrix reorganization in a subdomain of the cer in u cells. consis- tently, em analysis of the microcephaly relevant cerebral brain organoid model ( ) showed that zikv-induced er reorganization from perinuclear stacked rough er sheets to a perinuclear, circular region of convoluted smooth er tubules (figure ). these results are consistent with zikv induction of a perinuclear tubular matrix. er localization of zikv ns b and ns b structural proteins we then labeled cells for zikv ns proteins ns b and ns b to assess their relationship to the zikv- induced tubular matrix. d sted analysis showed a predominant distribution of both ns b and ns b to the cer and more particularly to the dense zikv-induced crescent-shaped tubular matrix in ermoxgfp transfected u cells ( figure a ). while ns b is predominantly associated with the dsrna-positive cer, ns b labeling extended throughout the cer as well as to the per ( figure a ). to quantify this, we identified ns b-positive and ns b-positive er segments and determined their overlap with total er, cer and per ( figure a ). while ns b was present at high levels on both per and cer segments, ns b was enriched in the cer relative to the per and presented a similar er distribution to dsrna ( figure a ). the majority (> %) of dsrna-labeled puncta were associated with ns b or ns b, consistent with the presence of both these ns proteins in the zikv-induced tubular matrix. in contrast, a minority of ns b (~ %) and ns b (~ %) spots overlapped with dsrna spots ( figure b ). in the dsrna-positive cer, the highly punctate ns b labeling differed from a more reticular ns b label- ing. these two zikv ns proteins therefore exhibit distinct distributions within the zikv-induced tubular matrix when not associated with dsrna replication complexes ( figure b ). together with the differential distribution of ns b and ns b within the er as a whole ( figure a ), these results highlight that these two zikv ns proteins do not associate exclusively with replication factories and suggest that following synthesis of the zikv polyprotein, ns b and ns b undergo distinct biosyn- thetic pathways before reuniting in er-associated replication complexes. are required by non-deep learning methods; and ) provide the ability to move beyond simple classification to inspect discriminative regions (i.e. subregions of the er within each cell). we therefore applied deep neural networks to identify and distinguish the morphological fea- tures of the er of zikv-infected cells. a pipeline outlining our approach is shown in figure a . we train a cnn using d frames (each representing a single z-frame) from d sted volumes of er- figure . er distribution of zikv ns b and ns b proteins. a) mock-or zikv-infected ermoxgfp (red) stably transfected u cells were immunostained for zikv ns b or ns b (green) and dsrna (white). merged images show ns b or ns b (green) overlaid with the cer, per or dsrna-positive er (white). graphs show quantification of volume percent of ns b, ns b and dsrna er regions relative to total er, cer or per. b) mock-or zikv-infected ermoxgfp stably transfected u cells were immunostained for zikv ns b or ns b (green) and dsrna (red). white squares show rois of adjacent zoomed images in which white arrowheads show colocalization between ns protein and dsrna puncta. graphs show percent of dsrna puncta overlapping ns b or ns b puncta (left) and percent of ns b or ns b puncta overlapping dsrna puncta. cells per biological replicate (n= ) with each dot representing a cell. anova with post-hoc tukey hsd: *=p< . , **=p< . , and ***=p< . . error bars represent sem. all images are maximum projections from d sted stacks. scale bar= microns. roi scale bar = microns. rate of networks when dealing with small target datasets. certain filters (combinations of weights) learned on the first dataset (i.e. imagenet) may still be useful for classifying a second dataset (i.e. sted); as a result, less weight updates are needed before achieving good performance. us- ing a pretrained vgg as our base model we obtained a % boost in test accuracy, compared against a random weight initialization. using ermoxgfp labelled er alone, the cnn was able to distinguish between zikv-and mock-infected cells with an % accuracy ( figure b, figure . deep learning classification pipeline: pretrained convolutional neural network accurately predicts labels of d frames from d sted volumes. a) leave-one-out cross validation is successively applied to each cell. this prevents information from d frames leaking between training, validation and test sets. during training, network uses d frames from cells (specifically, for training and for validation). the trained cnn then predicts a class label (i.e. zikv-infected or mock-infected) to each d frame of the remaining test cell. class activation maps are also generated for each d frame belonging to the test set. b) cnn performance reported on a cell basis and across d frames. normalized confusion matrices report the total number of predicted labels (zikv-infected or mock-infected) over the total number of ground truth labels. for example, % of all mock-infected d frames were predicted correctly by the cnn (top right). predicted cell labels correspond to the majority label of predicted frame labels for each cell (top left). when excluding frames beyond the cell with reduced ermoxgfp signal, performance metrics increase both in terms of cell label predictions (bottom left) and individual frame label predictions (bottom right). the remaining cell. this process is outlined in figure a , and is repeatedly applied using each cell show poor accuracy to predict class label (supp fig ) . when considering those frames containing ermoxgfp signal intensity greater than the median, we achieved % accuracy and % sensitiv- ity. on a per frame basis, accuracy for all frames was % and sensitivity % that increased to % and %, respectively, when considering frames expressing ermoxgfp greater than median this suggests that the cams used to identify both zikv-and mock-infected cells correspond to high er density regions localized over the cell (see figure b ) and that vgg is using differences in the er label (ermoxgfp) to identify slices as either zikv-or mock-infected. figure b, left) . conversely, regions identified by the thresholded mock cam have increased er density for tn compared to tp cells ( figure b, right) . we then calculated the average euclidean figure c ) and the precise nature of the features that the cnn uses to discriminate between zikv-and mock-infected cells remains to be determined. cam localization analysis shows that the neural network uses the same cer region that we have observed to be modified upon zikv infection. deep learning therefore has the ability to identify the er morpho- logical changes associated with zikv infection. zikv infection is characterized by re-organization of the er to create replication factories and con- voluted er membranes involved in viral replication, whose ultrastructure has been elegantly char- given roi, er density is defined as total ermoxgfp intensity within the roi divided by ermoxgfp area inside the roi. er density for each roi defined by the cam is then normalized by the er density of the whole cell. a) er density of rois defined by cam thresholds from - % with increments of % is compared across subgroups: zikv-infected d frames correctly predicted to be infected (true positives); mock-infected d frames correctly predicted to be uninfected (true negatives); zikv-infected d frames incorrectly predicted to be uninfected (false negatives); mock-infected d frames incorrectly predicted to be infected (false positives). b) er densities of % cams rois compared across subgroups. c) euclidean distances between center of mass of % cams rois and weighted center of mass of ermoxgfp signal. ( , ). the er is a morphologically complex organelle, containing smooth er tubules and ribosome- studded rough er sheets identified ultrastructurally by em since over years ( networks that correspond to previously described per tubular matrices ( ). while we were unable to detect er sheets by super-resolution analysis of cultured u cells, em of brain organoids shows the transformation of er sheets to convoluted membranes upon zikv infection. this suggests that organoid structures present more highly developed er structures than cultured cells; application of d live cell super-resolution analysis ( ) to this model of the developing fetal brain, composed of a heterogenous population of cell types, may lead to better definition of complex er structures and their dynamic transitions in response to stress, such as viral infection. nevertheless, the fixed cell d sted analysis applied here demonstrates that convoluted membranes associated with zikv replication derive from tubular matrix reorganization in the cer. the zikv-induced cer-localized, high er density tubular matrices are enriched for dsrna. as formance for d super-resolution microscopy data sets that will be of service to other researchers applying deep learning approaches to super-resolution microscopy. the interpretability of artificial intelligence is an evolving field and we believe that interpretable methods, such as grad-cam ( ), are important tools for the understanding of deep neural net- works applied to exploratory data sets. this approach has now allowed us to identify features of discriminatory regions, and has not, to our knowledge, been applied to subcellular morphology, figure . network performance analysis a) performance metrics are reported across predictions of frame labels and cell labels, where cell labels correspond to the majority label of predicted frame labels for each cell. results are reported using a given selection criteria: using all frames (rows , ), using only frames with normalized ermoxgfp signal greater than mean normalized ermoxgfp signal (rows , ) or greater than median normalized ermoxgfp signal (rows , ). mean and median thresholds are computed on a cell basis. the rug plot (above x-axis) visualises distribution of z-frames. b) accuracy reported across corrected z-frames, z= is where normalized ermoxgfp intensity sum is maximal. c) normalized ermoxgfp intensity sum plotted against corrected z-frame. dashed lines indicate the median (orange) and mean (red) normalized ermoxgfp intensity sum computed across all frames. microtubules and the endoplasmic reticulum are highly inter- dependent structures rough sheets and smooth tubules mechanisms determining the morphology of the peripheral er a class of membrane proteins shaping the tubular endoplasmic reticulum dynamic nanoscale morphology of the er surveyed by sted microscopy reticulon and climp- control nanodomain organization of pe- ripheral er tubules increased spatiotemporal reso- lution reveals highly dynamic dense tubular matrices in the peripheral er architecture and biogenesis of plus-strand rna virus replication fac- tories rig-i-like receptors: cytoplasmic sensors for non-self rna molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) hostile takeover: hijacking of endoplasmic reticulum function by t ss and t ss effectors creates a niche for intracellular pathogens endoplasmic reticulum: the favorite intracellular niche for pathogen-endoplasmic-reticulum interactions: in through the out door immunolocalization of the dengue virus nonstructural glycoprotein ns suggests a role in viral rna replication human coronavirus: host-pathogen interaction zika virus associated with meningoencephalitis . fauci as, morens dm. zika virus in the americas-yet another arbovirus threat ultrastructural characterization of zika virus replication factories cytoarchitecture of zika virus infection in human neuroblastoma and aedes albopictus cell lines virus in human fetal neural progenitors persists long term with partial cytopathic and limited molecular aspects of dengue virus replication genome sequence of a zika virus strain isolated from the serum of an infected patient in thailand in rewiring cellular networks by members of the flaviviridae family the host protein reticulon is utilized by flaviviruses to facilitate membrane remodelling virus perturbs mitochondrial morphodynamics to dampen innate immune responses. cell host microbe composition and three- dimensional architecture of the dengue virus replication and assembly sites zika ns b is a crucial factor recruiting ns to the er and activating its protease activity a palette of fluorescent proteins optimized for diverse cellular environments ligand-induced redistribution of a human kdel receptor from the golgi complex to the endoplasmic reticulum a conserved er targeting motif in three families of lipid binding proteins and in opi p binds vap using brain organoids to understand zika virus- induced microcephaly imagenet classification with deep convolutional neural networks very deep convolutional networks for large-scale image recognition. arxiv preprint arxiv rethinking model scaling for convolutional neural networks skin lesion analysis toward melanoma detection: a challenge at the international symposium on biomedical imaging (isbi), hosted by the international skin imaging collaboration (isic) a dataset for breast cancer histopathological image classification learning deep features for discriminative computer vision and pattern recognition evaluating cnn-based semantic food segmentation across illuminants a novel segmentation framework for uveal melanoma in magnetic resonance imaging based on class acti- vation maps coronavirus infection, er stress, apoptosis and innate immunity. front micro- biol studies on the endoplasmic reticulum. i. its identification in cells in situ applying systems-level spectral imaging and analysis to reveal the organelle interactome atlastin en- doplasmic reticulum-shaping proteins facilitate zika virus replication er-shaping atlastin proteins act as central hubs to promote flavivirus replication and virion assembly crystal structure of unlinked ns b-ns protease from zika virus flaviviral ns b, chameleon and jack-in-the-box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention zika virus ns a and ns b proteins dereg- ulate akt-mtor signaling in human fetal neural stem cells to inhibit neurogenesis and induce knowledge transfer for melanoma screening with deep learning texture analysis for muscu- lar dystrophy classification in mri with improved class activation mapping. pattern recognition breast cancer histology images classification: training from scratch or transfer learn- ing? ict express grad-cam: visual explanations from deep networks via gradient-based localization form follows function: the importance of endoplas- mic reticulum shape defining host-pathogen interactions employing an artificial intelligence workflow. elife sars- coronavirus replication is supported by a reticulovesicular network of modified endoplasmic retic- ulum ca + signaling machinery is present at intercellular junctions and structures associated with junction turnover in rat sertoli cells key: cord- -ewnf cz authors: srivastava, mayank; zhang, ying; chen, jian; sirohi, devika; miller, andrew; zhang, yang; chen, zhilu; lu, haojie; xu, jianqing; kuhn, richard j.; andy tao, w. title: chemical proteomics tracks virus entry and uncovers ncam as zika virus receptor date: - - journal: nat commun doi: . /s - - -y sha: doc_id: cord_uid: ewnf cz the outbreak of zika virus (zikv) in created worldwide health emergency which demand urgent research efforts on understanding the virus biology and developing therapeutic strategies. here, we present a time-resolved chemical proteomic strategy to track the early-stage entry of zikv into host cells. zikv was labeled on its surface with a chemical probe, which carries a photocrosslinker to covalently link virus-interacting proteins in living cells on uv exposure at different time points, and a biotin tag for subsequent enrichment and mass spectrometric identification of the receptor or other host proteins critical for virus internalization. we identified neural cell adhesion molecule (ncam ) as a potential zikv receptor and further validated it through overexpression, knockout, and inhibition of ncam in vero cells and human glioblastoma cells u- mg. collectively, the strategy can serve as a universal tool to map virus entry pathways and uncover key interacting proteins. z ika virus (zikv) has been the focus of immense investigation since the recent epidemic. while studies on zikv structure revealed its structural similarity to other virus relatives within the family flaviviridae, some notable differences such as the asn glycosylation site present on each of the e proteins may affect receptor interactions, antibody response, and downstream biology of the virus , . for the past several years, efforts have been made to understand fundamental biology of zikv infection, including the use of animal models to evaluate their immune response to the virus invasion , as well as potential therapeutics against zikv infection [ ] [ ] [ ] . however, unanswered questions remain regarding molecular mechanisms of host restriction and immune evasion . identification of direct interactors of zikv during its entry into host cells could not only suggest the molecular pathways manipulated by the virus, but also provide immense opportunity to develop antivirals by offering new potential drug targets. owing to the transient nature of these interactions and the extreme rapidness in the flavivirus entry in general, identification of dynamic interactors of virus is a formidable task. our understanding of zikv internalization and cellular trafficking would greatly benefit from a systematic, temporal characterization of major proteins involved in the dynamic virus entry. real-time fluorescence microscopy has been used to study the transport, acidification, and fusion of single virus , . the movement of single virus demonstrated an intriguing and dynamic process. the molecular information, in particular protein machinery involved in the process, is typically limited to labeled molecules in the amazing technique. on the other hand, affinity and chemical proteomics studies identified virusinteracting proteins [ ] [ ] [ ] . the molecular mechanisms and dynamic virus-host interactions responsible for the internalization of zikv, however, have remained unresolved. a systematic quantitative measurement of temporal changes in virus-protein interactions may prove extremely valuable for the identification of host molecules as potential therapeutic targets. we have previously used chemical proteomics strategies and modified a nanoparticle, polyamidoamine generation dendrimer, to understand the endocytic pathways of a nanoparticle followed by a recent study to investigate the entry of salmonella into host cells . here, we expand the concept and hypothesize that chemical modification of zikv would not significantly affect its infectivity and would allow us to track the virus entry into living cells and identify virus-interacting proteins by mass spectrometry (ms), revealing the spatiotemporal distribution of the key proteins involved in the pathways for zikv entry and trafficking. synthesis and characterization of zikv-labeling probe. we devised and synthesized a multifunctional chemical probe ( fig. a; supplementary figs. - ) bearing a labeling group that conjugates the probe to the zikv surface, a photo-reactive group that allows for covalent crosslinking of zikv proteins to interacting host cell proteins upon uv exposure, and an isolation tag of biotin for purifying the interacting proteins for quantitative ms analysis, thus facilitating the investigation of host-pathogen interactomes in a time-resolved manner (fig. b) . we chose the maleimide group to label the virus through its specific conjugation with thiol groups on the virus surface proteins at physiological condition to form a stable thioether linkage. as sulfhydryls thiols are present in most proteins but are not as abundant as primary amines, we expected limited labeling on cysteine residues would have a minimal labeling effect on the zikv activity. according to the structure of mature zikv determined by cryoelectron microscopy by our group , there are cysteines in zikv e ( in the ectodomain, in the transmembrane domain) and no cysteine in m protein (supplementary fig. a ; cysteine residues are highlighted as gray spheres in the structures). in addition, zikv, like other flaviviruses and enveloped viruses in general, is quite unstable and prone to undergo structural changes under external influence . considering the virus stability and infectivity, we preferred minimal labeling of virus through the maleimide-thiol conjugation under mild conditions at neutral ph. moreover, the three functionalities are separated by a polyethylene glycol (peg)-like linker to improve water solubility, while offering the flexibility for efficient crosslinking and enrichment. the labeling was first examined with a standard protein and then with intact zikv. bovine serum albumin (bsa) was incubated with mm of reagent in phosphate buffer ph at °c and the labeled protein was enriched on streptavidin beads and analyzed by sds-page. the labeling efficiency was estimated to be - % ( supplementary fig. b ). after labeling, modified zikv was lysed, and the labeled zikv surface proteins were purified on streptavidin beads and assessed by silver stain and western blotting using the g antibody against the e protein ( supplementary fig. c ). we further analyzed the captured proteins using ms. multiple unique peptides from e protein were identified by ms ( supplementary fig. ). we did not identify any peptide from virus membrane (m) protein, capsid (c), or any of the nonstructural proteins of zikv, further confirming the exclusive tagging of virus surface with the reagent. this result is primarily owing to the membrane impermeable attribute of the chemical proteomic probe imparted by the pegylated linkers. finally, we examined the effect of labeling purified zikv with several concentrations and labeling time points by the plague assay. no loss of infectivity was observed under labeling conditions for mm reagent concentration ( supplementary fig. d ). hence, we concluded that the minimal labeling of virus achieved by cysteine-reactive maleimide group does not perturb the infectivity of the virus. chemical proteomics to track the early-stage entry of zikv. we used the labeled zikv to infect vero cells and interacting proteins were crosslinked at fixed time points to identify the virus-host factors and elucidate the virus entry mechanism (fig. c) . flavivirus are quite promiscuous in their selection of receptors for entry to different cells. the complex entry mechanism might involve multiple receptor interactions to help virus internalize. though some previous studies have identified axl, a tam family tyrosine kinase, as a putative receptor for zikv , some conflicting evidence including ours suggests that the virus might employ multiple different classes of receptors for entry , . furthermore, while most of the viruses are believed to enter cells by clathrin-mediated endocytosis, there is no evidence suggesting the absence of any parallel mode of virus entry. the complexity of the flavivirus entry mechanism hints at the presence of varied virus-protein interactions after membrane recruitment of host cellular proteins to initiate viral infection. in order to identify zikv receptors, we allowed the virus to attach to the cells at °c for h, followed by uv photo crosslinking on ice. for the virus entry, we chose and min according to a previous study that indicates the membrane fusion of a similar flavivirus at s post binding, during its entry into vero cells . after crosslinking proteins at designated time points of attachment or entry, cells were harvested and proteins were extracted, followed by the enrichment using avidin beads. we reasoned that the chemical proteomic probe on the virus surface only crosslinks with proteins in direct contact with zikv, which can subsequently withstand vigorous washing conditions to remove nonspecifically bound proteins. the tryptic peptides derived from enriched samples were then analyzed by nanoflow hplc coupled to high-resolution ms. proteins were identified by a shotgun proteomic strategy and quantitated using the label-free method to measure their relative abundance across three time points and to distinguish crosslinked proteins from nonspecifically bound proteins. in total, we identified around crosslinked proteins across three time points, out of which more than proteins are previously implicated in virus infection ( fig. a and supplementary data ). the principal component analysis (pca) shows that all biological replicates are tightly clustered together and each time point is well-separated, meaning the samples are clustered by the nature of sample but not the batch (fig. b) . the pca and heatmap analysis also indicates that distinctive proteins were crosslinked at three different time points, suggesting that the strategy was able to reveal the temporal distribution of the interacting proteins crosslinked with zikv during the virus' early entry (fig. b, c) . gene ontology (go) analysis, as expected, indicated proteins annotated as membrane and extracellular region were significantly overrepresented in the crosslinked proteins across all time points (fig. d) . to further investigate whether the strategy was also capable of correlating spatial information with the virus crosslinked proteins, we performed the string analysis to determine whether there is statistical overrepresentation of specific genes or proteins in the sample at specific time points and identify proteins specific at the attachment or cellular entry stages ( supplementary fig. ). notably, we identified crosslinked proteins at min, of which were membrane proteins, such as c qbp, cd , itga , letm , ncam , rack , and slc a . complement c q binding protein is a key host factor for efficient respiratory syncytial virus (rsv) production . the tetraspanin cd facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases . e-syt proteins were discovered to impact the formation of virus-induced syncytia during hsv- infection . integrin α directly interacts with hepatitis e virus (hev) and plays a key role in cellular attachment and entry of nehev . receptor of activated protein c kinase (rack ) is important for lymphocystis disease virus entry and infection . f cell-surface antigen heavy chain (slc a ) was reported interacting with zikv ns b protein and was indicated as a candidate host factor for zikv infection . in this study, coimmunoprecipitation followed by western blotting of transduced cells verified that slc a specifically associates with zikv env (supplementary fig. a) . interestingly, neural cell adhesion molecule (ncam ), which was reported as a receptor for rabies virus , was identified at different time points and presents as a candidate receptor for zikv infection. previous analysis of the global proteomic changes that occurred during differentiation of labeled zikv was diluted in dmem and incubated with confluent cells for h at °c. in addition, cells were incubated with the labeled viruses in °c for fixed time points to allow virus entry. unbound viruses were removed and cells were directly exposed to uv light. cells were lysed and biotinylated proteins were captured on the avidin beads. proteins were digested on beads using sequential lys-c and trypsin digestion, and analyzed by lc-ms/ms. label-free quantitation was performed using maxquant to identify and quantify the crosslinked proteins. nature communications | https://doi.org/ . /s - - -y article hnpcs into neurons also revealed significant upregulation of ncam . moreover, a few other noticeable proteins crosslinked specifically at min include ap m and calml . ap m is the µ subunit of adaptor protein- (ap- ) complex that recognizes the tyrosine-rich sorting signals on the cytoplasmic tail of receptor proteins . our crosslinking experiment validates the complex's structural assembly on the membrane as ap m was selectively crosslinked at min of infection, while no other ap- subunits were observed. ap m has further been shown to modulate early-stage infectious entry of hcv, in its phosphorylated form . calml was discovered as a potential host factor for zikv infection . we further demonstrated that the strategy allowed us to identify clusters of proteins representing a temporal shift of zikv subcellular localizations and the functions of crosslinked proteins are highly correlate to the temporal information in many cases (fig. , supplementary data ). for example, at min, we observed certain proteins enriched such as stat , a key mediator of type-i interferon signaling. it has been reported that zikv suppresses the host immune response by inhibiting the type-i interferon signaling pathway . previous studies also have suggested the mechanism of blocking immune response by zikv as either the degradation of signal transducer and activator of transcription (stat ) , or antagonism of stat and stat phosphorylation . rack , which mediates the interactions between ifn receptor and stat , was also identified as a zikv-interacting protein at that time point. previously, a different class of virus was shown to interact with rack , thus initiating the dissociation of rack -stat complex and inhibition of interferon signaling by the virus . our study emphasizes the involvement of both stat and rack in the immune response elicited by the zikv infection, suggesting zikv might employ a similar approach to suppress the host immune response. we also identified an isoform of a key component of recycling endosomes, rab a. the role of rab a in transport of viral ribonucleoprotein or core proteins to plasma membrane for the generation of new virus particles of influenza and hepatitis c virus (hcv), respectively, is well documented . however, some viruses may also utilize the recycling endosomal pathway to evade lysosomal degradation. our study suggests the use of the recycling pathway by zikv after entry. besides rab a, we also observed rab c at min. previous studies have established the importance of rab c in the entry of zikv . the identification of rab c and rab a in our chemical proteomics experiment at a late time point of entry is consistent with the published data on japanese encephalitis virus (jev) , a neurovirulent pathogen from the flavivirus genus structurally similar to the zikv. other proteins identified of infection include hspa , also known as heat shock cognate (hsc ), known for its role in vesicle uncoating in the later stage of endocytosis . overall, more than direct interactors such as ap b and itgb were also identified as zikv interactors in previous work , further indicating the confidence of the identified interactors in our study. in addition, we examined temporally zikv-crosslinked proteins against protein components involved in major endocytic mechanisms for zikv internalization. prior studies showed zikv infection could be prevented by lysosomotropic agents which neutralize the normally acidic ph of endosomal compartments and was also blocked by chlorpromazine , , indicating the requirement of clathrin-mediated endocytosis and low ph for zikv infection. in our study, itgb , hspa , rab c, rack , and rab a were crosslinked, suggesting the clathrin-mediated pathway employed by the virus to infect vero cells. furthermore, identification of arcn , itga , flna, and flnc also suggests the utilization of a caveolar-mediated pathway by zikv for endocytosis. identification and verification of ncam as zikv receptor. finally, considering ncam was crosslinked at several time points, totally not detected in control samples and ncam is abundantly expressed in brain, we further examined whether ncam is a potential receptor for zikv infection leading to neurological disorders. to assess the physiological importance of ncam interaction with zikv, we first examined the surface expression of ncam on u- mg and vero cells. we found that ncam was highly expressed on the surface of u- mg and vero cells (fig. a) . the immunofluorescence image also revealed that ncam is located on the membrane of the transfected hek t cells (fig. b) . to validate the interaction between ncam and zikv env, we performed coimmunoprecipitation experiments in the context of zikv env and ncam -flag overexpression. immunoblot analyses revealed that ncam specifically interacted with the zikv env (fig. c ), but not with egfr, another transmembrane glycoprotein as a negative control (fig. d ). in addition, we found that the other host factor identified in our study, hspa , previously reported as a key host factor for zikv infection and zikv nonstructural protein stabilization , also bound to the zikv env protein directly ( supplementary fig. b ). we knocked out hspa in u- mg cells using two different crispr/cas sgrnas. the knockout efficiency by the sgrnas was confirmed with western blot analysis and the efficiency of sgrna is lower than that of sgrna ( supplementary fig. c ). we found that hspa depletion by sgrna in u- mg cells remarkably attenuated zikv infection ( supplementary fig. c ). to further assess the effect of ncam on zikv binding and entry, we employed ncam extracellular domain (ecd) protein and anti-ncam antibody to compete and block ncam binding, respectively, and examined whether it could lead to the reduction in zikv attachment and internalization. preincubation with ncam ecd protein, but not the control protein, remarkably reduced zikv binding (fig. f) and entry (fig. g) into u- mg cells. similarly, pretreatment of u- mg cells with the anti-ncam antibody also reduced zikv binding (fig. f) and entry (fig. g) . ncam ecd and antibody also inhibited zikv binding to vero cells ( supplementary fig. d ), but had no statistical difference on the zikv internalization ( supplementary fig. e ). in this study, we present a chemical proteomic approach in which virus was chemically tagged with a biocompatible probe, to reveal the virus-host interactome in real time. the work demonstrates how chemical proteomics can facilitate our understanding of molecular mechanisms and key players in virus infection. specific identification of virus-interacting proteins was made possible by the integration of several techniques: a multifunctional chemical probe that can achieve the labeling, crosslinking, and isolation steps, photo-reactive crosslinking that allows us to select designated time points and capture interacting proteins covalently to minimize loss during lysis and washing, and label-free ms-based quantitation. in particular, the analyses of proteomic data sets with or without virus infection at different time points enabled the extraction of temporal and spatial information during the virus infection, which provides a useful and universal tool to study any pathogen invasion in theory. compared with the previously reported mass spectrometric methods for the identification of receptors specific to glycoproteins , , our method offers the additional advantage of covalently linking proteins at different time points, thus serving the dual purpose of identification of receptors and other host factors involved at different stages of virus entry. the chemical proteomics strategy was applied to zikv and enabled us to identify multiple zikv-interacting proteins that indicate zikv subcellular localizations and potential entry mechanisms, among which a new zikv receptor was discovered and validated through virus attachment and entry assays. overexpression of ncam in hek t cells increased viral binding and entry. ncam depletion in u- mg cells remarkably inhibited zikv infection. inhibition of ncam receptor by ncam ecd and anti-ncam antibody reduced the zikv attachment and entry into u- mg cells. there was also % of reduction in zikv attachment to vero cells but no statistical significant reduction was observed for the zikv entry into vero cells. we reason that vero cells are less dependent on specific receptors for virus infection and therefore are commonly used as a model cell line for virus infection. on the other hand, zikv infection of u- mg cells are highly dependent on specific receptors. once a specific receptor, e.g., ncam , was blocked, significant reduction in attachment and entry into u- mg cells was observed. to exclude a general effect of ncam on other viruses binding and entry, we performed an inhibition assay using influenza a virus (iav) and dengue virus (denv- ). ncam ecd and anti-ncam antibody had minimal effects on iav binding and entry ( supplementary fig. f , left), but ncam ecd had significant effect on denv binding and anti-ncam antibody had minimal effects on denv- binding and entry ( supplementary fig. f , right), suggesting that ncam may affect the attachment of zikv or denv but not iav. to further confirm the receptor activity of ncam , we overexpressed ncam in hek t cells and the expression efficiency was validated with immunofluorescence imaging (fig. b) and western blotting assays ( supplementary fig. ), and then we performed binding and entry assays. heterologous expression of human ncam in t cells enhanced both zikv attachment (fig. h ) and internalization (fig. i) . to further demonstrate the requirement of ncam for infection by zikv, we knocked out ncam in u- mg cells using two different crispr/cas sgrnas. the knockout efficiency by the sgrnas was confirmed with flow cytometry staining and the efficiency of sgrna (green) is lower than that of sgrna (blue) (fig. j) . we found that ncam depletion by sgrna in u- mg cells remarkably attenuated zikv infection (fig. k, l) . this result supports our observation that ncam could be a potential receptor for zikv infection (supplementary fig. g) . the chemical proteomics strategy highlighted its unique feature that allowed us to track the virus movement in real time, which is challenging due to the highly dynamic nature of the process and the transient virus-host protein interactions. moreover, the crosslinking chemistry permits the identification of potential receptors which present analytical challenges to identify the interaction on cell membrane. lastly, this technology can be applied to relatively unstable enveloped viruses, owing to the minimal labeling by cysteine-reactive maleimide group. briefly, virus particles were precipitated from the media with % polyethylene glycol (peg) overnight at °c, pelleted at , × g for min at °c. resuspended particles were pelleted through a % sucrose cushion, resuspended in . ml nte buffer ( mm tris ph . , mm nacl, mm edta), and purified with a discontinuous gradient in % intervals from % to % k-tartrate, mm tris ph . , mm edta. mature virus was extracted from the gradient, concentrated, and buffer exchanged into nte buffer. plaque assay. the plaque assay was performed as described below. briefly, virus was diluted serially in the order of ten folds and incubated with monolayers of vero cells for h at room temperature. cells were layered with agarose and incubated at °c for days. plaques were counted following cell staining using neutral red. synthesis and purification of a multifunctional chemical probe. the viruslabeling chemical probe was synthesized on the rink-amide-am-resin ( - mesh) % dvb manually, using standard solid phase peptide synthesis approach (supplementary fig. ) . a % piperidine solution in dmf (n,n-dimethylformamide) was used to deprotect the fmoc ( -fluorenylmethoxycarbonyl) groups, while % tfa (trifluoroacetic acid) was used for boc (tert-butoxycarbonyl) group deprotection. hctu (o-( h- -chlorobenzotriazole- -yl)− , , , -tetramethyluronium hexafluorophosphate) was utilized as an activating agent for the carboxyl group on the incoming reactant, in presence of the base nmm ( methylmorpholine). the synthesis was performed on the µmol scale and using . -fold excess of the reagents compared with the resin. each step involved the deprotection of amine group, activation of carboxyl group followed by coupling reaction. the excess reagents were removed by thorough washing of beads by dmf. ninhydrin test was performed after each deprotection and coupling reaction. the synthesis was performed using the strategy, as previously described . mg ( µmol) of rink-amide-am-resin was added to the fritted reaction vessel. beads were conditioned with dmf for min. the solution was removed by filtration, and % piperidine in dmf was added to the beads for fmoc deprotection. the mixture was end-to-end rotated for min, and solution was removed followed by washing of beads with dmf. . , . , . , . , . , . , . , . , . , . , . , . , . the pure maleimide-biotin ( mg, . µmol) was dissolved in dmf and reacted with excess nhs-lc-diazirine (succinimidyl- -( , ′-azipentanamido) hexanoate) in phosphate buffer ph , for h at room temperature. the product was purified by directly injecting into the hplc and using similar conditions as described above. the virus-labeling chemical probe was obtained as a white powder ( . mg, . µmol, %) and characterized by maldi-tof, h and c nmr (supplementary fig. ) . (m, h), . - . (m, h), . - . (m, h), . (s, h) . maldi showed a peak at . m/z, corresponding to the m-n + h + . this is due to the loss of n from diazirine under maldi conditions. bsa labeling/virus labeling. fifty micrograms bsa or purified zikv was diluted to µl with pbs ph , and mixed with the labeling chemical probe in the final concentration of mm (supplementary fig. b-d) . the labeling was carried out by gentle end-to-end rotation in °c overnight. for the infection experiment, virus labeling was initiated a day before the cells reached < % confluency for infection. the reaction was quenched by adding three times excess of cysteine. virus infection and crosslinking of host proteins. vero cells were first grown in t- flasks in dmem supplemented with % fbs, then passaged to the cm plates and grown to < % confluency. cells were washed with cold pbs twice, and cooled down to °c. the labeled virus was diluted in dmem and added to the cells at moi of . cells were gently rocked for h in °c, to allow for virus attachment. for the receptor crosslinking, the unbound virus was removed, cells were washed once with cold pbs, and directly exposed to the uv light for min on ice. all the above operations were performed on ice and using cold pbs to minimize any virus entry. to understand the virus internalization mechanism, additionally virus was allowed to enter cells by incubation in °c for or min, following preattachment for an hour at °c. subsequent to uv photo crosslinking, cells were collected by scraping in pbs, and stored in − °c until further processing. as a control, cells treated with the labeling chemical probe and exposed to uv were included to account for random crosslinking. sample preparation for lc-ms/ms analysis. frozen cells were lysed in % sds, mm tris hcl ph . supplemented with protease inhibitor on ice, using sonication ( cycles for s each, with an interval of s). cell lysates were cleared by centrifugation at , rpm to pellet down cell debris, and supernatant were used for the biotin-neutravidin affinity purification. bicinchoninic acid assay (thermo fisher scientific) was performed for protein quantitation, and the lysates equivalent to mg protein for each sample were reduced and alkylated by boiling at °c, in mm tcep (tris( -carboxyethyl)phosphine) and mm caa (chloroacetamide) respectively. the lysates were then diluted to . % sds and rotated with µl preconditioned neutravidin beads slurry in °c overnight. the beads were washed with . % sds in mm tris (ph . ), m nacl, mm glycerol in mm tris (ph . ), and then transferred to the low protein binding eppendorf tubes, where they were further washed three times with mm ammonium bicarbonate (abc) buffer ph . two hundred microliters abc buffer was added to the beads, and proteins were digested on-bead at °c using µg lys-c for h and ng trypsin for h. the supernatant containing peptides was collected and beads were washed twice with µl abc buffer, further pooled with the supernatant. peptides were acidified and desalted using in-house stagetips with sdb-xc ( m). the peptides were dried in speedvac before subjecting to lc-ms/ ms analysis. lc-ms/ms analysis. the peptides were dissolved in . % formic acid and injected into easy-nlc (thermo fisher scientific). the peptides were separated on a cm in-house column ( µm od × µm id), packed with c resin ( . µm, Å, bischoff chromatography, leonberg, germany) and heated to °c with a column heater (analytical sales and services, flanders, new jersey). the mobile phase was comprised of . % formic acid in ultra-pure water (solvent a) and . % formic acid in % acetonitrile (solvent b), and the gradient used for separation was - % b over a linear min at a flow rate of nl/min. the easy-nlc was connected online to the ltq-orbitrap velos pro mass spectrometer (thermo fisher scientific) by a nanospray source. data acquisition was performed in the data-dependent mode, in which a full scan (range from m/z - with a resolution of , at m/z ) was followed by ms/ms scans of top intense ions (normalized collision energy %, automatic gain control e , maximum injection time ms) with a dynamic exclusion for s and dynamic list of . proteomic data analysis. raw files were processed with maxquant v . . . , and the label-free quantitation (lfq) was performed. the raw data were searched against uniprotkb (rhesus macaque) fasta database (version july , , entries) with andromeda search engine, using default parameters. the first peptide precursor mass tolerance was set at ppm, and ms/ms tolerance at . da. carbamidomethylation was set as a fixed modification for cysteines, while oxidation of methionine and acetylation at n-terminus were selected as variable modifications. enzyme specificity was set to trypsin with maximum two missed cleavages. the search was performed with % false discovery rate at both peptide and protein levels. the identifications were transferred from the sequenced peaks to the unidentified peaks of the same m/z within a time window of . min (match between runs) across samples. for lfq, initially, the lfq intensities were extracted from the maxquant output file. proteins with valid values in minimum two out of three replicates in at least one group were only considered, and values were imputed for all missing values based on normal distribution. significant proteins at different time points vs. control (fc ≥ , t test p ≤ : ) were considered as crosslinked proteins, which were further processed by homemade matlab program for functional analysis. for heatmap analysis, the expression value (ibaq) of total quantified proteins were clustered based on euclidean distances with average linkage using modified function clustergram in matlab (version r b), and heatmap was also visualized by matlab. the color shows that there are great changes from sample to sample, where red is upregulation, green is downregulation, black represents no change, and gray represents non available. each column in the graph represents an experiment condition, and each row corresponds to a gene. row of protein were normalized by maximum value of corresponding row. the rows and columns are displayed in the order given by the clustering output trees in the two dimensions. go enrichment analysis of the differentially expressed proteins were conducted according to the information from go databases, each bar in the figure denotes the enrichment score from different sample, and enrichment score was defined as −log (p). p value was calculated using the hypergeometric formula as below: n is the number of all identified proteins that can be connected with go analysis information. n is the number of differential proteins in n. m is the number of proteins that can be connected with a certain go term. m is the number of differential proteins with certain go term. if p value below . , we regard this go term as a significant enrichment of differential proteins. the information of protein-protein interaction of significant proteins were retrieved by string database, and visualized by cytoscape. viral attachment and entry assay. for antibody inhibition experiment, u- mg cells in twelve-well plate were preincubated with µg/ml anti-ncam antibody (cat. no. bd- , bd) or control isotype igg antibody (cat. no. bd- , bd) in dmem supplemented with % fbs for h at °c. cells were then incubated with purified zikv (moi = ) on ice for h in the presence of antibody. the supernatant was then removed and the cells were washed three times with cold pbs. cellular rna was extracted and purified for test the viral attachment. otherwise, prewarmed medium was added to the cells to initiate zikv internalization. the cells were incubated at °c for additional min and then cellular rna was extracted and purified. then rt-qpcr was performed to measure viral entry. for ncam protein inhibition experiment, purified zikv (pfu = ) were preincubated with µg ncam ecd (sino biological, cat. no. -h h) or control protein for h at °c. the viruses (moi = ) were then added to u- mg cells in twelve-well plate and incubated on ice for h. the supernatant was then removed and the cells were washed three times with cold pbs. cellular rna was extracted and purified for test the viral attachment. otherwise, prewarmed medium was added to the cells to initiate zikv internalization. the cells were incubated at °c for additional min and then cellular rna was extracted and purified. then rt-qpcr was performed to measure viral entry. for ncam overexpression experiment, control pcdna . plasmid or pcdna . -ncam plasmid were transfected to t cells separately. cells were then incubated with purified zikv (moi = ) on ice for h after h transfection. the supernatant was then removed and the cells were washed three times with cold pbs. the increase in viral attachment was measured with rt-qpcr. on the other hand, prewarmed medium was added to the cells to initiate zikv internalization. the cells were incubated at °c for additional min and then cellular rna was extracted and purified. then rt-qpcr was performed to measure internalized zikv rna. immunoprecipitation assay. for co-immunoprecipitation experiments, t cells ( × cells per cm dish) were transiently transfected with µg of pcdna . -ncam / pcdna . -hspa and pcmv-ns /pcmv-e separately for h using turbofect transfection reagent (thermo fisher scientific). cells were rinsed twice with cold pbs and were then transferred to clean tubes and lysed in cell lysis buffer for immunoprecipitation supplemented with % protease inhibitor cocktail (cat. no. p , sigma). cell lysates were incubated with piercetm protein a/g agarose (cat. no. , sigma) for h at °c and were then subjected to centrifugation at , × g for min at °c. the supernatant was transferred to a new tube and incubated with µl anti-flag m affinity gel (cat. no. a , sigma) overnight at °c. the sepharose samples were centrifuged, washed five times with cell lysis buffer and eluted using × flag peptide (cat. no. f , sigma). then all samples were boiled with sds loading buffer for min. flow cytometry experiments. surface expression of ncam was analysed in u- mg and vero cells by staining the cells with rabbit anti-ncam antibodies (pe) ( : , cat. no. -mm -p, sino biological) at room temperature for min. cells were washed three times with pbs supplemented with % fbs. all flow cytometry experiments were carried out using an lsrfortessa cell analyser (bd bioscience). samples were analysed using flowjo software version (treestar). crispr-cas knockout assay. oligos encoding sgrnas for generating knockout cells using crispr-cas were cloned into the lenticrisprv plasmid (addgene plasmid, cat. no. ) as previously described . the oligo sequences of the sgrnas targeting ncam and hspa are listed as follows. ncam -sgrna : aacgccaacatcgacgacgc; ncam -sgrna : acaccactgagatc cgctgc; hspa -sgrna : acagatgccaaacgtctgat; hspa -sgrna : ctagactgttaccaatgctg. lenticrisprv clones containing the guide sequences were sequenced, purified, and used for lentiviral production. to generate heterogeneous knockout cell populations, u- mg cells were infected with the lenticrisprv -derived lentivirus for h and were then reseeded into complete dmem containing µg/ml puromycin for days to select for transduced cells. surviving populations derived in this manner were propagated and expanded for using. rna extraction and real time-quantitative polymerase chain reaction (rt-qpcr). rna was isolated from mammalian cells (u- mg, vero, and hek t) using rneasy mini kit (qiagen, valencia, ca) and normalized based on total rna amount as determined by nanodrop™ / c spectrophotometer (thermo fisher scientific). rt-qpcr was performed using superscript iii platinum sybr green one-step qpcr kit w/rox (thermo fisher scientific) and analyzed on applied biosystems real-time pcr system. the samples were subjected to thermal cycling for min at °c, min at °c, and cycles of s at °c and min at °c, at which point data were collected, and this was followed by dissociation curve analysis. the ct values obtained were converted to the number of zikv rna molecules using a standard curve generated from in vitro-transcribed viral rna. zikv cdna clone, used for in vitro transcription, was kindly provided by shi. for the standard curve, the plasmid containing zikv cdna, was linearized by cla and viral rna was transcribed using t rna polymerase (new england biolabs). the dna template was digested by rnaasefree-dnaase enzymatic treatment for min at °c and the viral rna was subsequently purified by rneasy mini kit (qiagen). rna concentration and quality were determined by nanodrop and e copies of rna were serially diluted tenfold and subjected to thermal cycling as described above to obtain the standard curve and pcr efficiency. all pcr primers are listed as follows: zikv-f tgggaggtttgaagaggctg; zikv-r tctcaacatggcagcaagatct; gapdh-fctgggctacactgagcacc; gapdh-raagtggtcgttgag ggcaatg; denv-f aaggactagaggttagaggagac; denv-r ggcgttctgtgcctggaatgat; iav-f cgcacagagacttgaggatg; iav-r tgggtctccattcccattta. immunofluorescence. hek t cells (for transfection with ncam ) were seeded on cover slips in -well plate. cells were washed with pbs and fixed with . % paraformaldehyde (pfa) for min at room temperature. cells were again washed with pbs three times and blocked with % bsa in pbs for h. anti-ncam antibody in blocking solution was incubated with the cells for h at room temperature. cells were washed three times and incubated with anti-mouse fitc or anti-mouse alexa fluor for h at room temperature. dapi staining was performed for min, followed by final three pbs washes. cover slips were mounted on glass slide and images were captured using olympus ix fluorescence microscope with a x oil immersion objective. for detecting zikv, cells on slides were fixed with % pfa for min at room temperature, permeabilized with . % triton-x in pbs for min, and blocked with blocking buffer ( % bsa and % donkey serum diluted in pbs) for min. immunofluorescence analyses of zikv-infected cells were performed using a mouse anti-flavivirus envelop protein antibody ( : , clone d - g - - , millipore), with a alexa fluor donkey anti-mouse igg (h + l) ( : , ab , abcam) as the secondary antibody. all cells were mounted with prolongtm gold antifade with dapi (life technologies, p ) and imaged with a tissuefaxs flow-type tissue cytometer (tissuegnostics gmbh, vienna, austria). all statistical analyses of immunofluorescence staining present the results from at least cells per replicate, and data are shown as the mean ± s.e.m. structure of the thermally stable zika virus the . a resolution cryo-em structure of zika virus axl is not an indispensable factor for zika virus infection in mice neutralizing human antibodies prevent zika virus replication and fetal disease in mice a human antibody against zika virus crosslinks the e protein to prevent infection specificity, cross-reactivity, and function of antibodies elicited by zika virus infection zika virus: immune evasion mechanisms, currently available therapeutic regimens, and vaccines live cell imaging of viral entry dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells direct identification of ligand-receptor interactions on living cells and tissues hatric-based identification of receptors for orphan ligands an orthogonal proteomic survey uncovers novel zika virus host factors time-resolved proteomic visualization of dendrimer cellular entry and trafficking tracking pathogen infection by time-resolved chemical proteomics zika virus is not uniquely stable at physiological temperatures compared to other flaviviruses axl mediates zika virus entry in human glial cells and modulates innate immune responses axl promotes zika virus infection in astrocytes by antagonizing type i interferon signalling mitochondrial protein p /hapb /gc qr/c qbp is required for efficient respiratory syncytial virus production the tetraspanin cd facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases extended synaptotagmin interacts with herpes simplex virus glycoprotein m and negatively modulates virus-induced membrane fusion integrin alpha is involved in non-enveloped hepatitis e virus infection voltage-dependent anion channel protein (vdac ) and receptor of activated protein c kinase (rack ) act as functional receptors for lymphocystis disease virus infection the neural cell adhesion molecule is a receptor for rabies virus interaction of tyrosine-based sorting signals with clathrinassociated proteins ap- -associated protein kinase and cyclin g-associated kinase regulate hepatitis c virus entry and are potential drug targets a mouse model of zika virus pathogenesis zika virus targets human stat to inhibit type i interferon signaling zika virus antagonizes type i interferon responses during infection of human dendritic cells association of mumps virus v protein with rack results in dissociation of stat- from the alpha interferon receptor complex rab a is essential for transport of the influenza virus genome to the plasma membrane rab and rab are required for clathrin-dependent endocytosis of japanese encephalitis virus in bhk- cells zika virus dependence on host hsp provides a protective strategy against infection and disease global interactomics uncovers extensive organellar targeting by zika virus infection by zika viruses requires the transmembrane protein axl, endocytosis and low ph molecular characterization of staphylococcus aureus plasmids associated with strains isolated from various retail meats enhancing dengue virus maturation using a stable furin over-expressing cell line ucsf chimerax: meeting modern challenges in visualization and analysis the pride database and related tools and resources in : improving support for quantification data western blot. following overexpression of ncam in hek t, cells were lysed at h post transfection. the samples were boiled at °c in gel loading buffer and , -dithiothreitol (dtt) for min. the cell lysates were separated on the precast nupage - % bis-tris polyacrylamide gels (invitrogen) for min at constant voltage of v. a mops solution ( mm mops, mm tris-base, mm edta, . % sds) was used as a running buffer. the proteins were transferred onto polyvinylidene fluoride membranes in bicine-bis-tris transfer buffer containing % methanol, for min at a constant current of ma. the membrane was blocked with % bsa in tbst, and probed with anti-human ncam ( : , , cell signaling technology) for h at room temperature. following washings, anti-mouse igg hrp-conjugated secondary antibody ( : , s, cell signaling technology) was utilized for visualization ( supplementary fig. ). western blot detection of zikv env was performed using a rabbit anti-env antibody ( the matlab code used for functional analysis has been uploaded to pride partner repository with the dataset identifier pxd [http://proteomecentral. proteomexchange.org/cgi/getdataset?id=pxd ]. source data are provided with this paper. this project has been funded by nih grant r gm and nsf grant to w.a.t., r ai to r.j.k., and yfa to h.j.l. and y.z. we also thank the resource for biocomputing, visualization, and informatics at the university of california, san francisco for the creation of the zikv figures in supplementary fig. a using ucsf chimerax with support from nih r -gm and p -gm . m.s. and ying z. performed the initial experiments and analyzed the data. yang z. and h.j.l. analyzed the data. a.m., d.s., and r.k. provided zikv and initial experiments on the zikv characterization. j.c., j.q.x., and z.l.c. performed the validation experiments and analyzed the data. r.k. and w.a.t. designed the experiment. ying z. and w.a.t. wrote the paper. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -y.correspondence and requests for materials should be addressed to y.z., j.x., r.j.k. or w.a.t.peer review information nature communications thanks aurelie mousnier, andreas pichlmair and pei-yong shi for their contribution to the peer review of this work.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/ . /. key: cord- - s vh authors: delvecchio, rodrigo; higa, luiza m.; pezzuto, paula; valadão, ana luiza; garcez, patrícia p.; monteiro, fábio l.; loiola, erick c.; dias, andré a.; silva, fábio j. m.; aliota, matthew t.; caine, elizabeth a.; osorio, jorge e.; bellio, maria; o’connor, david h.; rehen, stevens; de aguiar, renato santana; savarino, andrea; campanati, loraine; tanuri, amilcar title: chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: s vh zika virus (zikv) infection in utero might lead to microcephaly and other congenital defects. since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. here we show that chloroquine exhibits antiviral activity against zikv in vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. we demonstrate that chloroquine reduces the number of zikv-infected cells in vitro, and inhibits virus production and cell death promoted by zikv infection without cytotoxic effects. in addition, chloroquine treatment partially reveres morphological changes induced by zikv infection in mouse neurospheres. zika virus (zikv) is an arthropod-borne virus, transmitted by aedes mosquitoes, that belongs to the flavivirus genus, which also includes other pathogens such as west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and dengue virus (denv). zika virus was first isolated from a sentinel monkey in the zika forest in uganda in [ ] . since then, zikv has been isolated from humans and mosquitoes throughout africa and southeast asian countries. phylogenetic analysis of the nonstructural protein encoding region has disclosed three zikv lineages: east african, vero cells (atcc, manassas, va, usa) are derived from the kidney of african green monkey and were grown in dmem high glucose (gibco, thermo fisher scientific, waltham, ma, usa) supplemented with % fetal bovine serum (fbs) (gibco). human brain microvascular endothelial cells (hbmec) were a kind gift from dr. julio scharfstein (federal university of rio de janeiro, rio de janeiro, brazil), and hbmec isolation was performed as previously described [ ] . these cells were cultured in dmem high glucose supplemented with % fbs. the c / cell line is derived from aedes albopictus. c / cells (atcc, manassas, va, usa) were grown in leibovitz l- medium (gibco) supplemented with . g/l tryptose phosphate broth (sigma aldrich, boston, ma, usa), mm glutamine (gibco), . % sodium bicarbonate (gibco), x non-essential amino acids (gibco) and % fbs. neural stem cells (nscs) were derived from human induced pluripotent stem cells (ipscs). ipscs were provided by the biobank of ipscs of the brazilian ministry of health (conep b- # . / - ). according to the supplier, fibroblasts were reprogrammed using the protocol developed by paulsen et al. [ ] , and transduced with the cytotune-ips sendai kit (thermo fisher scientific, waltham, ma, usa). ipscs presented a normal karyotype and the expression of pluripotency markers. these cells were cultured with e culture media (gibco) on a matrigel (bd biosciences, san jose, ca, usa) coated surface. ipsc colonies were manually passaged every - days until they reached %- % confluence and were maintained at • c in humidified air with % co . to produce nscs, human ipscs were exposed to the serum-free neural induction medium (gibco), containing neurobasal medium (gibco) and the pluripotent stem cell neural induction supplement (gibco), according to the manufacturer's protocol [ ] . briefly, the medium was changed every other day until day , when initial nscs are split and grown on neural expansion medium (advanced dmem/f and neurobasal medium ( : ) with neural induction supplement; gibco). nscs were used after four passages in neural expansion media. mouse central nervous system (cns) cells were harvested from swiss mouse embryos at embryo day (e ) and grown for h as free floating neurospheres in neurobasal culture media (gibco) supplemented with x b (gibco). chloroquine diphosphate was kindly supplied by farmanguinhos (fiocruz, rio de janeiro, brazil). the lyophilized powder was diluted in double distilled water to mm. the chloroquine solution was filtered through a . µm membrane and stored at − • c. zikv strain mr (uganda/africa, accession no.: nc . , kindly provided by dr. davis ferreira, federal university of rio de janeiro, rio de janeiro, brazil) was isolated from a rhesus monkey and injected intracerebrally on swiss mice for several passages [ ] and zikv br (recife/brazil, zikv pe/ , accession no: kx . , kindly provided by dr. marli tenório cordeiro, centro de pesquisas aggeu magalhães, recife, brazil) was isolated from a patient presenting classical symptoms of zikv infection [ ] . these viruses were propagated in vero and c / cells, respectively. briefly, the cells were infected with zikv at a multiplicity of infection (moi) of . and incubated at • c. after h, the inoculum was removed and replaced with dmem high-glucose (vero) and leibovitz l- (c / ) growth media supplemented with % fbs. after to days, the conditioned medium was harvested, centrifuged at × g, and sterile-filtered to remove cells and cellular debris. virus stocks were stored at − • c. virus titers were determined by plaque assay performed on vero cells. virus stocks or samples were serially diluted and adsorbed to confluent monolayers. after h, the inoculum was removed and cells were overlaid with semisolid medium constituted of alpha-mem (gibco) containing % carboxymethyl cellulose (sigma aldrich) and % fbs (gibco). cells were further incubated for days when cells were fixed in % formaldehyde. cells were stained with % crystal violet in % ethanol for plaque visualization. titers were expressed as plaque forming units (pfu) per milliliter. vero cells, hbmec, and nsc were infected with zikv mr or zikv br strain at an moi of . after h, the inoculum was removed and medium containing chloroquine ( . to µm) was added to the cells. after to days, cells were fixed with % paraformaldehyde (sigma aldrich) in phosphate buffered saline (pbs) for min at room temperature and washed with pbs. cells were permeabilized with . % triton x- (sigma aldrich) in pbs, washed with pbs, and blocked with pbs with % fbs. cells were incubated with g , a pan-flavivirus antibody raised against the zikv envelope e protein produced in g - - hybridoma (atcc), diluted : in pbs with % fbs. cells were labeled with donkey anti-mouse alexa fluor antibody (thermo scientific, waltham, ma, usa) diluted : in pbs with % fbs, and were analyzed by flow cytometry in a bd accuri c (becton, dickinson and company, franklin lakes, nj, usa) for zikv infection. vero, hbmec, and nsc were seeded on black -well plates with clear bottoms and infected with zikv mr or zikv br strain at an moi of for h. neurospheres were seeded on coverslips and infected with zikv mr with . × pfu after infection, the viral inoculum was removed and cells were incubated with medium containing chloroquine ( . to µm) for to days, depending on cell type. cells and neurospheres were fixed with % paraformaldehyde in pbs for min at room temperature. the fixative was removed and the samples were washed three times with pbs. blocking of unspecific binding of the antibody and permeabilization were performed with pbs supplemented with % bovine serum albumin (bsa, sigma aldrich) and . % triton x- for min at room temperature. incubation with anti-map antibody was performed following the manufacturer's instructions (abcam, cambridge, cambridgeshire, uk, # ; : ) and the g antibody was diluted : in pbs with % bsa and incubated with cells for h. after washing three times with pbs, cells were incubated with secondary antibodies coupled to alexa fluorochromes (thermo fisher scientific) for min and washed five times. coverslips with neurospheres were mounted with prolong gold mounting medium (thermo fisher scientific). samples were imaged using either leica sp or leica spe (leica biosystems, wetzlar, hesse, de) confocal microscopes and a nikon te (tokyo, japan) inverted microscope coupled to a leica dfc fx camera (leica biosystems, wetzlar, germany). vero, hbmec, and nsc were exposed to zikv mr at an moi of . after h, inoculum was removed and chloroquine-containing medium ( . to µm) was added to the cells. five days post-infection, µl of celltiter blue reagent (promega, madison, wi, usa) was added in each well, incubated for - h, and fluorescence was measured ( / nm), except for nscp cells when celltiter blue was added at three days post-infection. mean fluorescence intensity (mfi) and standard deviation were displayed. in order to calculate the half maximum effective concentration (ec ) that protects cells from death caused by zikv infection, mfi values from zikv mr control were subtracted from every condition and then these values were normalized over the mock control. these data were plotted and hill parameter sigmoidal regression was performed on sigma plot v. . (systat software inc., san jose, ca, usa). vero cells, hbmec, and nsc were incubated with medium containing chloroquine ( . to µm) for days (nsc) or days (vero and hbmec). celltiter blue reagent (promega) was added in each well, incubated for - h, and fluorescence was measured ( / nm). in order to calculate the % cytotoxicity concentration (cc ), mfi values were normalized over the untreated control. these data were plotted and hill parameter sigmoidal regression was performed on sigma plot v. . (systat software inc.). vero cells were inoculated with zikv mr at an moi of for h at • c. cells were washed three times with cold pbs to remove unbound virus and treated with µm chloroquine that was added at different time points: , . , , , and h post-infection. conditioned media were collected at h post-infection to analyze the production of virus particles through viral rna content or the amount of infectious virus particles. viral rna was extracted and quantitative reverse transcription polymerase chain reaction (rt-qpcr) was performed. the titer of infectious virus particles was determined by plaque assay. vero cells were infected with zikv mr or zikv br strain at an moi of for h at • c. virus input was washed three times with cold pbs and cells were treated with chloroquine ( . to µm) for h, and then the supernatant was collected and the rna was extracted and analyzed by relative quantification by rt-qpcr. the supernatant was also evaluated by plaque assay to quantitate the infectious virus particles. viral rna was extracted from µl supernatant of infected cells using qiamp minielute virus spin (qiagen, hilden, düsseldorf ,de), following the manufacturer's recommendations. zikv detection was performed using one step taqman rt-qpcr (thermo fisher scientific) on a real-time pcr system (applied biosystems) with primers and the probe described elsewhere [ ] . threshold cycle (ct) was determined and ∆ct (ct chloroquine treated − ct untreated) was calculated. the fold reduction of virus particles' release, including defective viral particles, were calculated by ∆ct . mean and standard deviation (sd) were calculated for each assay. one way analysis of variance (anova) was conducted using the non-parametric test (kruskal-wallis) followed by dunn's multiple comparisons test. a p-value of < . was considered significant. all analyses were performed on graphpad prism v. (graphpad software, san diego, ca, usa). the sample size is provided in the respective figure legends. we characterized the antiviral properties of chloroquine in vero cells, a model widely used to study viral infections. vero cells were infected with zikv mr at an moi of (i.e., pfu/cell) and were then treated for days with chloroquine in concentrations ranging from . to µm. viral infectivity was assessed using the g antibody, which detects flavivirus envelope e protein. we observed that chloroquine treatment decreased the number of zikv-infected cells in a dose-dependent manner. flow cytometry analysis showed a reduction of % and % in zikv-infected cells when cultures were treated with µm and µm chloroquine, respectively, compared to untreated infected cells ( figure a ). immunofluorescence staining corroborated these results ( figure b ) and additionally, chloroquine decreased the production of infectious ( figure c ) and total ( figure d ) virus particles, including defective viral particles, by zikv-infected cells. to confirm that viral inhibition is independent of chloroquine cytotoxicity, the viability of uninfected cells treated with chloroquine ( . to µm) for days was analyzed. chloroquine did not impact cell viability at concentrations of µm or lower ( figure e ). we further analyzed whether chloroquine treatment could protect vero cells from zikv infection as assessed by cell viability. chloroquine, ranging from . to µm, increased cell viability from % up to % ( figure f ). microcephaly cases and neurological disorders have only been associated with infection with strains of zikv from the asian lineage, detected in french polynesia and in the americas [ , , ] . to determine the inhibition spectrum of chloroquine against zikv infection, vero cells were infected with the brazilian isolate of the asian lineage (zikv br). chloroquine decreases the percentage of cells infected with the brazilian isolate from % to % and % at . and µm, respectively ( brazilian strain at an moi of and exposed to chloroquine for h. the supernatant was collected and viral rna was relatively quantified over the untreated infected control (b) or infectivity was analyzed by immunofluorescence with g antibody (c). the dashed line represents fold reduction on virus production of . data are represented as mean ± sd from two independent experiments. statistical significance was assessed by kruskal-wallis test and multiple comparisons with infected and untreated control corrected by dunn's test (* p < . ). inhibition of viral infection mediated by chloroquine can occur in both the early and later stages of the viral replication cycle [ , ] . to evaluate which step of the viral cycle was susceptible to inhibition, chloroquine was added to vero cells at different time points post-infection with zikv mr . the supernatant was collected h post-infection and virus production was evaluated by relative quantification of viral rna over the untreated control by rt-qpcr. virus titers were also determined by plaque assay in vero cells. incubation of vero cells with chloroquine at h postinfection had a greater impact on the production of zikv particles, decreasing viral rna -fold over the controls ( figure a ). the addition of chloroquine from min to h post-infection was able to reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. inhibition of viral infection mediated by chloroquine can occur in both the early and later stages of the viral replication cycle [ , ] . to evaluate which step of the viral cycle was susceptible to inhibition, chloroquine was added to vero cells at different time points post-infection with zikv mr . the supernatant was collected h post-infection and virus production was evaluated by relative quantification of viral rna over the untreated control by rt-qpcr. virus titers were also determined by plaque assay in vero cells. incubation of vero cells with chloroquine at h post-infection had a greater impact on the production of zikv particles, decreasing viral rna -fold over the controls ( figure a ). the addition of chloroquine from min to h post-infection was able to reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. reduce virus release - fold over untreated, infected-cells. however, chloroquine added at h post-infection had only a minor effect on viral production ( figure a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. considering that zikv infects hbmecs [ ] , we investigated whether chloroquine could inhibit viral infection of these cells. chloroquine reduced % and % of the number of zikv mr -infected hbmecs at and µm, respectively ( figure a,d) . these concentrations are non-cytotoxic ( figure b ), and protected approximately % of hbmecs from zikv infection as demonstrated by the increase in cell viability ( figure c ). considering that zikv infects hbmecs [ ] , we investigated whether chloroquine could inhibit viral infection of these cells. chloroquine reduced % and % of the number of zikv mr -infected hbmecs at and μm, respectively ( figure a,d) . these concentrations are non-cytotoxic ( figure b) , and protected approximately % of hbmecs from zikv infection as demonstrated by the increase in cell viability ( figure c ). neural stem cells are key cells in the process of corticogenesis, giving rise to the three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. depletion of the nsc pool is one of the main mechanisms responsible for primary microcephaly [ ] . to evaluate if chloroquine could protect these cells from zikv mr infection, nscs were exposed to up to μm chloroquine for days. chloroquine treatment decreased the number of nscs infected with zikv mr by %, and, through cell viability assessment protected % of nscs from zikv infection, without cytotoxicity effects ( figure a-d) . similar results were observed when nscs were infected with the brazilian strain ( figure e ). neural stem cells are key cells in the process of corticogenesis, giving rise to the three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. depletion of the nsc pool is one of the main mechanisms responsible for primary microcephaly [ ] . to evaluate if chloroquine could protect these cells from zikv mr infection, nscs were exposed to up to µm chloroquine for days. chloroquine treatment decreased the number of nscs infected with zikv mr by %, and, through cell viability assessment protected % of nscs from zikv infection, without cytotoxicity effects ( figure a-d) . similar results were observed when nscs were infected with the brazilian strain ( figure e ). neuroprogenitor-enriched neurospheres, when subjected to differentiation culture conditions, can generate neurons. our group recently demonstrated that zikv infection affected neurosphere size, neurite extension, and neuronal differentiation [ ] . as we previously observed, neurospheres infected with zikv strain mr showed convoluted and misshapen neurites. neurite extension was evaluated in chloroquine treated cultures by microtubule-associated protein (map ) staining and phase contrast microscopy and although many neurospheres were severely impacted by the infection, many others displayed the same general characteristics of mock-infected spheres indicating that chloroquine treatment rescued the neurite extension phenotype (figure a-c) . furthermore, zikv infection decreased when neurospheres were treated with . μm chloroquine, as evaluated by g staining (figure d-f) . data are represented as mean ± sd from two to three independent experiments. statistical analysis was performed with kruskal-wallis test and multiple comparisons with infected and untreated control corrected by dunn's test (* p < . ). neuroprogenitor-enriched neurospheres, when subjected to differentiation culture conditions, can generate neurons. our group recently demonstrated that zikv infection affected neurosphere size, neurite extension, and neuronal differentiation [ ] . as we previously observed, neurospheres infected with zikv strain mr showed convoluted and misshapen neurites. neurite extension was evaluated in chloroquine treated cultures by microtubule-associated protein (map ) staining and phase contrast microscopy and although many neurospheres were severely impacted by the infection, many others displayed the same general characteristics of mock-infected spheres indicating that chloroquine treatment rescued the neurite extension phenotype (figure a-c) . furthermore, zikv infection decreased when neurospheres were treated with . µm chloroquine, as evaluated by g staining (figure d-f) . chloroquine is known to be a non-specific antiviral agent, but its effect on the zika virus replication has not been evaluated yet. this is the first report of inhibitory effects of chloroquine on zikv replication, which, given the ongoing epidemics, may become interesting both for the scientific knowledge of the virus and for the clinical perspective. although zika virus was first identified in uganda in , from january to april , zikv transmission has been reported in countries and territories [ ] . the zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce zikv transmission and avoid the spread of the disease by travelers. moreover, a drug that blocks placental transfer of the virus could decrease the figure . chloroquine inhibits zikv infection in mouse neurospheres. mouse neurospheres were infected with zikv mr ( . × pfu and were treated with chloroquine for days. neurospheres were analyzed by phase contrast microscopy (a-c), and triple stained for envelope viral protein (green), microtubule-associated protein (map- , red), a neuron-specific protein, and dapi (blue) (d-f). chloroquine is known to be a non-specific antiviral agent, but its effect on the zika virus replication has not been evaluated yet. this is the first report of inhibitory effects of chloroquine on zikv replication, which, given the ongoing epidemics, may become interesting both for the scientific knowledge of the virus and for the clinical perspective. although zika virus was first identified in uganda in , from january to april , zikv transmission has been reported in countries and territories [ ] . the zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce zikv transmission and avoid the spread of the disease by travelers. moreover, a drug that blocks placental transfer of the virus could decrease the chance of vertical transmission in viremic pregnant women as was shown for hiv-infected pregnant women treated with antiretroviral therapy [ ] . here we demonstrated that chloroquine decreases the number of zikv-infected cells and protected cells from zikv infection as measured by cell viability at non-cytotoxic concentrations (figures , and ) . the ec or concentration of chloroquine that protected % of the cells from zikv infection assessed by cell viability, was . - . µm depending on the cell model and the cc ranged from . to . µm ( table ) . the values of ec obtained for zikv mr are lower than those obtained for denv inhibition (~ µm) and hiv inhibition ( µm) [ , ] . furthermore, we observed similar zikv inhibitory effects of chloroquine when tested on different zikv lineage infections (figures and ) , supporting the idea that chloroquine could help to manage recent infections caused by asian zikv lineage. although chloroquine has shown antiviral activity against a large spectrum of viruses in vitro, few clinical studies have been performed to evaluate chloroquine effects on patients with viral infections. two clinical trial studies of chloroquine have been conducted to assess chloroquine treatment in patients infected with denv [ , ] . one of the trials evaluated the benefits of chloroquine treatment for days in patients infected with denv and showed no reduction in the duration or intensity of denv viremia or nonstructural protein (ns ) antigenemia clearance [ ] . however, a trend towards a reduction in the number of dengue hemorrhagic fever cases was noticed in the chloroquine-treated group [ ] . a more recent clinical trial of chloroquine administration to denv-infected patients, also for days, showed that % of the patients in the chloroquine-treated group reported feeling less pain and showed improvement in the performance of daily chores during treatment [ ] . moreover, the symptoms returned after medication withdrawal. however, chloroquine treatment did not reduce the duration and intensity of the fever or duration of the disease [ ] . the antiviral effect of chloroquine may be insufficient to produce a decrease in viral load or improvement of the disease progression when chloroquine/hydroxychloroquine is used in monotherapy. however, chloroquine may produce a significant antiflaviviral effect when used in combination therapies, as recently shown in a clinical trial of hydroxychloroquine plus ribavirin and interferon alpha in individuals infected with hepatitis c virus (hcv) [ ] . in regard to the potential antiviral therapeutic combinations for zika, a freshly published screening of drugs already approved for other clinical indications has resulted in the identification of more than candidate drugs [ ] . of note, one of these is mefloquine, a compound related to chloroquine. in terms of safety for pregnant women, however, mefloquine is included in the b category, i.e., a drug for which the animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women. be that as it may, the aforementioned study corroborates our results using chloroquine, and provides new anti-zikv drugs that could be tested in combination with chloroquine. differing from mefloquine administration, the use of chloroquine during pregnancy was thoroughly evaluated and when prophylactic doses of chloroquine were administered for malaria ( mg/week), no increment in birth defects was observed [ ] . higher concentrations of chloroquine ( mg to mg/day) were administered to pregnant women with severe diseases, such as lupus or rheumatoid arthritis. in these studies, few cases of abortion and fetal toxicity were observed. however, fetal toxicity or death could not be discarded as direct consequences of the disease itself. in addition, all term deliveries resulted in healthy newborns [ , ] . chloroquine has been successfully tested in animal models. twice daily administration of chloroquine ( mg/kg) has been shown to increase the balb/c mice survival rate to %- % after infection with ebola virus (ebov) [ ] . in a c bl/ mice model for coronavirus infection, chloroquine ( mg/kg) protected % of -day-old suckling mice infected with human coronavirus oc (hcov-oc ) when administered to pregnant mice day prepartum [ ] . a survival rate of % was observed in balb/c mice infected with avian influenza h n virus treated with chloroquine at mg/kg/day [ ] . these results suggest that chloroquine has the potential to inhibit zikv in in vivo mouse studies. chloroquine is widely distributed to body tissues as well as its analogue hydroxychloroquine. the concentration of hydroxychloroquine in the brain is - times higher than in the plasma [ ] . the concentration of chloroquine in the plasma reached µm when a daily intake of mg was prescribed to arthritis patients [ ] . chloroquine is able to cross the placental barrier and is supposed to reach similar concentrations in both maternal and fetal plasma [ ] . concentrations of chloroquine, similar to the ec values calculated here (table ) , are achieved in the plasma in current chloroquine administration protocols and might reach the brain. different mechanisms for the chloroquine inhibition of viral infection have been described [ ] [ ] [ ] . we observed a strong reduction in the release of zikv particles when the drug was added at h post-infection (figure ) , suggesting a higher impact on early stages of infection, possibly during fusion of the envelope protein to the endosome membrane. chloroquine inhibits acidification of the endosome, consequently inhibiting the low ph-induced conformational changes required for the fusion of the envelope protein of flaviviruses with the endosomal membrane [ ] . chloroquine was also effective in decreasing virus release, although less pronouncedly and not statistically significant, when added after the early stages of virus infection (from . to h post-infection), suggesting that later stages of the zikv replication cycle might also be affected (figure ) . zikv was detected in the cerebrospinal fluid of zikv-infected adult patients that manifested meningoencephalitis, indicating that zikv invades the central nervous system through yet unknown mechanisms. transcytosis through the endothelial cells of the blood brain barrier is a known mechanism of viral access to the central nervous system [ , ] . here we demonstrated, by different methodologies, that chloroquine protects hbmec, an immortalized cell line widely used as in vitro model of the blood-brain barrier [ ] , from zikv infection (figure ) . recent studies showed that neural stem cells are highly permissive for zikv infection and one of the mechanisms proposed for the cause of microcephaly would be the depletion of the stem cell pool induced by zikv [ , , ] . our data showed that chloroquine inhibits the infection of human neural stem cells ( figure ). using the mouse neurospheres model to study neural stem cell differentiation into neurons, another process that might be disturbed in microcephaly, we observed that chloroquine inhibited the infection of neuronal progenitors and partially protected the ability of these cells to extend neurites ( figure ). the protective effect of chloroquine on stem cells and committed progenitors is potentially a groundbreaking feature of this compound, as it would be prescribed to women at childbearing age that are traveling to affected countries and women planning pregnancy in endemic areas. this would decrease the chances of infection and thus fetal damage, especially to the developing brain. our results suggest that the chloroquine concentrations inhibiting zikv replication in vitro may overlap the highest drug concentrations detected in humans [ ] . we therefore suggest that the therapeutic potential of chloroquine for zika be subjected to further study. zika virus. i. isolations and serological specificity genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia molecular evolution of zika virus during its emergence in the th century zika virus infection in pregnant women in rio de janeiro-preliminary report notes from the field: evidence of zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses -brazil zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? congenital zika virus infection: beyond neonatal microcephaly detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study zika virus impairs growth in human neurospheres and brain organoids zika virus infects human cortical neural progenitors and attenuates their growth zika virus outbreak on yap island, federated states of micronesia zika virus infection experimentally induced in a human volunteer guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study leparc-goffart, i. evidence of sexual transmission of zika virus male-to-male sexual transmission of zika virus -texas hydroxychloroquine and chloroquine retinopathy pregnancy outcome following first trimester exposure to chloroquine inhibition of human immunodeficiency virus infectivity by chloroquine in vitro inhibition of human influenza a virus replication by chloroquine chloroquine inhibits dengue virus type replication in vero cells but not in c / cells japanese encephalitis virus enters rat neuroblastoma cells via a ph-dependent, dynamin and caveola-mediated endocytosis pathway amodiaquine, an antimalarial drug, inhibits dengue virus type replication and infectivity bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells full genome sequence and sfrna interferon antagonist activity of zika virus from recife, brazil zika virus, french polynesia, south pacific endocytosis is a critical step in entry of subgroup b avian leukosis viruses characterization of herpes simplex virus-containing organelles by subcellular fractionation: role for organelle acidification in assembly of infectious particles type iii interferons produced by human placental trophoblasts confer protection against zika virus infection genetic causes of microcephaly and lessons for neuronal development the impact of african and brazilian zikv isolates on neuroprogenitors virus microcephaly and guillain-barré syndrome reduction of maternal-infant transmission of human immunodeficiency virus type with zidovudine treatment. pediatric aids clinical trials group protocol study group a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults chloroquine use improves dengue-related symptoms hydroxychloroquine augments early virological response to pegylated interferon plus ribavirin in genotype- chronic hepatitis c patients a screen of fda-approved drugs for inhibitors of zika virus infection safety of chloroquine in chemosuppression of malaria during pregnancy antimalarial drugs, systemic lupus erythematosus and pregnancy a systematic screen of fda-approved drugs for inhibitors of biological threat agents antiviral activity of chloroquine against human coronavirus oc infection in newborn mice anti-malaria drug chloroquine is highly effective in treating avian influenza a h n virus infection in an animal model recent developments in the understanding of the pharmacokinetics and mechanism of action of chloroquine dose refinements in long-term therapy of rheumatoid arthritis with antimalarials transfer of chloroquine and desethylchloroquine across the placenta and into milk in melanesian mothers mechanism of borna disease virus entry into cells weak bases affect late stages of mayaro virus replication cycle in vertebrate cells effects of chloroquine on viral infections: an old drug against today's diseases? rodenhuis-zybert, i.; wilschut, j. flavivirus cell entry and membrane fusion human immunodeficiency virus- uses the mannose- -phosphate receptor to cross the blood-brain barrier mechanism of west nile virus neuroinvasion: a critical appraisal brain-region-specific organoids using mini-bioreactors for modeling zikv exposure chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids acknowledgments: this work was supported by conselho nacional de desenvolvimento e pesquisa (cnpq), key: cord- - jyui ca authors: lai, zheng-zong; ho, yi-jung; lu, jeng-wei title: harringtonine inhibits zika virus infection through multiple mechanisms date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: jyui ca mosquito-borne zika virus (zikv) is a flavivirus that came under intense study from to for its well-known ability to cause congenital microcephaly in fetuses and neurological guillain–barré disease in adults. substantial research on screening antiviral agents against zikv and preventing zikv infection are globally underway, but food and drug administration (fda)-approved treatments are not available yet. compounds from chinese medicinal herbs may offer an opportunity for potential therapies for anti-zikv infection. in this study, we evaluated the antiviral efficacy of harringtonine against zikv. harringtonine possessed anti-zikv properties against the binding, entry, replication, and release stage through the virus life cycle. in addition, harringtonine have strong virucidal effects in zikv and exhibited prophylaxis antiviral ability prior zikv infection. the antiviral activity also observed in the treatment against japanese encephalitis reporter virus (rp -gfp strain). overall, this study demonstrated that harringtonine would be a favorable potential candidate for the development of anti-zikv infection therapies. zika virus (zikv) is a member of flavivirus genus of the flaviviridae family and is a mosquito-borne virus with positive single-stranded rna. other flaviviruses include yellow fever virus (yfv), dengue virus (denv), west nile virus (wnv), tick-borne encephalitis viruses (tbev), and japanese encephalitis virus (jev). like these, the genome size of zikv is approximately , nucleotides in length and encodes approximately amino acids, which translate a single polyprotein. the polyprotein produces three structural proteins (capsid, pre-membrane, and envelope) and seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) via viral and cellular proteolysis [ , ] . among these viral proteins, the envelope protein is responsible for viral entry and influences host attachment [ ] . however, the nonstructural proteins are related to viral rna replication and virion assembly [ ] . zikv was first discovered in rhesus macaques in ugandan forests in . the first outbreak of zikv occurred in on yap island in the western pacific ocean, followed by a large epidemic in central and south american countries in - [ ] . the spread of zikv has caused a global health concern. in addition to transmission by infected mosquitoes, zikv can also be transmitted the cytotoxicity profiles of harringtonine in non-infected vero cells were evaluated. vero cells were grown in fresh medium with raising concentrations of harringtonine in -well microplates; cytotoxicity was assessed for h using a cck- assay, which evaluated cell proliferation by measuring cellular metabolic activity. the cell viability of vero cells was approximately or % at harringtonine concentrations up to or nm after h treatment, respectively ( figure a ). to avoid drug-induced cell cytotoxicity of harringtonine treatment, this was limited to nm in subsequent antiviral experiments. to investigate the anti-zikv activity of harringtonine, we assessed the inhibition of virus infection in vero cells with moi = . under different concentrations of harringtonine for h. for this, vero cells monolayer were cultured in -well microplates overnight, infected with ffu zikv per well, and incubated with raising concentrations of harringtonine from , and nm for h. intracellular viral rna levels, protein expression levels and virus progeny in supernatants were respectively determined by rt-qpcr, western blotting and fluorescent focus assay (ffa). the dose-dependent anti-zikv activities of harringtonine were observed to decrease viral rna/protein production and progeny yield ( figure b-d) , indicating that virus propagation was suppressed. results of ifa assay also showed that harringtonine treatment inhibited zikv infection in a dose-dependent manner ( figure e ,f). taken together, our data indicated that harringtonine inhibited zikv infection by suppressing the production of zikv rna, viral protein and reducing virion yield in vitro. the experiments were carried out in triplicates, and the error bars represent standard deviation (a). vero cells were infected with zikv and were treated with different concentrations of harringtonine for h. the anti-zikv ability of harringtonine was analyzed by measuring viral rna levels (b). the cell supernatants were harvested, and virus titers were assessed by fluorecent focus assay (c). the intracellular protein expression of env was assessed by western blot (d). the severity of zikv infection was determined by ifa (e) and quantification of vero cells (f). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . scale bar: μm. to further evaluate the individual stages of the virus life cycle, a time of addition assay was performed ( figure a ). harringtonine ( nm) was administered at different stages of zikv infection with moi = . , and then the levels of zikv rna in cells, as well as virus titers in supernatants, were determined after h treatment. the zikv group and full-treatment group were, respectively, negative control and positive control. both levels of zikv rna production ( figure b ) and progeny yield ( figure c ) reduced at all processes of the virus life cycle. the strongest inhibition effect of harringtonine was observed at the post-treatment stage (approximately log inhibition of viral rna level and progeny yield), which suggested that the compound may mainly inhibit at the late stage of zikv infection. in addition, both levels of zikv rna and progeny yield were also suppressed at the co-treatment stage, which suggested that the compound may also affect at the early stage of zikv infection. in order to investigate the effect after zikv entry, vero cells were seeded and inoculated with zikv at moi = . for h absorption. then, harringtonine nm was added at , , and h after the inoculum removed. the viral rna level was detected after h incubation. based on the above data, we have determined that harringtonine effectively interferes at late stage of zikv infection ( figure d ). the viability was determined with a cell counting kit assay. the experiments were carried out in triplicates, and the error bars represent standard deviation (a). vero cells were infected with zikv and were treated with different concentrations of harringtonine for h. the anti-zikv ability of harringtonine was analyzed by measuring viral rna levels (b). the cell supernatants were harvested, and virus titers were assessed by fluorecent focus assay (c). the intracellular protein expression of env was assessed by western blot (d). the severity of zikv infection was determined by ifa (e) and quantification of vero cells (f). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . scale bar: µm. to further evaluate the individual stages of the virus life cycle, a time of addition assay was performed ( figure a ). harringtonine ( nm) was administered at different stages of zikv infection with moi = . , and then the levels of zikv rna in cells, as well as virus titers in supernatants, were determined after h treatment. the zikv group and full-treatment group were, respectively, negative control and positive control. both levels of zikv rna production ( figure b ) and progeny yield ( figure c ) reduced at all processes of the virus life cycle. the strongest inhibition effect of harringtonine was observed at the post-treatment stage (approximately log inhibition of viral rna level and progeny yield), which suggested that the compound may mainly inhibit at the late stage of zikv infection. in addition, both levels of zikv rna and progeny yield were also suppressed at the co-treatment stage, which suggested that the compound may also affect at the early stage of zikv infection. in order to investigate the effect after zikv entry, vero cells were seeded and inoculated with zikv at moi = . for h absorption. then, harringtonine nm was added at , , and h after the inoculum removed. the viral rna level was detected after h incubation. based on the above data, we have determined that harringtonine effectively interferes at late stage of zikv infection ( figure d ). . zikv rna levels were determined using rt-qpcr at different infection processes (b). virus titers in supernatants were determined using fluorescent focus assay (ffa) (c). the nm of harringtonine was added at , , and hpi (hour post infection). at hpi, the viral rna level was analyzed by rt-qpcr (d). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . our previous studies have shown that virus could bind onto cells, but could not enter into the cells at • c. thus, °c utilized to verify the effect of harringtonine on zikv absorption and then removed the inoculum and washed which could focus on the effect of virus binding. to further clarify the inhibitory process of harringtonine at the co-treatment stage, temperature difference-based binding assay and entry assay, replication, and release assay were conducted. the nm harringtonine was added during zikv infection (moi = . ) at °c, then the treated cells were washed, and fresh medium was added at °c. after h, rna and virus titer were detected and used to verify the effect of viral binding. furthermore, the cells were infected with zikv (moi = . ) at • c for h incubation, then the inoculum was removed, washed to replace with nm harringtonine, and incubated at • c for another hour. subsequently, nm harringtonine was replaced with fresh medium, which was used to investigate the effect of viral entry. the results demonstrated that harringtonine could inhibit zikv infection with moi = . through virus binding ( figure a ,b) and virus entry processes ( figure c,d) . besides, the cells were infected with zikv (moi = . ) at • c for h, and then incubated at • c for another hour. when zikv entered into the cell, the specified concentration of harringtonine was added. the rna level was used to verify whether harringtonine affected virus rna replication, and the viral progeny yield was used to further determine whether harringtonine influenced virus release. these results indicated that harringtonine also reduced the viral rna replication and virion release ( figure e ,f) by rt-qpcr and ffu assay. moreover, we also assessed whether harringtonine affects zikv stability. the zikv supernatant was added with the specified concentration of harringtonine for h incubation to verify the virucidal ability. then, the zikv supernatant was progressed -fold serial-diluted and ffa was applied to determine the reduction of virus stability. therefore, the above experiments could verify the effects of different stages. the results of harringtonine were to inhibit zikv stability at a concentration of and nm ( figure g ). we produced a timeline of time of binding, entry, replication, and release assays ( figure h ). interestingly, the antiviral effect of harringtonine was also observed even at the pre-treatment stage, when cells were pre-treated with harringtonine for h before virus infection (figure a,b) . the result of the analysis of molecular docking was used to measure the likelihood of harringtonine binding to the zikv envelope proteins, the highest patchdock score was ( figure a,b) . overall, the above evidence indicated that harringtonine possessed antiviral activities by not only disrupting viral replication, release but also blocking viral binding, entry, as well as exhibiting prophylactic effect before zikv infection. binding, entry, replication and release assays. the red line refers to the zikv absorption period and dotted blue line refers to the harringtonine administration period (h). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . . to further assess the antiviral activity of harringtonine against other virus, the japanese encephalitis reporter virus (rp -gfp strain) with moi = . was used. briefly, vero cells were seeded in -microplates overnight. cells were then infected with ffu rp -gfp virus in the presence of harringtonine at different concentrations for h. intracellular viral rna levels were determined using rt-qpcr. the inhibitory effect on viral infection was evaluated by observing virus fluorescent protein expression under an inverted fluorescence microscope. results showed that to nm harringtonine exhibited a dose-dependent anti-rp -gfp activity ( figure a-c) . consequently, harringtonine exhibited an antiviral potential to be used in the treatment of jev infection. to further assess the antiviral activity of harringtonine against other virus, the japanese encephalitis reporter virus (rp -gfp strain) with moi = . was used. briefly, vero cells were seeded in -microplates overnight. cells were then infected with ffu rp -gfp virus in the presence of harringtonine at different concentrations for h. intracellular viral rna levels were determined using rt-qpcr. the inhibitory effect on viral infection was evaluated by observing virus fluorescent protein expression under an inverted fluorescence microscope. results showed that to nm harringtonine exhibited a dose-dependent anti-rp -gfp activity ( figure a-c) . consequently, harringtonine exhibited an antiviral potential to be used in the treatment of jev infection. zikv infection in neonates with congenital microcephaly born from zikv-infected pregnant women was identified as an emerging health issue in brazil from to . to prevent the severe consequences of zikv infections and reduce zikv-induced neurological defects, an effective anti-zikv agent is required. compounds isolated from natural plants have been widely evaluated in many antiviral studies [ , ] . previous research on the natural cephalotaxus alkaloids harringtonine, homoharringtonine, and cephalotaxine majorly showed antitumor effects, such as antileukemic activity [ , , ] . cephalotaxus alkaloid antitumor effects were suggested via their inhibitory effects on protein synthesis and partly on dna synthesis [ , ] . however, further research on cephalotaxus alkaloids demonstrated antiviral effects against hepatitis b virus (hbv), bovine viral diarrhea virus (bvdv), chikv, vzv, foot and mouth disease virus (fmd), vesicular stomatitis virus (vsv), newcastle disease virus (ndv), and sars-cov- [ , , [ ] [ ] [ ] [ ] [ ] [ ] . in this study, the drug-induced cytotoxicity of harringtonine was first assessed in vero cells, the vero cells have been previously reported to be highly permissive for zikv growth and replication [ ] . the concentration of harringtonine used in this anti-zikv study was no more than nm to avoid drug-induced cytotoxicity and kept the cell viability of vero cells remained above % ( figure a ). this concentration of harringtonine was much lower than that used in the treatment of chikv with µm harringtonine in bhk cells [ ] . this distinction may be due to the different cell lines and experimental approaches used. a time of addition experiment was conducted to determine which stage of zikv life cycle was disrupted. harringtonine treatments exhibited anti-zikv effects on all virus life stages including co-treatment, post-treatment, and even pre-treatment ( figure ) . noticeably, post-treatment of harringtonine showed that the most potent inhibitory effects on intracellular viral rna level and viral progeny yield in supernatants. harringtonine also exhibited a prophylactical antiviral activity before zikv infection in vitro, suggesting that harringtonine probably entered and was retained in the cells and then exerted inhibitory effects. a previous study demonstrated that harringtonine decreased in chikv rna synthesis and protein production and also reduced viral progeny. furthermore, harringtonine also presented the prophylactical antiviral activity in chikv infection. [ ] . denv-infected cells revealed the increase of subpolysomal mrnas which might correlate to the repression of translation at the initiation stage [ ] . harringtonine, through a block following s subunit joining, could inhibit the initiation of translation, and be used to verify if denv infection could affect the translation elongation. denv infection could disrupt the host cell translation at the starting stage, but does not change the translation elongation [ ] . other studies reported that harringtonine inhibited protein synthesis by blocking poly(u)-directed polyphenylalanine synthesis and peptide bond formation [ ] , and interfered with large ribosome subunit [ ] . therefore, the decrease of viral protein expression might also relate to down-regulating protein synthesis. previous evidences indicated that harringtonine was an inhibitor of protein synthesis, which is most likely to inhibit the large ribosomal unit of eukaryotes, thereby inhibiting the translation of non-structural or structural proteins [ , ] . the above evidence implies that the antiviral activities of harringtonine might occur on host factors. harringtonine was effective against sindbis virus (sinv) and chikv and exhibits a dose-dependent inhibitory effect, but it is not effective in inhibiting the growth of encephalomyocarditis virus (emcv). therefore, the antiviral effect of harringtonine may be limited to related viruses transmitted by mosquitoes [ , ] . to further clarify the inhibitory activities of harringtonine that occurred at the co-treatment stage, binding assay and entry assay were performed. the results indicated that harringtonine could block both viral binding and entry into host cells based on the decrease in viral rna production ( figure a ,c) and virion progeny ( figure b,d) . harringtonine also revealed the virucidal ability which could destroy the virion stability ( figure g ). through molecular docking, harringtonine was predicted the binding affinity of the envelope of zikv, which might be the reason of harringtonine blocking the early stage of zikv infection and affecting the virion stability. compared to our previous study of cephalotaxine, harringtonine obviously possessed more complex mechanisms against zikv infection than cephalotaxine, in blocking virus binding, entry, stability and possessing prophylactical antiviral ability. the results mean that harringtonine treatment could be used in more complicated medical conditions against zikv infection. both of the in vivo and clinical treatments, these results proved the safety or lower drug toxicity of harringtonine [ ] [ ] [ ] . in our previous study, we also demonstrated that cephalotaxine exhibited anti-zikv and denv activity in vitro. although both harringtonine and cephalotaxine belong to cephalosporin isolates, they still have different anti-zikv abilities. first of all, the ec and cc of harringtonine are . nm and > µm, while the ec and cc of cephalotaxine are µm and > µm. the selectivity index (si) of harringtonine is . , while the si of cephalotaxine is . . second, both harringtonine and cephalotaxine have inhibitory effects after virus entry, because they have inhibitory effects in post-infection treatment. however, harringtonine revealed more effects on zikv binding and entry, whereas cephalotaxine did not observe the same effect. third, the virucidal assay showed that µm cephalotaxine reduced the infection ability of zikv by about %, but did not show more effects at a concentration of µm (data not shown) [ ] . however, the harringtonine has a better inhibitory effect ( . % at nm and . % at nm) in the virucidal assay, and it is dose-dependent. in this study, harringtonine possessed the ability against zikv infection during virus binding, entry, and virucidal assay. furthermore, the molecular docking showed that harringtonine could bind with zikv envelope protein ( figure ). the envelope protein was the most important structural protein of zikv and responded for virus binding and entry. based on the above new findings, harringtonine is more conducive to becoming a new candidate drug against zikv infection than cephalotaxine. despite these compounds sharing similar structures, their multiple biological and pharmacological activities may vary with different side chains [ ] . in this study, the anti-jev effects of harringtonine was also investigated and demonstrated that harringtonine possessed dose-dependent antiviral activities ( figure a -c). in conclusion, harringtonine was proved to suppress zikv and jev infection, and all of those evidence indicated that cephalotaxus alkaloids might possess the broad-spectrum anti-viral effects in flaviviruses through multiple mechanisms. african green monkey kidney cells (vero; atcc, ccl- ) was used in this study, as it is more permissive to zikv (pravabc ; genbank sequence accession: ku ) replication; vero cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) and antibiotics under a % co incubator at • c. harringtonine was purchased from chemfaces (catalog number: cfn ), dissolved in % dimethyl sulfoxide (dmso) as a stock of mm, and stored at − • c until use. green fluorescence protein-expressing japanese encephalitis reporter virus (rp -gfp strain) was kindly given to us by dr. lin ren-jye. the propagation and titration of zikv were performed using vero cells. virus titer was determined using the fluorescent focus assay (ffa). the propagation and titration of jev were conducted using c / mosquito cells (c / ; atcc, crl- ) and vero cells, respectively. cytotoxicity of harringtonine was determined using the cell counting kit (cck- , dojindo laboratories, kumamoto, japan). increasing concentrations of harringtonine with fresh medium were added to cells in -well microplates in triplicates and incubated at • c in a % co incubator. after a or h incubation period, the medium was replaced with µl of fresh medium containing µl of cck- reagent for h. the absorbance at nm was measured using an elisa reader (synergy ht, biotek, winooski, vt, usa). the cell viability values for treated cells were normalized with those of untreated cells. total rna including viral genomic rna was extracted from infected cells using total rna reagent (bioman, tri ), after that rna levels were measured using the one-step × rt-qpcr mix sybr green kit (bioman; catalog number: qrp ). rt-qpcr was performed on a roche lightcycler (roche applied science, indianapolis, in) at the following conditions: • c, min; • c, min; ( • c, s; • c s; • c s) for cycles. the primers used to detect zikv, jev and β-actin (internal control) were as follows: zikv: forward primer '-ttggtcatgatactgctgatgc- and reverse primer '-ccttccacaaagtccctattgc- ', jev: forward primer '-tccgtcaccatgccagtctt- ' and reverse primer '-gaggatgattctgtaagtatctaggtatagagccc- ', and b-actin: forward primer '-aggcaccagggcgtgat- ' and reverse primer '-gcccacataggaatccttc tgac- ' [ ] . data were analyzed using the -ct method. viral titers were performed by using ffa. briefly, the virus solution was serially diluted and added to monolayer vero cells. the medium was discarded after a h incubation time, and cells were overlaid under semisolid dmem containing . % methylcellulose for h. subsequently, assay of immunofluorescence was conducted and fluorescent viral foci were counted under an inverted fluorescence microscope (whited). results of viral titers were expressed as fluorescent focus units per ml (ffu/ml). cells cultured in -well plates were fixed with % formaldehyde at room temperature for h and were then permeabilized with an equal ratio of chilled methanol and acetone ( : ) for min. cells were then washed three times with phosphate-buffered saline (pbs) and blocked with % skimmed milk for h and were stained with anti-flavivirus envelope protein g primary antibody ( : dilution; produced in-house) for h. subsequently, cells were washed three times with pbs again, and alexa fluor -conjugated goat anti-mouse igg secondary antibody ( : dilution; jackson immunoresearch laboratories, inc.; west grove, pa, usa) was added at • c for another h incubation. stained cells were visualized using an inverted fluorescence microscope (olympus ckx , olympus, japan), as previously reported [ ] . total cell lysates were harvested by adding ripa lysis buffer supplemented with protease inhibitors. anti-flavivirus envelope protein g antibodies ( : dilution; produced in-house) and rabbit anti-β-actin polyclonal antibodies ( : dilution; finetest, wuhan, china, catalog number: fnab ) were used as primary antibodies. anti-mouse or anti-human horseradish peroxidase-conjugated antibodies were used as secondary antibodies. blots were developed by adding enhanced chemiluminescence (ecl) reagent. vero cell monolayers were cultured overnight in -well microplates. drug-containing medium ( nm of harringtonine) was added at different time points relative to the h period of cell infection with approximately ffu of zikv (moi = . ). for the pre-treatment group (pre), harringtonine was added h before the virus infection. for the co-treatment group (co), harringtonine was added at the beginning of the virus infection. for the post-treatment group (po), harringtonine was added after the h period of infection. for the full-duration treatment group (full), harringtonine was added throughout the infection. cells were washed twice with pbs in each stage. after h incubation, cells and supernatants in all groups were harvested. the levels of intracellular viral rna and virus titers from supernatants were determined by rt-qpcr and ffa, respectively, as previously described [ , , ] . for binding assay, zikv (moi = . ) were inoculated onto vero cell monolayers in culture medium with or without nm of harringtonine for h at • c. cells were then washed twice with pbs, and fresh medium was added for h incubation at • c in a % co incubator. the levels of intracellular viral rna and virus titers in supernatants were determined by rt-qpcr and ffa, respectively. for entry assay, fresh medium containing zikv (moi = . ) was added to vero cells at • c for h. cells were then washed twice with pbs, and fresh medium with or without nm of harringtonine was added for another h incubation at • c in a % co incubator. thereafter, cells were washed twice with pbs again before being added to fresh medium. after a h incubation period, the levels of intracellular viral rna and virus titers in supernatants were determined by rt-qpcr and ffa, respectively [ , ] . the cells were infected with zikv (moi = . ) at • c for h, and then incubated at • c for another hour. when zikv entered into the cell and added the specified concentration of harringtonine. after day incubation, the cells lysate were collected for viral rna replication assay by rt-qpcr and the supernatant was assessed by the ffu assay to determine the viral release [ , ] . zikv ( × ffu) was respectively mixed with harringtonine at , , , and nm at • c for h; virus titers were then determined by ffa [ ] . the zikv envelope proteins ( ire) crystal structure was obtained from the protein data bank (pdb). three-dimensional ligand structures of (chemspider id: ) was obtained from the chemspider database and converted into the pdb format using pymol software, as previously reported [ ] . patchdock was used in this project to conduct molecular docking analyses. finally, the conformations were then ranked according to docking scores [ ] . the data obtained from this study were statistically analyzed in triplicate and using graphpad prism . software (graphpad software inc., san diego, ca, usa), and values were expressed as mean ± standard deviation. the statistical analyses of data were calculated using a two-tailed student's t-test, where a p-value < . was considered significant. full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika virus: history, emergence, biology, and prospects for control structures of the zika virus envelope protein and its complex with a flavivirus broadly protective antibody pathogenesis of flavivirus infections: using and abusing the host cell zika virus outbreak on yap island, federated states of micronesia potential sexual transmission of zika virus risk of zika virus transmission by blood donations in brazil zika virus infection and the eye microcephaly and zika virus infection study links zika virus to guillain-barre syndrome structures of harringtonine, isoharringtonine, and homoharringtonine anti-varicella-zoster virus activity of cephalotaxine esters in vitro inhibition of chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression identification of inhibitory compounds against singapore grouper iridovirus infection by cell viability-based screening assay and droplet digital pcr comparative in vitro antitumor activity of homoharringtonine and harringtonine against clonogenic human tumor cells cephalotaxine inhibits zika infection by impeding viral replication and stability herb-target interaction network analysis helps to disclose molecular mechanism of traditional chinese medicine effect of feeding chinese herb medicine ageratum-liquid on intestinal bacterial translocations induced by h n aiv in mice new natural products in cancer chemotherapy molecular modes of action of cephalotaxine and homoharringtonine from the coniferous tree cephalotaxus hainanensis in human tumor cell lines inhibition of translation in eukaryotic systems by harringtonine harringtonine, an inhibitor of initiation of protein biosynthesis in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication inhibitory effects of homoharringtonine on foot and mouth disease virus in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo shedding light on the effect of natural anti-herpesvirus alkaloids on sars-cov- : a treatment option for covid- remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro comparative analysis of different cell systems for zika virus (zikv) propagation and evaluation of anti-zikv compounds in vitro flavivirus infection uncouples translation suppression from cellular stress responses u determines the species specificity of the a-site cleft antibiotics: the structures of tiamulin, homoharringtonine, and bruceantin bound to the ribosome specificity of protein synthesis inhibitors in the inhibition of encephalomyocarditis virus replication uptake, initial effects, and chemotherapeutic efficacy of harringtonine in murine leukemic cells sensitive and resistant to vincristine and other chemotherapeutic agents antitumor activities of harringtonine and homoharringtonine, cephalotaxus alkaloids which are active principles from plant by intraperitoneal and oral administration long survival in an elderly patient with acute myeloid leukaemia after treatment with harringtonine palmatine inhibits zika virus infection by disrupting virus binding, entry, and stability antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission micafungin is a novel anti-viral agent of chikungunya virus through multiple mechanisms suramin inhibits chikungunya virus entry and transmission patchdock and symmdock: servers for rigid and symmetric docking we wish to thank yen-mei lee and hsin-hsuen shen for their experimental assistance. the authors declare no conflict of interest.molecules , , key: cord- -fhruiw authors: jaeger, anna s.; weiler, andrea m.; moriarty, ryan v.; rybarczyk, sierra; o'connor, shelby l.; o'connor, david h.; seelig, davis m.; fritsch, michael k.; friedrich, thomas c.; aliota, matthew t. title: spondweni virus causes fetal harm in ifnar (-/-) mice and is transmitted by aedes aegypti mosquitoes date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: fhruiw spondweni virus (sponv) is the most closely related known flavivirus to zika virus (zikv). its pathogenic potential and vector specificity have not been well defined. sponv has been found predominantly in africa, but was recently detected in a pool of culex quinquefasciatus mosquitoes in haiti. here we show that sponv can cause significant fetal harm, including demise, comparable to zikv, in a mouse model of vertical transmission. following maternal inoculation, we detected infectious sponv in placentas and fetuses, along with significant fetal and placental histopathology, together suggesting vertical transmission. to test vector competence, we exposed aedes aegypti and culex quinquefasciatus mosquitoes to sponv-infected bloodmeals. aedes aegypti could efficiently transmit sponv, whereas culex quinquefasciatus could not. our results suggest that sponv has the same features that made zikv a public health risk. zika virus (zikv) was originally isolated over seventy years ago, and was thought to cause a mild, self-limiting, febrile illness (dick et al., ; simpson, ) . not until the outbreak in the americas in and was zikv identified as a cause of significant adverse pregnancy outcomes (johansson et al., ; melo et al., ) . before the definition of congenital zika syndrome (czs) in , gestational arbovirus infection was not associated with birth defects. spondweni virus (sponv) is the closest known relative to zikv, but whether sponv is an emerging threat to pregnant women and their babies is unknown. it was previously thought that sponv was geographically confined to africa and caused only mild disease in rare human infections, reminiscent of the consensus around zikv in the decades following its discovery, but recent data suggest that it may be spreading beyond africa (white et al., ). sponv may therefore be poised to harm pregnancies in new, immunologically naive populations. to do this, sponv would need to fulfill two major criteria: it would need to be vertically transmitted and cause fetal harm, and be transmitted between humans by the urban mosquito vector aedes aegypti, which is associated with large-scale outbreaks of related arboviruses. the first identification of sponv was thought to have occurred in in south africa (theiler and downs, ; wolfe et al., ) . however, it was later recognized that sponv was in fact isolated three years earlier in nigeria, but was misidentified at the time as a strain of zikv because of serological cross-reactivity (haddow et al., ; simpson, ; draper, ) . serological cross-reactivity with zikv and other flaviviruses likely still confounds accurate diagnostics today. as a result, only six well-documented clinical cases of draper, ) . it is likely that many infections have gone unrecognized-serosurveys have detected evidence of sponv infection in countries throughout sub-saharan africa (kokernot et al., a; kokernot et al., b; brottes et al., ; ardoin et al., ; wolfe sponv caused fetal harm, similar to what is observed from zikv infection in this model. vector competence experiments showed that ae. aegypti could transmit sponv when exposed to bloodmeal titers that approximate physiological titers, while cx. quinquefasciatus nonpregnant, mixed sex -to -week-old mice lacking type i interferon signaling (ifnar -/-) ar (this is the only strain used in these studies, so it will be referred to hereafter as sponv); or pfu of the highly pathogenic african-lineage zikv strain dak ar (zikv-dak) (jaeger et al., ) . since contemporary sponv isolates from haiti do not exist, we used the only available low-passage isolate, sponv strain sa ar . this strain is . % nucleotide identical with the sponv genome recovered from mosquitoes in haiti (genbank:mg ). serum was collected at , , and days post-inoculation (dpi) to confirm infection and determine the replication kinetics of sponv in nonpregnant ifnar -/- mice. we also collected and tested serum at , , and days from mice surviving sponv inoculation, because sustained vrna loads were observed with the ifnar -blocking mab model (salazar et al., ) . sponv viral titer in the serum peaked at dpi (fig. a) , and in surviving animals there was no detectable viremia at , , or dpi. higher serum titers were observed in animals inoculated with the lowest dose of sponv ( pfu). we postulate that this could be the result of higher inoculating doses causing a rapid initial rise in viremia, which in turn induces a more robust immune response, leading to more rapid clearance of virus from the serum, but confirmation will require further studies. zikv-dak viremia also peaked at dpi and reached significantly higher titers at dpi than either pfu of sponv or pfu zikv-dak. based on our preliminary experiments with sponv in nonpregnant animals, and the results from our past studies (jaeger et al., ), we chose this dose to minimize the potential confounding impacts of maternal illness on fetal outcomes. we collected serum samples from dams at and dpi to confirm maternal infection. all dams were productively infected, with detectable viremia for all groups by dpi (fig. a) . zikv-dak replicated to significantly higher titers at dpi as compared to sponv (student's t-test p-value = . , t = . , df = ). dams were monitored daily pregnancies and with uninfected counterparts. in general, fetuses appeared either grossly at the time of necropsy, we observed high rates of resorption from both zikv-dak-and sponv-infected pregnancies. resorption rates from zikv-dak-and sponv-infected pregnancies were not significantly different (zikv-dak: . % vs. sponv: . %, fisher's exact test, p = . ). resorption rates for both sponv and zikv-dak were significantly higher than pbs-inoculated controls (p < . ). despite significantly higher maternal viremia observed at dpi with zikv-dak-infected dams, the fact that resorption rates did not significantly differ between the two groups indicates that both zikv-dak and sponv have a propensity to harm the developing fetus that is independent of the amount of replication in maternal blood. surprisingly, and in contrast to the results described by to further characterize the range of pathogenic outcomes of congenital sponv infection and to assess differences between models, we repeated experiments by treating dams with inoculation with zikv-dak or sponv (sheehan et al., ) . this model has been used previously for assessing both zikv and sponv pathogenesis during pregnancy, but does confirm infection, and all dams were productively infected with sponv or zikv-dak following treatment with either dose of mab (fig. d) . maternal viremia did not significantly differ between treatment groups (sponv/ mg vs. sponv/ mg: p= . ; zikv/ mg vs. zikv/ mg: p= . ; one-way anova with tukey's correction for multiple comparisons). zikv- dak titers, however, were significantly higher than sponv titers (sponv/ mg vs. zikv/ mg: p= . ; sponv/ mg vs. zikv/ mg: p= . ). next, adhering to our previously established experimental timeline, dams were necropsied on e . to assess and compare fetal outcomes. at the time of necropsy, we observed no significant resorption from either zikv or sponv infected pregnancies, after either dose of mab (fig. e) , consistent with the results described by salazar et al. observed after e . virus challenge and e . or e . dam sacrifice (salazar et al., ) . resorption rates from zikv-dak-and sponv-infected pregnancies were not significantly different (fisher's exact test, p> . for all comparisons). it is possible that the differences in outcomes in these two models may be due to the closely related to both zikv and sponv and it is not known to cause adverse pregnancy outcomes in humans. to examine whether maternal denv- infection is sufficient to induce fetal resorption, we s.c. inoculated pregnant dams on e . with . x pfu of denv- . prior to studies in pregnant animals we confirmed that this route and dose would result in productive infection in nonpregnant animals (fig. a) . all dams were productively infected with denv- with detectable vrna loads at and dpi (fig. a) . importantly, fetuses continued to develop as examined on e . , and rates of resorption were not significantly exact test, p = . ) (fig. b) . these observations confirm that fetal harm was specifically associated with zikv-dak and sponv infection, but because denv- infected mice do not show clinical signs, we cannot exclude the possibility that the more severe fetal outcomes to begin to understand the potential for sponv to be vertically transmitted, a subset of placentas and fetuses were collected for plaque assay at time of necropsy from all virus treatment groups. from the ifnar +/tissues, infectious virus was detected in % of zikv- dak placentas and fetuses screened (fig. c) . virus was detected in all but one sponv placenta and % of fetuses (fig. c) . viral titers were significantly higher in sponv placentas than their corresponding fetuses (one-way anova with tukey's multiple comparisons; p < . ), as were zikv-dak placenta viral titers as compared to zikv-dak fetuses (p = . ). in addition, zikv placenta and fetal viral titers were significantly higher than sponv titers (p < . ). placental tissues from dams treated with anti-ifnar mab (fig. f) . antibody dose did not affect the viral titer present in fetuses or placentas after either sponv-or zikv-dak- inoculation (p > . for all comparisons; one-way anova with tukey's multiple comparisons). in general, fetal and placenta tissue titers were significantly higher in zikv- dak challenge groups as compared to sponv challenge groups, with a more significant difference in placenta tissue titers than fetal tissue titers (fig. f) . of note, infectious sponv was detected in fetuses from both mab treatment groups, which is in contrast to the placental tissues from denv- infected pregnancies were also screened for infectious virus via plaque assay. infectious virus was not detected in any of the screened fetal and placental tissues, further suggesting the specificity of fetal harm to zikv and sponv (fig. c) . to better understand the impact of in utero sponv exposure, tissues from the developing ifnar +/placenta and fetus were evaluated microscopically. in pbs-and denv-inoculated fetal blood spaces (fig. ) . in contrast, zikv-dak-and sponv-inoculated dams displayed varying degrees of placental pathology with severe effects predominantly observed in the the labyrinth zone, including vascular injury involving maternal and/or fetal vascular spaces, infarction (obstructed blood flow), necrosis, apoptosis, and hemorrhage (fig. ) . overall, the severity of the vascular injury in the labyrinth zone was similar between zikv-dak and in the fetuses, there was no significant microscopic pathology from pbs-and denv- inoculated dams. in contrast, fetuses from zikv-dak-and sponv-inoculated dams demonstrated varying degrees of pathology. in fetuses from the sponv-inoculated dams, fetal injury was evident as mild pulmonary inflammation and mild to moderate segmental necrosis of the brain and spinal cord (fig. ) . these data provide indirect evidence that vertical transmission did occur. pathologic findings were more widespread and severe in fetuses from zikv-dak-inoculated dams and included severe necrosis and inflammation of the lung, liver, kidney, brain, and spinal cord. because sponv rna was detected in a pool of cx. quinquefasciatus in haiti, we compared the relative abilities of ae. aegypti and cx. quinquefasciatus from florida to transmit sponv in the laboratory. sponv titers in naturally infected hosts-to which feeding mosquitoes might be exposed in nature-are undefined. therefore, we conducted our experiments with blood meal titers ranging from ~ - pfu/ml. we considered these doses to be physiologically relevant based on studies with denv ( % mosquito infectious doses = . - . viral cdna copies/ml) (duong et al., ) and zikv ( % mosquito infectious doses = . - . pfu/ml) (ciota et al., ) . to assess vector competence, mosquitoes were exposed to viremic bloodmeals via water-jacketed membrane feeder maintained at table ). ae. aegypti that had been exposed to we speculate that the difference in outcomes between these two models could be due to displayed the most severe histologic phenotype that corresponded with higher placenta and fetus titers in both pregnancy models (fig. ) . sponv histopathology was more following inoculation with sponv, zikv, or pbs, mice were sacrificed at e . . tissues were carefully dissected using sterile instruments that were changed between each mouse to minimize possible cross contamination. for all mice, each organ/neonate was evaluated grossly in situ, removed with sterile instruments, placed in a sterile culture dish, and further processed to assess viral burden and tissue distribution or banked for future assays. briefly, uterus was first removed, and then dissected to remove each individual conceptus (i.e, fetus and placenta when possible). fetuses and placentas were either collected in pbs supplemented with % fbs and penicillin/streptomycin (for plaque assays) or fixed in % pfa or % neutral buffered formalin for imaging. we characterized an embryo as in the resorption process if it met the following criteria: significant growth retardation compared to litter mates and controls accompanied by clearly evident developmental delay, i.e., morphology was ill defined; or visualization of a macroscopic plaque in the uterus (flores et al., ) . tissues were fixed in % paraformaldehyde for hours and transferred into cold, sterile dpbs until alcohol processed and embedded in paraffin. paraffin sections ( μm) were stained with hematoxylin and eosin (h&e). pathologists were blinded to gross pathological findings when tissue sections were evaluated microscopically. the degree of pathology at the maternal-fetal interface was rated on a scale of - : -no lesions (normal); -mild changes ( - focal lesions or - % of zone involved); -mild to moderate changes ( - focal lesions or - % of zone involved); -moderate to severe changes ( - focal lesions or - % of zone involved); -severe (> focal lesions or > % of zone involved). the final score was dependent upon the greater of two parameters (# of lesions or % zone involved). this was an identical scoring system to what we reported previously (jaeger et al., ). the final scores were determined as a consensus score of two independent pathologists. for each zone in the placenta (myometrium, decidua, junctional zone, labyrinth, and chorionic plate/membranes) a 'general' overall score was determined, a score for the amount of 'inflammation', and a score for direct 'vascular injury'. the 'general' score was based on an interpretation of the overall histopathologic findings in each placenta, which included features of necrosis, infarction, apoptosis, hemorrhage, thrombosis, mineralization, vascular injury, and inflammation. the 'inflammation' score quantified the amount of inflammation in that layer. the 'vascular injury' score assessed vascular wall injury (fibrinoid necrosis, endothelial swelling), dilatation of the vessels or spaces, necrosis, loss of vascular lumen diameter, and intraluminal thrombi. the myometrial layer representing the uterine therefore meaningful comparisons between strains could not be assessed. the decidual layer (maternal in origin), the junctional zone composed of fetal giant cells and spongiotrophoblast, and the labyrinth layer (the critical layer for gas and nutrient exchange between the fetal and maternal vascular systems) were scored. since the percentage of injured/pathologic labyrinth zone is a predictor of poor fetal outcome, we also independently scored the labyrinth zone based only on the percentage of fetal and maternal vascular injury/loss using the following scoring system: - %- (background); - %- (mild); - %- (moderate); - %- (moderate to severe); and > %- (severe). photomicrographs were obtained using a bright light microscope olympus bx and olympus bx (olympus inc., center valley, pa) with attached olympus dp digital camera (olympus inc.) and spot flex mp camera (spot imaging), and captured using commercially available image-analysis software (cellsens dimensionr, olympus inc. and spot software . ). all mosquitoes used in this study were maintained at the university of minnesota, twin cities as described (christensen and sutherland, ) vector competence studies mosquitoes were exposed to sponv-or zikv-infected bloodmeals via water-jacketed membrane feeder maintained at . °c (rutledge et al., ) . bloodmeals consisted of defibrinated sheep blood (hemostat laboratories, inc.) and fresh virus supernatant, yielding infectious bloodmeal titers ranging from ~ - pfu/ml. bloodmeal titer was determined after feeding. infection, dissemination, and transmission rates were determined for individual mosquitoes and sample sizes were chosen using long established procedures (aliota et al., fisher's exact test was used to determine differences in rates of normal vs. abnormal concepti. virus stock sequence data have been deposited in the sequence read archive (sra) with accession codes pending. the authors declare that all other data supporting the findings of this study are available within the article. compared to pbs controls (fisher's exact test). ***p < . ; **p < . . -------- culex pipiens and aedes triseriatus mosquito susceptibility to zika virus wolbachia reduces transmission of zika virus by aedes aegypti the wmel strain of wolbachia reduces transmission of chikungunya virus in aedes aegypti louis encephalitis virus in mice epidemiologic study of arboviruses in the arba-minch district of ethiopia isolations of arboviruses in the lagos area of nigeria, and a survey of antibodies to them in man and animals human to mosquito transmission of dengue viruses antiviral immunity backfires: pathogenic effects of type i interferon signaling in fetal development exsheathment and midgut penetration in aedes aegypti effects of zika virus strain and aedes mosquito species on zika virus. i. isolations and serological specificity infection via mosquito bite alters zika virus tissue tropism and replication kinetics in rhesus macaques asymptomatic humans transmit dengue virus to mosquitoes evidence of vertical transmission and co-circulation of chikungunya and dengue viruses in field populations of aedes aegypti (l.) from guerrero early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy a diversified portfolio virol , vi-viii genomic epidemiology reveals multiple introductions of zika virus into the united states dengue, urbanization and globalization: the unholy trinity of the (st) century genetic characterization of spondweni and zika viruses and susceptibility of geographically distinct strains of aedes aegypti culex quinquefasciatus (diptera: culicidae) to spondweni virus distinguishing between zika and spondweni viruses a gorilla adenovirus-based vaccine against zika virus induces durable immunity and confers protection in pregnancy zika virus infection in pregnant rhesus macaques causes placental dysfunction and immunopathology protective efficacy of nucleic acid vaccines against transmission of zika virus during pregnancy in mice gestational stage and ifn-λ signaling zika and the risk of microcephaly survey for antibodies against arthropod-borne viruses in the sera of indigenous residents of studies on arthropod-borne viruses of tongaland. viii. spondweni virus, an agent previously unknown, isolated from taeniorhynchus (mansonioides) uniformis survey for antibodies against arthropod-borne viruses in the sera of indigenous residents of the caprivi strip and bechuanaland protectorate quantitative genetics of aedes aegypti vector competence for dengue viruses: towards a new paradigm discovery and full genome characterization of two highly divergent simian immunodeficiency viruses infecting black-and-white colobus monkeys (colobus guereza a mouse model of zika virus pathogenesis shared and distinct functions of type i and type iii interferons zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria pathogenesis and sexual transmission of spondweni and zika viruses further isolations of the arboviruses from mosquitoes collected in tongaland, south africa, - isolation of spondweni virus from four species of culicine mosquitoes and a report of two laboratory infections with the virus the interaction of glutamic and aspartic acids with excitatory amino acid receptors in the mammalian central nervous system congenital zika virus infection: beyond neonatal microcephaly increased yellow fever virus infection and dissemination rates in aedes aegypti mosquitoes orally exposed to freshly grown virus virus infection during pregnancy in mice causes placental damage and fetal demise zika virus: following the path of dengue and chikungunya host and viral features of human dengue cases shape the population of infected and infectious aedes aegypti mosquitoes zika virus-related neurotropic flaviviruses infect human placental explants and cause fetal demise in mice no evidence of zika, dengue, or chikungunya virus infection in field-caught mosquitoes from the recife metropolitan region zika virus and birth defects--reviewing the evidence for causality vaccine mediated protection against zika virus-induced congenital disease studies on the feeding response of mosquitoes to nutritive solutions in a new membrane feeder dengue and zika virus cross-reactive human monoclonal antibodies protect against spondweni virus infection and pathogenesis in mice neutralizing human antibodies prevent zika virus replication and fetal disease in mice blocking monoclonal antibodies specific for mouse ifn-alpha/beta receptor subunit (ifnar- ) from mice immunized by in vivo hydrodynamic transfection zika virus infection in man the arthropod-borne viruses of vertebrates. an account of therockefeller foundation virus program zika virus and the nonmicrocephalic fetus: why we should still worry emerg spondweni virus infection in a foreign resident of upper volta the incidence of arthropod- borne viruses in a population of culicine mosquitoes in tongaland type i interferons instigate fetal demise after zika virus infection. sci immunol , analyzed data and drafted the manuscript o. developed and performed the deep sequencing pipeline declaration of interests ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work the authors acknowledge the university of minnesota, twin cities bsl program for facilities and neal heuss for support. we thank natalie benett for her contribution in mosquito the authors declare no competing financial interests. collected from mice during three independent replicates at , , and/or days post inoculation and titered via plaque assay. assay limit of detection was pfu. viremia peaked at dpi for all virus groups, with zikv-dak replicating to significantly higher titers at dpi than sponv (one-way anova). ****p < . ; ***p < . ; **p < . (b) survival curves of six-to eleven-week old ifnar -/mice s.c. inoculated with pfu of sponv, pfu of sponv, pfu zikv-dak, or a pbs control. sponv : n= ; sponv : n= , key: cord- -jmwn pdn authors: parvez, mohammad k.; parveen, shama title: evolution and emergence of pathogenic viruses: past, present, and future date: - - journal: intervirology doi: . / sha: doc_id: cord_uid: jmwn pdn incidences of emerging/re-emerging deadly viral infections have significantly affected human health despite extraordinary progress in the area of biomedical knowledge. the best examples are the recurring outbreaks of dengue and chikungunya fever in tropical and sub-tropical regions, the recent epidemic of zika in the americas and the caribbean, and the sars, mers, and influenza a outbreaks across the globe. the established natural reservoirs of human viruses are mainly farm animals, and, to a lesser extent, wild animals and arthropods. the intricate “host-pathogen-environment” relationship remains the key to understanding the emergence/re-emergence of pathogenic viruses. high population density, rampant constructions, poor sanitation, changing climate, and the introduction of anthropophilic vectors create selective pressure on host-pathogen reservoirs. nevertheless, the knowledge and understanding of such zoonoses and pathogen diversity in their known non-human reservoirs are very limited. prevention of arboviral infections using vector control methods has not been very successful. currently, new approaches to protect against food-borne infections, such as consuming only properly cooked meats and animal products, are the most effective control measures. though significant progress in controlling human immunodeficiency virus and hepatitis viruses has been achieved, the unpredictable nature of evolving viruses and the rare occasions of outbreaks severely hamper control and preventive modalities. incidences of emerging and/or re-emerging infections have significantly affected human health since ancient times [ ] . the emerging pathogens are defined as novel etiological agents that have been recently introduced in a population. the "spanish flu" responsible for tens of millions of casualties in the early twentieth century, was the most devastating natural calamity in human history [ ] . the flu pandemic returned in as "asian flu" and then in as "hong kong flu" that killed about three million people [ ] . the most recent re-emergence of influenza on this scale was in as "swine flu" that claimed , lives [ ] . on its pandemic course in / , a novel severe keywords emerging infections · novel viruses · viral outbreaks · virus evolution incidences of emerging/re-emerging deadly viral infections have significantly affected human health despite extraordinary progress in the area of biomedical knowledge. the best examples are the recurring outbreaks of dengue and chikungunya fever in tropical and sub-tropical regions, the recent epidemic of zika in the americas and the caribbean, and the sars, mers, and influenza a outbreaks across the globe. the established natural reservoirs of human viruses are mainly farm animals, and, to a lesser extent, wild animals and arthropods. the intricate "host-pathogen-environment" relationship remains the key to understanding the emergence/reemergence of pathogenic viruses. high population density, rampant constructions, poor sanitation, changing climate, and the introduction of anthropophilic vectors create selective pressure on host-pathogen reservoirs. nevertheless, the knowledge and understanding of such zoonoses and pathogen diversity in their known non-human reservoirs are very limited. prevention of arboviral infections using vector control methods has not been very successful. currently, new acute respiratory syndrome coronavirus (sars-cov) infected > , people, causing deaths in countries [ ] . actually, it was the discovery of the human immunodeficiency virus (hiv) in the early s that initiated a worldwide awareness of and research interest in emerging novel viral pathogens. over the past several decades, new outbreaks of infections have led to the discovery of a diverse array of highly pathogenic viruses mainly those belonging to the filoviridae, arenaviridae, bunyaviridae, paramyxoviridae, coronaviridae, flaviviridae, togaviridae and hepeviridae families. some examples include bk virus (bkv), jc virus (jcv), merkel cell polyomavirus (mcv or mcpyv), severe fever with thrombocytopenia syndrome virus (sftsv), hantavirus (htnv), and sin nombre virus (snv) [ ] [ ] [ ] . following the fatal cases of lujo virus in southern africa in [ ] , another arenavirus, the lassa virus (lasv), first reported in nigeria in , re-emerged in guinea, liberia, and mali in , in ghana in , and in benin in [ ] . human metapneumovirus (hmpv) was first identified in the netherlands in , and was subsequently linked to an acute lower respiratory tract infection in children, similar to respiratory syncytial virus (rsv). recently, in , a novel avian influenza a strain (h n ) of "bird flu" in china [ ] and the middle-east respiratory syndrome (mers)-cov have been identified [ ] . of note, while was threatened by the re-emergence of the ebola virus, / has been challenged with the resurgence of the zika virus (zikv) [ ] . despite substantial advancements in the understanding of the biology of pathogens, the breakthroughs in prevention, and their effects on public health and the global economy, the emergence of novel pandemic viruses remains an enduring puzzle. this review presents an update on the knowledge of important emerging/re-emerging viral infections worldwide, discussing their possible origin, evolution, natural reservoirs, human adaptations, and risk factors ( fig. ). of the known viruses that infect humans, about % perpetuate naturally in non-human "reservoirs," largely farm mammals and poultry and, to a lesser extent, in wild animals and arthropods [ ] . it is estimated that zoonotic infectious agents constitute about % of the known human pathogens and up to % of "emerging" human pathogens [ ] [ ] [ ] . we have, however, limited knowledge of such zoonosis and the diversity of these viruses in their known reservoirs. the data on some of the domestic mammals harboring dozens of virus species is limited and we have insufficient knowledge about the wild animals that are estimated to host thousands of virus species [ ] . the deadly outbreak of mers-cov was linked to its zoonotic origin because of its close genetic homology to bat cov, but not to any other known hcov [ ] . current data shows that bats harbour the greatest diversity of covs, which vary from species-to-species and region-toregion [ ] . the lyssaviruses of rhabdoviridae are zoonotic human pathogens that cause fatal encephalitic disease. in addition, european bat lyssavirus type- /- (eblv- /- ), bokeloh bat lyssavirus (bblv) [ ] , and australian bat lyssaviruses (ablv) have been implicated in human fatalities [ ] [ ] [ ] . rabies virus (rabv), the archetypal lyssavirus, is historically one of the most dreaded viruses, with zoonotic potential in dogs, cats, and ferrets, and includes other domestic and wild mammals [ ] . globally, several mammalian species, including the deer mouse, rice rat, and cotton rat are recognized as potential reservoir of htnv [ ] . in china, the htnv and seoul virus (seov) are zoonotically linked to the striped field mouse and the norway rat, respectively. also in china, the fugong virus (fugv), a novel htnv has recently been identified in small oriental voles [ ] . htnvs have also been detected in shrews and bats, but their link to human illness remains to be established. moreover, in addition to humans and pigs, there is a growing chain of mammalian hosts for the hepatitis e virus (hev) that includes deer, boar, mongoose, rabbit, rat, goat, camel, bat, ferret, moose, etc. [ , ] . likewise, the highly prevalent torque teno virus (ttv), of the newly established virus family, anelloviridae, also infects pigs, cows, sheep, cats, dogs, and chickens [ ] . arboviruses like dengue virus (denv), chikungunya virus (chikv), zikv, and west nile virus (wnv) are the arthropod-borne viruses that have re-emerged in many tropical and subtropical regions in the last decades. notably, zikv was known as a neglected tropical disease confined to africa and asia until an outbreak was reported in on yap island and in / on islands in the pacific, thus expanding its geographical territory [ , ] . wnv remains the most important mosquito-borne encephalitis pathogen in north america, involving culex sp. and the american robin in its transmission cycle. since its emergence in the west in , it has undergone adaptive genetic changes as it spreads throughout north america [ ] . furthermore, crimean-congo hemorrhagic fever virus (cchfv) is considered to be one of the major emerging diseases spreading to and within the european nations, following the expanding distribution of anthropophilic ticks [ ] . every year, > , cases of cchfv, due to human-tohuman transmission, are reported in southeastern eu-rope, including turkey. sftsv, a previously unidentified tick-borne bunyavirus, has recently emerged in china with fatality rates as high as % [ ] . while dna viruses are believed to have been evolving and diversifying for millions of years [ ] , most rna viruses are likely to have a much more recent evolution and "human-adaptation" for only thousands of years [ , ] . owing to their error-prone polymerase/reverse-transcriptase (approx. - /site/replication cycle) that seems to operate at optimal fidelity, rna viruses exist as more genetically diversified populations than dna viruses. nevertheless, of the hitherto recognized species of human rna viruses consisting of genera and families, only a minority has adapted to humans [ ] . in contrast, of the known dna virus species with genera and families, nearly % are adapted to human hosts [ ] . in the human-adaptation process, the viral genetic mutations, re-assortment or virus-host genetic recombination might lead to the establishment of stable virus lineages in human populations. it is, therefore, quite possible that such human-adapted viruses could circulate asymptomatically and remain undetected until their novel clinical manifestations are noticed. to understand this further, a recently isolated hev genotype from a chronic hepatitis e patient containing a recombinant virus-host rna genome was shown to infect cultured human, pig, and deer hepatocytes [ ] . it is assumed that there is a pool of human virus species still to be discovered. the composition of such a viral pool is dynamic, changing over time, i.e., while some virus species tend to become extinct, others continue to evolve in their natural hosts. more commonly, new species arise as a result of jumps from one host to another, thus crossing the species barrier [ ] . humans are, therefore, no more than "incidental" or "spillover" hosts for pathogens. however, only a minority of such viruses are capable of persisting in certain human populations (endemics) or spreading across populations (epidemics) in the absence of a reservoir. differential host factors like age, health, physiology, nutritional status, exposure history, simultaneous infection with > pathogen, immunocompetence, and genetics are determinants to human susceptibility to an infection. the field of phylodynamics, combining a modeling framework for host, epidemiological, and molecular data, especially for rna viruses, shows particular promise for parvez understanding the patterns of viral evolution during epidemics [ , ] . moreover, our underappreciated aspect of growing human populations, global changes in land usage, and the introduction of anthropophilic vectors create selective pressure on hosts and reservoirs. for example, both wnv and chikv evolved rapidly after being introduced to new locations and encountering new vectors. when aedes albopictus rather than a. aegypti became the main vector of chikv in the indian subcontinent after the - epidemic, the same viral strain spread rapidly, and the subsequent mutants seemed to circulate and persist more efficiently [ ] . a unique molecular signature of the chikv epidemic was a single amino acid substitution (a v) in the envelope protein (e ). this mutation was identified in % of the strains reported during the later phase, and was associated with a high epidemic potential for chikv [ ] . zikv, initially known to be transmitted by mosquitoes, was recently reported as being transmitted, albeit more rarely, via sexual contact, saliva, breast milk, blood transfusions, and from mother to child [ , ] . the key to understanding the emergence/re-emergence of novel viruses is to know the intricate "hostpathogen-environment" relationship in the evolution of pathogens. while the emergence of infectious diseases in naive regions is caused primarily by the movement of pathogens via trade and travel, local emergence is driven by a combination of environmental and social change. notably, virus transmission rates are often higher in dense than in sparse populations, and the spread is often greatly enhanced by air travel or migration. pathogens introduced into novel regions often cause explosive epidemics followed by a declining incidence whereas those that emerge locally due to land usage or social changes usually show consistent increases. a recent example is the emergence of zikv in brazil in [ , ] . phylogenetic studies suggested that zikv from the pacific islands outbreak in / was probably introduced into brazil during the fifa world cup or the fia world endurance championship auto racing series. the dispersal of the virus in brazil resulted in an explosive epidemic of zika fever, and the infection spread to other countries due to frequent travel. while most human infections are known to have zoonotic origins, it is certain that alterations in the environment, due to industrialization and urbanization, are an important but completely neglected factor. though the origins of most of the human viruses are not known so far, the great majority can be categorized as "crowd diseases" that require a relatively high host-density to persist [ ] . notably, the recent outbreaks of h n , hcov, hendra virus, nipah virus, and mers-cov suggest that the asia-pacific region is the global hot-spot for the emergence of novel rna viruses. in this case, an order-of-magnitude estimation of such event per years is broadly consistent with human demographic history [ ] . notably, rna viruses are known to incorporate drastic mutations in their genome, an example being the large duplication events in the g protein gene of rsv. two such remarkable events were the -bp duplication in group b rsv in argentina in and the -bp duplication in group a rsv in canada in [ , ] . these new genotypes of rsv with their duplications, known as ba and on , respectively, spread to different geographical regions across the globe due to immunologically naive travellers [ ] [ ] [ ] [ ] . the mathematics of these spreading events is well known today, and a sophisticated array of computational and mathematical models can be used to accurately back-predict such events. an example of this is the first case-clusters of the sars outbreak and the subsequent global spread, including the country-by-country distribution of human cases [ , ] . recent investigations have evaluated the transmission dynamics of zikv infection using mathematical models [ ] . we may not ignore that wild animals constitute an important but poorly understood reservoir for known and undiscovered human pathogens, including viruses. furthermore, the relative importance of an animal species as a source of human infection is a function of the prevalence of zoonotic agents in that species and the probability of close contact (direct or indirect) with susceptible humans. clearly, these factors vary geographically, and changes in patterns of human and animal disease will continue to result from socio-economic and ecological changes at the human-to-animal interface. if risk is a function of frequency of contact and a probability of successfully adapting to human hosts, viruses acquired from non-human primates might already be better adapted to successful transmission than those from other mammals, like bovine, porcine, feline, and rodent mammals. for example, sars-cov has been detected in masked-palm or gem-faced civet cats which are sold at emergence of pathogenic viruses intervirology ; : - doi: . / chinese wildlife markets [ , ] . like hev, many enteric viruses (e.g., jcv) are found worldwide in high concentrations in urban sewage, and lead to water contamination in countries with poor resources [ ] . recurring epidemics caused by emerging viruses have necessitated the formulation of effective control measures, but the zoonotic transmission of many of these viruses has contributed significantly towards the challenges associated with their prevention and control. for instance, the prevention of arthropod-borne infections (e.g., denv, chikv, and zikv) using vector control methods has only been partially successful [ ] . preventing mosquitos breeding can be mediated by avoiding the accumulation of rain water in endemic regions. other preventive measures like the use of repellent creams and clothes that cover the body should be employed to avoid mosquito bites. insectidal sprays should be utilized in endemic regions to control the mosquito population. additionally, prevention of the transmission of other zoonotic viruses should also be addressed in a systematic manner to combat the infection of humans with these pathogens. it is now well known that many emerging human viruses can be transmitted via contact with infected animals and the consumption of animal products, including freshwater and seafood products. the new approach to food safety and to protecting humans against foodborne health risks is the most effective measure to control the human illnesses associated with animal diseases. however, the vast majority of human infections are not reported to clinicians, and so such etiological agents remain unidentified and continue to contaminate the population. as discussed, poultry are high-risk birds for emerging novel influenza a strains. pork is another potential source of chronic hev. in a public health care initiative, the french health authority has recommended to cook pig-liver sausages prior to consumption [ ] . precautions and proper care should therefore be taken when selecting, purchasing, and hunting high-risk animals and when cooking meat. the current biosecurity measures appear to have been generally more successful for constraining bacterial diseases, but less effective for viral diseases. surveillance of the emerging human viruses must include livestock, wild animal, and potential arthropod vectors as well as their environments at an international level of coordination. furthermore, individuals like livestock herders, zookeepers, hunters, rangers, and veterinarians working with reservoir or high-risk animals must take hygienic measures. notably, many viruses that affect the respiratory tract, like influenza, rsv, mers-cov, sars-cov, and hmpv are transmitted by respiratory secretions and aerosol droplets. effective measures for preventing their transmission in a community and their nosocomial spread are the adoption of good practices like frequent hand-washing and avoiding direct contact with patients [ ] . in addition, generating awareness in the general public about various aspects of these diseases will also assist in the better management and control of these emerging viral infections. the overall balance between exposure and evolution as driving forces of human virus diversity is difficult to assess accurately, particularly for viruses that are not known to have adapted to humans and that exist primarily in animal reservoirs. despite landmark advances in understanding the nature and biology of many pathogenic viruses, there is limited knowledge on emerging novel viruses, their potential reservoirs, and their modes of transmission. for example, although the person-to-person transmission of bkv is known, no zoonotic or other origin has yet been established. likewise, though mcv is found in respiratory secretions, healthy skin sheddings and gastrointestinal tissues, its precise mode of transmission still remains undetermined. the perpetual nature of the emergence and transmission of infectious diseases therefore poses an ongoing challenge which is volatile and ever-changing. however, despite incidences of infection and pathogenicity, for many viruses like cchfv and zikv, there is no vaccine prophylaxis or therapeutic intervention available at present. the recurring post-monsoon outbreaks of dengue and chikungunya fever and their control in the indian subcontinent remain challenging. the complexity of the co-circulation of denv, chikv, and zikv in many regions makes differential diagnosis difficult due to overlapping clinical manifestations and the partial cross-reactivity of antibodies [ ] [ ] [ ] [ ] . these challenges include a requirement for constant surveillance, prompt and efficient diagnosis, and development of new therapies. there is a further need for devoted research, not only to develop counter-measures but also to understand the basic biology of new viral pathogens and human susceptibilities. while advances in biomedical technologies have enabled us to prospectively identify pathogenic inter-species viruses and assess their transmissibility, the environmental release of several potential "laboratory-adapted" viruses is considered to be a big threat. parvez the frequent emergence or re-emergence of deadly viral infections has significantly affected human health despite extraordinary progress in biomedical knowledge. high population density, rampant constructions, poor sanitation, changing climate, and the introduction of anthropophilic vectors create selective pressure on hosts and pathogen reservoirs. although we know that a substantial fraction of well-identified mammalian viruses is responsible for human etiology, there are large numbers of evolving viruses waiting to infect and adapt. the unpredictable nature of novel infections, the rare occasions of outbreaks, the small numbers of confirmed cases, the asymptomatic occurrence, and occurrence in remote areas severely hamper the assessment of control and preventive modalities. efficient vaccines are already available against important human viruses, like hbv and human papillomavirus. even though drug resistance in hbv and hiv has accelerated alarmingly, new generations of broad-spectrum anti-viral agents as combination regimens hold promise. moreover, specific geographical regions or interfaces between humans, livestock, wildlife, and the environment should be targets for intense surveillance. in this way, further extensive research will surely enhance our understanding and capacity for predicting new pandemics and preparing control measures in advance. the origin of plagues: old and new death from pandemic influenza during the first world war: a perspective from personal and anecdotal evidence estimated global mortality associated with the first months of pandemic influenza a h n virus circulation: a modelling study who: summary of probable sars cases with onset of illness from new human papovavirus (b.k.) isolated from urine after renal transplantation cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy clonal integration of a polyomavirus in human merkel cell carcinoma nosocomial outbreak of novel arenavirus infection, southern who: lassa fever fact sheet analysis of the clinical characteristics and treatment of two patients with avian influenza virus (h n ) a novel coronavirus capable of lethal human infections: an emerging picture zika virus diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence risk factors for human disease emergence host range and emerging and re-emerging pathogens drivers, dynamics, and control of emerging vectorborne zoonotic diseases overviews of pathogen emergence: which pathogens emerge, when and why? curr top microbiol detection of a novel human coronavirus by realtime reverse-transcription polymerase chain reaction update on sars research and other possibly zoonotic coronaviruses molecular epidemiology of bat lyssaviruses in europe potential exposure to australian bat lyssavirus a human case of encephalitis due to a lyssavirus recently identified in fruit bats australian bat lyssavirus infection: a second human case, with a long incubation period the challenge of new and emerging lyssaviruses cdc: rodents in the united states that carry hantavirus fugong virus, a novel hantavirus harbored by the small oriental vole ( eothenomys eleusis ) in china zoonotic origin of hepatitis e hepatitis e as a zoonosis human anelloviruses: an update of molecular, epidemiological and clinical aspects zika virus outbreak on yap island, federated states of micronesia paho: regional zika epidemiological update (americas) west nile virus population genetics and evolution the impact of crimean-congo hemorrhagic fever virus on public health fever with thrombocytopenia associated with a novel bunyavirus in china reconstructing the origins of human hepatitis viruses family level phylogenies reveal modes of macroevolution in rna viruses origins of major human infectious diseases the diversity of human viruses crossspecies infections of cultured cells by hepatitis e virus and discovery of an infectious virushost recombinant unifying the epidemiological and evolutionary dynamics of pathogens the evolution and emergence of rna viruses sequential adaptive mutations enhance efficient vector switching by chikungunya virus and its epidemic emergence a single mutation in chikungunya virus affects vector specificity and epidemic potential an updated review of zika virus major changes in the g protein of human respiratory syncytial virus isolates introduced by a duplication of nucleotides genetic variability of human respiratory syncytial virus a strains circulating in ontario: a novel genotype with a nucleotide g gene duplication cocirculation of -bp duplication group a and -bp duplication group b respiratory syncytial virus (rsv) strains in riyadh, saudi arabia during genetic diversity and evolutionary insights of respiratory syncytial virus a on genotype: global and local transmission dynamics ten years of global evolution of the human respiratory syncytial virus ba genotype with a -nucleotide duplication in the g protein gene genetic variability in the g protein gene of group a and b respiratory syncytial viruses from india epidemiology, transmission dynamics and control of sars: the - epidemic forecast and control of epidemics in a globalized world transmission dynamics of zika virus in island populations: a modeling analysis of the bats are natural reservoirs of sars-like coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china potential transmission of human polyomaviruses through the gastrointestinal tract after exposure to virions or viral dna chikungunya virus: recent advances in epidemiology, host pathogen interaction and vaccine strategies chronic hepatitis e infection: risks and controls risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review phylogenetic and molecular clock analysis of dengue serotype and from new occurrence of co-infection with dengue viruses during emergence of chikungunya virus in indian subcontinent after years: a review molecular characterization of dengue and chikungunya virus strains circulating in new delhi, india the authors declare no conflicts of interest. key: cord- - xk authors: mutso, margit; st john, james a.; ling, zheng lung; burt, felicity j.; poo, yee suan; liu, xiang; Žusinaite, eva; grau, georges e.; hueston, linda; merits, andres; king, nicholas j.c.; ekberg, jenny a.k.; mahalingam, suresh title: basic insights into zika virus infection of neuroglial and brain endothelial cells date: - - journal: j gen virol doi: . /jgv. . sha: doc_id: cord_uid: xk zika virus (zikv) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and guillain–barré syndrome. the pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood–brain barrier and via peripheral (including cranial) nerves. cellular responses to infection are also poorly understood. this study characterizes the in vitro infection of laboratory-adapted zikv african mr and two asian strains of ( ) brain endothelial cells (hcmec/d cell line) and ( ) olfactory ensheathing cells (oecs) (the neuroglia populating cranial nerve i and the olfactory bulb; both human and mouse oec lines) in comparison to kidney epithelial cells (vero cells, in which zikv infection is well characterized). readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. although not as high as in vero cells, viral titres exceeded ( ) plaque-forming units (p.f.u.) ml(− ) in the endothelial/neuroglial cell types, except hoecs. despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. immunolabelling of infected cells revealed localization of the zikv envelope and ns proteins in the cytoplasm; ns staining overlapped with that of dsrna replication intermediate and the endoplasmic reticulum (er). infected oecs and endothelial cells produced high levels of pro-inflammatory chemokines. nevertheless, zikv was also able to establish persistent infection in hoec and hcmec/d cells. taken together, these results provide basic insights into zikv infection of endothelial and neuroglial cells and will form the basis for further study of zikv disease mechanisms. zika virus (zikv) is a recently emerged mosquito-borne virus belonging to the genus flavivirus, family flaviviridae. the virus was initially isolated from a rhesus monkey in the zika forest near entebbe, uganda, during yellow fever investigations in and was subsequently isolated from wild-caught aedes spp. mosquitoes collected in uganda in [ ] . the recent emergence of this virus as a cause of larger outbreaks of disease was first reported in when an outbreak of zikv was identified on yap island, federated states of micronesia, in the southwestern pacific ocean [ ] . three-quarters of the population of yap island were estimated to be infected during the outbreak, with the majority of the patients presenting with mild disease [ ] . in october the virus was identified as the cause of an outbreak of dengue-like illness in french polynesia, located in the south pacific [ , ] . thousands of suspected cases of zikv infection were reported during the outbreak, with most patients presenting with mild disease, fever, arthralgia, maculopapular rash and conjunctivitis. during these outbreaks, an increase of neurological complications in the form of guillain-barré syndrome (gbs) in zikvinfected patients and microcephaly associated with zikv infection during pregnancy were noted [ , ] . the pathogenesis of disease caused by zikv, including the mechanisms of neuroinvasion and host cell responses to infection, are currently not clearly delineated. the pathway by which zikv gains access to the central nervous system (cns) is unknown. the mechanism of neuroinvasion may involve multiple routes, as is seen with other neurotropic flaviviruses, such as west nile virus, for which hypotheses of both haematogenous and transneural entry have been proposed [ , ] . olfactory ensheathing cells (oecs), the glial cells of the primary olfactory nervous system, are found in the olfactory nerve and bulb, and have crucial roles in the regeneration of olfactory axons, which occurs throughout life. transneuronal transmission of neurotropic virus such as rabies virus [ ] has been shown to involve the olfactory system, but it remains unknown whether other neurotropic viruses such as zikv can enter the cns via this path. further, whilst it is known that oecs are highly phagocytic cells that can phagocytose microorganisms and be pathogen hosts [ ] [ ] [ ] [ ] , their roles in virus dissemination or as immunoregulatory cells in vivo are not clearly defined. another potential neuro-invasion model for microorganisms that has been proposed is crossing the bloodbrain barrier (bbb). the bbb prevents virus circulating in blood from entering the brain. the human cerebral microvascular endothelial cell line (hcmec/d ) is a stable, easily grown bbb cellular model used in a wide range of research areas, including passage of infectious micro-organisms across the bbb [ ] [ ] [ ] [ ] [ ] . there are reports showing that hcmec/d cells are susceptible to zikv infection, leading to the speculation that zikv has the ability to cross the bbb [ , ] . in the present study, the permissiveness of human and mouse neuroglial cells, including oecs and hcmec/ d s, to zikv strains belonging to asian genotypes and the highly adapted mr was investigated. we show that brain endothelial cells and neuroglial cells are permissive for zikv infection, may potentially serve as a persistent reservoir of infection, and could contribute to cns inflammation through the production of pro-inflammatory chemokines. immortalized mouse olfactory ensheathing cells (moecs) [ ] and human olfactory ensheathing cells (hoecs) [ ] were maintained in dulbecco's modified eagle's medium (dmem) with % foetal calf serum (fcs). vero cells (african green monkey epithelial cells; atcc, ccl- ) were maintained in dmem with % fcs. human brain endothelial cells (hcmec/d ) [ ] were maintained in endothelial cell growth basal medium (ebm- medium) with % fcs, . µm hydrocortisone, μg ml − ascorbic acid, % chemically defined lipid concentrate, mm hepes and ng ml − basic fibroblast growth factor. all cells were cultured at °c in a humidified atmosphere of % co and were passaged at -day intervals after attaining - % confluency. three zikv strains were used. (i) mr (uganda), an african genotype, is a rhesus monkey origin virus isolate from the centers for disease control and prevention (cdc), which is strongly adapted to mice. while mr does not represent the natural african lineage due to it being highly adapted, it nevertheless has been used in many other studies, thus allowing a good comparison with those studies. (ii) beh , an asian genotype, derived from an infectious clone based on the sequence of the brazilian strain [ ] . (iii) prvabc , an asian genotype, is a human isolate from a puerto rico patient [ ] and was kindly provided by dr jill carr, flinders university, australia. these viruses were propagated and plaque-titred in vero cells. viral titres were determined by plaque assay on vero cells. briefly, vero cells were seeded in -well plates. tenfold dilutions of each virus stock were prepared in culture medium and µl of each dilution was added to the cells. cells were incubated with virus dilution for h at °c and then the inoculum was removed and the cells were overlaid with ml of dmem supplemented with % fcs containing . % carboxymethyl cellulose (sigma life science). cells were incubated at °c for days prior to staining with crystal violet fixing solution ( . % crystal violet in % ethanol and . % formaldehyde). plaques were counted and the virus titre expressed as plaque-forming units (p.f.u.) ml − . all multiplicity of infection (m.o.i.) values used in subsequent experiments were calculated based on virus titres in vero cells. multistep growth curves were performed using moec, hoec, hcmec/d and vero cell lines. briefly, cells were seeded in -well plates and infected with zikv at an m.o.i. of . in serum-free media. cells were incubated for h, the inoculum was removed and the cells were washed twice with phosphate-buffered saline (pbs). the cells were overlaid with . ml of complete media and incubated at °c. cell culture media were collected at , . , , , , , and days post-infection (p.i.). at each time point, all media were removed and replaced with fresh medium. virus titres were determined by plaque assay. cells were cultured on glass coverslips, infected at an m.o.i. of in serum-free medium for h, washed once with pbs and covered with complete growth medium. mock-infected cells were used as controls. at indicated time points, the cells were washed with pbs, fixed with % paraformaldehyde and permeabilized with . % triton x- for min at room temperature. fixed cells were washed with pbs and blocked with % fcs in pbs. cells were immunolabelled using the following primary antibodies: rabbit anti-ns (obtained in-house) at : dilution [ ] , mouse anti-flavivirus group antigen antibody, clone d - g - - (millipore, usa), monoclonal anti-dsrna antibody (english and scientific consulting; szirak, hungary) or golgi marker mouse anti- k (abcam, usa) at : dilution for h or pdi-fitc (thermo fisher, usa). following the removal of primary antibodies, cells were washed and treated with the respective anti-rabbit/mouse alexa- / / -conjugated goat antibodies (life technologies); ′, ′-diamidino- -phenylindole (dapi) (life technologies) was used to counterstain nuclei. slides were mounted using slowfade gold reagent. immunofluorescence images were obtained and analysed using a nikon a r+ confocal microscope. images were analysed using imaris software. to determine the infectivity of zikv in the selected cell lines, confluent monolayers of each cell type were infected at an m.o.i. of . samples were harvested at h post-infection (p.i.) and stained with live/dead cell viability dye (life technologies). cells were fixed in % paraformaldehyde in pbs at room temperature for min, washed twice with facs buffer (pbs with % fbs) and permeabilized using . % tween in facs buffer for min at °c. cells were labelled with rabbit anti-ns primary antibody, diluted : in . % tween in facs buffer (diluent) for min on ice and washed twice in diluent. the primary antibody was detected using anti-rabbit alexa antibody in . % tween in facs buffer. samples were examined using the bd lsr fortessa cell analyser and the resulting data were analysed with flowjo software. the live and ns -positive population of each cell line was determined and used as an indication of the efficiency of viral infection. the viability of infected cells was determined using a cell proliferation assay (mtt, sigma-aldrich, usa). briefly, -well plates were seeded with × cells per well. the cells were infected with mr or beh strain at an m.o.i. of . after h the media was replaced with µl of complete medium. at , , or days p.i. µl of mtt reagent ( mg ml − in pbs) was added into each well; cells were incubated for to h until a purple precipitate become visible. the medium was removed, the precipitates were dissolved by adding µl of dmso and absorbance was measured at nm. mouse and human oecs and hcmec/d cells were infected as described above. infected cultures were maintained for months, with : dilution of the culture when reaching - % confluency. the cell lines obtained using this procedure are referred to as moec_mr , hoec_ mr , hcmec/d _mr , moec_beh , hoec_ beh , hcmec/d _beh , moec_prvabc , hoec_prvabc and hcmec/d _prvabc . to analyse the presence of zikv rna in moec_mr , hoec_ mr , hcmec/d _mr , moec_beh , hoec_ beh , hcmec/d _beh , moec_prvabc , hoec_prvabc and hcmec/d _prvabc cells, the cells were seeded in six-well plates at a density of approximately cells/well. after h incubation, cells were washed twice with pbs and replaced with ml of fresh media. after another h incubation cells were collected and total rna was extracted for determination of virus genome copy number. to analyse the growth of zikv-infected cells, hoec, hoec_mr , hoec_prvabc , hcmec/d , hcmec/ d _mr and hcmec/d _prvabc cells were seeded in six-well plates at a density of cells/well. day , and post-seeding total cell numbers in the well were counted using the standard haemocytometer cell counting method according to protocol. briefly, cells were trypsinized and collected into ml medium to obtain a cell suspension. fifty microlitres of cell suspension was mixed with µl of . % trypan blue solution and was applied into the haemocytometer chamber. the total cell number was counted using the following formula: cells/ml=(total cells counted/number of boxes counted)×dilution factor× ×total sample volume. total rna were extracted from persistently infected cells using the rneasy kit (qiagen, germany) according to the manufacturer's protocol. the obtained rna was reversetranscribed using random nanomer primers and moloney murine leukaemia virus reverse transcriptase (sigma-aldrich). serial dilutions of pcci-sp zikv plasmid [ ] were used to generate a gene copy standard curve. viral genome copy number was determined by rt-qpcr using ssoadvanced universal probes supermix (bio-rad) in a . µl reaction and primers targeting the ns region (mr , ′ aaat acac atac caaa acaa agtggt and ′ tcca ctcc ctct ctgg tgttg) or envelope region (prvabc and beh , ′ agatgtcggccctg-gagttc and ′ ttgccacaccgtccttgagg). all reactions were performed in -well plates using a bio-rad cfx touch real-time pcr detection system. the samples were amplified using the following conditions: °c for min, and cycles of °c for s, °c for s and °c for s. a dissociation curve was acquired using cfx manager software and used to determine the specificity of the amplified products. the copy numbers of the amplified products were interpolated from the standard curve using graphpad prism software. media collected from infected hoec and hcmec/d cells were assessed for ccl , ccl , ccl , ccl and cxcl using enzyme-linked immunosorbent assay (elisa) (r and d systems) as specified by the manufacturer. all statistical analyses were performed with graphpad prism software. statistical differences were analysed using two-way analysis of variance (anova) with bonferroni's post hoc and one-way anova with a tukey's post hoc test. to characterize infection by different zikv strains (mr , prvabc and beh ), brain endothelial cells (hcmec/d ) and neuroglial olfactory ensheathing cells (hoec and moec) were infected at an m.o.i. of . . vero cells, for which zikv infection is well characterized, were used as a positive control. although not reaching titres as high as those in vero cells ( fig. a ; peaked ~ p.f.u. ml − at days p.i.), all zikv strains replicated in brain endothelial cells and neuroglial cells, with the highest titres being observed in hcmec/d ( fig. b ; peaked ~ p.f.u. ml − at days p.i.) followed by moec ( fig. c ; peaked around p.f.u. ml − at days p.i.). in hoec cells, titres clearly above the detection limit were only observed for the mr strain ( fig. d ; peaked ~ p.f.u. ml − at days p.i.). these observations show that both african and asian zikv strains are able to infect and replicate in neuroglial and brain endothelial cells. with the exception of moec cells, mr appeared to be the fastest replicating zikv strain, with titres that were consistently higher at days p.i. compared to prvabc and beh (fig. a,b d) . consistent with this observation, mr also induced more prominent cytopathic effect (cpe) in vero cells than beh : the viability of mr -infected cells was reduced to ~ % by days p.i., while, in contrast, the viability of beh -infected cells remained at > % even at days p.i. (fig. a, b) . in contrast to vero cells, both mr and beh strains had no significant effect on the viability of neuroglial or brain endothelial cells (fig. a, b) . combined, these findings suggest that neuroglial and brain endothelial cells may harbour an intrinsic mechanism of suppressing or controlling infection, viral replication and virus-induced cytotoxicity. we next determined zikv infection efficiency in different neuroglial cell lines by detecting the percentage of cells that were positive for zikv non-structural protein (ns ). the viral growth curve revealed that in vero cells the release of new viral particles only occurred after h p.i. (fig. s , available in the online version of this article) and the highest levels of ns were observed at - h p.i. (fig. ) . therefore, infected hoec and hcmec/d cells were harvested at h p.i.; at this time point only primarily infected cells had detectable expression of ns . flow cytometry revealed that the infection efficiency of all zikv strains in the neuroglial cells (hoec) and endothelial cells (hcmec/d ) was much lower than could be counted from the m.o.i. calculated based on titres obtained using vero cells. unlike in the growth curve experiment (fig. d) , all three strains performed similarly in hoec cells and infected approximately % of the total cell numbers (fig. ) . interestingly, in hcmec/ d cells both asian strains showed significantly higher infectivity (~ % infected cells) compared to the mr strain (~ . % of infected cells). these results clearly show a much lower susceptibility to virus infection in neuroglial and brain endothelial cells compared to non-neural vero cells; this finding is consistent with the observed lower growth efficiency (fig. ) . furthermore, there is no clear correlation between efficiency of infection and efficiency of replication (virion production) in neuroglial and brain endothelial cells. thus, mr has the lowest infection efficiency in hcmec/d cells (fig. ) and yet it produced more virions than any of the asian strains (fig. b) . these data, again, may also suggest that intrinsic mechanism(s) responsible for reduced viral entry or/and replication and/ or virion formation/release are present in neuroglial and endothelial cells. to study the intracellular localization of zikv ns and envelope proteins, zikv-infected vero cells were examined by immunofluorescence assay (ifa). viral proteins were visualized at , , , , and h p.i. (fig. ) . at h p.i., the ns and envelope proteins were distributed evenly in the cell cytoplasm. the highest levels of envelope and ns protein were detected at h p.i. and h p.i. at these time points, localization of both ns and envelope protein appeared to be in cytoplasmic membranes that may represent endoplasmic reticulum (er) and/or golgi apparatus. ns , albeit at reduced levels, was also detected at both and h p.i (fig. ) . at h, there were signs of cell death (fig. ) , an observation consistent with the results from the viability assay (fig. ) . next, hcmec/d cells infected with different zikv isolates were examined by ifa at h p.i. using antibodies against golgi apparatus ( k protein), er membranes (pdi), ns and dsrna that served as a marker for viral rna replication. consistent with previous observations ns was localized in the cytoplasm (fig. a, b) . the widespread punctate staining pattern of dsrna overlapped in some areas with ns staining and showed clear co-localization with er membrane (fig. b) , a site of likely localization of zikv replication complexes. overlap of ns was also observed with golgi marker, but not to the same extent as for er marker (fig. a) . the ifa results obtained for zikv infected vero, hoec and moec cells (fig. s ) were almost identical to those obtained for hcmec/d cells (fig. ) . to examine the virus persistence in neuroglia and human brain endothelial cell cultures, the viral rna copy numbers in hcmec/d , hoec and moec cells infected with mr , prvabc and beh for months were quantified using rt-qpcr (fig. ) . the viral genome copy numbers were high in mr -and prvabc -infected hcmec/d and hoec cells. for beh the viral copy number was only high in hcmec/d cells. beh was also detected in moec cells, albeit at low levels. release of infectious virions was only detected for hcmec/d infected with any of the zikv strains (data not shown). these data indicated that all three cell lines can become persistently infected. furthermore, at least hcmec/d cell line produced zikv virions after months of infection. analysis of the percentage of ns -positive cells using staining with anti-ns antibody and facs analysis revealed that ns levels were only above the detection limit in some of the cells; the percentage of ns positive cells showed large variation (from - %) depending on cell line and clone. a correlation between virus titre and the percentage of ns -positive cells in the culture was also observed. these data indicate that cells do not harbour zikv in a uniform manner in persistently infected cultures and contain a variable percentage of cells in which the presence of zikv cannot be revealed. mtt assays did not reveal any cytotoxic effect of zikv acute infection in brain endothelial and neuroglial cells (fig. ) . interestingly, all these cells still produced infectious virions, in some cases even at days p.i. (fig. b-d) , and, at least in the case of hcmec/d cells, continued to do so months later. to understand the effect of zikv persistence in these cells, we compared the proliferation of hcmec/d and hoec cells that had been preinfected for months with zikv-mr or zikv-prvabc with the growth of the same type of uninfected cells over a day period. it was found that the proliferation of hcmec/d cell lines infected with either zikv strain was clearly lower than that of uninfected cells, and the differences become prominent at day and further increased by day after the cells had been seeded (fig. a) . similarly, the proliferation of hoec_mr cells was slow. in addition, these cells exhibited poor attachment, with the result being a loss of ~ % of cells observed by day . in contrast, persistent infection of hoec cells by prvabc had no detectable effect on cell proliferation (fig. b) . pro-inflammatory chemokines were determined in hcmec/ d and hoec cell cultures infected with zikv mr and prvabc at an m.o.i. of . at h p.i. cell culture media were collected, clarified by centrifugation and used for the detection of ccl , ccl , ccl , ccl and cxcl using elisa. all chemokines were significantly upregulated in the samples from infected cultures compared to mock-infected cultures (fig. ) . in particular, all these chemokines were produced at slightly higher levels in mr -infected cells compared to cells infected with prvabc ; however, these differences did not reach statistical significance. the recent outbreak of zikv associated with neurological complications and microcephaly in newborns has stimulated a significant amount of research to investigate cell tropism, to develop suitable in vitro and in vivo models and to understand the mechanisms of zikv-induced pathology. both asian and african strains of zikv have been shown to replicate in a variety of human cell types, including epidermal, neural and placental cells, although there are differences in replication efficiencies, the type of cellular immune response induced and the induction of apoptosis and autophagy [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the african strain mr is very heavily adapted for mouse neurons and multiple studies confirm a higher infectivity and replication rate for mr using in vitro models compared to strains belonging to asian genotype. a direct in vitro comparison of the infectivity of neural stem cells and cellular responses induced by strains belonging to african and asian genotypes suggested that the african strain had a higher infection rate and induced stronger anti-viral responses compared with the strain belonging to the asian genotype [ ] . although neurological complications have been more evident with the recent outbreak of zikv in south america, caused by the asian genotype, it is unclear if there are strain differences in neuropathology and it is possible that the disease associated with strains of african genotype are less frequently confirmed and characterized. in the present study, the permissiveness of human and mouse oec and hcmec/ d cells to african mr and asian zikv, the kinetics of viral growth, the localization of virus proteins and dsrna in the neural cells, and zikv persistence as well as cytokine responses were investigated. as expected, vero cells supported high levels of viral replication. however, zikv also replicated very efficiently in hcmec/d cells, which is in accordance with other studies [ , ] . it is suggested that this efficient zikv replication in endothelial cells is due to the specific receptor axl present in these cells [ ] and that a potential mechanism for zikv invasion of the cns via the bbb involves affecting brain capillary permeability [ ] . zikv infection and replication has previously been shown for neuroglia within the cns radial glia [ ] , myelinating oligodendrocytes [ ] and astrocytes [ ] , but never for oecs, the neuroglia of the primary olfactory nervous system. oecs surround and support the axons of olfactory sensory neurons as they extend from the olfactory mucosa into the olfactory bulb in the brain, forming the olfactory nerve. oecs are also present in the outer layer of the olfactory bulb termed the nerve fibre layer, where they are thought to mediate the organization of primary olfactory axons to their correct bulbar targets [ ] [ ] [ ] [ ] . the olfactory nerve is a route by which certain bacteria and viruses can reach the brain [ , [ ] [ ] [ ] . thus, oecs provide a potential route for access of pathogens and consequently have also been shown to be the main innate immune cells in the olfactory nerve [ ] . in this study, we clearly showed the permissiveness of this cell type to support zikv infection. compared to vero or hcmec/d , viral titres were lower in hoec and moec, suggesting reduced replication efficiency in these cells. despite the detection of reasonably high viral titres in the growth medium of neuroglial cells, the facs analysis of infected cells suggested that only a small percentage of the cells contained detectable amounts of zikv ns . nevertheless, ifa using antibody against dsrna, a marker for viral genome replication, confirmed the permissiveness of the cell lines for zikv rna replication. the localization of ns in the cytoplasm is classical for flaviviruses. colocalization of dsrna and ns with er membrane marker indicates that zikv replication complexes are, as expected, localized at er membranes. identifying other proteins interacting with ns and the role of these complexes in the coordination of replication steps and pathways could provide a target for impeding or interrupting viral replication. the infection of vero cells resulted in readily visible cpe from to days p.i.; this coincided with a decline in cell viability. interestingly, no cpe was detected in neuroglial cells. some studies have given insights into the possibility of persistent zikv infection. zikv was shown to persist in primary human foetal neural progenitors for at least month with no activation of cytokine responses despite some cell death occurring [ ] . recently, a study on sertoli cells showed that even after weeks, cells were producing virions, suggesting that sertoli cells are a major reservoir of virus [ ] . in vivo studies in rhesus monkeys provide evidence of zikv persistence in the cns and lymph nodes [ ] . mladinich et al. [ ] reported the presence of zikv in primary human brain microvascular endothelial cells and hcmec/d cultures at days p.i. without cpe development and suggested that these cells could be susceptible to persistent infection. here, we confirmed that zikv indeed develops persistent infection in hcmec/d cells; even after months of infection zikv rna is present in these cells at high copy numbers and production of infectious virions was observed. in contrast, no particle production was observed for persistently infected moec or hoec cells. there are two possible explanations for the absence of virus production in these cell lines. it is possible that these cells harbour defective but replicating viral genomes and lost the ability to produce plaques. it may also be that it was not possible to detect virus produced in these cells using standard assays (plaque assay) as the yield of infectious virus was below the detection level of the plaque assay. these results suggest that brain endothelial cells may act as a reservoir for zikv and a potential route for virus to reach to the brain. additionally, persistent infection could be detected after months in hoecs, albeit to a lesser extent and in a zikv strain/genotype-specific manner. the fact that the african strain, mr , is heavily adapted for neurons compared to recent strains of the asian genotype fig. . zikv can establish persistent infection in moec, hoec and hcmec/d cell cultures. moec, hoec and hcmec/d cell cultures were infected with three different zikv strains for months. after this, cells were collected, total rna was isolated and viral genome copy numbers were determined using rt-qpcr. the data represent the mean±sem from two independent experiments. nd, not detected. the dotted line indicates the detection limit of the assay ( viral rna copies µg − total rna). might explain the observed advantage of mr in these types of studies. mladnich et al. reported persistent zikv infection of primary human brain microvascular cells and suggested the involvement of the isg /ifn pathway in persistence, similar to that reported for hepatitis c virus [ ] . how zikv is able to sustain stable infection without cytotoxicity remains to be resolved. acutely infected cells produced high levels of proinflammatory chemokines, including ccl , ccl , ccl , ccl and cxcl , which could contribute to cns inflammation during zikv infection in vivo by promoting the recruitment of various leukocytes into the cns. cxcl (il- ) and ccl (mip- α) could potentially recruit neutrophils, whilst ccl (mip- β) may recruit monocytes and nk cells. in particular, ccl (mcp- ), induced by flavivirus infection in various cells [ ] [ ] [ ] , could drive the recruitment of bone marrow-derived ccr hi inflammatory monocytes into the cns in neuronal infection, as it does in other flavivirus models, to promote immunopathology [ , ] . in the case of foetal neuronal infection, however, the extent to which this would recruit foetal monocytes or even maternal inflammatory monocytes is unclear. later in infection sustained ccl production would likely drive maximal recruitment of the adaptive virus-specific t cell effectors that could clear virus. all these chemokines are chemoattractant for innate immune cells, consistent with their induction by flaviviruses early in infection, but each chemokine also has multiple functions in addition to chemoattraction [ , ] . given that many chemokines engage with multiple receptors, and all of the receptors for pro-inflammatory chemokines engage with multiple chemokines, it is presumably the combination of different receptors on individual cell types, activated by the differential concentration of each chemokine produced, that finally determines what cells migrate to the infected tissues and how each cell responds. overall, the growth kinetics analysis, replication efficiency studies, localization studies and cytokine expression studies suggest that neuroglial and brain endothelial cells are susceptible to zikv infection. specifically, the fact that both olfactory ensheathing neuroglial cells and brain microvascular cells could support zikv replication may relate to the ability of the virus to enter the cns via the olfactory nerve or via the bbb. the persistent infection detected without demonstrable cpe suggests that neuroglial cells and brain endothelial cells could provide a useful model for further investigation of intrinsic mechanism(s) responsible for reduced viral entry, rna replication and/or virion formation. use of these models can further promote our understanding of zikv-induced neuropathology. the authors declare that there are no conflicts of interest. zika virus. i. isolations and serological specificity zika virus outbreak on yap island, federated states of micronesia zika virus outside africa rapid spread of emerging zika virus in the pacific area zika virus infection complicated by guillain-barre syndrome--case report guillain-barré syndrome ( cases) occurring during a zika virus outbreak in french polynesia a critical role for induced igm in the protection against west nile virus infection mechanism of west nile virus neuroinvasion: a critical appraisal the olfactory nerve: a shortcut for influenza and other viral diseases into the central nervous system olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system phagocytosis of bacteria by olfactory ensheathing cells and schwann cells olfactory ensheathing cells: the primary innate immunocytes in the olfactory pathway to engulf apoptotic olfactory nerve debris burkholderia pseudomallei penetrates the brain via destruction of the olfactory and trigeminal nerves: implications for the pathogenesis of neurological melioidosis alteration of blood-brain barrier integrity by retroviral infection limitations of the hcmec/d cell line as a model for aβ clearance by the human blood-brain barrier interaction of factor h-binding protein of streptococcus suis with globotriaosylceramide promotes the development of meningitis zika virus persistently infects and is basolaterally released from primary human brain microvascular endothelial cells the hcmec/d cell line as a model of the human blood brain barrier axl-mediated productive infection of human endothelial cells by zika virus low-dose curcumin stimulates proliferation, migration and phagocytic activity of olfactory ensheathing cells reversibly immortalized human olfactory ensheathing glia from an elderly donor maintain neuroregenerative capacity the hcmec/d cell line as a model of the human blood brain barrier reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a brazilian zika virus isolate phylogeny of zika virus in western hemisphere the brazilian zika virus strain causes birth defects in experimental models the south pacific epidemic strain of zika virus replicates efficiently in human epithelial a cells leading to ifn-β production and apoptosis induction biology of zika virus infection in human skin cells zika virus infects human placental macrophages zika virus strains potentially display different infectious profiles in human neural cells zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells zika virus infects human cortical neural progenitors and attenuates their growth western zika virus in human fetal neural progenitors persists long term with partial cytopathic and limited immunogenic effects disruption of glial cell development by zika virus contributes to severe microcephalic newborn mice zika virus tropism and interactions in myelinating neural cell cultures: cns cells and myelin are preferentially affected characterisation of zika virus infection in primary human astrocytes olfactory ensheathing cells (oecs) and the treatment of cns injury: advantages and possible caveats crucial roles for olfactory ensheathing cells and olfactory mucosal cells in the repair of damaged neural tracts olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord olfactory ensheathing cell transplantation following spinal cord injury: hype or hope? herpes simplex virus- infects the olfactory bulb shortly following ocular infection and exhibits a long-term inflammatory profile in the form of effector and hsv- -specific t cells burkholderia pseudomallei rapidly infects the brain stem and spinal cord via the trigeminal nerve after intranasal inoculation murine olfactory bulb interneurons survive infection with a neurotropic coronavirus human sertoli cells support high levels of zika virus replication and persistence zika virus persistence in the central nervous system and lymph nodes of rhesus monkeys accelerated dendritic cell differentiation from migrating ly c(lo) bone marrow monocytes in early dermal west nile virus infection microarray analysis of gene expression in west nile virus-infected human retinal pigment epithelium ly c+ "inflammatory monocytes" are microglial precursors recruited in a pathogenic manner in west nile virus encephalitis therapeutic inflammatory monocyte modulation using immunemodifying microparticles beyond chemoattraction: multifunctionality of chemokine receptors in leukocytes the chemokine superfamily revisited five reasons to publish your next article with a microbiology society journal . the microbiology society is a not-for-profit organization. . we offer fast and rigorous peer review -average time to first decision is - weeks. . our journals have a global readership with subscriptions held in research institutions around the world. . % of our authors rate our submission process as 'excellent' or 'very good'. . your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord- -peqz okh authors: girard, marc; nelson, christopher b.; picot, valentina; gubler, duane j. title: arboviruses: a global public health threat date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: peqz okh a conference on «arboviruses, a global public health threat» was organized on june – , at the merieux foundation conference center in veyrier du lac, france, to review and raise awareness to the global public health threat of epidemic arboviruses, and to advance the discussion on the control and prevention of arboviral diseases. the presentations by scientists and public health officials from asia, the americas, europe and africa strengthened the notion that arboviral diseases of both humans and domestic animals are progressively becoming dominant public health problems in the world. the repeated occurrence of recent deadly epidemics strongly reinforces the call for action against these viral diseases, and the need for developing effective vaccines, drugs, vector control tools and strong prevention programs. declaring a dengue pandemic in the s was a sentinel call to action in the fight against a range of emerging arboviral diseases of humans [ , ] . the past years have seen a dramatic émergence/re-emergence of epidemic arboviral diseases [ , ] . the recent outbreak of neurological disorders and neonatal malformations associated with zika virus (zikv) infection in latin america { }, the yellow fever (yfv) epidemics in angola and brazil with importation to china [ ] , the ever-expanding west nile virus (wnv) epidemic in the americas [ ] , the recent emergence in east africa and global spread of chikungunya virus (chikv) [ ] , as well as the ongoing and expanding dengue virus (denv) pandemic in the tropics and subtropics [ ] have reinforced the call for action in the fight against emerging and re-emerging arboviral diseases. these epidemics underscore the urgency and need for integrated control and prevention of arboviral diseases, especially those transmitted by aedes mosquitoes in urban areas [ , ] . prevention and control strategies currently focused on vector control, including insecticide treatment, environmental management and social mobilization have not been effective in practice. it is widely recognized that no strategy alone can fully address the problem. however, some intervention tools have helped reduce the disease burden. for example, timely access to clinical services and appropriate care can reduce mortality dramatically [ ] , indoor residual spraying (irs) and indoor space spraying (iss) may be effective in reducing mosquito populations and exposure to arboviruses [ ] . in addition, personal protection, clinical diagnosis and management, laboratory-based surveillance and vaccination, can be effective [ ] . vaccines are available to protect against japanese encephalitis and yellow fever [ ] , and the first dengue vaccine, even though limited in its applications, was licensed in [ ] . dr duane gubler (duke-nus medical school, singapore) reminded the audience that the frequency and magnitude of the arboviral epidemics and the extent of their geographic spread have progressively increased over time, accelerating in the past years and now occurring globally in the tropics [ , ] . https://doi.org/ . /j.vaccine. . . - x/ as an illustration, denvs were found in the s in less than endemic countries and only a few thousand cases were reported each year. in contrast, in the virus had become endemic in countries, causing an estimated million yearly infections and million symptomatic cases [ ] . in the s, denv serotypes and could be found only in south-east asia. but in the early s, all four serotypes of denv had dramatically spread to to all regions of the tropics [ ] . similarly, a new strain of chikv emerged in east africa in , spreading to asia and then to the rest of the tropical world in years [ ] . and epidemic zikv emerged in the pacific and spread around the world in only years [ ] . all of these viruses are transmitted by the urban mosquito, aedes aegypti. west nile virus (wnv), transmitted primarily by bird feeding culex mosquitoes, was introduced to the western hemisphere for the first time in , rapidly spreading from the east coast of the usa to the rest of the country and to canada before invading the caribbean, central and south america [ ] . in , , cases of wnv encephalitis in horses and , cases in humans were reported in the usa. wnv is now enzootic in the region. dr joao bosco siqueiras (institute of tropical pathology and public health, goias, brazil) described another dramatic example, that of yellow fever, which is also transmitted by aedes aegypti. the mosquito is widely prevalent in the tropics including tropical america and most countries in subsaharan africa. in - yellow fever emerged and expanded into the south and southeastern parts of brazil, where yellow fever vaccination was not common. then, in - , it emerged in central brazil, infecting many travelers. cases of yellow fever were exported from brazil to europe, peru and the usa. the virus continued to spread in - into the areas of bahia, rio and sao paolo and was detected in municipalities, causing small urban epidemics [ ] . the death toll increased to persons in and in the first half of . in africa, yellow fever spread from angola to the democratic republic of congo in - , and emerged in nigeria and uganda in [ ] . more dramatically, cases were imported from angola to china, which is the first time in history that confirmed yellow fever was introduced to asia [ ]! as outlined by dr duane gubler, the new and worrisome aspect of emerging arbovirus epidemics is that they can occur in urban centers, as was observed with dengue fever, zika, chikungunya and yellow fever. the urban vectors are aedes mosquitoes, primarily aedes aegypti, which has spread around the world in past centuries, and secondarily aedes albopictus, whose spread began in the s [ ] . the emergence and spread of dengue, chikungunya and zika infections actually reflect the growing geographical spread of aedes spp across all continents. the fact that arboviral diseases have become a major threat to urban populations is the result of: ) population growth and urbanization, which results in crowding of humans, inadequate housing, waste management and accumulation of trash, including used automobile tires, plastics, tins, etc, creating ideal ecological conditions for urban aedes populations to thrive; ) the spread of aedes spp mosquitoes around the world and throughout the tropics and subtropics; ) the lack of effective vector control and infectious disease prevention; and ) the globalization of air transport, which facilitates the rapid spread of pathogens and the diseases they cause (more than billion passengers have travelled on air lines in the year ) [ , , , ] . as these trends will continue, epidemic arboviral diseases will increasingly threaten global, national and local political and economic security and could create a global public health emergency, similar to or even greater than the current sar-cov- pandemic. one should not forget that over . billion people live in areas exposed to urban aedes mosquitoes. dr duane gubler also noted that since arboviral diseases represent a major threat to urban populations, it is necessary to reassess surveillance strategies. currently, surveillance systems are based on passive reporting of loosely defined clinical syndromes with infrequent laboratory confirmation. he suggested that all at-risk countries should have an enhanced passive disease surveillance system based on well-defined case definitions supported by serological and virological laboratory testing. three major arbovirus pathologies should be monitored: systemic febrile syndrome, haemorrhagic disease syndrome, and meningo-encephalitis syndrome. an active laboratory-based syndromic surveillance network is also badly needed. weekly reports by local physicians, as done in brazil, where they have turned out to be most useful, should also be encouraged. however, as discussed by dr elizabeth hunsperger (cdc, atlanta, usa), differential diagnosis in the clinical setting may be difficult, especially outside of seasonal disease, as mild and asymptomatic infections are common. as an example, laboratory testing for dengue is important in order to distinguish the disease from other febrile illnesses such as malaria, leptospirosis or influenza, other arbovirus diseases such as chikungunya, zika, west nile, japanese encephalitis or yellow fever, and even from typhoid/paratyphoid (salmonella typhi or salmonella paratyphi spp bacteria). recently, who has recommended the serotesting of individuals in dengue vaccination settings to screen out those with no evidence of past infection. each of theses objectives requires different test characteristics. standard dengue diagnostics assays include rt-pcr, virus neutralization assays, immunoassays to detect denv ns antigen, and assays to detect anti-denv igms [ref] . the most sensitive and specific assays are rt-pcr and the ns elisa assay. low-cost point-ofcare (poc) rapid diagnostic tests (rdts) are currently not adequate for the clinical and vaccination settings, while elisa, rt-pcr and other strategies with much higher levels of performance are are too slow and costly. the latter tests are needed in public health surveillance and research settings. these diagnostic approaches are instrument-dependent and require appropriate facilities and highly trained technical staff to perform complex diagnostic tests, unfortunately none of which are available in resource-limited areas. dengue diagnostics will greatly benefit from a dengue poc test that meets the who assured criteria (affordable, sensitive, specific, rapid, user-friendly and delivered to those in need) [ ] . note that laboratory diagnosis of dengue can be achieved with a single serum specimen obtained during the febrile phase of the illness by testing for denv nucleic acid, non-structural protein (ns ) and anti-denv igms. the current poc rdts detect both anti-denv igms and iggs as well as the ns antigen. this has the potential to change the current situation in resource-limited areas and improve dengue clinical management. denv viremia occurs for up to days following the onset of fever, and anti-denv igms begin to appear around days after fever onset. detection of denv nucleic acid by rt-pcr is the most sensitive and specific means to confirm acute infection, but it may not necessarily reflect infectious virus as it only detects viral rna, which may persist in biologic fluids after infectious virus is cleared. immunoassays to detect denv ns antigen also provide acceptable levels of sensitivity and specificity. both these diagnostic approaches are instrument-dependent and require appropriate facilities to perform complex diagnostic tests. recently, it was found that apoprotein h (apo h) can be efficiently used to detect bacteria and viruses in blood samples. as described by dr francisco veas (french institute for dvelopment (ird), montpellier, france), beads coated with apo h can readily capture viruses with a glycoprotein-rich envelope such as influenza virus, ebola virus, denv and other arboviruses, or even hcv. the beads are easy to use, the process is very fast and shows high sensitivity: one can readily detect pfu of virus in ml of whole blood. dr rome buathong (bureau of epidemiology, dept of disease contrtol, thailand ministry of public health) reminded the audience that until the brazilian outbreak in - , zikv was a little known arbovirus presenting with a mild dengue-like illness without any major complications [ ] . it initially spread from sub-saharan africa to asia without detection. thus, although the virus was present in thailand, it was not detected until may , when a canadian traveler returning from thailand was diagnosed with the disease. a zikv surveillance program was launched in the country (from january to december ) revealing a yearly peak of zikv infection during the rainy season, occurring - weeks after the peak of dengue disease. a total of confirmed cases of zikv infection was recorded in thailand during this two year period. the age groups most affected were the - followed by the - year groups. a total of cases of zikv infection was recorded in pregnant women, leading to miscarriages and birth abnormalities, including cases of microcephaly. aedes mosquitoes in the country were found to be zikv positive, but other routes of transmission included blood transfusion and sexual intercourse. zikv rna could readily be detected from plasma, saliva and urine of infected patients. sexual transmission of zikv has been documented to occur up to three weeks after onset of illness in the male partner. shedding of the viral rna in semen has been well documented, but only % of semen samples that were pcr positive could be shown to contain infectious virus by cell culture. dr nikos vasilakis (university of texas medical branch) reviewed the explosive emergence of zikv in - in the south pacific and south america, with a focus on brazil where major clinical outcomes in pregnant women were common. the alarming numbers of zikv associated microcephaly and other gestational and neonatal complications brought the virus into the spotlight. increased reporting of guillain-barré syndrome (gbs) and other neurological manifestations was also associated with zikv infections. the first association between zikv infection and microcephaly was reported in in recife, brazil where three women infected with zikv during their pregnancy delivered babies with microcephaly. it was retrospectively discovered that neurological diseases, including microcephaly, in babies born to zikv-infected mothers, had been observed in french polynesia as early as . as reported by dr patricia brasil (oswaldo cruz foundation, rio do janeiro), a prospective cohort study for zikv infection in pregnant women and infants was initiated in rio de janeiro in early . zikv was recovered from the amniotic fluid and placenta of the pregnant women, and also from the cerebral spinal fluid of their babies. microcephaly was observed in %- % of the newborns, while structural birth defects such as hypertonicity, cardiac defects, ophtalmic abnormalities, elbow abnormality, and seizures were observed in up to . % of the newborns. a follow-up of brazilian babies born to mothers infected by zikv during pregnancy showed that up to % had abnormal hearing or other manifestations, and % showed signs of abnormal behavior. at months of age, only % of the babies displayed normal neurodevelopment. dr mauricio lacerda nogueira (faculty of medicine, sao jose do rio preto brazil) talked of the interplay between various flaviviruses. given that brazil is hyperendemic for arboviruses, there was concern that previous heterotypic flavivirus (e.g. dengue) exposure could exacerbate zika disease pathogenicity. denv antibodies (ab) bind to zikv. could they drive greater zikv replication through antibody-dependent enhancement (ade)? the phe-nomenom was initially described for denv, but it has also been found to exist for rabies virus, coxsackievirus b , coronaviruses, and even hiv. to answer the question, a cohort of healthy volunteers in vila toninho, brazil, was followed up from to , looking for evidence for a possible ade phenomenom. in spite of the fact that % of the cohort had denv ab at the start of the study, no significant clinical difference could be observed in zikv infections between denv ab positive and denv ab negative persons. thus, the epidemiological evidence in brazil did not support the hypothesis that previous exposure to denv infection could enhance zikv pathogenicity. by contrast, it has been reported that denv- and denv- ab might be protective against zikv infection. similarly, the presence of yfv ab has recently been associated with a better prognosis of zikv infection in pregnant women. other hypotheses, such as enhanced infectivity of zikv for aedes mosquitoes, have not been confirmed. interestingly, a a v mutation in the e gene of chikv has recently been described which is associated with enhanced infectivity of the virus for aedes albopictus mosquitoes. as discussed by dr nikos vasilakis, the most convincing explanation for the sudden aggressivity of zikv lies in the discovery by yuan et al [ ] in of a mutation in the prm gene of the brazilian strain of zikv, which could strongly contribute to the generation of fetal microcephaly. the mutation was also found in zikv strains from french polynesia, where numerous cases of microcephaly have been recorded, but not in virus strains from africa, where microcephaly has never been observed. dr marc lecuit (pasteur institute, paris), addressing the question of the cellular and molecular mechanisms of microcephaly, reminded the audience that, in contrast with chikv, which does not cross the placental barrier, zikv can readily penetrate and infect placental cells. inoculation of zikv in the brain of mouse embryos triggers an endoplasmic reticulum stress which perturbs the physiological unfolded protein response (upr) within apical cortical progenitor cells that control neurogenesis. sustained endoplasmic reticulum stress leads to apoptosis. thus, it is likely that, in pregnant women, zikv crosses the placental barrier, gets to the brain of the foetus and crosses the blood-brain barrier, then targets apical cortical progenitor cells, which leads to the blockade of upr and the arrest of neurogenesis, and, as an obvious consequence, to microcephaly. microcephaly has not been observed in africa; it appears to be a specific property of the brasilian zikv strain, and is most likely related to the prm mutation identified by yuan et al [ ] . dr michael gaunt (london school of hygiene and tropical medicine) reported a series of experiments done on zikv and denv to identify potential genetic determinants of ade. a amino acid long motif in the denv glycoprotein was identified which might be associated with ade. dr annelies wilder-smith (london school of hygiene and tropical medicine) reviewed the eu research program on zikv (the 'zikaplan'), which oversees institutional global partners with plans to set up a sustainable latin american research preparedness network for emerging diseases. a longitudinal cohort study of , subjects aged - is being followed for three years in different locations in brazil, to further refine the full spectrum and risk factors of congenital zika syndrome, including neurodevelopment milestones, mental retardation, etc, during the first years of life. also to be addressed is whether neurological problems associated with zikv infection, such as meningoencephalitis or acute zika myelitis, are the direct consequence of zikv infection, or are mediated by immune responses. novel zikv diagnostic tests will also be developed. dr louis lambrechts (pasteur institute, paris) reminded the audience that aedes aegypti, which is distributed throughout the tropical and sub-tropical world on all continents, serves as the major vector to transmit urban arboviruses, including the denvs, zikv, yfv, chikv, etc. whereas most female mosquitoes take only one blood meal a day, aedes aegypti females need multiple blood feedings every day, which makes them more efficient at transmitting epidemic disease. the comparison between aedes aegypti and ae albopictus also shows that, although ae albopictus is often more susceptible to infection by denv than ae aegypti, it appears to be significantly less efficient at transmitting the virus. in addition, factors such as the mosquito genotype, its geographical location, and the strain of arbovirus, influence the transmission dynamics of the disease. for example, zikv infection of ae aegypti mosquitoes seems to be influenced by the sequence of the ns protein of the virus. as reported by dr scott ritchie (james cook university, cairns, australia), the use of wolbachia infection of aedes mosquitoes has great potential to control the spread of the arboviruses they transmit. wolbachia-infected male aedes mosquitoes become sterile and, if sterile males are repeatedly introduced in a given area, the resident aedes populations can be suppressed. alternatively, wolbachia-infected male and female mosquitoes can be introduced together, which leads to a progressive replacement of the resident aedes population, which could then show a much reduced capacity at transmitting denv, zikv, chikv, yfv and possibly other viruses [ , ] . the use of wolbachia is recommended by the world mosquito program to eliminate dengue. a trial in australia has demonstrated that, shortly after release of wolbachia-infected aedes mosquitoes, over % of the mosquitoes population at the site became wolbachia-infected, and the transmission of denv was stopped [ ] . similar trials are being conducted in viet nam, indonesia and brazil. the fear that wolbachia infection of ae aegypti would lead to its replacement by ae albopictus seems unwarranted so far, but it requires further studies. dr joao bosco siqueira reported that brazil had suffered from a shortage of yellow fever vaccine. fortunately, the usual dose of the brazilian vaccine contains , pfu of attenuated yfv (strain d), when only , pfu is a sufficient protective dose [ ] . it has therefore, been possible to use fractional doses of vaccine with evidence of protection and relatively no difference in reported adverse events. faced with the spread of yellow fever throughout the country, the brazilian government has updated its vaccination policy and now recommends that the entire population should be vaccinated. as part of this effort, a change in schedule for children was implemented from a single dose at years of age to a dose schedule with the first dose administered at months and a second dose at years. this effort is complicated by inadequate vaccine supply and also false rumors that the vaccine would not be protective. fake news is unfortunately powerful and, as a result, vaccine refusal has been rising in the population! as reviewed by dr in-kyu yoon (international vaccine institute, south korea), dr christopher nelson (sanofipasteur, lyon, france) and dr annelies wilder-smith, major advances in dengue research have resulted in development of new vaccines that show promise for use in dengue prevention, such as sanofi pasteur's recently licensed cyd-tdv (dengvaxia Ò , sanofi pasteur, lyon, france) and six other dengue vaccine candidates in various phases of clinical trials. cyd-tdv is a tetravalent live attenuated chimeric vaccine consisting of a d yfv genome with the pre-membrane (pre-m) and envelope (e) genes from each of the four antigenically distinct denv serotypes. the vaccine has undergone large-scale phase clinical trials in asia and latin america [ , ] , demonstrating increasing efficacy with age and higher efficacy in baseline seropositives. efficacy was highest at preventing severe dengue and hospitalization, and moderate for overall dengue. it also varied with serotype, being higher ( - %) for denv- and - infection, and lower for denv- and - . notably, during the third year of the asian phase trial, an increased risk of dengue hospitalization was seen in children aged - years. efficacy of the vaccine was only % or less in - years old and % in - years old. the vaccine was licensed in with an age indication of - years (up to years of age in some countries). the cyd-tdv vaccine is now licensed in countries, and has been introduced into public immunization programs in endemic areas of the philippines and brazil. the vaccine showed high efficacy and good safety in seropositive persons in the - years age group, but a risk of severe dengue was observed in individuals who were naive for denv infection at the time they were vaccinated. it was hypothesized that cyd-tdv mimics primary infection among individuals with no prior dengue infection (previously dengue-unexposed, seronegatives) and a secondary-like infection among those with prior dengue infection (previously dengue-exposed, seropositives) [ , [ ] [ ] [ ] [ ] . a retrospective case-cohort study was undertakern to analyze vaccine safety among dengue seropositive and seronegative trial participants. this work used anti-ns results from month of follow-up that were obtained using an anti-ns igg elisa developed for this purpose. the data confirmed the substantial benefit of cyd-tdv vaccination in those who are dengue seropositive and aged years or older, reducing symptomatic dengue, hospitalized dengue and severe dengue by~ %. however, in individuals of any age without evidence of prior dengue infection, the vaccine elicited an increased risk of severe dengue, as announced by sanofi pasteur in november . the vaccine should therefore be administered only to dengue seropositive persons, which obviously implies the need for a pre-vaccination screening strategy [ ] . the who sage dengue working group reviewed these data and, in april , the sage committee recommended to vaccinate only those with evidence of past dengue infection (seropositives) or with medical documentation of past dengue infection. these results have implications for other dengue vaccine candidates in clinical development, and for immunization program policy and program implementation. two other dengue vaccine candidates are currently in phase clinical trials: tdv, developed by the us cdc and manufactured by takeda, and tv / , developed by the us nih. tdv is a tetravalent live attenuated chimeric vaccine that uses an attenuated denv- backbone with the pre-m and e genes from each of the other three denv serotypes. the tdv vaccine is undergoing phase trials in brazil, columbia, nicaragua, panama, sri lanka and thailand. preliminary phase data show the vaccine is safe and has good efficacy against denv- and- , but the results for denv- and - are uncertain because there were only a small number of cases of these serotypes [ ] . tv /tv is a tetravalent live attenuated vaccine which includes three denv serotypes (denv- , - , À ) that have undergone attenuation through direct mutagenesis (a base-pair deletion), while the denv- candidate consists of a denv- -denv- chimera. the butantan institute in brazil, which is the sponsor of butantan-dv (tv ), received regulatory approval in december to begin a phase trial in brasil. three other denv vaccines are in less advanced development. they are an inactivated vaccine, a dna vaccine, and a subunit (the e glycoprotein) vaccine. but the difficulties of developing effective and safe dengue vaccines are multiple, as seen in the cyd-tdv experience, and many issues still remain, including: . the vaccine should elicit equal protection against the four denv serotypes, which has been extremely difficult to achieve, in view of competition between strains and/or ade of some strains: the question has been raised of whether it really is a must to develop tetravalent vaccines? . whether antibodies are associated with protection or risk (ade) seems to depend on titer, homotypy versus heterotypy, etc. how can that be understood and controlled? . why does severity of the disease, especially that of the postvaccination adverse events, vary with age? and . what is the feasibility and what will be the cost of a necessary pre-vaccination screening? nevertheless, the substantial public health benefits of even a moderately effective dengue vaccine continue to drive all the efforts at developing dengue vaccines [ , ] , hoping that safety concerns may be adequately addressed. a large number of zika vaccine candidates are under development and have entered clinical trials, including: a mrna vaccine; a dna vaccine that encodes the zikv prm and e genes and is presently entering phase iib trials in the usa, brazil, and several countries in latin america (puerto rico, mexico, panama, peru, columbia..); a purified inactivated vaccine adjuvanted with alum, which was shown to be immunogenic after two doses by the im route in a phase clinical trial; and a chimeric measles virus vaccine that expresses the prm and e genes of zikv. as outlined by dr anna durbin (john hopkins bloomberg school of public health) many hurdles still remain before licensure of a safe and effective zikv vaccine can occur. the transmission of zikv has declined so much that it will be difficult to perform a phase efficacy study. in addition, clinical trial efficacy endpoints for a zikv vaccine are not well established: one can wonder if a vaccine will not have to fully prevent infection in order to prevent microcephaly, a very high bar for any vaccine indeed! dr georges thiry (sas senergues, france) reported the creation of the 'coalition for epidemic preparadness innovations' (cepi), which was launched in january in oslo, london and washington with the aim to prevent future epidemics by the development of new vaccines. three major targets have been selected for the manufacturing and testing of candidate vaccines: mers-cov, lassa fever virus and nipah virus. cepi is responsible for vaccine development from preclinical to phase a trial. several vaccines are being followed up: a lassa-vsv vaccine, developed by iavi; a lassa-dna vaccine; a lassa-measles (mv) vaccine, developed by themis; a mers-cov-mv vaccine, also developed by themis; and a nipah glycoprotein subunit vaccine with adjuvant. most phase studies have taken place in - and phase a trials are expected to follow in - . arboviruses are in the news and at the top of social, political and public health agendas. arbovirus epidemics will increasingly threaten global, national and local political and economic security and create a global public health emergency. not only is there an increased risk of epidemic arboviral diseases in rural areas, but there also is now an increased risk of urban epidemics. dengue epidemics have recently seen their amplitude increase dramatically: in , there were fold more reported cases of denv infection in brazil than during the preceding year, leading to deaths; during the same period, , dengue-infected persons had to be hospitalized in bangladesh, where some deaths occurred, and more than deaths from dengue were reported in the philippines! an estimated million dengue infections occur annually [ ] . the development of vaccines and vector control tools to prevent arbovirus diseases is actively being pursued [ , ] , and the possibility of developing a trivalent vaccine against zikv, chikv and wnv is even being entertained. many hurdles however, remain before licensure of a safe and effective zikv vaccine can occur, as outlined above. similarly, the difficulties at developing an effective and safe denv vaccine are multiple, and many questions still arise. what will be the cost of implementing pre-vaccination screening? whom to vaccinate in places where denv seropositivity in years of age turns out to be very low (only % in singapore for example)? the dramatic emergence and spread of epidemic arboviral diseases has made it necessary to reassess surveillance strategies. all at-risk countries should have an enhanced passive disease surveillance system based on well-defined case definitions and supported by serological and virological laboratory testing. an active laboratory syndromic surveillance system, such as was developed in the past by who for influenza or for poliomyelitis, is badly needed. it is hoped that such a system will be set up rapidly. but we also need more effective mosquito control tools to use in conjunction with vaccines, timely access to clinical serices and appropriate care. only by using an integrated approach to prevention and control will we be able to successfully reverse the trend of emergent epidemic arboviral diseases. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the xxth century dengue pandemic dengue and dengue hemorrhagic fever the global threat of emergent/reemergent vector-borne diseases. in: vector-borne diseases: understanding the environmental, human health, and ecological connections epidemic arboviral diseases: priorities fror research and public health increase in reported prevalence of microcephaly in infants born to women living in areas with confirmed zika virus transmission during the first trimester of pregnancy -brazil importation of yellow fever into china: assessing travel patterns the continuing spread of west nile virus in the western hemisphere the emergence of arthropod-borne viral diseases: a global prospective on dengue, chikungunya and zika fevers the global distribution and burden of dengue global strategy for dengue prevention and control community effectiveness of indoor spraying as a dengue vector control method: a systematic review dengue haemorrhagic fever: diagnosis, treatment, prevention and control who immunization, vaccines and biologicals. vaccine position papers efficacy and longterm safety of a dengue vaccine in regions of endemic disease yellow fever epidemiological update identifying global vulnerabilities to urban transmission of yellow fever virus aedes albopictus and the world trade in used tires, - : the shape of things to come urbanization and globalization: the unholy trinity of the st century the global distribution of the arbovirus vectors aedes aegypti and ae albopictus policy and practice. a guide to aid the selection of diagnostic tests isolation of zika virus from aedes aegypti mosquitoes in malaysia a single mutation in the prm protein of zika virus contributes to fetal microcephaly assessing the epidemiological impact of wolbachia deployment for dengue control using wolbachia for dengue control: insights from modelling the awed trial (applying wolbachia to eliminate dengue) to assess the efficacy of wolbachia-infected mosquito deployments in yogyakarta, indonesia: study protocol for a cluster randomised controlled trial immunogenicity of fractional-dose vaccine during a yellow fever outbreak -final report benefits and risks of the sanofi-pasteur dengue vaccine: modeling optimal deployment dengue vaccine: hypotheses to understand cyd-tdvinduced protection the long-term safety, public health impact, and cost-effectiveness of routine vaccination with a recombinant, live-attenuated dengue vaccine (dengvaxia): a model comparison study effect of serostatus on dengue vaccine safety and efficacy efficacy of a tetravalent dengue vaccine in healthy children and adolescents beyond efficacy: the full public health impact of vaccines estimating the full public health value of vaccination the organizing committee of the meeting included the following scientists: drs duane j gubler, jacques louis, christopher b nelson, mauricio nogueira, valentina picot, usa thisiyakorn, in-kyu yoon, and mrs cindy grasso.moderators and presenters were drs duane gubler, usa thisiyakorn, joao bosco siqueira, mauricio lacerda nogueira, christopher nelson, rome buathong, nikos vasilakis, michael gaunt, patricia brasil, marc lecuit, anna durbin, georges thiry, annelies wilder-smith, louis lambrechts, scott ritchie, in-kyu yoon, and elizabeth hunsperger. key: cord- -gcfx authors: ianevski, aleksandr; zusinaite, eva; kuivanen, suvi; strand, mårten; lysvand, hilde; teppor, mona; kakkola, laura; paavilainen, henrik; laajala, mira; kallio-kokko, hannimari; valkonen, miia; kantele, anu; telling, kaidi; lutsar, irja; letjuka, pille; metelitsa, natalja; oksenych, valentyn; bjørås, magnar; nordbø, svein arne; dumpis, uga; vitkauskiene, astra; Öhrmalm, christina; bondeson, kåre; bergqvist, anders; aittokallio, tero; cox, rebecca j.; evander, magnus; hukkanen, veijo; marjomaki, varpu; julkunen, ilkka; vapalahti, olli; tenson, tanel; merits, andres; kainov, denis title: novel activities of safe-in-human broad-spectrum antiviral agents date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: gcfx according to the who, there is an urgent need for better control of viral diseases. re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified compounds that target at least three viral diseases. we tested of these compounds against eight different rna and dna viruses. we found novel activities for dalbavancin against echovirus , ezetimibe against human immunodeficiency virus and zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rift valley fever virus. thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases. dozens of viruses, such as fluav, hsv- , vzv, cmv and nov, constantly infect human population and represent substantial public health and economic burden (dalys and collaborators, ; disease et al., ) . emerging and re-emerging viruses, such as ebov, marv, lasv, chikv, zikv, denv, rvfv, mers-and sars-cov, surface from natural reservoirs approximately one each year and also represent global threats (howard and fletcher, ; who, ) . according to who, there is an urgent need for better control of these viruses, including drug-resistant and vaccine immunity escaping viral strains (bekerman and einav, ; de clercq and li, ) . antiviral drugs and vaccines are the most powerful tools to combat viral diseases. most drugs and vaccines, however, selectively target a single virus, thereby providing a "one drug-one bug" solution. by contrast, broad-spectrum antivirals (bsas) can cover multiple viruses and genotypes and reduce the likelihood of development of resistance. therefore, some bsas can be used for rapid management of new or drug-resistant viral strains, for treatment of viral co-infections reducing therapy complexity, as well as a first-line treatment or the prophylaxis of acute virus infections. thus, to overcome time and cost issues associated with the development of virus-specific drugs and vaccines, the development of bsas should be prioritized (bekerman and einav, ) . nucleotide and nucleoside analogues are excellent examples of bsas. they inhibit transcription and/or replication of different rna and dna viruses (de clercq, ) . in particular, valaciclovir inhibits replication of different herpesviruses and hbv (laube et al., ; vere hodge and field, ) . cidofovir and its lipid conjugate brincidofovir also inhibit replication of dsdna viruses, such as herpesviruses, adv, bkv, and hpv (andrei et al., ) . ribavirin blocks viral rna synthesis of fluav, hcv and rsv (hong and cameron, ) . favipiravir and bcx also inhibit replication of different rna viruses (mckimm-breschkin et al., ) . however, viruses are able to develop resistance to some of these nucleotide and nucleoside analogues. other examples of bsa agents include inhibitors of cellular pathways, which are exploited by different viruses for efficient viral replication (debing et al., ) . these agents overcome the problem of antiviral drug resistance. for example, lipid-lowering statins (atorvastatin, lovastatin, simvastatin, and fluvastatin) inhibit cellular hmg-coa reductase and attenuate replication of some enveloped viruses (bernal et al., ; enserink, ) . anti-malaria quinolones (chloroquine and hydroxychloroquine) inhibit acidification of endosomes, which is an essential process for uncoating of ssrna viruses (al-bari, ). anticancer kinase inhibitors dasatinib, imatinib, gefitinib, nilotinib, erlotinib and sunitinib impair intracellular viral trafficking and exert bsa effects (bekerman et al., ; schor and einav, ) . the anti-duchenne muscular dystrophy agent, alisporivir, targets cellular cyclophilin and inhibits the folding of hcv, hiv, mers-and sars-cov proteins, and, therefore, prevents formation of infectious virus particles (boldescu et al., ; de wilde et al., ; soriano et al., ) . thus, both host-directed antivirals and nucleotide/nucleoside analogues could possess bsa activity. here, we hypothesised that some of the identified safe-in-human bsas could possess novel antiviral activities and, therefore, could be used for treatment of many different viral infections. to prove this hypothesis, we reviewed safe-in-man approved, investigational and experimental antiviral agents. we identified compounds that target at least three viral diseases. we tested of the compounds against different viruses and found novel activities for of these agents. we conclude that the spectrum of antiviral activities for existing bsa agents could be expanded towards other viral diseases. information on the viruses and associated human diseases is summarized in table s . information on approved, investigational and experimental safe-in-human antivirals is summarized in tables s -s . this information was extracted from drugbank ( ), clinical trials websites ( ) and pubmed. information on approved, investigational, and experimental antivirals, which target ≥ viral diseases, is summarized in table s . eye diagrams and interaction network plots were created with javascript library d .js v ( ). a structural similarity plot for the drugs was constructed and visualized using a c-spade web application (ravikumar et al., ) . the compounds used in this study, their suppliers and catalogue numbers are summarized in table s . to obtain mm stock solutions compounds were dissolved in % dimethyl sulfoxide (dmso, sigma-aldrich) or milli-q water. the solutions were stored at − °c until use. bhk- cells (baby hamster kidney fibroblasts) were grown in glasgow's minimal essential medium (gmem) containing . % fetal bovine serum (fbs; gibco, paisley, uk), % tryptose phosphate broth (tpb), mm hepes, u/ml penicillin and . mg/ml streptomycin (penstrep, lonza basel, switzerland). ach- cells, a model for chronic hiv- infection, which possesses a single integrated copy of the provirus hiv- strain lai (nih aids reagent program), were grown in rpmi- medium supplemented with % fbs and penstrep. madin-darby canine kidney epithelial (mdck) cells, human embryonic kidney cells (hek t) and african green monkey kidney epithelial cells (vero) were grown in dulbecco modified eagle's medium (dmem; sigma-aldrich, st. louis, mo, usa) supplemented with mm l-glutamine (lonza; basel, switzerland), u/ml penstrep and % fbs. human telomerase reverse transcriptase-immortalized retinal pigment (rpe) cells were grown in dmem-f medium supplemented with u/ml penstrep, mm l-glutamine, % fbs, and . % sodium bicarbonate (sigma-aldrich). tzm-bl cells were grown in dmem supplemented with % heat-inactivated fbs and penstrep. human lung adenocarcinoma epithelial cells, a , were cultured in dmem containing . g/l nahco , mm hepes (euroclone, milan, italy), penstrep, and % fetal bovine serum (fbs, gibco) at °c. all cell lines were grown in humidified incubator at °c in the presence of % co . human influenza a/wsn/ (h n ) virus was generated using eight-plasmid reverse genetics system in hek and vero-e cells, as described previously (hoffmann et al., ) . ev strain was propagated in a monolayer of vero cells, as described earlier (myllynen et al., ) . hsv- was amplified in vero cells, as described previously (nygardas et al., ) . zikv fb-gwuh- strain was cultured in vero e cells, as described earlier (driggers et al., ) . for production of hiv- , × ach- cells were seeded in ml of full culture media, and hiv- production was induced by the addition of nm phorbol -myristate -acetate (viira et al., ) . the induced cells were incubated for h, and subsequently, the hiv- containing media was collected and filtered. the amount of hiv- in the stock was estimated by quantification of p protein in the media. quantity of p was measured using a reference recombinant purified p protein and anti-p -elisa, which was developed in-house. chikv- sg-nanoluc strain was generated from icdna clone of picres representing lr opy strain belonging to east/central/ south african genotype (utt et al., ) . rrv- sg-nanoluc strain derived from rrv-t strain (jupille et al., ) . sequence encoding nanoluc protein was placed between non-structural and structural regions of chikv or rrv genomes under the control of native subgenomic promoters of the viruses. to ensure expression of viral structural proteins, a copy of subgenomic promoter (residues − to + for chikv and residues − to + for rrv, positions given with respect to start site of subgenomic rna) was inserted immediately downstream of sequence encoding for nanoluc. recombinant rvfv expressing the far-red fluorescent protein katushka instead of the deleted nss protein (rrvfvΔnss::katushka) was used in this study (islam et al., ) . the virus stocks were stored at − °c. all the experiments with viruses were performed in compliance with the guidelines of the national authorities using appropriate biosafety laboratories under appropriate ethical and safety approvals. fluav virus was titrated in mdck cells using plaque assay as described previously (denisova et al., ) . ev titers were determined by plaque assay on a cells, as described earlier (marjomaki et al., ) . zikv titers were determined by plaque assay on vero-e cells, as described earlier (kuivanen et al., ) . hsv- titers were determined by plaque titration in vero cells in the presence of human immunoglobulin g ( μg/ml), as described earlier (paavilainen et al., ) . chikv- sg-nanoluc and rrv- sg-nanoluc were titrated in bhk- cells using plaque assay, as described previously (oo et al., ; taylor et al., ) . for testing the viability and death of compound-treated mock-, fluav-, ev -, chikv- sg-nanoluc-, rrv- sg-nanoluc-, zikv-and hsv -infected cells, approximately × rpe cells were seeded in each well of a -well plate. the cells were grown for h in cell growth medium. the media was replaced with virus growth medium (vgm) containing . % bsa, mm l-glutamine, . % nahco , and μg/ml l- -tosylamido- -phenylethyl chloromethyl ketone-trypsin (tpck)-trypsin (sigma-aldrich) in dmem-f . in the case of zikv, the media was replaced with dmem-f medium supplemented with u/ ml penstrep, mm l-glutamine, % fbs, and . % sodium bicarbonate. the compounds were added to the cells in three-fold dilutions at seven different concentrations starting from μm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. when virus induced a cytopathic effect in cells (typically - hpi), celltox green express cytotoxicity reagent (ctxg, : dilution in the assay well, promega, madison, wi, usa) was added in vgm and the fluorescence was measured with a pherastar fs plate reader (bmg labtech, ortenberg, germany) or hidex sense microplate reader (hidex oy, turku, finland). the media was removed from the cells and stored at − °c. celltiter-glo viability reagent (ctg, promega, madison, wi, usa) containing firefly luciferase and luciferin was added ( μl per well). the luminescence was measured with a pherastar fs plate reader. for testing virus titers in compound-treated and non-treated fluav-, ev -, zikv-and hsv -infected cells the media was collected, serially diluted in pbs, and added to mdck (fluav), a (ev ), and vero-e (zikv and hsv- ) cells. the media was changed, and the cells were overlaid with plaque assay media. the cells were fixed, and viral titers were calculated. the titers were expressed as plaque-forming units per ml (pfu/ml). the presence of chikv- sg-nanoluc and rrv- sg-nanoluc in the media from non-or drug-treated rpe cells was evaluated by passaging the viruses in fresh rpe cells. the viruses expressed nano-luciferase from viral promoter, which was measured from lysed cells h post infection using renilla luciferase assay (promega, madison, wi, usa). for testing compound toxicity and efficacy against hiv- , approximately × tzm-bl cells were seeded in each well of a -well plate. tzm-bl cells express firefly luciferase under control of hiv- ltr promoter allowing quantitation of the viral infection (tat-protein expression by integrated hiv- provirus) using firefly luciferase assay. the cells were grown for h in cell growth medium. compounds were added to the cells in three-fold dilutions at seven different concentrations starting from μm. no compounds were added to the control wells. the cells were infected with hiv- (media that contained ng of hiv- p was used per well) or mock. at hpi, celltox green express cytotoxicity reagent (ctxg, : dilution in the assay well) was added and the fluorescence was measured with a pherastar fs plate reader. the media was removed from the cells, the cells were lysed, and firefly luciferase activity was measured using the luciferase assay system (promega, madison, wi, usa) and pherastar fs plate reader. to determine the efficacy of compounds against rvfv, the fluorescent intensity of individual infectious cell foci was quantified. briefly, a cells ( × /well) were grown in -well black plates with transparent bottoms (greiner bio-one international). compounds were serially diluted in dmso in two-fold steps from μm to . μm and mixed with pfu of rrvfvΔnss::katushka virus in a total volume of μl medium (moi, . ). the final concentration of dmso in the assay was %. the growth medium was removed from the wells and μl of compound and virus mixture was added to the cells for each compound concentration. at hpi the medium was removed, and cells were fixed with % paraformaldehyde (pfa) for h and washed with phosphate-buffered saline (pbs). μl pbs was added to each well and the plate was analyzed in the trophos plate runner hd (trophos, roche group) to count the number of virus infected cells per well, by identifying all individual cells expressing the far-red fluorescent protein katushka (islam et al., ) . the toxicity of compounds was analysed using ctg assay. the half-maximal cytotoxic concentration (cc ) and the halfmaximal effective concentration (ec ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version . a (graphpad software la jolla, ca, usa) or using hill curve fit with sigmastat . software (spss inc., chicago, il, usa). the relative effectiveness of drugs were quantified as the selectivity indexes (si = cc /ec ). there is limited information available on approved, investigational and experimental bsas. to identify all potential bsas, we reviewed approved, investigational and experimental safe-in-human antiviral agents in drug bank, clinical trials websites and pubmed, respectively. first, we searched for drugs, which have been approved for treatment of viral diseases in human. by excluding vaccines and discontinued drugs, we identified active antiviral substances. these compounds target viral, human and unknown factors and inhibit viruses (table s ). the approved antivirals include neutralizing antibodies, interferons, antisense oligonucleotide, and small molecules. only of the molecules target viruses or host factors associated with ≥ viral diseases. fig. shows bsas and other approved antiviral drugs linked to viral and host targets through viruses they inhibit. next, we reviewed all investigational antivirals and their safety profiles. by excluding vaccines from the search parameters, we found active compounds. these compounds target viral, human and unknown factors and inhibit viruses (table s ). the drug candidates include neutralizing antibodies, antisense oligonucleotides, interferons, enzyme and small molecules. only of the small molecules target ≥ viral diseases. fig. shows these and other investigational antiviral agents linked to cellular and host factors through targeted viruses. next, we searched for drugs, which have been approved for treatment of non-viral diseases, but for which antiviral activity has been reported. we found active compounds, which are all small-molecules. these compounds target viral, human and unknown factors, and inhibit human viruses (table s ) . thirty-nine of the antiviral agents target ≥ viral diseases. fig. shows these and other experimental antiviral agents connected to their host and viral targets through viruses they inhibit. altogether, we identified antiviral agents with available safety information in humans. these agents target host, viral and unknown factors and inhibit different viruses, belonging to viral families. fifty-nine agents have bsa activity (table s ). these agents target of the reported viruses (except andv, emcv and rabv) (fig. s ). structural analysis of bsa revealed that several drugs (such as, acyclovir, famciclovir and valacyclovir; imatinib and disatinib; minocyclin and doxycycline; ritonavir and lopinavir; hydroxychloroquine and chloroquine; esomeprazole and omeprazole) have similar scaffolds (fig. s ) . these drugs target a limited number of host and viral factors (fig. s ). in particular, statins (fluvastatin, lovastatin and simvastatin) target human hmg-coa; dalbavancin, oritavancin, teicoplanin and telavancin target human cyp a ; acyclovir, valacyclovir, famciclovir, azacitidine, brincidofovir, cidofovir, foscarnet, trifluridine, and vidarabine inhibit viral dna polymerases; bcx , favipiravir and ribavirin inhibit viral rna polymerases; and lamivudine and tenofovir disoproxil inhibit viral reverse transcriptases. we hypothesized that approved, investigational and experimental antiviral agents, which target ≥ viral diseases, could inhibit other viral infections. as a proof of concept, we tested of the identified bsas against fluav, ev , chikv, rrv and hsv- infections in rpe cells (alisporivir, cyt , sunitinib and thymalfasin were excluded; table s ). we also tested of the bsas against zikv infection in rpe cells (alisporivir, cyt , sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded). we evaluated viability and death of virus/mock-infected cells using ctg and ctxg assays, respectively. the ctg assay quantifies atp, an indicator of metabolically active living cells, whereas ctxg assay uses fluorescent cyanine dye that stains the dna of dead cells. after initial screening we found several hits, which kept infected cells alive or rescued infected cells from virus-mediated death. these hits are gemcitabine, gefitinib and vibarabine (fluav); gemcitabine, pirlindole dibucaine, fluoxetine and dalbavancin (ev ); gemcitabine, imatinib, ivermectin, lopinavir, lovastatin, ezetimibe, fluoxetine, bcx , chloroquine and hydroxychloroquine (zikv); chloroquine and mycophenolic acid (chikv); chloroquine, mycophenolic acid, dibucaine and itraconazole (rrv); as well as -azacitidine, gemcitabine, trifluridine and vidarabine (hsv- ). we repeated the ctg and ctxg assays with selected compounds and titrated fluav, ev , zikv and hsv- produced in drug-treated and non-treated cells. the presence of chikv and rrv viruses in the media collected from non-or drug-treated rpe cells was evaluated by infecting fresh rpe cells and measuring reporter protein expression from viral promoter (nanoluciferase activity). these experiments confirmed antiviral activity of gemcitabine against fluav (fig. s ) ; gemcitabine, pirlindole, dibucaine, fluoxetine, and dalbavancin against ev (fig. s ) ; gemcitabine, lopinavir, ezetimibe, bcx , chloroquine, and hydroxychloroquine against zikv (fig. s ) ; chloroquine and mycophenolic acid against chikv (fig. s ) ; mycophenolic acid against rrv (fig. s ) ; and gemcitabine against hsv- (fig. s ) . importantly, dalbavancin and ezetimibe demonstrated novel antiviral activities against ev and zikv, respectively. in particular, they rescued infected rpe cells from virus-mediated cell death, and lowered production of infectious virus particles without detectable cytotoxicity (table ) . we also examined toxicity and antiviral activity of of the bsa agents (excluding alisporivir, cyt , sunitinib and thymalfasin) against hiv- infection in tzm-bl cells. our primary screen identified ezetimibe, minocycline and rapamycin as anti-hiv- agents. validation experiment confirmed all three hits (fig. s , table ). interestingly, ezetimibe is a novel inhibitor, whereas minocycline and rapamycin are known anti-hiv- agents (heredia et al., ; singh et al., ) . in addition, we examined antiviral activity and toxicity of of the bsa agents (alisporivir, cyt , sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded) against rvfv expressing the far-red fluorescent protein katushka in a cells. our screen identified azacitidine, bortezamibe, cyclosporine, doxycycline, ezetimibe, fluoxetine, gefitinibe, minocycline, oritavancin, ritonavir and topotecan. azacitidine, cyclosporine, minocycline, oritavancin, ritonavirand and bortezamib remained after excluding compounds with si < ( fig. s ; table ). interestingly, azacitidine, cyclosporine, minocycline, oritavancin and ritonavirand are novel, whereas bortezamib is a known inhibitor of rvfv infection (keck et al., ) . thus, we tested several known bsa agents against (−)ssrna, (+) ssrna, ssrna-rt and dsdna viruses and identified novel activities for dalbavancin against ev , ezetimibe against zikv and hiv- , as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rvfv. fig. shows known, validated and novel interactions between bsas and viruses. several approved and investigational agents, as well as safe in man chemical probes, were discovered for potential treatment of various viral diseases (bekerman and einav, ; de clercq, ; de clercq and li, ) . re-purposing such therapeutics from one viral disease to another could save resources and time needed for development of novel drugs. in this study, we tested approved, investigational and experimental bsa agents against fluav, rvfv, ev , zikv, chikv, rrv, hiv- and hsv- in vitro. we identified novel antiviral activities for dalbavancin (against ev ), ezetimibe (against hiv- and zikv), azacitidine, cyclosporine, minocycline, oritavancin and ritonavir (against rvfv) (fig. ) . dalbavancin is a lipoglycopeptide antibiotic, which is approved by the fda for the treatment of acute bacterial skin infections caused by staphylococcus aureus and streptococcus pyogenes. dalbavancin also inhibits mers-cov and sars-cov infections. it binds cathepsin l in the late endosomes/lysosomes and blocks the entry of ebov (zhou et al., ) . our study demonstrates that it also inhibits replication of ev , which also enters the cells via an endocytic route (krieger et al., ) . ezetimibe is an fda-approved medication that lowers plasma cholesterol by decreasing its absorption in the small intestine. ezetimibe also inhibits hbv and hdv infections by impairing viral entry mediated by pres -specific receptor hntcp (blanchet et al., ; lempp and urban, ; monrroy-bravo et al., ) . our study shows that ezetimibe also inhibited hiv- and zikv infections. however, the entry of these viruses into the cell is mediated by other receptors (hamel et al., ; wilen et al., ) . most probably, the anti-hiv- , ev , as well as hcv action of ezetimibe is associated with depletion of cholesterol which is required for entry of these viruses into the host cells (sainz et al., ) . importantly, this drug was successfully tested in combination with antiretroviral therapeutics to lower cholesterol levels in serum of hiv-infected patients (saeedi et al., ; wohl et al., ) . the question remains whether ezetimibe alone reduces hiv titers in these patients. azacitidine is a chemical analogue of cytidine, which is used in the treatment of myelodysplastic syndrome. it also inhibits fluav, adv, hiv- and hiv- replication by blocking viral rna or dna synthesis (beach et al., ; rawson et al., ) . cyclosporine is an immunosuppressive agent used for the treatment of rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome and keratoconjunctivitis sicca. in addition, cyclosporine is used to prevent graft rejection in organ transplant recipients. cyclosporine also inhibits hcv, fluav, wnv and zikv replication through blocking interaction of cellular cyclophilins with viral proteins and attenuating viral rna synthesis (barrows et al., ; firpi et al., ; qing et al., ) . minocycline is a broad-spectrum antibiotic and antiviral agent, which possesses activity against denv, hiv- and wnv (leela et al., ; quick et al., ; singh et al., ) . oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections (zhou et al., ) . ritonavir is an antiretroviral medication. it has also antiviral activity against mers-cov (chan et al., ) . we showed that azacitidine, cyclosporine, minocycline, oritavancin and ritonavir are active against rvfv. we also confirmed previously reported activities of chloroquine against chikv and zikv, mycophenolic acid against chikv and rrv, fluoxetine, pirlindole and dibucaine against ev , bcx , lopinavir and hydroxichloroquine against zikv, as well as gemcitabine against ev , fluav, zikv and hsv- (cao et al., ; delogu and de lamballerie, ; delvecchio et al., ; denisova et al., ; julander et al., ; kang et al., ; khan et al., ; kuivanen et al., ; lee et al., ; soderholm et al., ; ulferts et al., ; yuan et al., ) (fig. ) . the number of compounds with novel and confirmed antiviral properties may have been higher if we had used other cell lines, other viruses and viral strains, concentration ranges and purity of compounds, as well as endpoint measurements. also testing other compounds, which target < viral diseases, could reveal novel antiviral properties of these compounds and increase the number of potential bsas. for conducting this properly, a harmonized antiviral bioactivity data annotation, standardization, curation, and intra-resource integration is needed. excellent antiviral profiles from cell-line-based assays might not be reflected in vivo because systemic mechanisms may compensate the blocked target effect. therefore, the identification of bsa targets that are essential for viral replication but redundant for the cell is critical for reducing putative toxicities associated with blocking cellular pathways. thus, follow-up mechanistic studies and in vivo experiments are needed to validate our in vitro results. in conclusion, repositioning of safe-in-man agents from one viral disease to another could play a pivotal role in development of broadly acting antivirals. our study demonstrated the potential value of such approach with some examples, such as dalbavancin, ezetimibe, azacitidine, cyclosporine, minocycline, oritavancin and ritonavir, confirming a principle that "rich becomes richer". effective bsa treatment may shortly become available, pending the results of further pre-clinical studies and clinical trials. the most effective and tolerable compounds will expand the available therapeutics for the treatment of viral diseases. some of these compounds could be used as first-line therapeutics to combat emerging and re-emerging viral threats, having global impact by improving preparedness and the protection of the general population from viral epidemics and pandemics. the authors declare no financial and non-financial competing interests. half-maximal cytotoxic concentration (cc ), the half-maximal effective concentration (ec ) and minimal selectivity indexes (si) for selected broad-spectrum antivirals. ctxg -celltox green express cytotoxicity assay, ctg -celltiter-glo viability assay, otherplaque assay or reporter gene expression assay, n.a.not available. fig. . the interaction network between viruses and bsas, which are safe in man. drug-like shapes represent antiviral agents. blue spheres represent viruses. the diameter of spheres corresponds to the number of interactions between the viruses and the drugs. novel interactions between bsas and viruses are shown in red, validatedin blue, and known -in grey. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases insights into the mechanism of action of cidofovir and other acyclic nucleoside phosphonates against polyoma-and papillomaviruses and non-viral induced neoplasia a screen of fda-approved drugs for inhibitors of zika virus infection novel inhibitors of human immunodeficiency virus type infectivity infectious disease. combating emerging viral threats anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects statins in hiv-infected patients: potential beneficial effects and clinical use use of fda approved therapeutics with hntcp metabolic inhibitory properties to impair the hdv lifecycle broad-spectrum agents for flaviviral infections: dengue, zika and beyond inhibition of autophagy limits vertical transmission of zika virus in pregnant mice treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset clinical trial resources global, regional, and national disability-adjusted life-years (dalys) for diseases and injuries and healthy life expectancy (hale) for countries and territories, - : a systematic analysis for the global burden of disease study curious (old and new) antiviral nucleoside analogues with intriguing therapeutic potential approved antiviral drugs over the past years alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model the future of antivirals: broad-spectrum inhibitors chikungunya disease and chloroquine treatment chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection global, regional, and national incidence, prevalence, and years lived with disability for diseases and injuries for countries, - : a systematic analysis for the global burden of disease study zika virus infection with prolonged maternal viremia and fetal brain abnormalities infectious disease. old drugs losing effectiveness against flu; could statins fill gap? cyclosporine suppresses hepatitis c virus in vitro and increases the chance of a sustained virological response after liver transplantation biology of zika virus infection in human skin cells targeting of mtor catalytic site inhibits multiple steps of the hiv- lifecycle and suppresses hiv- viremia in humanized mice a dna transfection system for generation of influenza a virus from eight plasmids pleiotropic mechanisms of ribavirin antiviral activities emerging virus diseases: can we ever expect the unexpected? anti-rift valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir- , a broadacting antiviral compound efficacy of the broad-spectrum antiviral compound bcx against zika virus in cell culture and in a mouse model mutations in nsp and pe are critical determinants of ross river virus-induced musculoskeletal inflammatory disease in a mouse model synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses characterizing the effect of bortezomib on rift valley fever virus multiplication cellular impdh enzyme activity is a potential target for the inhibition of chikungunya virus replication and virus induced apoptosis in cultured mammalian cells echovirus entry into polarized caco- cells depends on dynamin, cholesterol, and cellular factors associated with macropinocytosis obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism valaciclovir for chronic hepatitis b virus infection after lung transplantation gemcitabine, a broadspectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion drug repurposing of minocycline against dengue virus infection inhibitors of hepatitis b virus attachment and entry internalization of echovirus in caveolae prevention and treatment of respiratory viral infections: presentations on antivirals, traditional therapies and host-directed interventions at the th isirv antiviral group conference effect of ezetimibe in hcv viral load after liver transplantation a novel open and infectious form of echovirus a herpes simplex virus-derived replicative vector expressing lif limits experimental demyelinating disease and modulates autoimmunity deciphering the potential of baicalin as an antiviral agent for chikungunya virus infection inhibition of clinical pathogenic herpes simplex virus strains with enzymatically created sirna pools cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral ns protein minocycline has anti-inflammatory effects and reduces cytotoxicity in an ex vivo spinal cord slice culture model of west nile virus infection c-spade: a web-tool for interactive analysis and visualization of drug screening experiments through compound-specific bioactivity dendrograms synergistic reduction of hiv- infectivity by -azacytidine and inhibitors of ribonucleotide reductase lipid lowering efficacy and safety of ezetimibe combined with rosuvastatin compared with titrating rosuvastatin monotherapy in hiv-positive patients identification of the niemann-pick c -like cholesterol absorption receptor as a new hepatitis c virus entry factor repurposing of kinase inhibitors as broad-spectrum antiviral drugs minocycline attenuates hiv- infection and suppresses chronic immune activation in humanized nod/ltsz-scidil- rgamma(null) mice immuno-modulating properties of saliphenylhalamide, sns- , obatoclax, and gemcitabine directly acting antivirals against hepatitis c virus effects of an in-frame deletion of the k gene locus from the genome of ross river virus screening of a library of fda-approved drugs identifies several enterovirus replication inhibitors that target viral protein c versatile transreplication systems for chikungunya virus allow functional analysis and tagging of every replicase protein antiviral agents for herpes simplex virus design, discovery, modelling, synthesis, and biological evaluation of novel and small, low toxicity s-triazine derivatives as hiv- non-nucleoside reverse transcriptase inhibitors who publishes list of top emerging diseases likely to cause major epidemics hiv: cell binding and entry. cold spring harb ezetimibe alone reduces low-density lipoprotein cholesterol in hiv-infected patients receiving combination antiretroviral therapy structure-based discovery of clinically approved drugs as zika virus ns b-ns protease inhibitors that potently inhibit zika virus infection in vitro and in vivo glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) this study was supported by the european regional development fund, the mobilitas pluss project mobtt (to denis e. kainov), jane and aatos erkko foundation grant # (to the vejo hukkanen). we thank qiuwei abdullah pan from the department of gastroenterology and hepatology, erasmus mc, university medical center rotterdam for advice. we thank ritva kajander for the hsv- culture. supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -tu xrt x authors: li, cui; gao, fei; yu, lei; wang, ruoke; jiang, yisheng; shi, xuanling; yin, chibiao; tang, xiaoping; zhang, fuchun; xu, zhiheng; zhang, linqi title: a single injection of human neutralizing antibody protects against zika virus infection and microcephaly in developing mouse embryos date: - - journal: cell rep doi: . /j.celrep. . . sha: doc_id: cord_uid: tu xrt x zika virus (zikv) is a mosquito-transmitted flavivirus that is generally benign in humans. however, an emergent strain of zikv has become widespread, causing severe pre- and post-natal neurological defects. there is now an urgent need for prophylactic and therapeutic agents. to address this, we investigated six human monoclonal antibodies with zikv epitope specificity and neutralizing activity in mouse models of zikv infection and microcephaly. a single intraperitoneal injection of these antibodies conveyed distinct levels of adult and in utero protection from zikv infection, which closely mirrored their respective in vitro neutralizing activities. one antibody, zk b , showed the most potent neutralization activity, completely protected uninfected mice, and markedly reduced tissue pathology in infected mice. thus, zk b is a promising candidate for the development of antibody-based interventions and informs the rational design of zikv vaccine. zika virus (zikv) is a mosquitotransmitted flavivirus that can cause severe neurological defects in humans. li et al. have identified a human monoclonal antibody capable of protection against zikv infection and related diseases when tested in mouse models. this antibody serves as a promising candidate for clinical development against zikv. the recent, widespread neurological deficits caused by an emergent strain of zika virus (zikv) have caught the world off guard wikan and smith, ) . zikv was first identified in the forests of uganda, and infection was generally benign in humans (dick et al., ) . however, this new strain of zikv is far more virulent and causes a range of clinical anomalies rasmussen et al., ; wikan and smith, ) . most notable are microcephaly and other congenital defects in infants born to mothers infected with zikv during pregnancy (mlakar et al., ; petersen et al., ; rasmussen et al., ; wikan and smith, ) . although the exact mech-anism of neuropathogenesis remains uncertain, clinical abnormalities have been linked to the aberrant development and loss of neural progenitor cells (npcs) (cugola et al., ; gabriel et al., ; garcez et al., ; li et al., a li et al., , b tang et al., ) . the contemporary strain of zikv has enhanced replication capacity and a specialized tropism for npcs (cugola et al., ; dang et al., ; garcez et al., ; li et al., b; tang et al., ) , although other types of cells are susceptible (tabata et al., ; weisblum et al., ) . the infection inhibits npc proliferation and differentiation and can trigger apoptosis or autophagy. critically, the highest rates of birth defects occur in pregnant mothers who are infected during their first and second trimesters. this is presumably because, during the early stages of gestation, npcs have a greater susceptibility to zikv infection, and there is more viral transfer across the placental barrier (mlakar et al., ; petersen et al., ; rasmussen et al., ; wikan and smith, ) . to fully protect the developing fetuses, an intervention must occur before this period or, ideally, prior to infection (marston et al., ) . neutralizing antibodies are the essential mediator of immunity against viral infection (burton and hangartner, ; corti and lanzavecchia, ) . for zikv and other flaviviruses, human neutralizing monoclonal antibodies target the surface envelope glycoprotein (e) that facilitates infection (dejnirattisai et al., ; fernandez et al., ; fibriansah et al., ; heinz and stiasny, ; magnani et al., ; pierson and diamond, ; pierson and graham, ; robbiani et al., ; rogers et al., ; sapparapu et al., ; stettler et al., ; wang et al., wang et al., , b zhao et al., ) . we previously reported on a panel of monoclonal antibodies (mabs) derived from the longitudinal samples of a zikv-convalescent individual and characterized their neutralizing activities, epitope specificities, and development timeline over the course of infection . we also reported on mouse models of zikv infection and microcephaly, with enhanced specificity for neurological infection figure . experimental design to evaluate the prophylactic potential of six human mabs against zikv infection in developing fetuses and ag mice (a) timeline for mab injection, zikv inoculation, infant delivery, and the monitoring of a complementary set of clinical, virological, and neuropathological outcomes from embryonic day . to p . the six mabs tested are shown alongside their ic values and epitope specificities. zikv-inoculated fetuses and neonates are indicated by zikv + in red; those left unexposed are indicated by zikv À in blue. the cartoon mice on p include zikv + (small and large indicated in red) and zikv À (large indicated in blue); the size representations reflect potential body weight outcomes. each mab was tested, and the outcomes were monitored in three littermate neonates of three pregnant mice on p and p . (b) different levels of protection conferred by the six mabs, shown with the number (n) of neonates monitored for survival in each mab treatment group. (c) the body weight of neonates among the different mab groups. zikv + neonates indicated in red are presented as a percentage of the blue zikv À littermates. the zk b -treated group had no discernable differences between the zikv + and zikv À littermates and is, therefore, shown as a single red/blue checkered bar. the number of neonates for each analysis is indicated above the respective mab, with zikv + in red and zikv À in blue. (legend continued on next page) using a contemporary zikv asian strain (gz ). in the model of microcephaly, the virus was inoculated directly into the lateral ventricles of the fetal mouse brain (li et al., a) . zikv replicated in the fetal brain, with preferential infection of npcs. infection resulted in cell-cycle arrest, differentiation defects, and a large number of cell deaths, as well as clinical presentations of microcephaly (li et al., a) . here, we use the mouse models of zikv infection and microcephaly to analyze the in vivo protective activities of six human mabs and compare the findings with our reported in vitro neutralization activity, as measured by plaque reduction neutralization test (prnt). our results offer compelling evidence that the in vivo protection is directly associated with in vitro neutralization. one antibody, zk b , provides complete protection in pre-exposure treatment and partial protection in post-exposure therapy, with markedly reduced tissue pathology. we believe that zk b is a promising candidate for the development of antibody-based interventions and informs vaccine design specific to zikv infection. in vivo protection correlates with in vitro neutralizing activity of mabs we evaluated and compared the protective potential of six representative human mabs following the protocol highlighted in figure a . all these (zk - , zk - , zk f , zk c , zk c , and zk b ) were isolated by our team. two additional zikv domain iii (diii)-specific mabs (zv and z ) isolated by others were included for comparative analysis (robbiani et al., ; zhao et al., ) . a human antibody, middle east respiratory syndrome- (mers- ), targeting the middle east respiratory syndrome coronavirus (mers-cov), was used as a negative control. our mabs were initially isolated from the peripheral b cells of a convalescent chinese man who acquired zikv while traveling to venezuela during the outbreak in . specimens were derived from sequential blood samples collected on day (zk - ), day (zk - ), day (zk f ), or day (zk c , zk c , and zk b ) after symptom onset. they targeted the zikv e with varying degrees of binding and neutralizing activities and epitope specificities . for example, zk b and zk c were strictly zikv specific and demonstrated the most potent neutralizing activities, as measured by prnt. of these, zk b recognized diii, while zk c was specific for domain i/domain ii (di/dii). in comparison, zk - , zk - , zk f , and zk c were much less potent against zikv but cross-neutralized other members of the flavivirus family, such as dengue virus serotypes denv and denv . on the zikv envelope, they targeted either di/dii (zk - , zk - , and zk f ) or di/dii/diii (zk c ). in terms of genetic analyses, these antibodies demonstrated diverse heavy-chain variable regions. two, zk - and zk f , fell into the immunoglobulinheavy variable (ighv) - family, while zk - and zk c belonged to the ighv - family. finally, zk b and zk c belonged to the ighv - and ighv - families, respectively. to test prophylactic activity against zikv infection, mg/kg of each mab was administered intraperitoneally to a group of three pregnant mice on embryonic day . ( figure a ). the following day (e . ), the developing littermate fetuses in each pregnant mouse were either left untouched (zikv À ; figure a , blue) or inoculated with plaque-forming units (pfus) of zikv asian strain gz directly into the lateral ventricles of the brain (zikv + ; figure a , red). the neonates were closely monitored after delivery and analyzed for a complementary set of clinical, virological, and neuropathological outcomes on post-natal day (p) and for survival up to p . as shown in figure b , the levels of protection conferred by the different mabs varied considerably but clearly correlated with their neutralizing activities, as measured by half-maximal inhibitory concentrations (ics ) in the prnt . the most potently neutralizing mab, zk b , conferred complete protection. survival rates of infected fetuses were as high as % on p . the next potent, zk c , however, conferred only partial protection with median survival of days. the two antibodies (zv and z ) isolated by others, together with the rest of the mabs isolated by us, offered negligible protection and were virtually similar to the isotype control mers- . we also assessed impact on developing body weights and found weights measured on p closely correlated with the neutralizing activity. the zikv + neonates treated with the most potent mabs, zk b and zk c , had weight gain similar to that of their zikv À littermates. the zikv + neonates treated with zv , z , and the remaining mabs failed to do so. the control zikv + mers- group had the greatest loss in body weight. next, we studied whether the protection pattern observed in the pregnant mice also occurred in non-pregnant mice. we administered mg of each mab (zk - , zk - , zk f , zk c , zk c , zk b , zv , and z ) or isotype control mers- to a group of four -to -week-old ag mice via the intraperitoneal (i.p.) route. the following day, animals were challenged with pfus of zikv asian strain gz via the i.p. route. survival rates, body weight, and viral rna copies in the blood were monitored for up to days ( figure d ). consistent with the outcomes for pregnant mice, the level of protection in nonpregnant mice correlated with antibody neutralizing activities as measured by half-maximal inhibitory concentrations (ics ) in the prnt ( figure e ). for example, the most potent and protective mab in pregnant mice, zk b , provided complete protection in non-pregnant mice, with survival rates of % days after the challenge. the next potent and protective, zk c , conferred only partial protection, with median survival of days. the rest of the tested mabs, including the diii-specific ones (zv and z ) isolated by others (robbiani et al., ; zhao et al., ) , offered even lower levels of protection. zk - was virtually identical to the isotype control mers- . (d-g) shown here: (d) timeline for mab injection, zikv inoculation, and monitoring for (e) survival, (f) body weight, and (g) blood zikv rna up to days in ag mice. the body weight and zikv rna in the whole blood derived from a single measurement showed distinct results among the study groups. the number of animals used in each group was four. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. similarly, changes in body weight measured over the -day follow-up also correlated with the neutralizing activities. the zikv + mice treated with zk b maintained relatively stable body weight throughout the study, although there was a noticeable decline after the first blood sample collection on day (figure f ). in contrast, zikv + mice treated with zk c lost substantial body weight beginning on day post-challenge. animals treated with the remaining mabs had severe and rapid losses in body weight that coincided with a clinical deterioration (figure e) . lastly, for zikv + mice treated with zk b , blood levels of viral rna were indistinguishable from those measured in uninfected animals on both day ( . ± . versus . ± . ) and day ( . ± . versus . ± . ) after challenge ( figure g ). however, rna levels measured in zk c -treated mice were, on average, log greater on day ( . ± . versus . ± . ) and more than logs greater on day ( . ± . versus . ± . ), compared to zk b . the remaining mabs failed to suppress viral replication. the measured zikv rna load on day ranged from . to . logs higher than that of zk b , depending on the mab used ( figure g ). taken together, these results demonstrated that the protective patterns of each mab were similar in both pregnant and non-pregnant mice and closely mirrored their respective in vitro neutralizing activities. the protective effects of each mab on body weight and overall survival mirrored their respective virological and neuropathological outcomes in treated mice. for instance, rna loads measured in zikv + neonates treated with zk b were suppressed in the blood ( . ± . log copies per milliliter) and the brain ( . ± . log copies per gram) to levels indistinguishable from those in zikv À littermates. in fact, zk b was able to reduce the zikv rna by . logs in the blood and . logs in the brain, relative to that in the zikv + mers- group (figures a and b ). in comparison, zk c was equally effective at suppressing replication in blood ( . ± . log copies per milliliter) but only moderately protective in the brain ( . ± . log copies per gram). the mabs zv and z only moderately suppressed zikv replication in the blood ( . ± . and . ± . log copies per milliliter, respectively) and the brain ( . ± . and . ± . log copies per gram, respectively). none of the remaining mabs notably suppressed replication in either blood or brain tissue. all of the zikv + neonates treated with these mabs had high levels of zikv rna, which were similar to levels in the zikv + mers- control group and significantly higher than those in their zikv À littermates (figures a and b ). importantly, low levels of zikv rna were associated with the healthier development of the neonatal brains. of the six representative mabs selected for in-depth neuropathological study, zikv + mice treated with either zk b or zk c maintained normal brain growth and structure. there were no notable differences in cerebral size, cortical thickness, or lateral ventricle area between these zikv + neonates and their zikv À littermates ( figures d- h ). however, zikv + mice treated with zv and z received only moderate protection, while zk f and mers- failed to provide any protection. low protection was measured by reduced cerebral size, thinner cortices, and enlarged lateral ventricles in zikv + neonates compared to their zikv À littermates ( figures d- h ). it is interesting to note that the e-specific human immunoglobulin g (igg) levels in the brain were higher in mice treated with zk b and zk c compared to the other mab groups. in particular, the levels of zk b ( . ± . mg/kg, which is translated into . ± . mg/ml) in zikv + neonates were well above the ic neutralization values measured in the prnt, whereas igg levels in the other mab groups measured less than or similar to those in the negative control pbs group ( figures a and c) . these results indicate that the tested mabs were able to transport across both the maternal-fetal placental barrier and the primitive blood-brain barrier of developing fetuses (saunders et al., ) . we also conducted immunohistochemical analyses of brain sections collected from p neonates in each group. as shown in figure a , in zk b and zk c groups, no zikv-infected cells (green) or apoptotic cells (cas + , red) were detected in the cortices of either zikv + neonates or their zikv À littermates. the mature neurons (neun; figure a , gray) and nuclei (dapi; figure a , blue) appeared healthy, with a tightly packed structure throughout the tissue sections. in distinct contrast, a large number of zikv-positive cells were identified throughout the cortices of the zikv + neonates in zv , z , zk f , and mers- groups. this high prevalence of infection was associated with significant cell death ( figure a , red) and degradation of the cortical structure ( figure a , gray and blue). we also quantitatively assessed the protective activity of zk b and zk c using marker cells from multiple tissue sections. as shown in figures b and c , treatment with zk b and zk c in zikv + neonates reduced zikv infection and cell death to levels indistinguishable from those of their zikv À littermates. furthermore, in the zk b and zk c groups, counts of mature neurons were equivalent between zikv + neonates and their zikv À littermates ( figure d ). however, for the zikv + neonates treated with either zv , z , zk f , or mers- , zikv-positive cell and apoptotic cell counts were as high as , cells and , cells, on average, per square-millimeter tissue section, respectively ( figures b and c) . counts of mature neurons were also significantly reduced in these groups ( figure d ). these results support the enhanced therapeutic activity of zk b and zk c at the cellular and tissue levels relative to zv , zv , zk f , and mers- and also offer an explanation for their impressive in vivo protection of developing fetuses. as zk b demonstrated the greatest prophylactic efficacy against zikv infection, we went on to evaluate its treatment efficacy after zikv infection of the developing fetuses. specifically, after the littermate fetuses were either left untouched (zikv À ) or inoculated with pfus of zikv gz in the lateral ventricle (zikv + ) on e . , we administered mg/kg zk b i.p. to the pregnant mice. this was done either on the same day (e . , day ), day later (e . , day ), or days later (e . , day ). the littermate neonates were closely monitored after delivery and analyzed for the same outcomes measured in the prevention experiments ( figure a ). as shown in figure a , delaying treatment from day to either day or day reduced the survival percentages. treatment on day offered the most effective treatment, with a median survival of about days. this is signif-icantly higher than that of mice treated on either day ( days) or day ( days). the survival advantage conferred by treatment on day corresponded with a clear reduction of viral load in the brain ( figure d ) and less damage to the cortical thickness and lateral ventricles (figures f and g) . however, the effect was less obvious when measured by body weight or each red dot represents a zikv + ; each blue dot represents a zikv À neonate. the red dots with blue coating represent no discernable differences between zikv + and zikv À littermates in zk b -and zk c -treated groups. the numbers of neonates for each mab analyses are indicated above each group, with zikv + in red and zikv À in blue. in (g) and (h), each dot represents the mean value of at least two slices from one neonate. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. blood serum viral loads on p (figures b and c ). considering that, in our mouse model, the brain is the initial and active site of viral replication, we expected that antibody treatment effects would be more sensitive and immediate in this organ and would be apparent before effects on other organs and body weight as a whole. this hypothesis was supported by immunohistochemical analyses of brain sections derived from p neonates. as shown in figure a , zikv-positive cells (green) were only sporadically detected in the day- treatment group. however, they were detected throughout the cortices if treatment was delayed to day or day . a greater level of infection was associated with increased cell death ( figure a , red) and cortical abnormalities ( figure a , gray and blue). in particular, the tightly packed and well-organized structure of matured neurons in the cortices became loosely connected and disorganized. furthermore, a quantitative assessment of marker cells derived from multiple each red dot represents a zikv + neonate; each blue dot represents a zikv À littermate. red dots with blue coating represent no discernable differences between zikv + and zikv À littermates in zk b -and zk c treated groups. each dot represents the mean value of at least two slices from one neonate analyzed. the numbers of neonates for each analysis are indicated above each group, with zikv + in red and zikv À in blue. scale bar, mm. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . . tissue sections supported the evidence that the time of treatment administration impacted treatment efficacy. for instance, in the zikv + neonate group, treatment on day significantly suppressed the number of zikv-positive cells to ± and apoptotic cells to ± per square millimeter of tissue section. treatment on day , however, allowed widespread cell infection and cell death counts as high as , ± , and ± per square-millimeter tissue section, respectively ( figures b and c ). however, although tissue samples from all three temporal treatment groups showed structural abnormalities in the cortices, there were no obvious reductions in the total number of mature neurons ( figure d ). treatment with the isotype mers- control was worst among all groups, with no signs of figure . evidence of therapeutic potential of zk b against zikv infection in developing fetuses (a) decreased survival when treatment administration is delayed from day (e . ) to day (e . ) or to day (e . ). the number (n) of neonates monitored for survival is indicated. (b-d) after treatment with zk b on day , day , or day , we analyzed zikv + neonates (indicated in red) and zikv À littermates (indicated in blue) at p for (b) body weight, (c) zikv rna copies in the blood, and (d) zivk rna copies in the brain. the number of neonates analyzed for each treatment time point is indicated above each group. (e-g) shown here: (e) the representative coronal sections of the neonatal brains at p was visualized with nissl staining and analyzed for (f) cortex thickness and (g) ventricular area. in (f) and (g), each dot represents the value of one slice. the total numbers of slices analyzed for zikv À , day , day , day , and mers- were , , , , and , respectively. the number of neonates analyzed for each treatment time point is indicated above each group. scale bar, mm. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. protection (figures and ) . these results highlight the critical role of zk b in attenuating disease progression by inhibiting cell infection and death at the cellular and tissue levels. furthermore, the time of administration relative to infection impacts efficacy. treatment has the greatest protective effect when initiated immediately after infection. we report here the systematic analyses of the prophylactic and therapeutic potential of a panel of human mabs against zikv infection in a mouse model of microcephaly and non-pregnant ag mice. we showed that different mabs demonstrated distinct protective activity against zikv infection and that the major determinant of efficacy is their intrinsic neutralizing activities. a single i.p. injection of pregnant and non-pregnant mice with the most potent mab, zk b , provided developing fetuses and adult animals with a complete protection against zikv infection. treatment with zk b markedly reduced fetal infection and tissue pathology and significantly delayed mortality. thus, zk b is a promising candidate for the development of antibody-based intervention and informs rational design of zikv vaccine. two unique aspects of our study are worth highlighting here. one is based in the tested mabs that are all derived from the same zikv convalescent individual at different time points in the course of natural infection. each has distinct neutralizing activity, epitope specificity, and lineage ancestry . this diversity not only allows us to pinpoint the major determinants of protection in the mouse model but also provides evidence for when protective potential arises during the course of a natural infection in humans. our results clearly show that neutralizing activity, rather than other features, is the critical biomarker for protection against zikv infection, although the role of fc-mediated antiviral functions still need further investigation (dejnirattisai et al., ; pierson and graham, ; sapparapu et al., ; stettler et al., ). among the mabs tested here, zk b is the most potent, and its epitope is located in the lateral ridge region within the diii of e . mabs that target diii with potent neutralizing activity have also been isolated by other groups, derived from either infected humans or mice, and have been shown to be effective in various models of zikv pathogenesis (fernandez et al., ; magnani et al., ; robbiani et al., ; stettler et al., ; wang et al., b; zhao et al., ) . some weakly neutralizing mabs have also been reported against the same domain (robbiani et al., ; sapparapu et al., ; stettler et al., ; zhao et al., ) . it would be hard to determine whether this convergence on diii is a purely random event or whether diii epitopes are somehow more exposed and, therefore, more accessible by the mabs. it would be interesting to compare the exact epitopes of the highly potent mabs to see whether the vulnerable spots are widely spread or confined to restricted regions on the diii. this information would undoubtedly contribute to our understanding of the immune recognition mechanisms and assist in the rational design of vaccines against zikv infection. similar to other reported diii-specific mabs, zk b was isolated late after the onset of symptoms ( days), when the production of diii-specific antibodies had become more prevalent the total numbers of slices analyzed for zikv À , day , day , day , and mers- were , , , , and in (b) and (d), and , , , , and in (c), respectively. the total number of neonates analyzed is indicated above each group. all data are presented as mean ± sem. **p < . ; ***p < . ; ns, not significant. . however, why the diii-specific response seems to be delayed relative to di/ii-specific responses, and whether a vaccine could induce the preferred diii-specific response ahead of those targeting di/ii, requires more in-depth study. nevertheless, the potent prophylactic and therapeutic activities demonstrated by zk b and other diii-specific mabs will serve as the key criteria for future antibody-based intervention against zikv infection. the other unique aspect of our study was the mouse model of microcephaly, in which the virus was directly inoculated into the lateral ventricles of the fetal brain. despite the surgical sophistication required with this technique, the model has been standardized with remarkable reproducibility and captures the phenotypic features that are key to zikv infection in humans (li et al., a wang et al., a; yuan et al., ) . direct inoculation eliminates the need for immune-deficient pregnant mice and allows for control over the developmental stage at which infection occurs. as a result, both pathogenesis and intervention strategies can be evaluated with more precision. critically, it is likely the most stringent model for brain infection, as it tests the immediate protective activity of mabs at the injection site, as well as their capacity in preventing subsequent dissemination throughout the body. in this regard, the levels of protection conveyed by zk b are, indeed, remarkable. furthermore, direct inoculation on e . also allows for the study of immediate and long-term impacts of zikv infection in the developing fetuses. in this study, neonates were followed up to p , but this could have been extended to investigate the long-term sequelae of zikv infection, be they physiological, cognitive, or behavioral. this feature is complementary to the existing model, in which zikv infection was initiated earlier in embryonic development and was frequently associated with fetal demise before or shortly after delivery (fernandez et al., ; morrison and diamond, ; sapparapu et al., ) . in the future, it would be interesting to test the panel of mabs identified here in the early infection model in order to evaluate their protective potential. in conclusion, our study provides compelling evidence that the protective potential of tested mabs against zikv infection in pregnant and non-pregnant mice was directly associated with their neutralizing activities measured in the prnt. the most potent neutralizing antibody, zk b , demonstrated impressive prophylactic and therapeutic activities and, therefore, could serve as a promising candidate for antibody-based interventions and inform rational vaccine design against zikv infection. we previously reported on the isolation and characterization of a panel of human mabs from longitudinal samples of a zikv convalescent individual and showed their distinctions in neutralizing activities, epitope specificities, and time of development during nature infection . we also reported on one of the most stringent mouse models of microcephaly, in which a contemporary zikv asian strain is inoculated directly into the lateral ventricles of the fetal brains (li et al., a) . the present study combines these methodologies in order to evaluate the protective potential of six representative human mabs against zikv infection and microcephaly. using a single i.p. injection of mabs in pregnant mice prior to zikv infection, we showed that these antibodies convey distinct levels of protection to the developing fetuses and newborns and that these levels closely mirrored their respective in vitro neutralizing activities. the most potent neutralizing antibody tested, zk b , provided a complete protection from zikv infection in pre-exposure treatment and partial protection in post-exposure therapy, as measured by markedly reduced tissue pathology. the tested mabs (zk b , zk c , zk f , zk c , zk - , and zk - ) targeted the zikv e and were initially isolated from a zikv convalescent chinese traveler visiting venezuela during the viral outbreak in . the details of their neutralizing activity, epitope specificity, lineage ancestry, and the time of isolation during natural infection were previously reported . two zikv diii-specific mabs, zv and z , were isolated by others (robbiani et al., ; zhao et al., ) and used here for comparative analyses. all mabs were in the human igg backbone, manufactured in f cells (atcc) by transient transfection, and purified by affinity chromatography using protein a agarose (thermo scientific). the mab concentration was determined using the bca protein assay kit (thermo scientific). we previously isolated the human mab mers- , which targets against the mers-cov and was used as a negative control (jiang et al., ) . approximately mg/kg of each tested mab or isotype control mers- was administered i.p. to a group of three pregnant mice at e . . all icr pregnant mice were purchased from beijing vital river laboratory animal technology. the experimental protocol and procedure were approved by the institutional animal care and use committee at tsinghua university ( -zlq ). the pregnant mice were kept in separate cages and maintained on a -hr/ -hr light/dark cycle throughout the experiment. for each tested mab, a group of three pregnant mice were included, and their littermate neonates were monitored for a complementary set of clinical, virological, and neuropathological outcomes on p and for survival up to p ( figure a) . specifically, the first (i) and second (ii) groups of littermate neonates were sacrificed on p . brain and blood samples were immediately collected, processed, and frozen at À c until use. group i was used to measure for viral loads in the blood serum and the brain, as well as human igg in the brain. group ii was used for blood serum viral loads, brain size, and section analysis. the third (iii) group littermate neonates were monitored for body weight at p and for survival up to p . non-pregnant c bl/ mice deficient in interferon (ifn)a, -b, and -g receptors (ag mice) were purchased from institute pasteur of shanghai, chinese academy of sciences. the mice were bred and maintained in a pathogenfree animal facility at tsinghua university. specifically, mg of each tested mab (zk - , zk - , zk f , zk c , zk c , zk b , zv , and z ) or isotype control mers- was administered to a group of four -to -weekold ag mice via the i.p. route. the following day, the animals were challenged with pfus of zikv (gz strain) via i.p. injection and monitored for survival, body weight, and viral rna copies in the blood up to days. on day and day after challenge, blood was collected from each animal for viral load measurement. microinjection of zikv into the brain ml zikv asian strain gz (genbank: ku and virus stock concentration . pfu/ml) was injected into the right side of the lateral ventricle of the embryonic mouse brains at e . , as described previously (li et al., a) . approximately half of the littermate neonates were injected with zikv (zikv + ), while the rest remained untouched (zikv À ). gz was derived from the same chinese traveler from whom the tested mabs were isolated . phylogenetic analysis of the complete coding sequences indicated that gz was tightly clustered with those circulating in the americas and belonged to the asian lineage, including those identified from french polynesia in . the whole blood ( ml) and right brain of the neonates were collected on p , immediately transferred to an eppendorf tube containing lysis buffer (qiagen), and kept in À c until use. each brain sample was weighted and homogenized using a stainless steel blender (next advance). total rna from the homogenized brain and the whole blood was extracted using an rneasy mini kit ( , qiagen) and reverse-transcribed into cdna using an iscript cdna synthesis kit ( - , bio-rad). as previously described, viral rna copies were quantified through taqman qpcr amplification of zikv envelope genes and expressed as log viral rna copies per gram for the brain or per millimeter for the blood samples calculated against the standard curve. the sequences for the primers and probes used for the analysis are shown as follows: zikv-fccgctgcccaacacaag, zikv-r ccactaa cgttcttttgcagacat, zikv-probe agcctaccttgacaagcartcagac actcaa ( fam, tamra). the neonatal right brains were collected on p and immediately frozen at À c until use. the frozen brain samples were weighted and homogenized with a stainless steel blender (next advance) before being mixed with ml pbs and allowed to stand at À c for hr. the tested mabs, suspended in the pbs, were collected by centrifugation at , g for min and quantified by serial dilution and application to elisa plates pre-coated with recombinant full-length e glycoprotein of zikv (gz , ku ), as previously described . bound mabs were detected using goat antihuman igg (fc specific) conjugated with horseradish peroxidase (promega, madison, wi, usa) and , , , -tetramethylbenzidine (tmb) substrate (cwbio, beijing, china). the mabs levels in the neonatal brains were calculated against a curve that was standardized using the corresponding mab suspended in pbs and expressed as milligrams per kilogram or micrograms per milliliter. brains were fixed in % pfa, dehydrated in % sucrose, and frozen in tissuefreezing medium before being sliced into -mm-thick tissue sections. for nissl staining, brain sections were stained with . % toluidine blue for min; dehydrated, in turn, by %, %, and % ethanol ( s, twice for each); and then hyalinized by xylene for more than min. for immunohistochemical staining, sections were blocked for hr at room temperature (rt) and incubated with the primary antibody for one night at c. after three rounds of extensive washing with phosphate-buffered saline with . % tween- (pbst), the secondary antibody was added and incubated at rt for hr. the section was then washed once more with pbst. the tissue sections were then imaged on an lsm (carl zeiss) confocal microscope and analyzed with imaris and imagej, as described previously (li et al., a) . the antibodies used for immunohistochemical analysis were caspase- (abcam, ab , : , ) and neun (abcam, ab , : , ). zikv serum ( : , ) was derived from the same convalescent patient. nuclei were stained with dapi (invitrogen). for experiments involving pregnant and non-pregnant mice, - mice were included in each assessment group to ensure representation and consistency of the data. all data were analyzed using prism software (graphpad). statistical evaluation was performed by student's unpaired t test. data were presented as mean ± sem. *p < . ; **p < . ; and ***p < . . broadly neutralizing antibodies to hiv and their role in vaccine design broadly neutralizing antiviral antibodies the brazilian zika virus strain causes birth defects in experimental models zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor tlr a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus zika virus. i. isolations and serological specificity antibody-mediated neutralization of flaviviruses: a reductionist view a dynamic landscape for antibody binding modulates antibody-mediated neutralization of west nile virus human antibodies to the dengue virus e-dimer epitope have therapeutic activity against zika virus infection a highly potent human antibody neutralizes dengue virus serotype by binding across three surface proteins recent zika virus isolates induce premature differentiation of neural progenitors in human brain organoids zika virus impairs growth in human neurospheres and brain organoids flaviviruses and their antigenic structure potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein zika virus disrupts neural progenitor development and leads to microcephaly in mice zika virus infects neural progenitors in the adult mouse brain and alters proliferation -hydroxycholesterol protects host against zika virus infection and its associated microcephaly in a mouse model neutralizing human monoclonal antibodies prevent zika virus infection in macaques considerations for developing a zika virus vaccine zika virus associated with microcephaly animal models of zika virus infection, pathogenesis, and immunity zika virus molecular mechanisms of antibodymediated neutralisation of flavivirus infection zika virus: immunity and vaccine development zika virus and birth defects-reviewing the evidence for causality recurrent potent human neutralizing antibodies to zika virus in brazil and mexico zika virus activates de novo and cross-reactive memory b cell responses in dengue-experienced donors neutralizing human antibodies prevent zika virus replication and fetal disease in mice the rights and wrongs of blood-brain barrier permeability studies: a walk through years of history specificity, crossreactivity, and function of antibodies elicited by zika virus infection zika virus targets different primary human placental cells, suggesting two routes for vertical transmission zika virus infects human cortical neural progenitors and attenuates their growth molecular determinants of human neutralizing antibodies isolated from a patient infected with zika virus transfer of convalescent serum to pregnant mice prevents zika virus infection and microcephaly in offspring a human bi-specific antibody against zika virus with high therapeutic potential zika virus infects early-and midgestation human maternal decidual tissues, inducing distinct innate tissue responses in the maternal-fetal interface zika virus: history of a newly emerging arbovirus delineating antibody recognition against zika virus during natural infection a single mutation in the prm protein of zika virus contributes to fetal microcephaly excretion of infectious zika virus in urine structural basis of zika virus-specific antibody protection we are grateful to the zikv convalescent patient for donating his blood samples and drs. jiang wang, wenxin hong, and lingzhai zhao for providing the patient with treatment and care. we thank drs. cheng-feng qin and gong cheng for providing the zika virus isolate gz and for assisting with evaluations of antibody protective activity in ag mice. we also thank ms. angela fan for editing the manuscript. all authors contributed to the present study, including intellectual input, results analysis, and actual writing and editing of the manuscript. specifically, c.l., f.g., r.w., y.j., l.y., and x.s. performed the experiments. c.y., x.t., f.z., z.x., and l.z. designed the study, and c.l., f.g., z.x., and l.z. analyzed the data and wrote the paper. patents have been filed for the isolated antibodies, and they are all available for collaboration in research and development through material transfer agreement. key: cord- -gmw gl r authors: saiz, juan-carlos; de oya, nereida jiménez; blázquez, ana-belén; escribano-romero, estela; martín-acebes, miguel a. title: host-directed antivirals: a realistic alternative to fight zika virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gmw gl r zika virus (zikv), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the americas in , where it has been responsible for a threat to global health, causing a great social and sanitary alarm due to its increased virulence, rapid spread, and an association with severe neurological and ophthalmological complications. currently, no specific antiviral therapy against zikv is available, and treatments are palliative and mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration. nevertheless, lately, search for antivirals has been a major aim in zikv investigations. to do so, screening of libraries from different sources, testing of natural compounds, and repurposing of drugs with known antiviral activity have allowed the identification of several antiviral candidates directed to both viral (structural proteins and enzymes) and cellular elements. here, we present an updated review of current knowledge about anti-zikv strategies, focusing on host-directed antivirals as a realistic alternative to combat zikv infection. since the beginning of the st century, a number of infectious disease threats have emerged that demand a global response. among them, severe acute respiratory syndrome virus, avian influenza in humans, pandemic influenza a (h n ), middle east respiratory syndrome coronavirus, chikungunya virus, and ebola virus have been the most threatening ones. nonetheless, the emergency of a vector-borne virus, zika virus (zikv), which is responsible for congenital malformations and other neurological and ophthalmological disorders, was hard to predict. zikv is a mosquito-borne virus belonging to the spondweni serocomplex in the genus flavivirus of the family flaviviridae [ ] . the virus has been isolated from various mosquito species, although it seems that the natural transmission vectors are mosquitoes of the genus aedes [ , ] . besides mosquito bites, viral direct human-to-human transmission can occur perinatally, sexually, and through breastfeeding and blood transfusion [ ] . the zikv genome is a single-stranded rna molecule (≈ . kb) of positive polarity encoding a single open reading frame (orf) flanked by two untranslated regions at the and ends [ ] . zikv was first isolated from the serum of a monkey in , and one year later from aedes africanus mosquitoes caught in the same area, the zika forest [ ] . until it was detected in asia in the s, the virus had been confined to africa. later on, human outbreaks were reported in the pacific islands, micronesia in and, then, in french polynesia in [ ] . the natural course of zikv infection was usually asymptomatic or produce a relatively mild illness and an uneventful recovery [ ] , hence, the virus was considered an almost neglected pathogen until its recent introduction into the americas in , when it became a threat to global health, showing increased virulence, rapid spread, since the recent outbreak in in the americas, a quite high number of possible antiviral candidates are being tested in vitro and in vivo. however, until now, no specific therapy has been approved against any flavivirus [ ] , including zikv [ ] , and, thus, current treatments are mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration [ ] . nevertheless, it should be noted that some commonly used drugs, such as acetylsalicylic acid, are contraindicated in zikv-infected patients, since they increase the risk of internal bleeding, and other arboviruses (dengue or chikungunya viruses) that can co-infect the patients may produce hemorrhages [ ] . due to the natural course of zikv infection, which is usually asymptomatic or produce a relatively mild illness and an uneventful recovery, when facing anti-zikv strategies, a very important point to take into account is the main target population that would benefit from it, namely immunocompromised patients and pregnant women and their fetuses [ ] . in this sense, only for some of the tested drugs their safety profiles are known [ ] . however, in cases of food and drug administration (fda) (https://www.drugs.com/) category b compounds (animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women), or even in those of category c (animal reproduction studies have shown an adverse effect on the fetus and there are no adequate and well-controlled studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), or d (there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), their use in pregnancy can be contemplated if the potential benefit outweighs the risks. even more, some of the assayed compounds cross the placenta and, thus, can also benefit the fetus. nonetheless, if used, this should be done in an individualized way, conditioning dosage and timings, and always under a clinician's control where the patient is informed of the pros and cons. current search for zikv antivirals is being conducted with different approaches: by screening of compounds libraries; by the repurposing of drugs of known active efficacy against other diseases now in use in clinical practice, many of which display broad-spectrum activity; and by testing natural products. two different strategies can be applied when pursuing for antivirals, those searching for compounds directed to viral targets (direct-acting antivirals) and those aimed to target cellular components needed for the viral life cycle (host-directed antivirals). among the virus-directed drugs tested [ , ] are those acting against the viral rna-dependent rna polymerase (non-structural protein (ns )) catalytic domain, including nucleoside analogs and polymerase inhibitors; the methyltransferase catalytic domain of the ns responsible for transferring the mrna cap; the ns b-ns trypsin-like serine protease needed for proper processing of the viral polyprotein; and the ns helicase. the crystal structures of all these proteins have already been resolved and will certainly help to find new antivirals [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the same way, structures from other viral proteins are also available that could help to design zikv therapeutic alternatives, such as those of the capsid c protein [ ], whose destabilization may impair zikv multiplication, the ns [ , ], an immuno-modulator, or the envelope glycoprotein [ - ], which mediates cell binding and endosomal fusion, constitutes a major target for neutralizing antibodies, and could be also the target for virucidal compounds [ ] . on the other hand, it has also been reported that passive transfer of neutralizing antibodies to pregnant mice suppresses zikv multiplication, inhibits cell death, reduces the number of progenitor neuronal cells, and prevents microcephaly [ , ] . likewise, administration of monoclonal antibodies (mabs) recognizing the domain iii of the zikv-e protein protect mice of lethal zikv challenge [ , ] and other mabs are able to bind and neutralize zikv, including those directed against the e dimer epitope [ ] . human polyclonal antibodies produced in transchromosomal bovines also protect mice from zikv lethal infection, eliminated zikv induced tissue damage in the brain and testes, and protected against testicular atrophy [ ] . thus, administration of therapeutic antibodies seems to also be a potential strategy against zikv. nevertheless, it should be noted that, although still controvertial in the case of zikv infection [ ] , the well-known antibody dependent enhancement effect (ade) [ ] , of which dengue virus (denv) is the prototypic model, may potentiate the risk of disease exacerbation. flaviviruses have small rna genomes (around . kb in length) and thus require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently [ ] . this offers an opportunity to search for host targets as therapeutic tools that, in many instances, as they are shared by different members of the flaviviridae family, can be envisaged as pan-flaviviral antivirals [ ] [ ] [ ] . this strategy can be directed to host factors implicated in infection, pathogenesis, and in the immune response, as it has been shown for denv and the west nile virus (wnv) [ ] . in addition, their effect would be less prone to the emergence of mutants that will escape their action, as often occurs with drugs targeting viral components. consequently, this kind of approach could ideally lead to the discovery of broad spectrum antivirals that could provide low cost but effective tools for the control of flaviviral threats. different approaches are being used to identify potential host factors as therapeutic targets against flaviviruses including the analyses of transcript levels (e.g., next generation rna sequencing) for altered expression patterns during infection, proteome changes, kinases activities variations, and protein-rna interactions (e.g., two-hybrid screenings and affinity chromatography). likewise, functional analysis can be applied by overexpressing cdnas or by rnai-mediated loss of function screens using dsrna, sirna, or shrna libraries, although it should be noted that in some cases downregulation is inefficient and some genes have redundant functions [ ] . replicons may also be used to specifically assay replication activity [ , ] . theoretically, host-acting antivirals can be directed to any molecule or pathway implicated in the different steps of the viral life cycle, from early events (binding, entry, and fusion), to the formation of the replication complex, and the viral maturation and egress. the first step of zikv infection is its binding to the cellular receptor ( figure ). several molecules have been proposed as a zikv receptor (members of the tyro /axl/mer (tam) family of receptor tyrosine kinases, t-cell immunoglobulin and mucin domain (tim) and dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)) that are expressed in different neuronal and non-neuronal permissive cell types. these molecules are also receptors for other viruses, including flaviviruses such as denv and wnv, regulate several cellular activities (adhesion, migration, proliferation, and survival, release of inflammatory cytokines, antigen uptake, and signaling), and play important roles in the host's response to infection [ ] . however, elimination of a known receptor does not necessarily result in complete protection from viral infection, since flaviviruses use different receptors and, thus, there is always redundancy and alternatives. for instances, inhibiting, downregulating, knocking-down, or ablating axl, although in some cases they reduce zikv infection, they do not completely abolish it, pointing to the use of different cell surface receptors on different cell types [ ] [ ] [ ] [ ] . different molecules have been shown to inhibit zikv infection at the entry step ( figure ). r (an axl kinase inhibitor) and myd (an axl decoy receptor) compromises, but do not completely abolish, zikv infection of glial cells [ ] . r , as well as cabozantinib, an inhibitor of axl phosphorylation, that are currently in clinical trials for anticancer activities, significantly impairs zikv infection of human endothelial cells in a dose-dependent manner by affecting a post-binding step [ ] . likewise, curcumin, a widely used food additive and herbal supplement, reduces zikv infection in cell culture inhibiting cell binding while maintaining viral rna integrity [ ] , as does suramin, an anti-parasitic that interferes with attachment to host cells and with virion biogenesis by affecting glycosylation and maturation [ , ] . once zikv binds to the cell receptor, like other flaviviruses, it is internalized through clathrin-mediated endocytosis and transported to the endosomes with the involvement of cellular actin and microtubules to establish a productive infection ( figure ) [ ] . after internalization, to start translation and replication, the viral genome is released inside the cytoplasm by fusing the viral envelope with the membranes of the cellular endosomes, a process triggered by acidic ph inside them [ , ] . nanchangmycin, an insecticide and antibacterial polyether, inhibits zikv multiplication and, although the exact mechanism of action has not been completely elucidated, it probably targets axl and blocks clathrin-mediated endocytosis [ ] . acid endosomal ph triggers rapid conformational changes on viral envelope protein that result in its fusion with endosomal membrane in a ph-dependent manner, thus allowing nucleocapsid release to the cytoplasm for genome uncoating ( figure ). the optimal ph for conformational rearrangements and viral fusion is . - . , and these processes are likely dependent on the presence of cholesterol and specific lipids in the target membrane [ ] . these processes can be potentially druggable, and in fact, arbidol, a broad-spectrum antiviral and immunomodulatory use for human influenza a and b infections, inhibits zikv multiplication in cell culture probably because it intercalates into membrane lipids leading to the inhibition of membrane fusion between virus particles and plasma membranes, and between virus particles and the membranes of endosomes [ ] . chlorpromazine, an antipsychotic drug that also inhibits clathrin-mediated endocytosis, reduced zikv infection, confirming the requirement for clathrin-mediated endocytosis of zikv [ ] . in addition, -hydroxycholesterol ( hc) is increased in zikv-infected human embryonic cells and brain organoids, and reduces viremia and viral loads without affecting viral binding, but blocking internalization and suppressing viral and cell membranes fusion [ ] . even more, hc reduces mortality and prevents microcephaly in zikv-infected mice, and also decreases viral loads in the urine and serum of treated non-human infected primates [ ] . daptomycin, a lipopeptide antibiotic that inserts into cell membranes rich in phosphatidylglycerol, which suggests an effect on late endosomal membranes enriched in this lipid, has also been described as a zikv inhibitor [ ] . the dependence on endosomal acidification for zikv infection also provides a host target suitable for antiviral intervention. for instance, obatoclax (or gx - ), an anti-neoplastic and pro-apoptotic inhibitor of the bcl- that targets cellular mcl- , impairs zikv endocytic uptake by reducing the ph of the endosomal vesicles in cell culture, and thereby most likely inhibits viral fusion [ , ] . however, obatoclax, which presents a low solubility, has not produced satisfactory results in clinical trials for hematological and myeloid diseases. saliphenylhalamide (saliphe), which targets vacuolar adenosine triphosphatase enzyme (atpase) and blocks the acidification of endosomes, inhibits zikv multiplication in human retinal pigment epithelial cells [ ] that are natural targets for zikv infection [ ] . similar results were found by adcock et al. ( ) with saliphe using a different screening [ ] ; however, they reported that, contrary to that described by others [ ] , other compounds that interfere with the endocytic pathway, such as dynasore, that blocks clathrin-mediated endocytosis, or monensin, a cation transporter, were either toxic for the cells used or did not show any anti-zikv activity, as neither did chloroquine (cq). these contradictory results are probably explained by the different methodologies, cell types, and, to a lower extent, viral strains used to analyze the antiviral activities of the compounds and suggest that compounds showing different activities should be carefully evaluated before going further with investigations. in this line, and contrary to above mentioned report [ ] , cq, an fda-approved anti-inflammatory -aminoquinoline and an autophagy inhibitor widely used as an anti-malaria drug that is administered to pregnant women at risk of exposure to plasmodium parasites, was shown to have anti-zikv activity in different cell types (vero cells, human brain microvascular endothelial cells (hbmecs), and human neural stem cells (nscs)), affecting early stages of the viral life cycle, possibly by raising the endosomal ph and inhibiting the fusion of the envelope protein to the endosomal membrane [ , ] . cq has been shown to reduce placental and fetal zikv infection [ ] , and also attenuate zikv-associated morbidity and mortality in mice and protect the fetus from microcephaly [ ] . even more, cq attenuated vertical transmission in zikv-infected pregnant interferon signaling-competent swiss jim lambert (sjl) mice, significantly reducing fetal brain viral loads [ ] . similarly, cq, and other lysosomotropic agents (ammonium chloride, bafilomycin a , quinacrine, mefloquine, and n-tert-butyl isoquine (gsk )) that neutralize the acidic ph of endosomal compartments, block infection of a human fibroblast cell line and vero cells [ , ] . additionally, by medicinal chemistry-driven approaches, a series of new , -bis(trifluoromethyl)quinoline and n-( -(arylmethylimino)ethyl)- -chloroquinolin- -amine derivatives have been proved to inhibit zikv replication in vitro with a higher potency than chloroquine or mefloquine [ , ] . more recently, by screening fda-approved drugs using a cell-based assay, it has been shown that amodiaquine, another antimalarial drug, also has anti-zikv activity in cell culture by targeting early events of the viral replication cycle [ ] . niclosamide, a category b antihelmintic drug approved by fda, was capable of inhibiting zikv infection, and although its antiflaviviral effect has been associated to its ability to neutralize endolysosomal ph and interfere with ph-dependent membrane fusion, in the case of zikv, it seems that it was affecting other post-entry steps [ ] . in addition, recently, it has been reported that niclosamide decreases zikv production, partially restores differentiation, and prevents apoptosis in human induced nscs; even more, it can partially rescue zikv-induced microcephaly and attenuate infection in a developed humanized zikv-infected embryo model in vivo [ ] . likewise, tenovin- , which represses cell growth and induces apoptosis in cells expressing p by inhibiting the protein-deacetylating activities of sirt and sirt and, thus, affects endosome functions, potently inhibits zikv infection in primary placental fibroblast cells [ ] . iron salt ferric ammonium citrate (fac) also inhibits zikv infection through inducing viral fusion and blocking endosomal viral release by promoting liposome aggregation and intracellular vesicle fusion [ ] . overall, these studies evidence the potential of targeting viral entry to combat zikv. once zikv-rna is released from the endosomes in the cytoplasm, it acts as mrna to synthesize the negative-strand viral rna that directs positive-strand rna synthesis (figure ) [ ] . silvestrol, a natural compound isolated from the plant aglaia foveolata that it is known to inhibit the asp-glu-ala-asp (dead)-box rna helicase eukaryotic initiation factor- a (eif a) required to unwind structured -untranslated regions and thus impairing rna translation, exerts a significant inhibition of zikv replication in a cells and primary human hepatocytes [ ] . n-( -hydroxyphenyl) retinamide (fenretinide or -hpr), an activator of retinoid receptors that inhibits the proliferation of cancer cells and can induce apoptosis, inhibits zikv in cell culture and significantly reduces both serum viremia and brain viral burden in mice by decreasing the rate of viral rna synthesis, though not via direct inhibition of the activity of the viral replicase [ ] . zikv relies on polyamines for both translation and transcription [ ] , so that, drugs targeting the polyamine biosynthetic pathway, such as difluoromethylornithine (dfmo or eflornithine), an fda-approved drug that is used to treat trypanosomiasis, hirsutism, and some cancers, as well as diethylnorspermine (denspm) limit viral replication in bhk- cells [ ] . zikv replication and particle morphogenesis take place associated with a virus-induced organelle-like structure derived from the membrane of the endoplasmic reticulum (er) (figure ) [ ] . de novo synthesized positive strand-rna, once packaged, form enveloped immature virions in the er, enter the secretory pathway and, then, in the trans-golgi network, the prm is cleaved before the virus is released from the infected cell ( figure ) [ , ] . er-membrane multiprotein complexes, such as the oligosaccharyltransferase (ost) complex, have been reported to be critical host factors for flavivirus multiplication. in this regard, it has been shown that the n-linked glycosylation inhibitor- (ngi- ) chemical modulator of the ost complex blocks zikv-rna replication in different cell types [ ] . similarly, the host er-associated signal peptidase (spase) is an essential, membrane-bound serine protease complex involved in cleavage of the signal peptides of newly synthesized secretory and membrane proteins at the er and also for processing of the flavivirus prm and e structural proteins [ ] . it has also been reported that cavinafungin, an alaninal-containing lipopeptide of fungal origin, potently inhibits growth of zikv-infected cells [ ] . nitazoxanide, a broad-spectrum antiviral agent approved by the fda as an antiprotozoan and with potential activity against several viruses in clinical trials (rotavirus and norovirus gastroenteritis, chronic hepatitis b, chronic hepatitis c, and influenza), also inhibits virus infection targeting a post-attachment step, most likely virus genome replication [ ] . likewise, brefeldin a, a penicillium sp. product that inhibits protein transport from the er to the golgi apparatus, inhibits zikv multiplication [ ] , as does emetine, an anti-protozoal agent that inhibits both zikv ns polymerase activity and disrupts lysosomal function [ ] . zikv infection leads to cell-death by inducing host caspase- and neuronal apoptosis during its propagation [ ] . thereby, bithionol, a caspase inhibitor, inhibits zikv strains of different geographical origin in vero cells and human astrocytes [ ] . similarly, by using a drug repurposing screening of over molecules, it was found that emricasan, a pan-caspase inhibitor that restrains zikv-induced increases in caspase- activity and is currently in phase clinical trials in chronic hepatitis c virus (hcv)-infected patients, protected human cortical neural progenitor cells (npc) in both monolayer and three-dimensional organoid cultures, showing neuroprotective activity without suppression of viral replication [ ] . additionally, bortezomib, a dipeptide boronate proteasome inhibitor approved for treatment of multiple myeloma and mantle cell non-hodgkin's lymphoma that regulates the bcl- family of proteins, has also been described as a zikv inhibitor [ ] . similarly, different cyclin-dependent kinase (cdk) inhibitors, such as (alphas)- -(acetylamino)-alpha-methyl-n-( -( -methylethyl)- -thiazolyl)benzeneacetamide (pha- ), reduced zikv-infection and propagation [ ] . however, cdk inhibitors should not be suitable for the treatment of pregnant women but could be useful for the treatment of other non-pregnant patients, preventing the complications associated with zikv infection. the need for specific host lipids for flavivirus replication and particle envelopment make lipid metabolism a potential target for an antiviral search [ , ] , and, even though manipulating a major metabolic pathway such as lipid biosynthesis can be envisaged as a dangerous antiviral approach due to the undesirable effects that could be detrimental for the host, current use of drugs such as ibuprofen and aspirin (cyclooxygenase- (cox- ) inhibitors) or statins ( -hidroxi- -metil-glutaril-coa (hmg-coa) reductase inhibitors) highlights the feasibility of lipid-based therapeutics [ , ] . accordingly, inhibition of key enzymes involved in fatty acid synthesis, such as acetyl-coa carboxylase (acc) [ ] , and fatty acid synthase (fasn) [ ] [ ] [ ] , are potential targets for anti-zikv therapy. in this line, we have reported that nordihydroguaiaretic acid (ndga) and its derivative tetra-o-methyl nordihydroguaiaretic (m n or terameprocol), two compounds that disturb the lipid metabolism probably by interfering with the sterol regulatory element-binding proteins (srebp) pathway, inhibit the infection of zikv and wnv, likely by impairing viral replication, as did other structurally unrelated inhibitors of the srebp pathway, such as -[(diethylamino)methyl]-n-[ -( -methoxyphenyl)ethyl]-n-( r)- -pyrrolidinyl-benzamide dihydrochloride (pf- ) and fatostatin [ ] . in the same way, the dependence on cholesterol for different processes during flavivirus infection also provides a suitable target for antiviral strategies. as mentioned above, hc reduces viremia and viral loads in vitro, and also reduces mortality and prevent microcephaly in mice, and decreases viral loads in the urine and serum in non-human infected primates [ ] . lovastatin and mevastatin are hypolipidemic agents (hmg-coa inhibitors) belonging to the family of statins that are widely used for lowering cholesterol in patients with hypercholesterolemia and have been previously shown to present antiviral activity against dengue and hepatitis c viruses. both agents have been proposed as therapeutic candidates against zikv [ ] . in fact, lovastatin attenuates nervous injury in animal models of gbs [ ] . likewise, imipramine, an fda-approved antidepressant, inhibits zikv-rna replication and virion production in human skin fibroblasts, probably by interfering with intracellular cholesterol transport [ ] . regarding sphingolipid metabolism, which has been involved in flavivirus infection [ ] , treatment with the neutral sphingomyelinase inhibitor gw reduced zikv production by affecting viral morphogenesis [ ] as described for other flaviviruses [ ] . finally, since adenosine monophosphate-activated protein kinase (ampk) is a master regulator of lipid metabolism, its activation by pf- or metformin reduced zikv infection by impairing viral replication [ , ] . thus, targeting lipid metabolism could provide therapeutic alternatives for the discovery of host-directed antivirals against zikv. the ns protein is the viral rna-dependent rna polymerase responsible for the rna synthesis that also inhibits interferon (ifn) signaling by acting over the signal transducer and activator of transcription (stat ) protein [ ] , being, thus, a major target for antiviral design. besides the proven antiviral activities of different nucleosides analogs and inhibitors of the zikv-ns [ ] , several inhibitors of the biosynthesis of nucleosides (purines and pyrimidines) also impair zikv replication (figure ). ribavirin is an inhibitor of the inosine monophosphate dehydrogenase (impdh) with antiviral activity to several rna viruses [ ] , but its mechanism of action is not entirely clear. it may act as a guanosine synthesis inhibitor, a viral cap synthesis inhibitor, a viral rna mutagen, and as an inducer of lethal mutagenesis [ ] [ ] [ ] . by using a cell-based assay, no antiviral activity of the drug was initially observed [ ] but, later on, it was reported that although no activity against zikv was detected in vero cells, the drug did inhibit virus multiplication in human cell lines, including liver huh- and rhabdomyosarcoma (rd) cells [ ] . further studies have confirmed an inhibitory activity of ribavirin against zikv strains of different geographical origin in various types of cells, such as human neural progenitor cells (hnpcs), human dermal fibroblasts (hdfs), human lung adenocarcinoma cells (a ), and even in vero cells [ ] [ ] [ ] . still more, the drug was shown to abrogate viremia in zikv-infected stat- -deficient mice [ ] , which lack type i ifn signaling, are highly sensitive to zikv infection, and exhibit a lethal outcome. two other inhibitors of impdh, merimepodib (mmpd or vx- ) [ ] and mycophenolic acid (mpa) [ , , ] also inhibit zikv-rna replication in different cell types, including huh- cells, human cervical placental cells, and neural stem and primary amnion cells. however, other authors [ ] have described that mpa have little effect on zikv replication and showed significant cell toxicity. likewise, azathioprine, another inhibitor of purine synthesis and immunosuppressant, impaired zikv replication in hela and jeg cells [ ] ; nonetheless, its use in pregnant women is not recommended. the above described contradictory results stress again the differences that drug treatments may have as a consequence of the different viral strains, cell types, and methodologies used to assess them. as with the inhibitors of purine biosynthesis, compounds inhibiting the synthesis of pyrimidines have also effect on zikv replication (figure ). so that, the virus was highly susceptible to brequinar and cid treatments in cell culture [ ] . however, it should be noted that it has been reported that brequinar, as well as dd , antiviral activity may not be due to pyrimidine deprivation, but rather to the induction of the cellular immune response [ , ] . similarly, other inhibitors of the pyrimidine synthesis, such as gemcitabine, an activator of cellular caspases [ , ] , and, although with a lower efficiency probably due to its lower solubility, -azauridine and finasteride, a -azasteroid analog of testosterone that inhibit type ii and type iii α-reductase and is being tested for benign prostatic hyperplasia and male pattern baldness, reduce zikv replication [ , ] . several other compounds have been shown to have anti-zikv activity by inhibiting viral entry and/or rna synthesis, although their mechanisms of action have not yet been fully elucidated. among them are antiparasitics such as ivermectin (used mainly against worms infections) and pyrimethamine (a folic acid antagonist that inhibits the dihydrofolate reductase and, thus, dna and rna synthesis, is classified as a pregnancy category c, and was initially used to treat malaria and now toxoplasmosis and cystoisosporiasis) [ ] ; antibiotics such as azithromycin that prevents infection, replication, and virus-mediated cell dead [ ] , and kitasamycin (a natural product from streptomyces narbonensis that inhibits protein biosynthesis) [ ] ; drugs used to prevent chemotherapy-induced nausea and vomiting as palonosetron (a fda-approved -ht antagonist) [ ] ; antidepressants like sertraline (a selective serotonin reuptake inhibitor) [ ] and cyclosporine (that is also use for rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome, and in organ transplants, is believed to lower the activity of t-cells, and is currently in clinical trials for tis possible use in ameliorate neuronal cellular damage) [ ] . similarly, after chemical screening, it was found that hippeastrine hydrobromide (hh), an active component of traditional chinese medicine, and amodiaquine dihydrochloride dihydrate (aq), an fda-approved drug for treatment of malaria, inhibit zikv infection of human pluripotent stem cell-derived cortical npcs and in adult mouse brain in vivo even when the infection was already ongoing but, again, their mechanisms of action are not known [ ] . besides drugs that act against host targets directly implicated in the viral cycle, there are compounds that can prevent undesirable effects of zikv infection. in this regard, zikv infection leads to massive neuronal damage, especially of neural progenitor cells, and neurodegeneration [ ] [ ] [ ] , via both direct replication in neuronal cells and possibly through increased excitotoxicity via over activation of n-methyl-d-aspartate receptor (nmdar)-dependent neuronal excitotoxicity in nearby cells. memantine, a pregnancy category b fda-approved drug widely used to treat patients with alzheimer's disease, as well as other nmdar blockers (dizocilpine, agmatine sulfate, or ifenprodil), prevents neuronal damage and death and intraocular pressure increase induced by zikv infection in infected mice, but it does not affect virus replication, pointing to its possible use to prevent or minimize zikv-related microcephaly during pregnancy [ ] . ebselen (ebs), an antioxidant that reduces oxidative stress and improves histopathological features in a testicular injury study model and is currently in clinical trials for various diseases, showed minor effects in reducing zikv progeny production and viral e protein expression and on overall survival and viremia level of challenged ag mice; however, it should be noted that ebs reduced some zikv-induced effects, such as testicular oxidative stress, leucocyte infiltration, and production of pro-inflammatory response, whereas, in a model of male-to-female mouse sperm transfer, the drug improved testicular pathology and prevented the sexual transmission of zikv [ ] . ifns play a key role in the elimination of pathogens and they are release upon the activation of the innate immune response by infecting viruses. in this way, zikv infection induces ifn signaling pathways and further activates cytoplasmic retinoic acid inducible gene protein (rig )-like receptors (rlrs) and several type i and iii ifn-stimulated genes, driving to the subsequent activation of the janus kinase (jak)/stat innate immune pathway that confer resistance to zikv infection [ ] . different studies showed that ifn-α, ifn-β, and ifn-γ inhibit zikv replication in cell culture [ , , ] , and that treatment of pregnant mice with ifn-λ reduced zikv infection [ ] . in addition, ifitm and ifitm , which are interferon-induced transmembrane proteins, impair early stages of zikv infection. even more, ifitm prevents zikv-induced cell death [ ] . likewise, it has been reported that an interferon-activating small molecule ( -( -fluorophenyl)- -( -isopropyl- , , -thiadiazol- -yl)- , -ihydrochromeno [ , -c] pyrrole- , -dione (avc) strongly inhibits replication of zikv in cell culture [ ] . however, it is also known that the virus is capable of evading type i ifn responses by acting over the jak/stat signaling pathway [ , [ ] [ ] [ ] , and that type i ifns might be mediators of pregnancy complications, including spontaneous abortions and growth restriction [ ] . in any case, use of ifn against zikv, alone or in combination with other antivirals, deserve further studies. by screening a library of known human micrornas (mirnas), small, noncoding rnas (sncrnas) that modulate gene expression post-transcriptionally and regulate a broad range of cellular processes, several mirnas were found to inhibit zikv by increasing the capability of infected cells to respond to infection through the interferon-based innate immune pathway [ ] . another alternative is intervening over epigenetic regulation by using epigenetics modulators. for instance, histone h k methyltransferases (ezh and ezh ) suppress gene transcription and it has been shown that inhibitors such as -[( s)-butan- -yl]-n-[( , -dimethyl- -oxo- h-pyridin- -yl)methyl]- -methyl- -( -piperazin- -ylpyridin- -yl)indole- -carboxamide (gsk- ) reduce zikv multiplication in cell culture through the activation of cellular antiviral and immune responses [ ] . in any case, further studies are needed to evaluate the potential therapeutic capability of these immunomodulators against zikv infection. a great effort is being lately made to find compounds to fight zikv infection by applying different approaches, from repurposing of drugs with known antiviral activity to the screening of bioactive molecules from different libraries, as well as natural products. however, most of the already tested drugs have been found to inhibit viral replication in vitro, and only a few have been tested in vivo. hence, since, in many instances, the results will be difficult to extrapolate to humans, it would be hard for most of the tested antivirals to complete the entire drug development pipeline. in addition, it should be remarked that many drugs could have untoward effects and, thus, careful evaluation should be conducted before using them in clinical practice, as the main target populations for anti-zikv therapy will be pregnant women and patients with other medical complications. many of the already tested drugs are directed against viral structural and enzymatic proteins, including, for instance, anticancer and anti-inflammatory molecules, antibiotics, and antiparasitics; however, it is well known that this approach can easily lead to the appearance of resistance. since flaviviruses require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently, this offers an opportunity to search for host targets as therapeutic tools that, in many instances, can be broad spectrum agents, and which effect would be less prone to the emergence of mutants that will escape their action. because of that, and even though manipulating host metabolic pathways can be seen as dangerous due to the undesirable effects that could be detrimental for the host, its success for other diseases make of them a realistic option for the treatment of zikv infection. phylogeny of the genus flavivirus potential of selected senegalese aedes spp. mosquitoes (diptera: culicidae) to transmit zika virus zika virus zika virus: the latest newcomer full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika virus. i. isolations and serological specificity zika virus outbreak on yap island, federated states of micronesia neurological manifestations of zika virus infection the history of zika virus detection of zika virus in urine zika virus tissue and blood compartmentalization in acute infection of rhesus macaques zika virus pathogenesis and tissue tropism zika virus infection damages the testes in mice zika virus infection of rhesus macaques leads to viral persistence in multiple tissues zika virus: what have we learnt since the start of the recent epidemic? front who vaccine pipeline tracker editorial overview: antivirals and resistance: advances and challenges ahead the race to find antivirals for zika virus consequences of zika virus infection during fetal stage and pregnancy safe drugs: an update advances in developing therapies to combat zika virus: current knowledge and future perspectives crystal structure of zika virus ns b-ns protease in complex with a boronate inhibitor structure of the ns helicase from zika virus crystal structure of unlinked ns b-ns protease from zika virus crystal structure of zika virus ns rna-dependent rna polymerase structures of ns methyltransferase from zika virus the crystal structure of zika virus ns reveals conserved drug targets antibody-dependent enhancement and zika: real threat or phantom menace? front pathogenic exploitation of fc activity pathogenesis of flavivirus infections: using and abusing the host cell role of host cell factors in flavivirus infection: implications for pathogenesis and development of antiviral drugs broad-spectrum agents for flaviviral infections: dengue, zika and beyond targeting host factors to treat west nile and dengue viral infections zika virus replicons for drug discovery establishment and application of flavivirus replicons probing molecular insights into zika virus (-)host interactions zika virus cell tropism in the developing human brain and inhibition by azithromycin genetic ablation of axl does not protect human neural progenitor cells and cerebral organoids from zika virus infection axl mediates zika virus entry in human glial cells and modulates innate immune responses axl is not an indispensable factor for zika virus infection in mice axl-mediated productive infection of human endothelial cells by zika virus curcumin inhibits zika and chikungunya virus infection by inhibiting cell binding polysulfonate suramin inhibits zika virus infection suramin inhibits zika virus replication by interfering with virus attachment and release of infectious particles molecular mechanisms of flavivirus membrane fusion acid-dependent viral entry screening bioactives reveals nanchangmycin as a broad spectrum antiviral active against zika virus lipids and flaviviruses, present and future perspectives for the control of dengue, zika, and west nile viruses arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses infection by zika viruses requires the transmembrane protein axl, endocytosis and low ph -hydroxycholesterol protects host against zika virus infection and its associated microcephaly in a mouse model a screen of fda-approved drugs for inhibitors of zika virus infection obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments evaluation of anti-zika virus activities of broad-spectrum antivirals and nih clinical collection compounds using a cell-based, high-throughput screen assay chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models antiviral activities of selected antimalarials against dengue virus type and zika virus inhibition of autophagy limits vertical transmission of zika virus in pregnant mice fda-approved drug, prevents zika virus infection and its associated congenital microcephaly in mice repurposing of the anti-malaria drug chloroquine for zika virus treatment and prophylaxis -(arylmethylimino)ethyl)- -chloroquinolin- -amine derivatives, synthesized by thermal and ultrasonic means, are endowed with anti-zika virus activity (trifluoromethyl)quinoline analogs show improved anti-zika virus activity, compared to mefloquine the antimalarial drug amodiaquine possesses anti-zika virus activities identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen niclosamide rescues microcephaly in a humanized in vivo model of zika infection using human induced neural stem cells antiviral effects of ferric ammonium citrate inhibition of zika virus replication by silvestrol antiviral activity of n-( -hydroxyphenyl) retinamide ( -hpr) against zika virus interferon-induced spermidine-spermine acetyltransferase and polyamine depletion restrict zika and chikungunya viruses inhibition of polyamine biosynthesis is a broad-spectrum strategy against rna viruses a structural perspective of the flavivirus life cycle post-translational regulation and modifications of flavivirus structural proteins a small-molecule oligosaccharyltransferase inhibitor with pan-flaviviral activity a crispr screen defines a signal peptide processing pathway required by flaviviruses the natural product cavinafungin selectively interferes with zika and dengue virus replication by inhibition of the host signal peptidase pediatric drug nitazoxanide: a potential choice for control of zika identification of novel antiviral of fungus-derived brefeldin a against dengue viruses emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry zika virus infects human cortical neural progenitors and attenuates their growth bithionol blocks pathogenicity of bacterial toxins, ricin, and zika virus targeting host lipid synthesis and metabolism to inhibit dengue and hepatitis c viruses the structure of mammalian cyclooxygenases present status of statin therapy modification of the host cell lipid metabolism induced by hypolipidemic drugs targeting the acetyl coenzyme a carboxylase impairs west nile virus replication dengue virus nonstructural protein redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis west nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol- -phosphate lipids dengue virus infection perturbs lipid homeostasis in infected mosquito cells antiviral activity of nordihydroguaiaretic acid and its derivative tetra-o-methyl nordihydroguaiaretic acid against west nile virus and zika virus zika antiviral chemotherapy: identification of drugs and promising starting points for drug discovery from an fda-approved library lovastatin attenuates nerve injury in an animal model of guillain-barre syndrome imipramine inhibits chikungunya virus replication in human skin fibroblasts through interference with intracellular cholesterol trafficking zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase- inhibitor gw the composition of west nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis direct activation of adenosine monophosphate-activated protein kinase (ampk) by pf- inhibits flavivirus infection through modification of host cell lipid metabolism suppression of zika virus infection and replication in endothelial cells and astrocytes by pka inhibitor pki - zika virus targets human stat to inhibit type i interferon signaling ribavirin-current status of a broad spectrum antiviral agent broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx- : a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon rna virus error catastrophe: direct molecular test by using ribavirin extinction of hepatitis c virus by ribavirin in hepatoma cells involves lethal mutagenesis efficacy of the broad-spectrum antiviral compound bcx against zika virus in cell culture and in a mouse model in vitro susceptibility of geographically and temporally distinct zika viruses to favipiravir and ribavirin ribavirin inhibits zika virus (zikv) replication in vitro and suppresses viremia in zikv-infected stat -deficient mice favipiravir and ribavirin inhibit replication of asian and african strains of zika virus in different cell models an impdh inhibitor, suppresses replication of zika virus and other emerging viral pathogens a sensitive virus yield assay for evaluation of antivirals against zika virus inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity discovery of a broad-spectrum antiviral compound that inhibits pyrimidine biosynthesis and establishes a type interferon-independent antiviral state high-content screening in hpsc-neural progenitors identifies drug candidates that inhibit zika virus infection in fetal-like organoids and adult brain the brazilian zika virus strain causes birth defects in experimental models zika virus impairs growth in human neurospheres and brain organoids zika virus disrupts neural progenitor development and leads to microcephaly in mice n-methyl-d-aspartate (nmda) receptor blockade prevents neuronal death induced by zika virus infection ebselen alleviates testicular pathology in mice with zika virus infection and prevents its sexual transmission zika virus infectious cell culture system and the in vitro prophylactic effect of interferons type iii interferons produced by human placental trophoblasts confer protection against zika virus infection gestational stage and ifn-lambda signaling regulate zikv infection in utero the ifitms inhibit zika virus replication a novel agonist of the trif pathway induces a cellular state refractory to replication of zika, chikungunya, and dengue viruses zika virus inhibits type-i interferon production and downstream signaling zika virus antagonizes type i interferon responses during infection of human dendritic cells axl promotes zika virus infection in astrocytes by antagonizing type i interferon signalling type i interferons instigate fetal demise after zika virus infection a microrna screen identifies the wnt signaling pathway as a regulator of the interferon response during flavivirus infection inhibitors of the histone methyltransferases ezh / induce a potent antiviral state and suppress infection by diverse viral pathogens key: cord- - k f tw authors: parker, elaine l.; silverstein, rachel b.; verma, sonam; mysorekar, indira u. title: viral-immune cell interactions at the maternal-fetal interface in human pregnancy date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: k f tw the human decidua and placenta form a distinct environment distinguished for its promotion of immunotolerance to infiltrating semiallogeneic trophoblast cells to enable successful pregnancy. the maternal-fetal interface also successfully precludes transmission of most pathogens. this barrier function occurs in conjunction with a diverse influx of decidual immune cells including natural killer cells, macrophages and t cells. however, several viruses, among other microorganisms, manage to escape destruction by the host adaptive and innate immune system, leading to congenital infection and adverse pregnancy outcomes. in this review, we describe mechanisms of pathogenicity of two such viral pathogens, human cytomegalovirus (hcmv) and zika virus (zikv) at the maternal-fetal interface. host decidual immune cell responses to these specific pathogens will be considered, along with their interactions with other cell types and the ways in which these immune cells may both facilitate and limit infection at different stages of pregnancy. neither hcmv nor zikv naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. here, we will highlight new approaches using placenta-on-a-chip and organoids models that are providing functional and physiologically relevant ways to study viral-host interaction at the maternal-fetal interface. pregnancy is a unique immunological phenomenon in which the semiallogenic fetus is able to grow in the maternal uterine environment. in order for a successful pregnancy to occur, healthy placentation is necessary to create an environment that is protective for the developing fetus and promotes growth. how immune balance is maintained by maternal and fetal cells to promote the survival of the genetically distinct fetus, while preventing infection by a large number of pathogens, is yet to be fully elucidated ( ) . this little understood enigma has been the subject of interest and research for decades ( ) . fertilization leads to the creation of single celled embryo which undergoes several successive divisions to form a blastocyst. the blastocyst is made up of two types of cells: the outer trophoblast or trophoectoderm (te) layer forming the placenta and chorion, and the inner layer or inner cell mass (icm) forming the embryo proper and amnion ( ) . the decidua underlying the embryo is called the decidua basalis, which composes the maternal side of the placenta. the maternal-fetal interface is made up of the maternal decidua and fetally-derived placenta. during implantation, the blastocyst attaches to the decidualized endometrium and the outer layer of the blastocyst differentiates into different lineages. the te gives rise to cytotrophoblast cells (ctbs) which follow villous and extravillous pathways to form the placenta. in the villous pathway, the mononuclear ctbs fuse, creating multinucleated syncytiotrophoblasts (stbs) that establish floating villi (fv). the fv are surrounded by maternal blood, with stbs aiding the provision of nutrients by enabling gas exchange and exchange of secreted pregnancy-related hormones (human chorionic gonadotropin, hcg, human placental lactogen, hpl) at the maternal-fetal interface. furthermore, ctbs act as anchoring villi for the attachment of the embryo to the uterus. the ctbs present in the cell column of the anchoring villi follow the extravillous pathway and differentiate into interstitial (ictbs) and endovascular extravillous trophoblast cells (ectbs). the ictbs further invade up to the inner third of the myometrium and ectbs remodel the spiral arteries in low resistance high blood flow to provide nutrients to the developing embryo ( ) ( ) ( ) ( ) . the invasion of trophoblast cells at the maternal-fetal interface occurs in the presence of a large population of maternal immune cells ( ) . this includes % decidual natural killer (dnk) cells, %- % macrophages, %- % t cells and . % dendritic cells ( ) ( ) ( ) . the abundance of decidual cytotoxic t cells and macrophages can vary through the course of pregnancy ( ) . the abundance of nk cells in the decidua during the first trimester, and through the pregnancy (albeit at lower abundance), implicates them as an essential element in both the promotion of an immunotolerant environment and the control of pathogenic infection during pregnancy ( figure ) . thus, the paradoxical maternal-fetal interface is admired for both its immunotolerance to semiallogeneic trophoblastic invasion (leading to a successful pregnancy) while remaining remarkably resilient to pathogenic infections. nevertheless, several pathogen, termed torch pathogens (described below), successfully cross the placental barrier and cause devastating infection in the developing fetus ( ) . in this review, we will look at the interactions between decidual immune cells and specific viral torch pathogens and review known mechanisms which may enable viral pathogenesis within the placental environment. torch is an acronym defining some of the most common infections associated with vertical transmission. initially described in , this group contained just pathogens; toxoplasmosis, rubella, cytomegalovirus (cmv) and herpes simplex type and ( ) . since then this group has been broadened to comprise a host of other infections including listeria monocytogenes, syphilis, varicella zoster virus, human immunodeficiency virus (hiv), enteroviruses and parvovirus b ( ) . most recently, following the zika virus (zikv) epidemic in south america resulting in observed congenital anomalies, this group has been further expanded to include zikv, with some suggesting renaming this group "torchz" ( ) . the mechanism by which these "torchz" pathogens are able to circumvent typical clearance by groups of immune cells (e.g. nk cells, macrophages and others) has been studied by many groups over the last few decades in order to elucidate not only routes of pathogenicity but also roles of immune cells within this immune-privileged environment ( ) . it remains to be proven whether the new emerging viral threat by sars-cov which causes covid- including in pregnant women, will be included in this group of vertically transmitted pathogens ( ) . in this review, we will focus on maternal and fetal macrophages, t cells, and nk cells and their relationship with each virus. we will focus on the viruses human cytomegalovirus (hcmv) and zikv, which are known causes of adverse pregnancy outcomes and delve into how they interact with various decidual immune cells to promote their survival and replication. we will examine the timings of pregnancy that appear to be most permissive to pathogenic infection by these viruses and we will look at the role of various immune cells in this context ( figure ). in the early first term decidua, %- % of resident leukocytes are t cells with approximately %- % of these t cells being cd + (t helper cells) and %- % being cd + (cytotoxic) t cells ( ) ( ) ( ) . further studies have estimated the decidual cd + population to be comprised of about % activated memory cd dim t cells and % cd + cd bright foxp + treg cells. unlike the peripheral circulation, the decidua has a higher ratio of cd + t cells to cd + t cells and an overall higher number of cd + t cells ( ) . in addition, approximately % of the decidual cd + population are effector-memory t cells with reduced perforin and granzyme b in comparison to their peripheral counterparts ( ) . one study published in described a small percentage of cd + t cells found in uncomplicated term decidua to be viral specific. though these populations of viral specific cd + t cells were . % and . % in the decidual basalis and decidual parietalis respectively, they demonstrated that this was higher than that seen in peripheral blood and postulated a role for their presence in the decidua as one of immunoprotection for the fetus. this study could not conclude upon the origin of these t cells, and whether they were recruited from the periphery or activated in the decidua. in addition, more work remains to be done to establish whether these virus specific cd + t cells exist early in pregnancy ( ) . another study has described the presence of a small population of cd + hla-g+ t cells which are thought to acquire hla-g through trogocytosis from decidual dendritic cells. it is thought that these t cells promote immunotolerance at the maternal-fetal interface, and they have been shown to be downregulated in pathologies such as preeclampsia (pe) ( ) . therefore, it appears t cells play specific roles in immunity and tolerance. to this end we will look at the role that various populations of t cells may play in either enabling or preventing infection by torch pathogens at the maternal-fetal interface. macrophages constitute - % of all leukocytes in the first trimester decidua and play an important role in tissue remodeling, angiogenesis, host defense and immunotolerance ( ) . macrophages are considered a key link between adaptive and innate immunity, communicating to other immune cells and modulating their activity ( , ) . these cells are therefore vital throughout pregnancy, adapting their phenotype to address the changing requirements of the evolving decidua ( ) . tissue resident decidual macrophages are thought to be recruited from monocytes in the peripheral circulation ( ) . distinct subtypes of macrophages have been shown to be present in first-trimester decidual tissue exhibiting immunomodulatory, proinflammatory, and tissue remodeling phenotypes and play key roles in protective immunity as well as fetal tolerance ( ) . decidual macrophages are known for their highly immunosuppressive phenotype at the maternal-fetal interface, expressing cd , dc-sign and tim- among other receptor markers ( , ) . in addition to these maternally derived macrophages exist fetal-derived macrophages called hofbauer cells (hcs), which sit in the stroma of the chorionic villi ( ) . these hcs are resident in close proximity to fetal vessels and trophoblast cells from the first trimester until birth. hcs could serve as a portal of entry for pathogens from the infected mother ( ) . initially during implantation, they appear to have an inflammatory m phenotype which has both microbicidal activity and promotes a cell-mediated th cytokine response. later, they shift to a mixture of both m and m phenotypes following trophoblastic invasion and remodeling ( , ) . several studies have implicated hcs in host viral interactions. here, we look at the reciprocal interactions between hcs, maternal macrophages, and hcmv and zikv. the nk cell population in the peripheral circulation is predominately made up of cd dim cd + cells, which are believed to have a more cytotoxic phenotype ( ) . approximately % of the peripheral circulation is constituted by cd bright nk cells, which have a more immunotolerant phenotype ( ) . in the decidua, these nk cell proportions are reversed; - % of the total lymphocytes are cd bright cd - ( ) . research has demonstrated a number of dnk subsets within the cd +cd -population. it is believed that this distinct immunotolerant population is fundamental to the maintenance of a successful pregnancy, with research postulating both an ability to enable the semiallogenic fetus to thrive while at the same time responding to pathogenic infections. these nk cells reside in the decidua basalis close to invading evts and express specific receptors (e.g. kir receptors, cd /nkg a, ilt ) to activate or inhibit evt function ( ) . this large population of dnk cells are known to be sustained during the first and second trimester, with their numbers declining toward term ( , ) . despite the unique immunotolerant phenotype demonstrated by dnk cells, it is evident that this cell population displays a high level of plasticity, gaining cytotoxic function in the presence of specific pathogens ( ) . one way by which this happens is through activation of dnk cell cytotoxcity via killer cell ig-like receptor ds (kir ds ). reduced expression of this receptor has been associated with adverse pregnancy outcomes such as miscarriages and fetal growth restriction and individuals with increased kir ds expression have shown better outcomes post-viral infections ( ) . we will explore further the role that nk cells play in specific viral infections in pregnancy torch pathogens hcmv human cytomegalovirus (hcmv) was first described in by margaret smith, who replicated a virus from two newborn babies who had died from cytomegalic inclusion disease (cid) ( ) . what we now know as hcmv first came to the attention of ribbert et al. in , where intranuclear inclusions within large cells were noted in renal and parotid gland cells of stillborn fetuses. these inclusions, often described as 'owl's eye inclusions', were noted to be surrounded by a clear halo ( ) . hcmv was identified in the s when smith, weller and rowe isolated and cultured hcmv from salivary glands, adenoid tissue and liver biopsies respectively ( , ) . mechanisms of vertical transmission of hcmv can either be transplacental during gestation or transvaginal during parturition; additionally, there is some evidence for breastmilk transmission ( ) . hcmv infection is most likely to occur in the third trimester, demonstrating a % risk of mother to child transmission in the first trimester compared to a % risk in the third trimester ( ) ( ) ( ) . congenital hcmv has been estimated to affect - in every , live births, with % of hcmv positive infants suffering neurological consequences from birth ( ) . hcmv infection during pregnancy therefore poses a substantial risk to the developing fetus, leading to congenital disease including cerebral abnormalities such as periventricular calcifications, microcephaly, visual impairment, sensorineural hearing loss, neurodevelopmental delay and hepatomegaly ( ) . congenital hcmv affects , - , pregnancies annually in the united states and accounts for % of all incidents of pediatric sensorineural hearing loss ( ) ( ) ( ) . it is estimated that the burden of morbidity associated with congenital hcmv infection is greater than that of other common congenital pediatric conditions such as down's syndrome or fetal alcohol syndrome ( ) ( ) ( ) . hcmv is also associated with intrauterine growth restriction and miscarriage. there is a great need to understand maternal immunity pathways involved in hcmv infection to develop effective vaccines ( ) . hcmv is associated with asymptomatic infection of most of the world's population and subclinical illness in pregnant mothers. in the us, an estimated % of unexposed pregnant women experience primary infection during pregnancy, resulting in congenital infection in % of cases from this population ( , ( ) ( ) ( ) ( ) ( ) . however, vertical transmission of hcmv is not only seen in mothers with primary infection but also igg seropositive mothers, who exhibit a % rate of congenital hcmv infection. mechanisms of infection have been studied through analysis of placental tissue from all three trimesters of human gestation. in placental tissues from those suffering from hcmv, necrosis and oedema has been noted associated with severity of congenital disease symptoms. it has also been noted that hcmv infection is often associated with bacterial coinfection with a potentially pathogenic synergism ( ) . hcmv resides in the chorionic villi, specifically infecting ctbs, stbs and hcs. it is believed that the ability to travel between stbs in the decidua is key to hcmv pathogenesis ( ) . many studies have explored the role of the adaptive and innate immune system in hcmv infection. below we review established interactions between hcmv and immune cells (figure ). hcmv's ability to infect different populations of macrophages has been demonstrated by several studies. hcmv has been shown to be sequestered by hcs, with placentas from confirmed cases of hcmv infection demonstrating significant hyperplasia of this cell population ( ) ( ) ( ) . a study investigating vaccine development showed that when neutralizing antibodies are produced against hcmv, rates of hcs infection are decreased ( ) . a different study utilizing placental explants showed hcmv-igg immune complexes to undergo fc receptor mediated transcytosis as a mechanism to traverse the syncytium to ctbs. hcmv is then taken up by hcs in the placental villi ( ) . furthermore, another study by loenen et al., supports the idea that hcmv genes are able to increase fcr expression on infected cells ( ) . another study suggested that hcmv replication in stbs is upregulated in the presence of macrophages ( ) by analyzing hcmv replication in stbs alone or when infected stbs were cultured with uninfected placental macrophages. this study also demonstrated elevated levels of hcmv viral titres in co-cultured supernatants when compared to those from stbs cultured alone. this demonstrates that not only do macrophages have the capacity to be infected by hcmv, but also that they may amplify hcmv infection of surrounding cells in the decidua. some studies have depicted a role for latently infected maternal decidual macrophages in congenital hcmv infection, describing how microbial infections or insults in the placenta may reactivate these macrophages and in turn reactivate hcmv infection ( ) ( ) ( ) . the maternal-fetal interface is unique in respect to allogenic interactions with cd + t cells. evts are known to invade the decidua, evading destruction despite the intrinsic ability of cd + t cells to recognize foreign antigen via mhc class i molecules. as discussed previously, one mechanism by which evts are believed to evade cd + t cell recognition is through a lack of expression of hla-a and hla-b, which are key to cd + cytotoxic activity. during pregnancy, many viruses have been shown to upregulate maternal cd + t cell activity, leading to migration of highly differentiated effector memory t cells to the decidua. despite many descriptions regarding the role of t cells in hcmv infection in the fetus and the mother, there are few studies identifying their tissue specific role at the maternal-fetal interface ( ) . hcmv is thought to limit cd + t cell activity through restriction of mhc class ii expression on apcs, which in turn may prevent activation of cd + and cd + t cells ( ) . this is thought to be mediated through the hcmv protein gpus , which may degrade mhc class ii glycoproteins or disrupt downstream ciita/jak/stat signaling pathways ( ) . crespo et al., in demonstrated that hcmv did not induce a significant difference in hla-g expression on either jeg- cells or primary evts. hla-g expression has been associated with immunotolerance, and therefore its persistence despite infection may act to protect infected trophoblast cells from cytotoxic destruction ( ) . studies looking at the role of t cells in viral infection at the maternal-fetal interface demonstrated lower t-cell numbers and response in mothers who vertically transmitted hcmv to their offspring when compared to infected mothers who did not transmit hcmv, potentially suggesting an active role for t cells in vertical hcmv transmission ( ) . more specifically, a reduction in the number of cd +cd ra +ifn-g+ treg cells and cd +cd ra+ifn-g+ t cells in mothers who transmitted hcmv to their fetus was noted when contrasted with mothers who were hcmv positive but did not transmit the infection. there was also a measurable blunted t cell response in hcmv infected mothers who vertically transmitted infection, compared to infected mothers who did not transmit the virus ( , ) . in infected mothers, hcmv virus specific t cells have been shown to be elevated in the final trimester when compared to uninfected mothers ( ) . congenital hcmv infection risk is highest for the fetus in the third trimester, with a % transmission risk compared to a % risk in first trimester ( ) . this is despite the abundance of immune cells, specifically nk cells, in early pregnancy. dnk cells exposed to hcmv infected decidual fibroblasts are known to alter their phenotype to express higher levels of activating receptors (such as nkg d and cd /nkg c or nkg e). uniquely, utilizing in vitro studies, it was noted that decidual nk cells had targeted cytotoxic activity against hcmv infected autologous decidual fibroblasts and heterologous uninfected fibroblast cells, but appeared to spare trophoblast cells ( , ) . this demonstrates a clear cytotoxic effector response by decidual nk cells to hcmv, switching from their typically immunotolerant phenotype with high levels of inhibitory receptor expression (cd /nkg a, lir- , kirs), to a cytotoxic phenotype ( , ) . this group also studied the interaction between dnk and hcmv-infected cells using hcmv positive and hcmv negative decidual villous explants. this investigation revealed through fluorescent staining of dnk cells that colocalisation of dnk cells to cells throughout the hcmv positive explant occurred, including synaptic connections which was not seen in hcmv negative explants. this was thought to suggest that the dnk cells were unable to connect with uninfected trophoblasts. this also demonstrates that dnk cells are able to localize and target hcmv infected cells while sparing fetal derived semiallogenic trophoblast cells ( ) . dnk cells are unique in their function, both contributing to immunotolerance at the maternal-fetal interface, thereby enabling invasive trophoblastic activity, as well as controlling pathogenic infection ( ) . this is thought to be mediated by secretion of specific cytokines ( , ( ) ( ) ( ) ( ) . the relatively limited vertical transmission of hcmv during the first trimester of pregnancy, when the population of nk cells is abundant, has led many to speculate about the role nk cells may play in hcmv control ( ) . tilburgs and colleagues have recently demonstrated distinct cytotoxic responses in dnk cells to hcmv in first trimester versus at term wherein term pregnancy dnks harbor reduced efficacy in responding to hcmv-infected cells ( ) . siewera et al., suggested that dnk cells undergo a phenotypic transformation to acquire cytotoxic function in the presence of hcmv-infected cells ( ) . this study proved, through antibody mediated abrogation of the fas ligand (fasl) and tumor necrosis factor-related apoptosis-induced ligand (trail) on dnk cells, that death of hcmv infected cells is not initiated by dnk cells through these death receptor-ligand pathways. however, this study demonstrated that dnk cells form immunological synapses with hcmv infected fibroblasts, enabling the delivery of perforin/granzyme for cellular destruction. furthermore, the ability of dnk cells to degranulate in the presence of hcmv infected fibroblasts was demonstrated to be through high levels of cd a expression, a key cell surface molecule in the mechanism of lytic granule release. dnk cells have also been found to secrete higher quantities of granulysin when compared to peripheral blood nk cells. upon incubation with infected fibroblast cells, it was noted that cd bright nk cells decreased from . % to %, while there was an elevation in cd expression by nk cells, denoting a transformation to a cytotoxic phenotype. hcmv infected cells have been noted to upregulate expression of natural cytotoxicity receptor (ncrs) nkp by almost -fold on dnk cells as well as increasing expression of nkg c. ncrs are associated with activation of the cytotoxic profile of nk cells. accompanying this was a reduction in nkg a, kir dl , kir dl , and ilt receptor expression, receptors aligned with nk effector inhibitory function ( ) . activating dnk cell receptors such as kir ds , kir ds , kir ds and kir ds have been correlated with antiviral activity ( ) . a study by crespo et al., demonstrated an increased population of kir ds + nk cells in the decidua, suggesting an increased activating dnk cell capability in response to hla-c , and thereby increased cytotoxic potential. these cells also displayed higher levels of cytolytic molecules when compared to peripheral nk cells. this study demonstrated that kir ds + dnk cells showed increased cytotoxicity to hcmv infected decidual stromal cells (dscs) positive for hla-c when compared to kir ds -dnk cells. this was not the case for infected jeg- and primary evt cells, which did not appear to initiate degranulation or cytokine secretion from dnk cells. despite this, a reduction in the number of infected evts in the presence of co-cultured dnk cells was noted, suggesting that dnk may be clearing virus infected evts by other means ( ) . hcmv has been seen to reduce expression of mhc class i, thereby potentially evading cd + t cell destruction ( ) ( ) ( ) . one study reports an initial reduction in hla-c expression on evts in hcmv infection. the possible reason for this is not clear, however this study suggests it could prevent inhibition of nk cells through the hla-c/kir dl route, with an additional suggestion of potentially other unknown ligands being upregulated for activation of kir ds , leading to cytotoxic action against infected cells ( ) . another study showed that the potential effect of dnk cell activation on t-cell activation could be mediated via an upregulation in hla-dr expression upon exposure to hcmv infected fibroblasts ( ) . therefore, dnk cells may play a role in congenital hcmv infection by potentially protecting the first trimester fetus from infection via activation of t cell function. collectively, these studies indicate varied interactions between dnk cells and hcmv, with many routes by which hcmv may evade clearance as well as a number of ways through which dnk cells may be activated in the presence of hcmv infected cells. additionally, dnk cells are seen specifically to modulate activity in the context of t cell activation. zika virus (zikv) was first isolated in in zika forest, uganda, from infected rhesus monkey serum during epidemiological yellow fever research ( , ) . however, the first case of human infection was not reported until , when three patients presented with jaundice and were later confirmed to have rising levels of zika antibodies ( ) . initially, zikv was associated with innocuous prodromal illness on the african and indian subcontinents transmitted by the aedes aegypti mosquito, leading to an asymptomatic or self-limiting course of infection ( ) . in , a mild disseminated infection was identified to be zikv in over % of the population of the island of yap ( ) . concerns regarding human zikv infections were not aroused until when incidences of neurological deficits associated with zikv infection were first described, with almost , recorded infections noted in french polynesia ( , ) . shortly following this in , a zikv epidemic began in south america where not only were neurological deficits such as guillain-barreś yndrome seen, but also spontaneous abortion and congenital malformations such as microcephaly in infants from infected mothers ( ) . by the end of the epidemic in brazil, there were more than , notifications of zikv cases ( ) . estimates for infants born with congenital zika syndrome (czs) after the - epidemic ranged from to in every , births ( ) . the threat of a zikv epidemic lingers, with who reporting countries affected by aedes aegypti mosquitoes, therefore carrying the potential for zikv infection and transmission ( ) . zikv demonstrated continuing global epidemic capacity in india in ( ) . zikv belongs to the flavivirus family alongside west nile virus, dengue virus, and yellow fever virus. zikv is an enveloped and icosahedral virus with a nonsegmented, . kb single stranded positive sense rna genomes ( ) . this virus is composed of several proteins, categorized as three structural (capsid, pre-membrane and envelope) and seven nonstructural proteins. the seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) are essential for viral replication and assembly, as well as being responsible for the pathogenicity of the virus by binding to transcription and restriction factors ( ) . the biggest risk of congenital zikv infection is for mothers infected during their first trimester ( ) . zikv infection demonstrates wide tissue tropism, with zikv successfully infecting the central nervous system, blood, retinal, genital and reproductive tissues including placenta ( ) ( ) ( ) ( ) ( ) . zikv was thought to be exclusively arthropod transmitted until cases of human-human transmission emerged in nonendemic regions, illustrating a role for sexual transmission ( ) ( ) ( ) . the presence of zikv rna has also been found in breast milk of zikv infected mothers ( ) ( ) ( ) . however, there are reports which suggest that vertical transmission of zikv by breastmilk does not occur in most cases, which suggests the possibility that breastmilk does not have a high enough viral load to infect the newborn ( , ) . despite some knowledge regarding zikv pathogenesis, its mechanism of infection in placental immune cell types remains limited ( ) ( ) ( ) ( ) . histopathology of zikv infected placentae has shown zikv infection in first trimester villous stromal tissue cells, which includes immune cells in the chorionic villi ( , , ) . uniquely, zikv was also found to infect ctbs, endothelial cells, fibroblasts and hc in chorionic villi, as well as amniotic epithelial cells and trophoblast progenitor cells ( , ( ) ( ) ( ) ( ) ( ) ( ) (figure ). similar to hcmv, zikv has also been shown to infect hcs and ctbs ( , , ) . during the first trimester of pregnancy, zikv infects hcs, entering the fetal blood stream in order to reside in the placenta. zikv uses hcs as a "trojan horse". this strategy is utilized by several viruses in order to cross the blood brain barrier, where the virus infects leukocytes, leading to them being carried across barriers and thereby enabling the propagation and spread of infection ( ) ( ) ( ) . hcs have been associated with zikv spread to the fetus through the "trojan horse" route ( ) . the presence of zikv-specific antigen was demonstrated in hcs in confirmed maternal infection. multiple studies suggest hcs are a crucial step in vertical transmission of zikv to fetal cells, demonstrating that hcs are preferentially infected when compared to ctbs ( , , ) . infection of hcs with zikv is thought to propagate infection through hyperplasia and proliferation of these cells, leading to persistence of this hc population into later trimesters ( , , ) . a study performed on first trimester fetal and maternal tissue showed that zikv can replicate in different cell types, such as decidual fibroblasts and macrophages. it can also infect trophoblasts and hcs as well as umbilical cord mesenchymal stem cells, suggesting that the route of zikv infection may move from the decidua basalis to the anchoring villi ( ) . a study performed using blood from + asian zikv infected pregnant women shows that cd + monocytes are the primary target of zikv infection. these monocytes are resistant to change in m phenotype and downregulate type ifn signaling, which induces the expression of different host genes involved in pregnancy complications ( ) . in the decidua basalis, zikv infects evts, macrophages and stromal cells. zikv also targets proliferative ctbs in the anchoring villi, however is unlikely to infect stbs due to ifnl-mediated antiviral defense mechanisms ( ) . zikv achieves replication within macrophages through fcr, tlr and dc-sign receptors ( , ) . in vitro studies have demonstrated zikv infection to be augmented in hcs by igg from prior flavivirus exposure through antibody dependent enhancement (ade) ( ) . there remain many gaps in knowledge regarding the role for macrophages targeted and infected by zikv. a study performed using decidual and chorionic villous tissue from early and mid-gestation human pregnancy shows that zikv appears to elevate type i and iii ifn expression, which does not occur in hcmv infection ( ) . studies looking at the interaction between zikv and t cells in humans are scarce although zikv infection has been demonstrated to activate both cd and cd t cells ( ) with specific increases in vd tcr+ cells which have been implicated in recurrent miscarriages but not associated with zikv-induced fetal complications. there have not been notable studies looking specifically at t cell zikv communication at the human maternal-fetal interface ( ) . a recent study examined peripheral t cell responses of confirmed cases of zikv infection that had been stimulated with pooled zikv peptides from all viral components ( ) . this study demonstrated responses from both cd + and cd + t cells to both structural and nonstructural zikv components. however, this study particularly showed that cd + t cells exhibited a strong response to nonstructural proteins ns , ns and ns , and cd + t cells a strong response to cap and env proteins. this response was demonstrated by marked ifn-g production from both cell subtypes indicating cell activation ( ) . another case looking at a zikv infected individual from the united states demonstrated interactions between the zikv ns and env proteins with cd + and cd + t cells, respectively. ( ) . furthermore, in a different study, cd + t cells of two a b d c fibroblasts occurs through fcgr and results in increased expression of some interferons, such as ifna, and decreased expression of others, such as ifn-g and ifnb. infection is associated with increased secretion of proinflammatory cytokines such as ip- , il- , and mcp- . ns viral proteins are thought to downregulate interferon-stimulated genes (isgs) and reduce interferon signaling via stat degradation, while the viral proteins ns and ns b inhibit ifn signaling by downregulating tbk . (c) cd + t cells exhibit a strong response to nonstructural ns , ns , and ns zikv proteins, while cd + t cells respond to cap and env zikv proteins. in both cases, response to zikv proteins was characterized by increased ifn-g production. (d) systemic dnk cells exhibit increased activation, including increased ifng production and cd a expression when incubated with zikv infected monocytes. frontiers in immunology | www.frontiersin.org october | volume | article zikv infected individuals showed activity in response to nonstructural proteins (ns , ns and ns ). consistently, cd + t cells were seen to raise activity against the structural protein env ( , ) . these studies demonstrate consistency in the response of cd + t cells and cd + t cells to zikv proteins, revealing cd + t cells to specifically respond to particular nonstructural proteins and cd + t cells to react to structural proteins, particularly cap and env ( , , ) . several studies have looked at the ability of denv-specific cd + and cd + t cells to be stimulated by the presence of zikv peptides in humans ( , ) . these studies showed viral epitopes for specific peptides located in similar regions and structurally conserved across flaviviruses; however, they displayed differences in their sequences ( ) . nonetheless, these studies indicated cross-reactivity between the viruses regarding their cd + and cd + t cell activity. one study demonstrated that cd + and cd + t cells from denv positive donors reacted to zikv viral peptides, resulting in an upregulation of ifng secreting cells. this group also showed that stimulation with zikv peptides for those in acute phase of zikv infection resulted in recruitment of elevated levels of cd + ifn-g+ t cells ( ) . a recent transcriptomics study investigated transcriptional signatures in cd /cd t cells, b, and nk cells and plasmacytoid dendritic cells in patients (nonpregnant) infected with zikv ( ) . interestingly, they did not note significance transcriptional changes in nk or cd t cells in a zikv infected background but noted significance alterations in pdcs. whether pregnancy plus zikv infection would affect the immune cell transcriptome in humans remains to be determined. studies specifically analyzing interactions between dnk cells and zikv in humans once again are lacking. however, studies have looked at zikv and its communication with peripheral nk cells. one such study postulated crosstalk between monocytes and nk cells in zikv infected patients. the activation of nk cells was associated with the presence of monocytes, which induced expression of ifn-g and cd a, key markers of nk cell function. depletion of monocytes in the peripheral blood reduced the levels of these markers and thus the activation of nk cells ( ) . there are few studies showing the interaction between zikv with nk cells. glasner et al., showed that zikv infection led to activation of mhc class i, which was somehow not sensed by dnk cells and their activating receptors, allowing the virus to escape nk cell-mediated killing. mhc class i expression is triggered through the ifn-b pathway via activation of rigi-irf ( ) . however, the mechanism by which nk cells may promote an immunosuppressive environment in the face of zikv infection is not clear. some studies have indicated that interactions between other aspects of the innate immune system and nk cells may be at play in zikv pathogenesis. there are several studies suggesting that pathogenesis of zikv is not mediated through decidual immune cells alone but rather conducted, at least in part, through the activation of interferonstimulated genes (isgs), which in turn leads to activation of innate host cell immunity ( ) . isgs act to specifically target viral replication. multiple studies have indicated zikv stimulation of interferons (ifn) to vary depending on the type of ifn. while type i and iii ifns have been shown to be inhibited by zikv, specifically the ns component of the pathogen, type ii ifns have been shown to be upregulated by the virus ( , ) . one study demonstrated that when type iii ifns were upregulated, specifically ifn-l , trophoblast cells were infected with zikv at a lower rate. further, ns , ns a and ns b have been demonstrated to inhibit ifn type i response. this leads to suppression of the tank binding kinase (tbk )/irf and jak-stat pathway, which in turn results in reduced activation of innate immune responses ( ) . interferon induced transmembrane protein (ifitm ) and ifitm specifically are isgs which act as restriction factors to inhibit zikv replication. the mechanism by which the inhibition and activation of innate immunity impacts the recruitment of innate immune cells to the site of infection is not clear. little is known about the role of nk cells in human zikv infection. one study has noted interactions between tlr , cd and ifitm , postulating that the restriction of zikv is associated with inhibitory activity of ifitm , potentially through activation of nk cells ( , ) . another group looking at isgs showed that viperin played a role in zikv pathogenesis, with data revealing that when viperin levels were high, zikv mrna levels were low and vice versa ( ) . ns is seen to target directly the akt-mtor pathway, leading to reduced signaling from this pathway and subsequent activation of autophagy in host cells ( ) . zikv has been shown to co-opt the autophagy pathway for post-rna replication capacity and survival ( , ) . importantly, the ns b-ns protease activity of zikv can be blocked by an inhibitor of autophagy, hydroxychloroquine (hcq) ( ) . hcq is an fda approved drug considered safe to use during pregnancy and could serve as an effective treatment for preventing zikv congenital syndrome ( ) . the relationship between zikv infected cells and attenuated ifn production has been extensively reported, leading to questions regarding the mechanism underlying this association. it has been proposed that zikv may infect cells through ade of infection. many cells express the fcg receptor, and it is thought that viral particles may complex with antibodies and thereby enter into cells via fcg receptors ( ) . host cells (such as trophoblasts and fibroblasts) infected with zikv demonstrate innate immune system activation with a rise in specific ifns (e.g. ifn-a), but falling levels of others such as ifn-l and ifn-b ( ) . the elevated levels of proinflammatory cytokines and chemokines, namely il- , mcp- and ip- which are linked to recruitment of immune cells such as monocytes and t cells ( ) . zikv has been shown in multiple studies to downregulate type i ifn signaling and to be active in suppression of antiviral signaling. zikv nonstructural proteins ns and ns b inhibit ifn signaling by downregulating levels of tbk . however, ns b downregulates the jak-stat pathway and inhibits apoptosis of zikv, and hence inhibits innate antiviral responses ( ) . one study specifically has implicated the role of the nonstructural zikv protein ns in promoting zikv propagation by targeting stat for degradation, thereby reducing isg levels ( ) . this is thought to promote viral replication through a dampened host innate immune cell response. there remains much to be elucidated in terms of zikv infection in human pregnancy. new studies are identifying metabolic reprogramming pathways underpinning innate immune responses to zikv which opens additional avenues of investigation ( ) . we refer readers to recent reviews highlighting zikv-immune interactions in adverse pregnancy outcomes ( ) and ade ( ) . the limited availability of placental tissues during early pregnancy has always been a challenge for the reproductive biologists, hampering the study of placental physiology and cell to cell interactions. in vitro cell line models can often be biologically distinct and therefore unable to demonstrate enough similarity to replicate the conditions of human pregnancy. in addition, the use of cell line models can fail to reproduce the complexity of the number of cell types and cell interactions present within the decidua. therefore, functional in vitro d models being are developed, for example placenta-on-a chip and organoid cultures, which can mimic in vivo conditions and would be useful to understand the mechanisms of viral host interactions. the 'placenta-on-a-chip' is a microfluidics model utilizing human trophoblast cells (bewo) and fetal derived cells (huvecs and hpvecs) ( , ) . these cell lines are cultured and separated by a semipermeable membrane within flow conditions with the purpose of understanding placental mechanisms and barrier function ( ) . recent reports have described the faithfulness of placenta-on-a-chip model to in vivo placental conditions ( ) . for example, glucose transport using a placenta-on-a-chip model was demonstrated by lee et al., and blundell et al., highlighting significant similarity to in vivo glucose transport in the human placenta ( , ) . placenta-on-a-chip models have also been used to investigate the transport of heparin and anti-hyperglycemic agents such as glyburide using bewo and human placental villous endothelial cells ( ) . recently, the transport of the xenobiotic compound caffeine across the placenta has been studied using this model system, providing new insights into the extent of caffeine transfer from mother to fetus ( ) . bacterial infections have also been studied using this model. zhu et al., showed that in the presence of escherichia coli (e. coli), trophoblast cells (bewo) activated the circulating macrophages on the "maternal" side of the chip to secrete several inflammatory cytokines that mimicked in vivo conditions during pregnancy ( ) . the impact of common environmental exposures such as titanium dioxide nanoparticles (tio -nps) has also been studied using this d placental model showing a series of different placental responses (barrier permeability, oxidative stress, cell apoptosis, and maternal immune cells behavior ( ) . they showed placental barrier permeability and maternal immune cells to be influenced by even low concentration of nps ( ) . therefore, this simple in vitro model can prove useful in understanding the environmental exposure of nps during pregnancy and can help in a range of biological studies ( ) . recent studies report generation of an organ-on-a-chip model, wherein decidualized human endometrial stromal cells and macrophage cell lines are co-cultured in a microfluidic device and shown to inhibit secretion of tnf-a in response to lps stimulation ( ) . these devices have also been used to determine the impact of cytokine secretion by dnk cells on the migration of primary trophoblast cells. these studies illustrate the functionality of microfluidic organ on chip devices to elucidate importance of maternal immune cells in the placenta ( ) . thus, the use of fetal membrane on organ-ona-chip provides a suitable model to explore the impact of pathogenic infections during pregnancy ( , ) . the use of in vitro trophoblast organoids as a d culture model also provides a new tool to understand the mechanism of implantation at the maternal-fetal interface. recent studies have shown the characterization of these organoids derived from sttrimester ctbs ( to weeks) and suggest their resemblance to primary trophoblast cells ( ) ( ) ( ) . due to similarity with the placental architecture, these organoids could be used to study physiological, metabolic and hormonal changes that occur during pregnancy. the viruses we highlighted in this review, hcmv and zikv, do not naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. in particular, there remains a paucity of understanding zikv-immune cell interactions during pregnancy. thus, the employment of placenta-on-a-chip or organ-on-a-chip, and organoid models will be pivotal in providing functional and physiologically relevant ways to study the interaction of immune cells at the maternal-fetal interface with viral pathogens that affect pregnancy. both hcmv and zikv can be sequestered into fetal macrophages. hcmv implicates hcs in the potential infection of other decidual cells, leading to the promotion of hcmv transcytosis in trophoblasts. zikv preferentially infects hcs, persisting in this cell population and potentially mediating infection of other fetal-derived cells. more poignant is the suggestion that decidual macrophages may mediate reactivation of hcmv by acting as a latent reservoir for infection. these studies collectively indicate a central role for macrophages in the pathogenesis of torch viruses. dnk cells have been seen to alter their phenotype to express higher levels of various activating receptors when in the presence of infected decidual fibroblast cells. they also are known for their plasticity in the face of specific pathogens, acquiring more cytotoxic function. kir dl /hla-c has been identified as a mechanism by which dnk cells are activated and display cytoxicity toward hcmv infected cells. it has also been suggested that dnk cell activation may trigger activation of t-cells through upregulating hla-dr expression on infected fibroblast cells. zikv viral components demonstrate capacity to elicit strong responses from peripheral cd + and cd + t cells, with ns , ns , and ns being associated with cd + stimulation whereas cap and env proteins being associated with cd +. we also see in this review the importance of interferon stimulating genes in the restriction of zikv replication. thus, the implications and outcomes of viral interactions with immune cells at the maternal-fetal interface are varied. we see the importance of the host immune response and recognize the importance of studying mechanisms of pathogenesis in detail to enable targeted therapeutic interventions including vaccines to mitigate the adverse outcomes of viral infections during pregnancy ( figure ) . finally, we posit that better understanding of the immunological underpinnings of infections at the maternal fetal interface can support the inclusion of pregnant women in trials testing vaccines and therapeutics to compact existing and emerging viral infections. ep, sv, and im wrote the manuscript. rs generated the figures. all authors contributed to the article and approved the submitted version. this work was funded in part by a grant from the national institutes for health/national institute for child health and development r hd to im. rs was supported by a marc ustar fellowship. regulatory t cells and the immune pathogenesis of prenatal infection immune mechanisms at the maternal-fetal interface: perspectives and challenges what is the placenta? defective implantation and placentation: laying the blueprint for pregnancy complications the effects of oxygen concentration and gestational age on extravillous trophoblast outgrowth in a human first trimester villous explant model single-cell reconstruction of the early maternal-fetal interface in humans maternal allo-recognition of the fetus inflammation and pregnancy: the role of the immune system at the implantation site granulated lymphocytes of pregnancy the unique properties of uterine nk cells decidual leucocyte populations in early to late gestation normal human pregnancy congenital viral infection: traversing the uterine-placental interface the torch complex-perinatal infections associated with toxoplasma and rubella, cytomegol-and herpes simplex viruses zika virus -reigniting the torch sars-cov and pregnancy: an invisible enemy? t cell behavior at the maternal-fetal interface fetal-maternal hla-c mismatch is associated with decidual t cell activation and induction of functional t regulatory cells foxp + regulatory t cells and t helper , t helper , and t helper cells in human early pregnancy decidua expression of nk cell receptors on decidual t cells in human pregnancy human decidual tissue contains differentiated cd + effectormemory t cells with unique properties the possible role of virus-specific cd (+) memory t cells in decidual tissue expansion of cd (+) hla-g(+) t cell in human pregnancy is impaired in pre-eclampsia the contribution of macrophages to normal and pathological pregnancies endometrial nk cells are special immature cells that await pregnancy human decidual macrophages suppress ifn-gamma production by t cells through costimulatory b -h :pd- signaling in early pregnancy role of macrophages in pregnancy and related complications monocyte and macrophage heterogeneity two unique human decidual macrophage populations macrophages at the fetal-maternal interface express markers of alternative activation and are induced by m-csf and il- tim- regulates innate immune cells to induce fetomaternal tolerance hofbauer cells: placental macrophages of fetal origin the phenotype of human placental macrophages and its variation with gestational age macrophage activation and polarization v-atpase upregulation during early pregnancy: a possible link to establishment of an inflammatory response during preimplantation period of pregnancy natural killer cells and pregnancy cd bright natural killer (nk) cells: an important nk cell subset the role of decidual immune cells on human pregnancy features of human decidual nk cells in healthy pregnancy and during viral infection expression of kir ds by decidual natural killer cells increases their ability to control placental hcmv infection propagation in tissue cultures of a cytopathogenic virus from human salivary gland virus (sgv) disease the history of cytomegalovirus and its diseases human cytomegalovirus: taking the strain the pathogenesis of human cytomegalovirus treatment of congenital cytomegalovirus infection: implications for future therapeutic strategies intrauterine transmission and clinical outcome of pregnancies with primary cytomegalovirus infection in relation to gestational age increased risk of cytomegalovirus transmission in utero during late gestation hiv in pregnant women and their offspring: evidence for late transmission the march towards a vaccine for congenital cmv: rationale and models cytomegalovirus viral and antibody correlates in young children cytomegalovirus shedding and delayed sensorineural hearing loss: results from longitudinal follow-up of children with congenital infection congenital cytomegalovirus (cmv) infection as a cause of permanent bilateral hearing loss: a quantitative assessment washing our hands of the congenital cytomegalovirus disease epidemic congenital cytomegalovirus infection: audiologic outcome newborn hearing screening-a silent revolution advancing our understanding of protective maternal immunity as a guide for development of vaccines to reduce congenital cytomegalovirus infections primary cytomegalovirus infection in pregnancy. incidence, transmission to fetus, and clinical outcome cytomegalovirus seroconversion rates and risk factors: implications for congenital cmv awareness of and behaviors related to child-to-mother transmission of cytomegalovirus review and meta-analysis of the epidemiology of congenital cytomegalovirus (cmv) infection cytomegalovirus seroprevalence and childhood sources of infection: a population-based study among pre-adolescents in the united states models of vertical cytomegalovirus (cmv) transmission and pathogenesis human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis histologic correlates of viral and bacterial infection of the placenta associated with severe morbidity and mortality in the newborn modeling of human cytomegalovirus maternal-fetal transmission in a novel decidual organ culture characterization of the fetal inflammatory response to cytomegalovirus placentitis. an immunohistochemical study cytomegalovirus vaccines: current status and future prospects maternal antibodies enhance or prevent cytomegalovirus infection in the placenta by neonatal fc receptor-mediated transcytosis immune evasion by human cytomegalovirus: lessons in immunology and cell biology placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin- and transforming growth factor-beta cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells reactivation of latent human cytomegalovirus in cd (+) monocytes is differentiation dependent intra-abdominal bacterial infection reactivates latent pulmonary cytomegalovirus in immunocompetent mice cd + effector t cells at the fetal-maternal interface, balancing fetal tolerance and antiviral immunity lymphoproliferative response in primary human cytomegalovirus (hcmv) infection is delayed in hcmv transmitter mothers development of human cytomegalovirus-specific t cell immunity during primary infection of pregnant women and its correlation with virus transmission to the fetus kinetics of effector functions and phenotype of virus-specific and gammadelta t lymphocytes in primary human cytomegalovirus infection during pregnancy cytomegalovirus sero positivity dramatically alters the maternal cd + t cell repertoire and leads to the accumulation of highly differentiated memory cells during human pregnancy the human decidual nk-cell response to virus infection: what can we learn from circulating nk lymphocytes? human cytomegalovirus infection elicits new decidual natural killer cell effector functions cytotoxic potential of decidual nk cells and cd + t cells awakened by infections human decidual natural killer cells are a unique nk cell subset with immunomodulatory potential decidual nk cells regulate key developmental processes at the human fetal-maternal interface tgfbeta promotes conversion of cd + peripheral blood nk cells into cd -nk cells with similarities to decidual nk cells critical and differential roles of nkp -and nkp -activating receptors expressed by uterine nk cells in early pregnancy human term pregnancy decidual nk cells generate distinct cytotoxic responses human cytomegalovirus immunity and immune evasion viral immune evasion: lessons in mhc class i antigen presentation kir ds -mediated activation overrides nkg a-mediated inhibition in hla-c c -negative individuals zika virus: new clinical syndromes and its emergence in the western hemisphere maternal-fetal transmission of the zika virus: an intriguing interplay zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria immune response to dengue and zika zika virus outbreak on yap island, federated states of micronesia zika virus in the americas: early epidemiological and genetic findings women's reproductive health knowledge, attitudes and practices in relation to the zika virus outbreak in northeast brazil the zika virus epidemic in brazil: from discovery to future implications countries and territories with current or previous zika virus transmission zika virus disease in india -update molecular biology of flaviviruses zika virus infection during pregnancy and congenital abnormalities zika virus pathogenesis and tissue tropism prolonged detection of zika virus in vaginal secretions and whole blood presence of zika virus in conjunctival fluid zika virus in the female genital tract zika virus infects human cortical neural progenitors and attenuates their growth probable non-vector-borne transmission of zika virus zika virus: history, epidemiology, transmission, and clinical presentation cross-reactive dengue virus-specific cd (+) t cells protect against zika virus during pregnancy zika virus shedding in human milk during lactation: an unlikely source of infection? evidence for mother-to-child transmission of zika virus through breast milk infectious zika viral particles in breastmilk breast milk transmission of flaviviruses in the context of zika virus: a systematic review transmission of zika virus through breast milk and other breastfeeding-related bodily-fluids: a systematic review zika virus infects human placental macrophages zika virus targets different primary human placental cells, suggesting two routes for vertical transmission zika virus infection of hofbauer cells maternal-fetal interplay in zika virus infection and adverse perinatal outcomes zika virus infection of first-trimester human placentas: utility of an explant model of replication to evaluate correlates of immune protection ex vivo syncytiotrophoblast of placentae from women with zika virus infection has altered tight junction protein expression and increased paracellular permeability zika virus productively infects primary human placenta-specific macrophages viral infection, proliferation, and hyperplasia of hofbauer cells and absence of inflammation characterize the placental pathology of fetuses with congenital zika virus infection host and viral mechanisms of congenital zika syndrome equine herpesvirus type enhances viral replication in cd a+ monocytic cells upon adhesion to endothelial cells osteopontin facilitates west nile virus neuroinvasion via neutrophil "trojan horse a trojan horse mechanism for the spread of visna virus in monocytes placental pathology of zika virus: viral infection of the placenta induces villous stromal macrophage (hofbauer cell) proliferation and hyperplasia zika virus reveals broad tissue and cell tropism during the first trimester of pregnancy asian zika virus strains target cd (+) blood monocytes and induce m -skewed immunosuppression during pregnancy zika virus infects early-and midgestation human maternal decidual tissues, inducing distinct innate tissue responses in the maternal-fetal interface host-virus interaction of zika virus in modulating disease pathogenesis enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity human zika infection induces a reduction of ifn-gamma producing cd tcells and a parallel expansion of effector vdelta t-cells protective to a t: the role of t cells during zika virus infection virologic, and immunologic characteristics of zika virus infection in a cohort of us patients: prolonged rna detection in whole blood ontogeny of the b-and t-cell response in a primary zika virus infection of a dengue-naive individual during the outbreak in miami, fl biphasic zika illness with rash and joint pain pericarditis associated with acute zika virus infection in a returning traveler cross-reactivity and anti-viral function of dengue capsid and ns -specific memory t cells toward zika virus structural influence on the dominance of virus-specific cd t cell epitopes in zika virus infection prior dengue virus exposure shapes t cell immunity to zika virus in humans immune-profiling of zikv-infected patients identifies a distinct function of plasmacytoid dendritic cells for immune cross-regulation zika virus infection preferentially counterbalances human peripheral monocyte and/or nk zika virus escapes nk cell detection by upregulating major histocompatibility complex class i molecules type iii interferons produced by human placental trophoblasts confer protection against zika virus infection selective activation of type ii interferon signaling by zika virus ns protein zika virus inhibits type-i interferon production and downstream signaling predicted protein interactions of ifitms may shed light on mechanisms of zika virus-induced microcephaly and host invasion zika virus evades interferon-mediated antiviral response through the co-operation of multiple nonstructural proteins in vitro zika virus ns a and ns b proteins deregulate akt-mtor signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy inhibition of autophagy limits vertical transmission of zika virus in pregnant mice differential and convergent utilization of autophagy components by positivestrand rna viruses hydroxychloroquine inhibits zika virus ns b-ns protease hiv- at the placenta: immune correlates of protection and infection zika virus targets human stat to inhibit type i interferon signaling metabolic reprogramming by zika virus provokes inflammation in human placenta cross-reactive antibodies during zika virus infection: protection, pathogenesis, and placental seeding placenta-on-achip: a novel platform to study the biology of the human placenta a microphysiological model of the human placental barrier drug transport across the human placenta: review of placenta-on-a-chip and previous approaches placental drug transport-on-a-chip: a microengineered in vitro model of transporter-mediated drug efflux in the human placental barrier placentaon-a-chip: in vitro study of caffeine transport across placental barrier using liquid chromatography mass spectrometry placental barrier-on-a-chip: modeling placental inflammatory responses to a d human placenta-on-achip model to probe nanoparticle exposure at the placental barrier decidual stromal cell-derived pge regulates macrophage responses to microbial threat a microfluidics assay to study invasion of human placental trophoblast cells instrumenting a fetal membrane on a chip as emerging technology for preterm birth research amnion membrane organon-chip: an innovative approach to study cellular interactions self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta trophoblast organoids as a model for maternal-fetal interactions during human placentation derivation of trophoblast stem cells from naive human pluripotent stem cells conflict of interest: im serves on the scientific advisory board of luca biologics.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © parker, silverstein, verma and mysorekar. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - evk g authors: röcker, annika e.; müller, janis a.; dietzel, erik; harms, mirja; krüger, franziska; heid, christian; sowislok, andrea; riber, camilla frich; kupke, alexandra; lippold, sina; von einem, jens; beer, judith; knöll, bernd; becker, stephan; schmidt-chanasit, jonas; otto, markus; vapalahti, olli; zelikin, alexander n.; bitan, gal; schrader, thomas; münch, jan title: the molecular tweezer clr inhibits ebola and zika virus infection date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: evk g ebola (ebov) and zika viruses (zikv) are responsible for recent global health threats. as no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer clr may inhibit ebov and zikv infection. this small molecule has previously been shown to inactivate hiv- and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. we found that clr indeed blocked infection of ebov and zikv in a dose-dependent manner. the tweezer inhibited infection of epidemic zikv strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. our findings show that clr is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent. zika virus (zikv) was first isolated in from a febrile rhesus macaque in the zika forest of uganda (dick et al., ) . since then, sporadic zikv infections occurred in africa and asia (haddow et al., ; hayes, ) . until , zikv infection usually has been associated with mild symptoms and thus, the virus had not been considered a threatening pathogen. however, since the recent outbreaks it is evident that zikv causes drastic birth defects, most prominently microcephaly (mlakar et al., ; rasmussen et al., ) , and is associated with neurological disorders, such as guillain-barré syndrome (cao-lormeau et al., ; krauer et al., ) . since , countries and territories reported ongoing zikv transmission (http://www. who.int/emergencies/zika-virus/en/). in brazil alone, . million persons had been infected (shuaib et al., ) , prompting the who to declare zikv a public health emergency of international concern. currently, there are no specific treatments or vaccines against zikv making development of effective preventive measures an urgent public health need (barrows et al., ; paixão et al., ) . zikv is transmitted mainly via mosquito bites but cases of sexual transmission also https have been reported (d'ortenzio et al., ; moreira et al., ; petersen et al., ) . like other members of the flaviviridae family (e.g. dengue virus), zika virions are surrounded by a host-membranederived lipid bilayer containing envelope glycoproteins responsible for cell entry (sirohi et al., ) . ebola virus (ebov) was discovered in near the ebola river in the former zaire. since then, outbreaks sporadically occurred in africa, with the most severe in , reaching epidemic proportions and a death toll of more than , people (centers for disease control and prevention (cdc), ) . ebov belongs to the family of filoviridae, forms enveloped filamentous infectious particles, and causes hemorrhagic fever, a rare and deadly disease with a high fatality rate. in addition to being a global health concern, the virus also is considered a potential biological threat agent. ebov likely is transmitted from infected bats to humans where it may spread through personal contact, contaminated objects, or sexual intercourse (mate et al., ; pandey et al., ) . noteworthy, even after recovery, ebov is found in some body fluids, including semen, up to several months (deen et al., ) . as for now, no licensed vaccine or medicine is available to prevent or manage future ebov outbreaks (sharma and ketki jangid, ) . molecular tweezers are novel drug candidates for the treatment of amyloidosis and related conditions. a lead compound, clr , has been used in many in vitro and in vivo studies to date schrader et al., ) . binding of clr to lysine residues in polypeptides disrupts noncovalent molecular interactions that are important for the abnormal self-assembly that leads to the formation of toxic oligomers and amyloid aggregates (schrader et al., ; sinha et al., ) . in fact, clr prevents assembly and promotes disaggregation of amyloid fibrils that are associated with neurodegenerative diseases ferreira et al., ; prabhudesai et al., ; richter et al., ) . therapeutic effects of clr have been demonstrated in animal models of parkinson's disease (lulla, ; prabhudesai et al., ; richter et al., ) , alzheimer's disease malik et al., ) , familial amyloidotic polyneuropathy (fap) (ferreira et al., ) , and desmin-related cardiomyopathy and was found to be safe in mice at doses -times higher than those showing therapeutic effects . we have recently established that clr also disrupted the formation of amyloid fibrils in semen (lump et al., ) , which enhance hiv- infection (münch et al., ; usmani et al., ) . surprisingly, clr also inhibited hiv- infection through a direct virusinactivating mechanism: the tweezer preferentially bound to raft-rich regions in the viral membrane resulting in the disruption of the lipid bilayer and loss of infectivity. in line with the membrane targeting activity, clr is inactive against non-enveloped viruses such as human adenovirus but destroyed other enveloped viruses, including hepatitis-c virus, human cytomegalovirus, and herpes simplex virus, demonstrating that clr represents a broadly active antiviral compound with prospects for microbicide or drug development (lump et al., ) . thus, we evaluated here whether clr might also block infection of re-emerging enveloped viruses. we show that clr indeed inhibits infection of replication-competent ebov and zikv as well as marburg, rabies, and sars corona virus (sars-cov) pseudoparticles. clr lost antiviral activity in the presence of serum, but remained active in the presence of semen, urine, saliva or cerebrospinal fluid. thus, this broadly active compound represents a promising lead for further development as a microbicide to protect from the sexual acquisition of zikv or ebov, or as a topically applied drug for the therapy of viral infections of the skin, mucosa or the respiratory tract. clr and clr were synthesized as described previously (fokkens et al., ; talbiersky et al., ) and . mm stock solutions were prepared in pbs. zikv envelope protein had been ordered from fitzgerald industries international, acton, usa. vero e cells were used for propagation and infection with zikv as described . tcid values (tissue culture infectious dose ; tcid /ml) were determined according to reed and muench, multiplied with a factor of . to obtain pfu/ml (plaque forming units) and, by normalizing to the number of cells, used to calculate the respective moi (multiplicity of infection). sw (human epithelial colon carcinoma) cells were kindly provided by ninel azoitei (center for internal medicine i, university of ulm, ulm, germany). hff (human foreskin fibroblasts), human glioblastoma (a ) or neuroglioma (h ) cells were kindly provided by jens von einem and karin danzer (ulm university medical center, ulm, germany). hepatic huh- cells were kindly provided by s. pöhlmann (göttingen, germany). the reporter cell line tzm-bl was obtained through the nih arrrp. virus stocks of r -tropic hiv- nl - th were generated by transient transfection of t cells as described (münch et al., ) . methods describing the effect of clr on pseudotyped lentiviral particles ( . .), ebola virus infection ( . .), the detection of zikv infection by a colorimetric mtt assay ( . .) or by cell-based zikv immunodetection assay ( . .), flow cytometry ( . .) and confocal microscopy ( . .) as well as the rna release assay ( . .) and the antiviral activity of clr in body fluids ( . ) can be found in the supplement. we have shown previously that clr inhibits hiv- infection by disrupting the viral membrane (lump et al., ) , suggesting that the antiviral activity of the tweezer is independent of the presence of the viral glycoproteins. to test this hypothesis, we generated luciferaseencoding retroviral particles harboring glycoproteins derived from ebov, the closely related filovirus marburg virus, rabies virus (rhabdoviridae) or sars-cov (coronaviridae). these pseudoparticles were exposed to clr and then added to huh- cells. clr , which lacks hydrophobic side arms and has no anti-hiv- activity (lump et al., ) , served as a negative control. infection rates were determined three days later by quantifying luciferase activity and showed that clr , in contrast to clr , efficiently blocked infection by all tested pseudoparticles (fig. a) . the half-maximal inhibitory concentrations (ic ) of clr against the four pseudoparticles were similar and ranged between . μm for rabies and . μm for marburg virus (fig. a) . these data corroborate our previous findings that clr targets the viral membrane rather than a viral glycoprotein (lump et al., ) . we tested next whether clr blocked infection by the replication-competent bsl pathogen ebov. vero e cells were inoculated with gfp-encoding ebov (ebihara et al., ; hoenen et al., ) that had been exposed to clr or clr . after days, the number of virus-induced plaques revealed that clr , but not clr , reduced the infectious titer in a dose-dependent manner with an ic of . μm (fig. b) . next, we determined the effect of clr on zikv infection. vero e cells were inoculated with the african zikv mr strain that was isolated in from a sentinel rhesus macaque (dick et al., ) in the absence or presence of clr or clr and viral replication was monitored by light microscopy. in the absence of clr , zikv caused a profound cytopathic effect (cpe) as evidenced by large plaques caused by detachment of cells. clr had no effect on this phenotype ( fig. a) . however, exposure to μm clr prevented formation of the virus-induced cpe entirely. to quantitatively assess the antiviral activity, we used a colorimetric mtt assay measuring the virally induced cpe (müller et al., ) . in the absence of compounds or in the presence of clr , zikv resulted in more than % dead (detached) cells. in contrast, clr suppressed cpe formation with an ic of . μm (fig. b ). the mtt assay allows an indirect measurement of zikv infection as the tetrazolium dye is metabolized only in living, non-infected cells. to evaluate the effect of clr on zikv more directly, infected cells were stained for the viral e protein and analyzed by confocal microscopy (schandock et al., ) . upon treatment with or μm of clr , e protein-specific fluorescence could not be detected, whereas clr had no effect, as expected ( fig. c ). lack of e protein expression in cells infected with clr -treated virus was confirmed by flow cytometry (fig. d, supplementary fig. s a ). of note, clr was not cytotoxic at concentrations active against zikv ( supplementary fig. s b ), and did not cause alterations in cell morphology (compare confocal images and dot plots of uninfected cells vs. μm clr exposed cells in fig. c and d). thus, clr prevents zikv infection of vero e cells. we hypothesized that if clr inhibited zikv infection by a similar mechanism to other enveloped viruses, its activity would be directed against the viral membrane (lump et al., ) . indeed, when clr was added to cells, rather than to the virus, and washed out prior to infection, no antiviral effect was observed in a cell-based zikv immunodetection assay (fig. a) . we performed next a time-course analysis in which zikv was exposed to μm clr for to s prior to infection. already after exposure for only s, clr reduced infection by ∼ %. infection declined further with increasing incubation time and was nearly at the level of uninfected cells after s incubation (fig. b) . in agreement with a direct activity against the , rhabdoviridae (rabies virus) or the coronaviridae family (sars-cov), were exposed to indicated concentrations of clr or clr and then used to infect huh- cells. after three days, infection rates were determined by quantifying firefly luciferase activity and subtracting background activity derived from uninfected cells. values represent % reporter gene activities ± sd derived from triplicate infections, normalized to values obtained for cells infected in the absence of tweezers. (b) analysis of replication competent ebov was performed using confluent vero e cells in -well plates. rgebov-egfp was preincubated with clr or clr and mixtures were added to the cells. after days, samples were analyzed by counting the number of plaques per well and calculating the corresponding plaque forming units per milliliter (pfu/ml) for each inhibitor and dilution. displayed values represent the mean of three independent experiments ± sd. ic values were calculated by graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (* denotes p < . ; ** denotes p < . ; *** denotes p < . ). light microscopy images of vero e cells infected with zikv mr in the absence or presence of μm clr or clr . images were taken days post infection (dpi). (b) zikv mr was incubated with . - μm clr or clr before these mixtures were used to infect vero e cells. after days, when significant cytopathic effects were visible, the number of adherent, viable cells were determined using the mtt assay (müller et al., ) . values represent mean ± sd of percentages derived from triplicate infections. ic was determined using graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (*** denotes p < . ) (c) confocal microscopy images of vero e cells infected with clr -or clr -treated zikv at day post infection. cells were stained for zikv e protein (green) and nuclei (hoechst, blue) and imaged using confocal microscopy. scale bar: μm. (d) flow cytometry of infected vero e cells. virus was pretreated with pbs, clr or clr and added to cells. h later, cells were fixed, permeabilized, and stained with an anti e protein antibody, and quantified using an alexa coupled secondary antibody. percentages indicate the fraction of protein e positive cells. ic, isotype control. a.e. röcker et al. antiviral research ( ) - virion, the ic of clr increased from . μm to . μm when a fold higher multiplicity of infection was applied (supplementary fig. s ). clr was shown to destroy hiv- membrane integrity through interaction with raft-rich regions in the retroviral envelope (lump et al., ) . in contrast to hiv- , the zikv particle is relatively densely packed with glycoproteins (sirohi et al., ) , which might hamper the interaction of the tweezer with the zikv membrane. to determine whether clr might inhibit zikv infection through direct interaction with glycoproteins, increasing amounts of recombinant viral e protein were titrated to clr , and then these solutions were assayed for anti-zikv activity. as shown in fig. c , even e-antigen concentrations of μg/ml did not affect the antiviral activity of clr , suggesting that the tweezer did not reduce zikv infectivity through binding to the viral e protein. interestingly, we also observed that elevated e-antigen concentrations of and μg/ml reduced infection in the absence of clr (fig. c) , which is likely due to the competition between the virion-associated e protein and the recombinant e protein for cellular receptors. to test whether clr interaction with the viral particle results in loss of membrane integrity, as was shown in the case of hiv- , (lump et al., ) , we measured viral rna genome release. sucrose cushion purified zikv was incubated with clr , clr , pbs or triton x- , as a positive control, for min at °c and the amount of total rna in the solution was determined fluorometrically. as expected, no viral for virus treatment, zikv was incubated with clr for min at room temperature before the mixtures were added to vero e cells. for cell treatment, clr was added directly to the cells; after h, the medium was replaced and the cells were infected with zikv mr . dpi, cell-based zikv immunodetection assay was performed. values represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (* denotes p < . ; *** denotes p < . ) (b) zikv was incubated for the indicated times with pbs or μm clr before the mixture was added to vero e cells. dpi, cell-based zikv immunodetection assay was performed. values represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare cells infected with clr -treated zikv to cells infected with zikv that had been incubated with pbs for the same time period (*** denotes p < . ). (c) indicated concentrations of zikv e protein were titrated to μm clr or pbs before zikv was added. mixtures were used to inoculate vero e cells. dpi, the number of adherent, viable cells were determined using the mtt assay (müller et al., ) . the values shown are mean ± sd from triplicate infections. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare cells treated with different concentrations of zikv e protein and clr to cells that had been treated with the same concentrations of e protein and pbs (*** denotes p < . ). (d) zikv mr was incubated with pbs, μm triton x- , . - μm clr or μm clr for min at °c. samples were inactivated by uv light of a laminar flow for min. then, μl of the samples were used to determine rna concentrations using the quantifluor ® rna system and a quantus fluorometer (promega). background values obtained from control samples using cell supernatant of uninfected cells were subtracted from the respective signals. rna levels of virus stock incubated with pbs were subtracted from these values. data points represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare samples treated with different clr concentrations, clr or triton x- to pbs-treated virus (*** denotes p < . ). a.e. röcker et al. antiviral research ( ) - rna was detectable when zikv was incubated with buffer or clr (fig. d ). in contrast, incubation with triton x- resulted in readily detectable viral rna suggesting that the detergent lysed the zikv particle. similarly, elevated amounts of rna were detected upon treatment of zikv with clr , demonstrating the physical destruction of the viral particle by the tweezer. of note, clr itself did not act as detergent but targets the viral membrane (lump et al., ) . next, we analyzed whether clr was active against epidemic zikv strains. the fb-gwuh- isolate was derived in from the fetus of a pregnant finnish tourist returning from south america (driggers et al., ) , and the prvabc- isolate represents the current american epidemic strain, isolated in from a puerto rican patient (lanciotti et al., ) . both zikv strains were exposed to clr upon infection of vero e cells. cell-based immunodetection assay and flow cytometry experiments demonstrated that the tweezer suppressed infection of both strains with ic values of . μm for fb-gwuh- ( fig. a and supplementary fig. s ) , and . μm against prvabc- (fig. b) , respectively. as zikv can be transmitted via sexual intercourse, we studied whether zikv infects cells derived from the anogenital region and if so, whether infection can be blocked by clr . zikv effectively infected cell lines derived from cervix (fig. a, supplementary fig. s ) , colon (fig. b, supplementary fig. s ) , and primary foreskin cells (fig. c, supplementary fig. s ). viral infectivity was suppressed by clr but not clr , with ic values in the μm range ( fig. and supplementary fig. s ). because zikv is neurotropic (cao-lormeau et al., ; mlakar et al., ) and clr has been shown previously to penetrate through the blood-brain barrier (bbb) when administered systemically richter et al., ) , we also tested whether clr blocked zikv infection of brain-derived cells. both a glioblastoma and h neuroglioma cells were susceptible to zikv infection and clr entirely abrogated infection at μm (fig. a) . finally, we confirmed these findings obtained in human cell lines using primary murine cerebellar neurons. zikv infected neuronal bystander cells, and this was blocked by clr (fig. b) . there was no apparent toxicity of clr in the primary neurons at this concentration. . . the antiviral effect of clr is abrogated by serum but not urine, saliva, semen, or csf its broad antiviral activity makes clr an interesting lead compound for antiviral prevention or treatment. for a systemic application, clr must retain antiviral activity in blood. to test whether this was the case, clr was diluted in human serum, and then exposed to zikv. as shown in fig. a , serum concentrations of . % and higher abrogated the anti-zikv activity of the tweezer. similarly, serum also abrogated the anti-hiv- activity of clr ( supplementary fig. s ), precluding its use as a systemically applied antiviral drug. in contrast, clr remained active and inhibited zikv infection in the presence of up to % of urine (fig. b) , saliva (fig. c ), cerebrospinal fluid (fig. e ) and % human semen (fig. d) . thus, clr inactivates zikv in the presence of body fluids associated with virus transmission, and could be administered locally to halt virus replication, or applied topically as a microbicide to prevent e.g. sexual virus transmission. thereafter, vero e cells were infected and days later, infection rates were determined via a cell-based zikv immunodetection assay. data points represent mean ± sd (n = ). ic values were determined with graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of compound (** denotes p < . ; *** denotes p < . ). zikv was incubated for min at °c with . - μm clr or clr . next, mixtures were used to inoculate hela (a), sw (b), or hff (c) cells. days later, infection rates were determined via quantification of the viral e protein using a cell-based zikv immunodetection assay. data points represent mean ± sem (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of compound (* denotes p < . ; ** denotes p < . ; *** denotes p < . ). a.e. röcker et al. antiviral research ( ) - . discussion our results establish clr as a broad-spectrum antiviral agent, which not only inhibits infection of established viral pathogens (lump et al., ) , but also of emerging viruses, such as ebov and zikv. we characterized here mainly the effect of clr on zikv, as there is currently no specific antiviral therapy nor a preventive vaccine available. clr blocked zikv infection of primary brain-derived murine cells (fig. b) as well as human cell lines derived from the anogenital region (fig. , supplementary fig. s ) and the brain (fig. a) with ic values between and μm. these values are in the same range as ic values obtained against ebov ( μm, fig. b ) and hiv- ( - μm) (lump et al., ) . several lines of evidence demonstrate that the antiviral activity of clr is directed against the zikv particle itself. first, treatment of cells with clr prior to infection had no antiviral effect (fig. a) . second, the ic of clr increased with increasing viral concentrations ( supplementary fig. s ) . third, the integrity of the zikv particle was lost upon clr treatment, as shown by the release of viral rna from clr -treated virions (fig. d) . these findings were in agreement with our previous observations that clr selectively disrupted viral membranes (lump et al., ) . interestingly, the selectivity stems from clr 's preferential interaction with membranes containing high levels of sphingomyelin and cholesterol, two lipids that are enriched in membranes of enveloped viruses, such as hiv- or ebov (bavari et al., ; brügger et al., ; chazal and gerlier, ; lorizate et al., ) . the selective interaction of clr with lipids that are enriched in the viral but not the cellular membrane may also explain its minimal effects on cell viability ferreira et al., ; lopes et al., ; prabhudesai et al., ; sinha et al., ) (fig. c , d, supplementary fig. s b ). clr is slightly cytotoxic at concentrations of ∼ mm, corresponding to selectivity indices of - , which is in a reasonable range for drug development (buschmann and mannhold, ; food and drug administration, ) . . clr inhibits infection of human and murine brain cells. (a) human glioblastoma (a ) or neuroglioma (h ) cells were infected with zikv in the presence or absence of . - μm clr . two days later, cells were stained for zikv e protein (green) and nuclei (with hoechst; blue) and imaged by confocal microscopy. scale bar: μm. (b) primary murine cerebellum cultures were infected with zikv mr that had been incubated with . - μm clr for min at °c. dpi, cultures were fixed, permeabilized and stained for the neuronal protein βiii tubulin (red), zikv e protein (green) and nuclei (with hoechst; blue). scale bar: μm. clr lost antiviral activity in the presence of serum, precluding application as a systemic antiviral drug. this finding was unexpected because clr has been found previously to be stable in mouse and human plasma, with a half-life of ∼ . h in mice after sc or iv injection . moreover, clr showed therapeutic effects in animal models of alzheimer's and parkinson's diseases malik et al., ; prabhudesai et al., ; richter et al., ) . these data suggest that the loss of antiviral activity in the presence of serum likely was due to binding of clr to serum proteins. the concentrations of clr required to prevent amyloid formation in vitro and in vivo are in the sub-micromolar range, which is - orders of magnitude lower than those needed to block viral infection. presumably, the vast majority of clr was bound to serum proteins and hence unavailable to act on the virus, whereas the minority of the tweezer remains free at concentrations allowing to exert anti-amyloid activity. this interpretation also is supported by the observation that clr retained anti-zikv activity in semen, urine, saliva, and csf. the total protein concentrations in these body fluids are ∼ - orders of magnitude lower than in serum (hu et al., ; vibhakar et al., ) . thus, a larger percentage of clr molecules likely are free in these biofluids and hence antivirally active. clr has been described previously as a novel bi-functional microbicide counteracting sexual hiv- transmission both through directly targeting virus infectivity and by inhibiting the infection-promoting activity of amyloids in semen (lump et al., ) . we show here that the tweezer also inhibits pathogenic zikv infection of cells derived from the anogenital region in the presence of semen. interestingly, both zikv and ebov are present in semen of infected individuals (atkinson et al., ; deen et al., ; moreira et al., ) and can be transmitted by sexual intercourse, similar to hiv- (d'ortenzio et al., ; mate et al., ; moreira et al., ) . thus, application of clr as a microbicide in the vaginal or rectal tracts might protect from acquiring different viral pathogens. interestingly, clr has been shown to penetrate the bbb richter et al., ) , where it may also block replication of neurotropic viruses such as zika or rabies (cao-lormeau et al., ; ludlow et al., ; mlakar et al., ) . however, it needs to be considered that clr may only block cell-free virus infection but not cell-to-cell viral spread, which may limit its utility as therapeutic agent. therefore, the effect of clr on cell-tocell viral transmission needs to be explored. another means of application of clr is topical, e.g., by directly administering clr on virus-infected body surfaces, such as the skin or mucous membranes, to treat, e.g., hsv-induced herpes labialis or genitalis. topical medications may also be inhalational, such as medications against flu or respiratory syncytial virus (rsv), or applied to the surface of tissues other than the . after min of incubation, the mixtures were used to infect vero e cells. days later, infection rates were determined via a cell-based zikv immunodetection assay. data points represent mean ± sem (n = ), except for csf and serum: mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of clr but in presence of the same respective body fluid concentration (* denotes p < . ; *** denotes p < . ). a.e. röcker et al. antiviral research ( ) - skin, such as eye drops applied to the conjunctiva, for example, in cmvinduced retinitis. to address the challenge of emerging and re-emerging viral pathogens, it is imperative to develop broad-spectrum classes of antiviral agents as the conventional one-bug-one-drug paradigm is insufficient (zhu et al., ) . current direct-acting antiviral drugs are highly successful but have a narrow spectrum of coverage and are only available against a very limited number of viral pathogens. moreover, drug development is slow and expensive, and it typically takes more than a decade to get market approval. currently, no specific antiviral treatment exists against most, if not all, emerging and re-emerging viruses. broad-spectrum antivirals may offer certain advantages as they reduce time and cost of drug development, allow off-label use, and could be applied even before a viral threat is diagnosed (bekerman and einav, ; zhu et al., ) . clr is broadly active against enveloped viruses as it disrupts lipid bilayers containing elevated levels of sphingomyelin and cholesterol (lump et al., ) , which are typically enriched in viral membranes (bavari et al., ; brügger et al., ; chan et al., ; chazal and gerlier, ; lorizate et al., ) . we have shown that clr inactivates major viral pathogens such as hiv- , hsv- , cmv, and hcv, as well as zikv and ebov. it is highly likely that clr also blocks other enveloped viruses, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) corona viruses, influenza or rsv. most notably, resistance development against clr is very unlikely to occur, as this would require changes in the lipid composition of the cellular and the viral membrane, which is difficult to envisage. in conclusion, clr represents a promising prototype of a broad-spectrum antiviral agent. all data were analyzed using graphpad prism version . for windows, graphpad software, la jolla california usa (www.graphpad. com). the significance level was calculated using one-way one-way analysis of variance (anova) (non-parametric, grouped), followed by bonferroni's multiple comparison test. p values of < . were considered significant (*, p < . **, p < . ***, p < . ). detection of zika virus in semen safety and pharmacological characterization of the molecular tweezer clr -a broad-spectrum inhibitor of amyloid proteins' toxicity protection of primary neurons and mouse brain from alzheimer's pathology by molecular tweezers a screen of fda-approved drugs for inhibitors of zika virus infection lipid raft microdomains combating emerging viral threats the hiv lipidome: a raft with an unusual composition drug selectivity: an evolving concept in medicinal chemistry guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study ebola outbreak in west africa -case counts retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides virus entry, assembly, budding, and membrane rafts. microbiol ebola rna persistence in semen of ebola virus disease survivors -final report zika virus (i). isolations and serological specificity evidence of sexual transmission of zika virus zika virus infection with prolonged maternal viremia and fetal brain abnormalities in vitro and in vivo characterization of recombinant ebola viruses expressing enhanced green fluorescent protein molecular tweezers targeting transthyretin amyloidosis a molecular tweezer for lysine and arginine guidance for industry; antiviral product development-conducting and submitting virology studies to the agency. department of health and human services genetic characterization of zika virus strains: geographic expansion of the asian lineage zika virus outside africa a novel ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals human body fluid proteome analysis zika virus infection as a cause of congenital brain abnormalities and guillain-barré syndrome: systematic review phylogeny of zika virus in western hemisphere molecular tweezers inhibit islet amyloid polypeptide assembly and toxicity by a new mechanism comparative lipidomics analysis of hiv- particles and their producer cell membrane in different cell lines neurotropic virus infections as the cause of immediate and delayed neuropathology neurotoxicity of the parkinson's disease-associated pesticide ziram is synuclein dependent a molecular tweezer antagonizes seminal amyloids and hiv infection using molecular tweezers to remodel abnormal protein self-assembly and inhibit the toxicity of amyloidogenic proteins molecular evidence of sexual transmission of ebola virus zika virus associated with microcephaly sexually acquired zika virus: a systematic review inactivation and environmental stability of zika virus development of a highthroughput colorimetric zika virus infection assay semen-derived amyloid fibrils drastically enhance hiv infection history, epidemiology, and clinical manifestations of zika: a systematic review strategies for containing ebola in west africa zika virus a novel "molecular tweezer" inhibitor of α-synuclein neurotoxicity in vitro and in vivo zika virus and birth defects-reviewing the evidence for causality a molecular tweezer ameliorates motor deficits in mice overexpressing α-synuclein macromolecular antiviral agents against zika, ebola, sars, and other pathogenic viruses molecular tweezers for lysine and arginine-powerful inhibitors of pathologic protein aggregation ebola vaccine: how far are we? re-emergence of zika virus: a review on pathogenesis, clinical manifestations, diagnosis, treatment, and prevention lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins the . a resolution cryo-em structure of zika virus molecular clip and tweezer introduce new mechanisms of enzyme inhibition direct visualization of hiv-enhancing endogenous amyloid fibrils in human semen salivary total protein levels and their correlation to dental caries inhibition of mutant αb crystallin-induced protein aggregation by a molecular tweezer broad-spectrum antiviral agents a.e.r. is funded by a fellowship of the landesgraduiertenförderung baden-württemberg and is part of the international graduate school in molecular medicine ulm. j.a.m. is indebted to the baden-württemberg stiftung for the financial support of this research project by the eliteprogramme for postdocs. authors declare to have no conflict of interest. supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -ec lu a authors: amorim, raquel; temzi, abdelkrim; griffin, bryan d.; mouland, andrew j. title: zika virus inhibits eif α-dependent stress granule assembly date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: ec lu a zika virus (zikv), a member of the flaviviridae family, is the most recent emerging arbovirus with pandemic potential. during infection, viruses trigger the host cell stress response, leading to changes in rna translation and the assembly of large aggregates of stalled translation preinitiation complexes, termed stress granules (sgs). several reports demonstrate that flaviviruses modulate the assembly of stress granules (sg). as an emerging pathogen, little is known however about how zikv modulates the host cell stress response. in this work, we investigate how zikv modulates sg assembly. we demonstrate that zikv negatively impacts sg assembly under oxidative stress conditions induced by sodium arsenite (ars), a treatment that leads to the phosphorylation of eif α. by contrast, no measurable difference in sg assembly was observed between mock and zikv-infected cells treated with sodium selenite (se) or pateamine a (pata), compounds that trigger eif α-independent sg assembly. interestingly, zikv infection markedly impaired the phosphorylation of eif α triggered in ars-treated infected cells, and the abrogation of sg assembly in zikv-infected cells is, at least in part, dependent on eif α dephosphorylation. these data demonstrate that zikv elicits mechanisms to counteract host anti-viral stress responses to promote a cellular environment propitious for viral replication. zika virus (zikv) is transmitted to humans primarily through mosquito bites, but there have also been cases of sexual, perinatal, and suspected blood transfusion transmission. it has been associated with fetal malformations and neurological disorders in adults. the rising concern about this pathogen led the world health organization to declare it as a public health emergency of international concern regarding neurological disorders. there is an urgent global scientific effort underway to better understand zikv biology and define interactions that occur between the virus and the host cell. we evaluated how zikv infection counteracts the assembly of dynamic aggregates of rna and proteins called stress plos neglected tropical diseases | https://doi.org/ . /journal.pntd. july , / a a a a a zika virus (zikv) is a positive-sense, single-stranded rna virus that belongs to the genus flavivirus of the family flaviviridae, which also includes yellow fever (yfv), west nile (wnv), dengue (denv) and japanese encephalitis viruses (jev) [ ] . the genome of zikv encodes a large polyprotein precursor that is co-and post-translationally processed by viral and cellular proteases into three structural proteins [capsid (c), precursor of membrane (prm), and envelope (e)] and seven nonstructural proteins [(ns , ns a, ns b, ns , ns a, ns b and ns )] that are involved in virus replication, which takes place in the cytoplasm of the host cell [ ] . like other flavivirus members, zikv relies mainly on arthropods such as mosquitoes or ticks for transmission and thus is classified as an arthropod-borne virus (arbovirus). the main arthropod vectors of zikv are aedes sp. mosquitoes (a. aegypti or a. albopictus) [ ] . along with the vector-borne transmission, other routes of zikv transmission have been demonstrated, including sexual transmission, transplacental and perinatal transmission and blood transfusion [ ] , raising the concern about the global spread of the disease. zikv was first isolated from a rhesus monkey in the zika forest (uganda) in [ ] . for more than years, zikv was rarely reported to cause disease in humans and was commonly associated with mild illness. in , there was an outbreak in the federated states of micronesia [ ] , followed by outbreaks in french polynesia in - , in which severe neurological complications were reported [ ] . since then, zikv is considered to be the most recent emerging arbovirus with pandemic potential [ ] . in , autochthonous transmission of zikv was confirmed in the northeastern region of brazil [ ] . a dramatic increase in reported cases of microcephaly in the affected brazilian regions suggested an association between zikv infection and fetal malformations [ ] and neurological disorders in adults, including guillain-barré syndrome and meningoencephalitis [ ] . in february , the world health organization declared a public health emergency of international concern regarding neurological disorders associated with the rapid emergence of zikv in oceania and the americas [ ] . in response to conditions of environmental stress, eukaryotic cells activate kinases (hri, gcn , pkr and perk) that phosphorylate eif α (eukaryotic initiation factor alpha) to ease cellular injury or, alternatively, to induce apoptosis. phosphorylation of eif α reduces global translation by impairing the formation of the ternary complex eif -gtp-trna met , allowing cells to conserve resources and to initiate a reconfiguration of gene expression to effectively manage stress conditions [ ] . protein synthesis arrest triggers the assembly of stress granules (sg), that are large ribonucleoprotein (mrnp) aggregates formed by stalled translation preinitiation complexes [ , ] . the major components of sg are untranslated mrnas, eukaryotic translation initiation factors (eif e, eif g, eif a, eif ), the s ribosomal subunit and rnabinding proteins such as the poly(a) binding protein (pabp), t-cell intracellular antigen (tia- ), tia- -related protein (tiar), and ras gtpase activating protein-binding protein (g bp ) [ ] . distinct cell host processes are interrupted or co-opted during viral infection, leading to the activation of cell stress responses on many levels. sg assembly lowers the cytosolic availability of components of the cellular translation machinery and functions as a platform that connects stress and antiviral innate responses, implying an overall antagonistic relationship between viruses and sgs [ ] . in this sense, viruses have evolved a plethora of strategies to guarantee their replication by preventing or blocking sg assembly in infected cells, for example by co-opting rna granule factors and/or blockage of activation of eif α kinases, such as pkr [ ] . cellular stress responses are essential in eliciting immune detection and in the cell's ability to shut down viral gene expression in response to viral infection. so far, little is known about how zikv modulates stress responses in infected cells. recently, it was shown that zikv infection triggers a potent repression of host cell translation initiation, while viral protein synthesis remains unaffected [ ] . the interplay between viral replication and the cellular stress response may contribute to the exacerbated pathogenesis seen in the current epidemic. elucidation of the interaction of viral components with host factors involved in sg assembly will provide new insight into the pathology of zikv infection. in this work, we investigated how zikv infection modulates sg assembly. green african monkey kidney (vero) (atcc) cells and human osteosarcoma-derived u os containing g bp -gfp (a kind gift from dr. paul anderson and nancy kedersha, harvard medical school [ ] ) cells were maintained at ˚c and % co atmosphere in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) (hyclone) and % penicillin/streptomycin (life technologies). cell viability was evaluated by trypan blue exclusion cytotoxicity assay [ ] to produce viral stocks, vero cells were infected with zikv strain prvabc / at a multiplicity of infection (moi) of . and incubated for days at ˚c. viral supernatants were then harvested, centrifuged at x g for minutes at ˚c and filtered on a μm syringe filter. viral titers were determined by plaque forming assay using culture media supplemented with carboxymethylcellulose (sigma) as described previously [ ] . a stock with a viral titer of x was used in the experiments. for immunofluorescence assays, . x vero or u os cells were seeded on mm diameter coverslips the day prior infection. for western blotting analysis, . x vero or u os cells were seeded in each well of a -well plate. then, cells were incubated for hour with zikv diluted in dmem at an moi of . [ ] . after this period, the viral inoculum was removed by aspiration and cells were incubated in complete culture media for the periods specified in each experiment. vero cells were seeded on mm coverslips and infected as described above. viral rna was labeled as described in [ ] . briefly, cells were treated for minutes with μg/ml actinomycin d (sigma) to block host cellular transcription. then, cells were transfected with mm -bromourudine -triphosphate (brutp) (sigma) using lipofectamine reagent (invitrogen). after hour, cells were fixed and processed for indirect immunofluorescence analysis. stress was induced using μm sodium ars (naaso ; sigma-aldrich) for goat anti-tiar (santa cruz biotechnology) was used for indirect immunofluorescence microscopy at a dilution of : ; rabbit anti-eif g (santa cruz biotechnologies) was used for indirect immunofluorescence at : ; mouse anti-zika ns (biofront technologies) was used at : for indirect immunofluorescence and : , for western blotting; mouse anti-brutp (enzo life sciences) was used for indirect immunofluorescence at : ; rabbit antiphospho eif α (ser ) (cell signaling technology) was used for indirect immunofluorescence and : and for western blotting at : , ; mouse anti-eif α (cell signaling technology) was used for western blotting at : , ; and mouse anti-actin (abcam) was used for western blotting at : , ; rabbit anti-gadd (thermo fisher scientific) was used for western blotting at : ; rabbit anti-perk antibody (cell signaling technology) was used for western blotting at : . horseradish peroxidase-conjugated secondary antibodies were purchased from rockland immunochemicals and used at : , , and alexafluor secondary antibodies were purchased from life technologies and used at : . cells were lysed in np lysis buffer ( mm tris ph . , mm nacl, . mm edta, . % np ). equal amounts of protein were separated by sds-page and transferred to a nitrocellulose membrane (bio-rad). blocking was performed using % nonfat milk in tris-buffered saline with . % tween (tbst) for hour at room temperature. membranes were probed with the indicated primary and appropriate horseradish peroxidase-conjugated secondary antibodies. for detection of total and phosphorylated forms of proteins, samples were run in duplicate gels and transferred to independent membranes for western blotting. membranes were probed for actin and protein levels were normalized in both membranes for the downstream densitometry analysis [ ] . proteins were detected using western lightning plus-ecl (perkinelmer). for quantitation, the pixel intensity of each band was determined using the imagej program (nih) and then normalized to the indicated control. cells were prepared for indirect immunofluorescence as described previously [ ] . briefly, cells were fixed in % paraformaldehyde and permeabilized with . % triton x- . to prevent nonspecific binding, the cells were blocked using roche blocking solution for minutes at room temperature. primary antibodies were applied followed by incubation with the appropriate secondary antibody in blocking solution. stained cells were mounted in prolong gold antifade reagent with dapi (life technologies). laser scanning confocal microscopy was performed using a leica dm b microscope equipped with a wavefx spinning disk confocal head (quorum technologies) using a x objective lens. images were acquired with a hamamatsu imageem em-charges coupled device (ccd) camera and collected as z-stacks that were rendered for image reconstruction using the imaris software (v. . . , bitplane, inc.). twenty-four hours after infection, vero cells were treated with μm ars for h, mm dtt for h or nm pata for h or u os cells were treated with mm se for h and then processed for immunofluorescence as described above. infected cells were identified by detection of viral protein ns or brutp labeled rna, and sg-positive cells were defined as having at least sg as determined by colocalized g bp and tiar or eif g and tiar puncta. at least cells were analyzed per condition in to fields in independent experiments and the data are presented as the percentage of cells containing sg. all experiments were performed in triplicate, and the data are presented as the mean ± standard deviation (sd). a p-value < . in a two-way anova test was considered statistically significant. graphpad prism (graphpad software inc.) was used to conduct statistical analyses and create graphs. zikv is a positive-strand rna virus that replicates in the cytoplasm but little is known about redistribution of host proteins in zikv infected cells. sequestering sg components to sites of viral replication is a strategy used by viruses to impair sg assembly in infected cells [ ] . to determine whether zikv replication altered the distribution of sg markers, vero cells were infected with zikv with an moi of . and , and hours after, nascent viral rna was labeled with brutp and detected by indirect immunofluorescence. in zikv-infected cells, viral protein or rna was not detectable to hpi. tiar was evenly distributed throughout mock-infected cells (fig a and b) , and eif g was distributed homogeneously in the cytoplasm (fig a and b) . however, in infected cells, tiar was still found in both the cytoplasm and nucleus but also concentrated in foci in the perinuclear region ( fig a and b) , colocalizing with the zikv rna ( fig a) and viral nonstructural protein, ns ( fig b) . no change in eif g distribution was observed between mock and infected cells (fig a and b) . these findings suggest that zikv infection induces the redistribution of tiar to sites of viral rna replication. cleavage of proteins that nucleate sg assembly has been reported to be a strategy employed by viruses to overcome cellular stress response [ ] . we next evaluated whether zikv replication alters the levels of sg markers. cells were mock infected or infected with zikv and at hpi cells lysates were collected and analyzed by sds-page followed by western blotting. no alteration was observed in the levels of g bp- , tiar and pabp between mock and infected cells ( fig c) . these results indicate that zikv infection does not induce changes in the levels of sg-nucleating proteins. several viruses, including many members of flaviviridae family, have the ability to modulate sg assembly to keep the cell environment favorable to their own replication [ , ]. we investigated whether zikv can interfere with the assembly of sg in infected cells. vero cells were infected with zikv or mock-infected and treated with sodium arsenite (ars) at hours post-infection to induce cellular stress. ars is an oxidative agent that rapidly induces sg assembly through phosphorylation of eif α [ ] . sg assembly was determined by indirect immunofluorescence of tiar and eif g and infected cells were identified by the presence of the viral protein ns . in the absence of stress, mock-infected (blue arrows) and zikv-infected cells (red arrows) exhibited sg assembly at a rate of . % and %, respectively (fig a, top panels and fig b) , indicating that zikv infection does not induce the assembly of sg. in mock-infected cells, ars treatment induced abundant sg assembly as expected, with . % of the cells presenting tiar and eif g co-localized in cytoplasmic puncta (fig a and b ). in contrast, zikv infected cells presented sg at a rate of only . % (fig a, bottom panel and fig b) . similar results were observed when u os cells were used in place of vero cells (s fig). these results indicate that zikv infection blocks the assembly of type i sgs. zikv does not block eif α-independent assembly of stress granules pateamine a (pata) is a natural product isolated from a marine sponge that disrupts the translation initiation by hyperactivating the eif a helicase and disrupting the eif f complex, leading to the assembly of sg in an eif α-independent manner [ ] . to test whether zikv infection was also capable of blocking pata-induced sgs, vero cells were mock-infected or infected with zikv and at hpi were treated with pata. sg assembly was determined by colocalized puncta of tiar and eif g and infected cells were identified by the presence of the viral protein ns . pata treatment, as expected, induced a robust sg assembly in . % of the mock-infected cells (fig a, top panels, and b). interestingly, zikv infection did not impair pata-induced sg assembly, as . % of the infected cells presented tiar and eif g puncta ( fig a, top panels, and b). sodium selenite (se) promotes the assembly of type ii sg that differ from canonical sgs in their morphology, composition and mechanism of assembly, mainly by disrupting the eif f complex formation through ebp [ ] . to test whether zikv infection alters se-induced sg assembly, u os cells (s fig) stably expressing gfp-g bp were mock-infected or infected with zikv and at hpi were treated with se. u os cells were used in place of vero cells due to the high toxicity of se to the latter ones. sg assembly was determined by colocalized puncta of tiar and g bp- and infected cells were identified by the presence of the viral protein ns . similarly to pata-induced sg, no significant difference was observed in the assembly of sg between mock and infected cells treated with se ( fig a, bottom panels, and b). these findings indicate that zikv infection blockage of sg assembly is eif α-dependent. many viruses modulate p-eif α levels during replication to assure viral protein synthesis and avoid cellular stress responses. for example, coronaviruses can induce gadd expression to enhance pp activity and consequently the dephosphorylation of eif α [ ], and herpesviruses encode a viral protein that mimics the function of gadd [ ] . we examined the phosphorylation status of eif α in zikv infected untreated or ars treated cells. protein lysates were analyzed by western blotting using an antibody specific for eif α phosphorylation at s . as shown in fig a and b , little phosphorylation of eif α was detected in mock-infected and untreated vero cells, with a slight increase in p-eif α in zikv-infected cells. as expected, high levels of eif α phosphorylation ( -fold increase) were observed in extracts of mock-infected cells treated with ars. however, in zikv-infected and ars treated cells, levels of eif α phosphorylation were zikv recruits tiar to sites of viral rna replication. vero cells were infected with zikv with an moi of . and a. at hpi, cells were treated with actinomycin d and the nascent viral rna was labeled with brutp and detected by immunofluorescence/laser scanning confocal microscopy (if/lscm) using a x objective lens; b. at hpi, cells were fixed and the viral protein ns was detected by immunofluorescence followed by confocal microscopy. representative of experiments; c. at hpi, cells were lysed and lysates were analyzed for g bp- , tiar, pabp, ns and β-actin by sds-page followed by western blotting. a. vero cells were infected with zikv with an moi of . or mock-infected and treated at hpi with μm ars for h to induce cellular stress. the sg markers tiar and eif g were observed by if/lscm and infected cells were identified by the consistently lower ( . -fold increase). zikv replication was confirmed by the detection of the viral protein ns in cell extracts. the amount of total eif α was similar under all conditions tested (fig a) , indicating that zikv replication does not alter its expression. to further confirm that zikv-infected cells exhibit lower levels of p-eif α under arsenite treatment, phosphorylation of eif α was also analyzed by if/lscm. as shown in fig c, phosphorylation of eif α is strongly induced in the cytoplasm of non-infected cells (blue arrows). in contrast, in zikvinfected cells, the phospho-eif α signal is visibly weaker (red arrow). upon arsenite treatment, the fluorescence intensity of p-eif α in infected cells was in average % lower in zikv-infected cells in comparison to mock-infected cells (fig d) . these results indicate that zikv infection impairs eif α phosphorylation triggered by oxidative stress. our findings show that zikv infection blocks sg assembly and phosphorylation of eif α triggered by ars, an hri activator. to investigate whether this blockage is dependent on the eif α kinase activated upon stress, vero cells were mock-infected or infected with zikv and at hpi were treated with dtt, an endoplasmic reticulum (er) stressor that activates perk. sg assembly was determined by tiar puncta and infected cells were identified by the presence of ns . dtt treatment induced sg assembly in . % of the mock-infected cells (fig a and b ). in contrast, only . % of zikv-infected cells presented sg (fig a and b) . the blockage of sg assembly correlates with lower levels of p-eif α upon dtt treated in zikvinfected cells (fig c, lane ) when compared to mock-infected cells (fig c, lane ) . interestingly, the activation of perk in dtt-treated cells, demonstrated by an increased perk mobility, was similar in mock and zikv-infected cells (fig c, compare lanes our results suggest that zikv infection might abrogate sg assembly by blocking eif α phosphorylation. to test this further, vero cells were infected with zikv and at hpi, the levels of gadd , a pp a cofactor, were evaluated. our results show that are gadd levels are significantly higher in zikv-infected cells as compared to uninfected cells (fig a and b) . to evaluate the role of gadd /pp a activity on zikv-infected cells, we treated cells with salubrinal and its derivative sal , small molecules that selectively inhibit the pp /gadd -mediated dephosphorylation of phospho-eif α [ , ] . vero cells were treated with μm of salubrinal or μm of sal for h prior to the addition of ars to the cells. the phosphorylation status of eif α was evaluated by western blotting analysis (fig c) . in cells treated with salubrinal prior to ars-induced stress, zikv-infected cells present higher levels of phospho-eif α as compared to mock-infected control (fig c, compare lanes and ) . similar results were obtained with sal [ ] (s fig). the assembly of sg in the distinct conditions was monitored by indirect immunofluorescence. sgs were induced in . ± . % of zikv-infected cells treated with ars. this value increased to . ± . % in cells that were treated with salubrinal prior to ars-induced stress and was not significantly different from mock-infected cells (fig d and e ). no significant difference was observed between control or salubrinal pre-presence of the viral protein ns . blue arrows: uninfected cells; red arrows: infected cells. b. at least cells in each condition were analyzed. cells with at least sg were considered positive. data are presented as mean ± sd from independent experiments. https://doi.org/ . /journal.pntd. .g zika virus inhibits stress granule assembly treated mock-infected cells (fig e) . hence, inhibiting eif α dephosphorylation reduces the ability of zikv infection to block ars-induced sg assembly. these results indicate that eif α dephosphorylation is differentially modulated during zikv replication and that this feature can contribute to zikv-mediated blockage of sg assembly. to further confirm the importance of modulating eif α for zikv replication, vero cells were infected with zikv and after hpi, salubrinal was added to culture media in increasing concentrations. after h, supernatants of each condition were collected and viral titer was determined by plaque forming assay and cells were lysed and lysates were processed by sds-page followed by western blotting. treatment of cells with salubrinal led to a dose-dependent decrease in the production of infectious particles released to the culture media (fig a, bar graph and b). cells treated with μm of salubrinal produce only . % of the infectious viral particles produced by control cells (fig a, bar graph and b ). salubrinal had no toxic effects on treated cells (fig a, line graph) . finally, a dose-dependent decrease in ns expression was observed in salubrinal treated cells (fig c) . the relationship between viruses and the cellular stress response is a multifaceted and complex phenomenon that depends on the structural and genetic characteristics of the virus and the host cell [ ] . infection by several types of rna and dna viruses results in changes in the cellular environment as viral replication co-opts several cellular pathways, including nutrient, energy and macromolecular synthesis, to produce infectious particles. in this process, viruses trigger the host cell stress response, which can lead to the assembly of sgs [ ] . since viral replication relies on the host translational machinery, most viruses suppress the stress response pathway and sg assembly at some point of their replicative cycle [ ] . interactions between stress proteins and viral components have been described in a large variety of experimental models at different stages of the viral lifecycle, depending on the type of virus and host cell [ , ] . zikv has emerged as a global public health threat over the last decade. many aspects of the molecular mechanisms involved in the pathogenesis of this emerging virus remain unclear and require further investigation. in this work, we described that zikv replication does not induce sg assembly in vero cells (fig ) . this contrasts with the results recently published by roth and colleagues [ ] that describe the assembly of sg-like structures on huh- cells infected with zikv. it is possible that those distinct findings are due to the usage of distinct cell lines. in our work, we also show that zikv and blocks sg assembly triggered by treatment of cells with ars (fig ) and dtt (fig ) . these finds are similar to the ones described recently by roth and colleagues, in which they describe that flaviviruses block sg assembly independently of the eif α kinase activated by stress [ ] . interestingly, during the review process of this manuscript, basu and colleagues [ ] reported that zikv-mediated blockage of sg assembly was specific for oxidative stress induced by arsenite. the reasons why these differences a. vero cells were infected with zikv with an moi of . or mockinfected and treated at hpi with nm pata for h to induce cellular stress. b. u os gfp-g bp -expressing cells were infected with zikv or mock-infected and treated at hpi with mm se for hours. sg assembly was determined by if/lscm staining for the sg markers tiar and eif g (vero cells) or g bp- and tiar (u os cells) and infected cells were identified by the presence of the viral protein ns . blue arrows: uninfected cells; red arrows: infected cells. c. at least cells in each condition were analyzed. cells with at least sg were considered positive. data are presented as mean ± sd from independent experiments. https://doi.org/ . /journal.pntd. .g a. vero cells were infected with zikv with an moi of . or mock-infected and treated at hpi with μm ars for h to induce cellular stress. lysates were analyzed for s -phospho(p)-eif α, eif α (total) and ns by sds-page followed by western blotting. b. densitometry quantification of p-eif α was determined by imagej analysis. values presented in the graph are normalized against the total amount of eif α in the cell lysate and represent fold change with the untreated mock-infected cells being arbitrarily set to . asterisks represent the statistically significant difference between mock and zikv-infected cells (two-way anova; p < . ). c. cellular stress was determined by if/lscm staining for sg markers, tiar and phosphor-eif α and infected cells were identified by the presence of the ns viral protein. were observed remain to be determined. several reports have shown that members of the flaviviridae family modulate sg assembly in infected cells. the ' stem loop from the viral minus strand of wnv and denv captures tia- and tiar to promote viral genome rna synthesis and inhibit sg assembly [ , ] . jev capsid protein interaction with caprin- leads to the sequestration of several sg components, such as g bp and usp , in the perinuclear region of infected cells, resulting in impairment of sg assembly [ ] and bovine viral diarrhea virus (bvdv) blocks ars-mediated sg assembly [ ] . tia and tiar are recruited to tick-borne encephalitis virus (tbev) sites of replication [ ] . finally, hepatitis c virus (hcv) replication leads to oscillating sg assembly/disassembly in infected cells through controlling the phosphorylation of eif α and co-opting tia- , tiar and g bp [ , ] . more recently, roth and colleagues [ ] demonstrated that denv and zikv uncouple translation suppression from the stress response by a mechanism that is yet to be identified. we demonstrate that zikv infection did not lead to a blockage in pata or se-induced sg (fig ) . the assembly of sg triggered by both molecules is independent of the phosphorylation of eif α, suggesting that zikv blocks stress granules assembly mainly via eif α signaling. interestingly, this does not seem to be a general feature of flaviviral infections, as it has been demonstrated by roth and colleagues that denv inhibits sg assembly induced by hippuristanol, an inhibitor of eif a rna binding [ ] . the phosphorylation of eif α is a key regulator of mrna translation initiation, and the level of phospho-eif α is modulated by the activities of kinases and phosphatases [ ] . oxidative stress induced by ars culminates on eif α phosphorylation by hri [ ], which prevents the recycling of the eif -gtp-trna met ternary complex, leading to polysome disassembly and consequent translational arrest and sg assembly [ ] . regulation of protein synthesis by eif α phosphorylation plays an important role in the cellular defense against viral infection, thus viruses evolved diverse strategies to prevent it. our results show that zikv attenuates eif α phosphorylation triggered by ars (fig ) and dtt ( fig ) and this ability is, at least in part, a consequence of modulating its dephosphorylation, as supported by the observation that treatment of cells with salubrinal reverses the zikvmediated blockage of sg assembly induced by ars (fig ) . similar to the finding of wang and colleagues using coronavirus [ ], we demonstrated that zikv infection induces a moderate increase in gadd expression (fig a and b) . recently, buchman and colleagues [ ] described a mechanism by which trehalose modulates p-eif α levels and stress granule assembly/disassembly by enhancing the expression of gadd and crep. the increase in the cellular levels of the pp phosphatase subunits could lead to faster dephosphorylation of p-eif α and disassembly of sgs, thereby rendering the cells able to recover more quickly from stress. it is possible that the enhanced levels of gadd found in zikv-infected cells play a similar role in response to stress. treatment of cells with salubrinal causes an accumulation of phospho-eif α through an inhibition of pp /gadd -mediated dephosphorylation of eif α without increasing eif α kinase activity [ ] . modulation of pp activity by viral infection was demonstrated for human cytomegalovirus [ ] , african swine fever virus [ ] , newcastle disease virus a. vero cells were infected with zikv with an moi of . or mock-infected and treated at hpi with mm dtt for h to induce cellular stress. the sg marker tiar was observed by if/lscm and infected cells were identified by the presence of ns . blue arrows: uninfected cells; red arrows: infected cells. b. at least cells in each condition were analyzed. cells with at least sg were considered positive. data are presented as mean ± sd from independent experiments. c. after dtt treatment, cells were lysed and lysates were analyzed for perk, s -phospho(p)-eif α, eif α (total) and actin by sds-page followed by western blotting. densitometry quantification of p-eif α was determined by imagej analysis. values presented are normalized against the total amount of eif α in the cell lysate and represent fold change with the untreated mock-infected cells being arbitrarily set to . https://doi.org/ . /journal.pntd. .g [ ] , papillomavirus [ ] and herpes simplex virus [ ] . icp . is a protein homologous to gadd encoded by hsv that is essential for hsv replication in some cell types. it binds cellular pp and promotes eif α dephosphorylation, ensuring viral replication despite activation of pkr [ ] . treatment of hsv-infected cells with salubrinal inhibits viral replication in a dose-dependent manner and leads to higher phospho-eif α levels [ , ] . similarly, our results demonstrate that zikv replication is severely impaired in salubrinal-treated cells (fig ) , indicating that zikv relies to some extent on eif α dephosphorylation for its replication. these findings are distinct from the model proposed by roth and colleagues [ ] , in which modulation of sg assembly in zikv-infected cells was independent of eif α dephosphorylation promoted by elevated gadd levels. cell type- fig . zikv modulates eif α dephosphorylation. a. vero cells were infected with zikv at an moi of . or mock-infected. at hpi, cells were lysed and cells were lysed and lysates were analyzed for gadd and actin by sds-page followed by western blotting. b. densitometry quantification of gadd and actin were determined by imagej analysis. values presented are normalized against the total amount of gadd in the cell lysate and represent fold change with the mock-infected cells being arbitrarily set to . c. vero cells were infected with zikv or mock-infected and treated at hpi with μm salubrinal for h to block the dephosphorylation of eif α and then treated with μm ars for h to induce cellular stress. lysates were analyzed for s -phospho(p)-eif α and eif α (total) by sds-page followed by western blotting. values of p-eif α fold change were normalized by the corresponding eif α levels of the same condition. d. vero cells were infected with zikv or mock-infected and at hpi were treated with μm salubrinal for h and then oxidative stress was induced by treatment with μm ars for h. sg assembly was determined by if/lscm staining for the sg markers tiar and eif g and infected cells were identified by the presence of the viral protein ns . blue arrows: uninfected cells; red arrows: infected cells.e. at least cells in each condition were analyzed. cells with at least sg were considered positive. data are presented as mean ± sd from independent experiments and asterisks represent the statistically significant difference between mock and zikv-infected cells (two-way anova; p < . ). https://doi.org/ . /journal.pntd. .g specificities can be responsible for those contrasting results. it remains to be determined whether treatment with salubrinal has secondary effects on the infected cells that could act in synergy with the pp /gadd inhibition and the mechanism by which zikv modulates this activity. in conclusion, our work provides new insights into the zikv biology by demonstrating that zikv inhibits sg assembly in a phospho-eif α dependent way. this ability may reflect one of the many strategies that zikv has evolved to control the host stress response and demonstrate that zikv elicits mechanisms to counteract host anti-viral stress responses to promote a cellular environment propitious for viral replication. elucidation of the interaction of viral components with host factors involved in sg assembly may provide new insights into the pathology of zikv infection and lead to the identification of novel targets for therapeutic intervention. a. u os cells were infected with zikv with an moi of . or mock-infected and treated at hpi with μm ars for h to induce cellular stress. the sg markers g bp- and eif g were observed by if/ lscm and infected cells were identified by the presence of the viral protein ns . blue arrows: uninfected cells; red arrows: infected cells. b. at least cells in each condition were analyzed. cells with at least sg were considered positive. data are presented as mean ± sd from independent experiments. c. u os cells were infected with zikv with an moi of . or mock-infected and treated at hpi with μm ars for h to induce cellular stress. lysates were analyzed for s -phospho(p)-eif α and eif α (total) by sds-page followed by western blotting. b. densitometry quantification of p-eif α was determined by imagej analysis. values presented in the graph are normalized against the total amount of eif α in the cell lysate and represent fold change with the untreated mock-infected cells being arbitrarily set to . asterisks represent the statistically significant difference between mock and zikv-infected cells (two-way anova; p < . ) (tif) s fig. eif α dephosphorylation modulated by zikv is inhibited by sal . vero cells were infected with zikv or mock-infected and treated at hpi with μm sal for h to block the dephosphorylation of eif α and then treated with μm ars for h to induce cellular stress. lysates were analyzed for s -phospho(p)-eif α and eif α (total) by sds-page followed by western blotting. values of p-eif α fold change were normalized by the corresponding eif α levels of the same condition. molecular biology of flaviviruses zika virus assessing the global threat from zika virus isolations and serological specificity genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia association between zika virus and microcephaly in french polynesia, - : a retrospective study emerging arboviruses in the pacific first report of autochthonous transmission of zika virus in brazil possible association between zika virus infection and microcephaly-brazil zika virus-associated neurological disorders: a review. brain: a journal of neurology emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations translational control in stress and apoptosis rna-binding proteins tia- and tiar link the phosphorylation of eif- α to the assembly of mammalian stress granules mammalian stress granules represent sites of accumulation of stalled translation initiation complexes visibly stressed: the role of eif , tia- , and stress granules in protein translation a selective inhibitor of eif al-pha dephosphorylation protects cells from er stress phosphorylation of eif alpha is a translational control mechanism regulating muscle stem cell quiescence and self-renewal adapting the stress response: viral subversion of the mtor signaling pathway eef and ras-gap sh domain-binding protein (g bp ) modulate stress granule assembly during hiv- infection arsenite-induced stress granule formation is inhibited by elevated levels of reduced glutathione in west nile virus-infected cells cell proteins tia- and tiar interact with the ' stem-loop of the west nile virus complementary minus-strand rna and facilitate virus replication the pestivirus n terminal protease npro redistributes to mitochondria and peroxisomes suggesting new sites for regulation of irf by npro the stress granule component tia- binds tick-borne encephalitis virus rna and is recruited to perinuclear sites of viral replication to inhibit viral translation hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection regulation of translation initiation in eukaryotes: mechanisms and biological targets modulation of p-eif α cellular levels and stress granule assembly/disassembly by trehalose cellular serine/threonine phosphatase activity during human cytomegalovirus infection the african swine fever virus dp l protein recruits the protein phosphatase catalytic subunit to dephosphorylate eif alpha and inhibits chop induction but is dispensable for these activities during virus infection regulation of de novo translation of host cells by manipulation of perk/pkr and gadd -pp activity during newcastle disease virus infection. the journal of general virology control of alpha subunit of eukaryotic translation initiation factor (eif alpha) phosphorylation by the human papillomavirus type e oncoprotein: implications for eif alpha-dependent gene expression and cell death. molecular and cellular biology icp . -dependent and-independent activities of salubrinal in herpes simplex virus- infected cells we would like to thank gary pignac-kobinger, nancy kedersha, paul anderson, jerry pelletier and colin crist for generous provision of cell lines, viruses and reagents, meijuan niu for technical help, and shringar rao and alessandro cinti for helpful comments on the manuscript. key: cord- -o d aa authors: yu, xi; zhang, liming; tong, liangqin; zhang, nana; wang, han; yang, yun; shi, mingyu; xiao, xiaoping; zhu, yibin; wang, penghua; ding, qiang; zhang, linqi; qin, chengfeng; cheng, gong title: broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, sars-cov- and other enveloped viruses date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: o d aa viruses are the major aetiological agents of acute and chronic severe human diseases that place a tremendous burden on global public health and economy; however, for most viruses, effective prophylactics and therapeutics are lacking, in particular, broad-spectrum antiviral agents. herein, we identified secreted bacterial lipases from a chromobacterium bacterium, named chromobacterium antiviral effector- (cbae- ) and cbae- , with a broad-spectrum virucidal activity against dengue virus (denv), zika virus (zikv), severe acute respiratory syndrome coronavirus (sars-cov- ), human immunodeficiency virus (hiv) and herpes simplex virus (hsv). the cbaes potently blocked viral infection in the extracellular milieu through their lipase activity. mechanistic studies showed that this lipase activity directly disrupted the viral envelope structure, thus inactivating infectivity. a mutation of cbae- in its lipase motif fully abrogated the virucidal ability. furthermore, cbae- presented low toxicity in vivo and in vitro, highlighting its potential as a broad-spectrum antiviral drug. genomic comparison, csp_bj shares either . % identity to chromobacterium haemolyticum ch -bl or . % identity to chromobacterium rhizoryzae jp - strain. oral supplementation of this bacteria in a. aegypti largely impaired mosquito permissiveness to denv (supplementary figure a and b) and zikv (supplementary figure c and d) , suggesting a close relationship between the identified csp_bj and csp_p strains. we next aimed to understand how csp_bj resists viral infection in mosquitoes. bacteria usually exploit many effectors, such as cellular components, metabolites or secreted proteins, to regulate their host immune or physiological status for effective colonization. we therefore identified the bacterial effector(s) that modulate infection of denv and zikv through differential fragmentation. in this experiment, we cultured csp_bj for hr at °c. the cell-free culture supernatant was collected by centrifugation and filtration through a . µm filter unit, whereas the cell lysates were generated by sonication. either the bacterial cell lysate or the culture supernatant was mixed with plaque-forming units (pfu) of denv or zikv and incubated for hr, and then the infectious viral particles were determined by a plaque forming assay ( figure a ). incubation of the culture supernatant but not the bacterial lysates resulted in significant suppression of denv ( figure b ) and zikv ( figure c ) infectivity in vero cells, indicating that an extracellular effector(s) secreted by csp_bj was responsible for viral inhibition. next, we investigated whether the effector(s) was a secreted protein, small peptide, lipid, polysaccharide or other metabolite. therefore, the culture supernatant was separated using a kda-cutoff filter (wu et al., ) . either the upper retentate (proteins and large peptides) or the lower liquid filtrate (small molecule compounds and short peptides) was mixed with the viruses for incubation in vero cells ( figure d ). intriguingly, inoculation of the retentate rather than the filtrate inhibited the infectivity of denv and zikv ( figure e and f), suggesting that the effector(s) might be a protein(s) secreted by csp_bj. subsequently, the protein components in the upper retentate were separated by sds-page and then identified by mass spectrometry (figure a ). the highly abundant proteins with secretable properties were selected, expressed and purified in an escherichia coli expression system ( figure b) . of all the proteins tested, the bacterial protein encoded by gene (accession: mt ) significantly impaired denv and zikv infection in vero cells ( figure c and d). we named this protein chromobacterium antiviral effector- (cbae- ). intriguingly, a cbae- homologue with . % amino acid identity named cbae- (accession: mt ) was further identified from csp_bj based on sequence comparison (supplementary figure a and b) . both effectors encoded a lipase domain with a typical gdsl motif (casas-godoy et al., ) . to further confirm the virucidal activity, both proteins were expressed and purified in e. coli ( figure e) . a serial concentration of recombinant proteins was mixed with pfu of denv or zikv for plaque assay in vero cells. the half-inhibitory concentration (ic ) of cbae- was . µg/ml for denv and . µg/ml for zikv ( figure f and g). however, the ic of cbae- for both flaviviruses was - times higher than that of cbae- , indicating a much more robust virucidal activity of cbae- against flaviviruses ( figure f and g). thus, we identified bacterial effectors with a high virucidal activity from a csp_bj bacterium. we next investigated the mechanisms by which these bacterial effectors resist viral infection. according to sequence analysis, cbaes contain a conserved lipase domain. lipases are a group of enzymes that catalyse the hydrolysis of the ester bond of glycerides into fatty acids and glycerol (casas-godoy et al., ) . we therefore assessed whether cbaes have lipase activity. in a plate degradation assay, both cbaes directly digested egg yolk lipoproteins and formed lytic halos whose diameters correlated with lipase activity ( figure a ). the sequence gdsl is the core motif of lipase activity (casas-godoy et al., ) . consistently, a s g mutation in this motif of cbae- fully disrupted its lipase activity ( figure a ), validating cbaes as secreted lipases of csp_bj. given their lipase activity, we hypothesized that cbaes might use their enzymatic activity to degrade viral lipid envelope, which may result in exposure of the viral rna (muller et al., ) . to address this hypothesis, serial concentrations of cbaes were incubated with × pfu of denv or zikv for hr at °c. the mixture was then treated with rnase a to evaluate the degradation of exposed viral genomic rna. compared to mock treatments in which the viruses were incubated with bsa, a significant reduction in viral rna was recorded by rt-qpcr when the viruses were treated with cbaes ( figure b and c), indicating that the lipase activity of cbaes directly disrupted the virion structure, thus resulting in viral genome release. consistent with these results, the s g mutant of cbae- that had no lipase activity completely failed to suppress both denv and zikv infection ( figure d and e), further indicating that the virucidal activity of cbaes is lipase-dependent. to validate that cbaes disrupt viral lipidic membranes, we incubated cbae- with purified zikv virions and processed the samples for transmission electron microscopy ( figure f ). the zikv particle typically has a diameter of - nm (hasan et al., ) and, in consistent with that, we observed intact viral particles in our control sample treated with µg/ml bsa ( figure f ). however, upon treatment with µg/ml cbae- in the same buffer as bsa solution, the integrity of zikv particles was fully disrupted ( figure f ). this is in agreement with our previous finding that treatment of cbaes resulted in viral genome release. since cbaes blocked viral infection in the extracellular milieu, we further assessed whether treatment of cbaes prior to viral inoculation could effectively block viral infection. we pre-treated vero cells with µg/ml of purified cbaes. subsequently, . moi of denv or zikv was used to challenge the cbaes-treated cells. pre-incubation with cbae- fully blocked the infectivity of both flaviviruses, whereas treatment with cbae- exhibited %- % inhibition ( figure f and g). since the cbaes showed a direct catalytic action on the viral lipid bilayer, we assessed their virucidal activity against other enveloped viruses. sars-cov- is a newly emerging coronavirus that causes the severe acute respiratory disease covid- . to date, no specific therapeutics are available against sars-cov- infection. we therefore assessed the virucidal effect of the cbaes on sars-cov- . in contrast to the large difference in virucidal activity against flavivirus infection, both cbaes presented a similar antiviral effect against a sars-cov- pseudovirus in hek- t-ace cells ( figure a ). consistently, infection with lowpassage sars-cov- in vero cells was also significantly suppressed by treatment with the cbaes ( figure b ). additionally, both cbaes showed broad-spectrum antiviral effects on hiv- pseudoviruses ( figure c , d and e), as well as hsv- ( figure f ). notably, compared to a hiv-neutralizing antibody (n ) with a near-pan neutralization breadth (huang et al., ) , cbaes were very potent with a much lower ic for hiv pseudovirus strains ( figure c, d and e). nonetheless, it is puzzling that the cbaes did not show any effect against infection with influenza a virus (iav) ( figure g ). the lipidic envelope of a viral particle is generally derived from the plasma membrane or the endoplasmic reticulum (er) membrane of a host cell. thus, cbaes can also act on host cell membranes and their cytotoxicity to host cells may be a major concern for developing a cbae-based broad-spectrum antiviral drug. generally, cbae- showed much higher toxicity figure d ). altogether, these data suggest that cbae- is much safer than cbae- to hosts, and thus could be a broad antiviral drug candidate. in this study, we identified two virucidal effectors with lipase activity, cbae- and cbae- , from the chromobacterium sp. csp_bj. both cbaes showed a potent virucidal activity against a variety of enveloped viruses including denv, zikv, sars-cov- , hiv- , and hsv- . notably, neither cbaes exhibited any effect against iav. the toxicity assessment showed that cbae- was much safer than cbae- to both human cells and mice. indeed, accumulating evidence indicates that certain lipases present a potent antiviral activity. either lipoprotein lipase or hepatic triglyceride lipase impaired hepatitis c virus (hcv) infection in human huh . cells through degrading virus-associated lipoproteins (shimizu et al., ) . a secreted phospholipase a (pla ) isolated from naja mossambica snake venom showed a potent virucidal activity against hcv, denv, and japanese encephalitis virus (jev); while the protein did not exhibit significant antiviral activity against sindbis virus (sinv), iav, middle east respiratory syndrome coronavirus (mers-cov) or hsv- (chen et al., ) . a secreted human pla has been shown to neutralize hiv- by degrading the viral membrane (kim et al., ) or by blocking viral entry into host cells rather than a lipase-mediated virucidal effect (fenard et al., ) , suggesting diverse virucidal mechanisms by pla . cbaes may inactivate viruses by their lipase activity that also damage cellular membranes; nonetheless, their specificity and affinity for the viral envelope could be significantly improved, thus further reducing their cytotoxicity and increasing virucidal efficacy. for example, the sars-cov- surface protein spike binds to human ace with high affinity (wang et al., ) , and thus soluble ace could be engineered with cbaes to greatly enhance its affinity and specificity for the viral envelope. given that human recombinant soluble ace can also inhibit sars-cov- infection in organoids (monteil et al., ) , it is possible that a soluble ace -cbae construction may have improved efficacy in blocking sars-cov- infections. viruses are the major aetiological agents of acute severe human diseases that impose a tremendous burden on the global public health and economy (ghosn et al., ; girard et al., ; wilder-smith et al., ) . given the fact that development of virus-specific vaccines and antiviral drugs is usually lengthy, broad-spectrum antiviral drugs could be crucial to prevent wide spread of new viral disease in a timely manner (schein, ) . cbae- , with a broad antiviral effect and low toxicity to hosts, could be a potential choice. our study provides a future avenue for the development of broad-spectrum antiviral drugs that might reduce the clinical burden caused by emerging viral diseases. human blood was collected from healthy donors who provided written informed consents. the collection of human blood samples and their use for mosquito feeding was approved by the local ethics committee at tsinghua university. eight-week-old female icr mice purchased from vital river laboratories in china were used for toxicity assay. the mice were maintained in a specific pathogen-free barrier facility at tsinghua university. the animal protocol used in this study was approved by the institutional animal care and use committee of tsinghua university and performed in accordance with their guidelines. aedes aegypti (the rockefeller strain) was maintained on a sugar solution in a low-temperature, illuminated incubator (model , thermo electron corporation) at °c and % humidity, according to standard rearing procedures (cheng et al., ) . vero cells, hek- t cells and a cells were maintained in dulbecco's modified eagle's medium ( - , gibco) supplemented with % heat-inactivated foetal bovine serum ( - , gibco) and % antibiotic-antimycotic ( - , invitrogen) in a humidified % (v/v) co incubator at °c. the vero, hek- t and a cell lines were purchased from the atcc (ccl- , crl- and ccl- , respectively). denv- (new guinea c strain), zikv (prvabc strain), hsv- , iav (h n pr strain) and sars-cov- were grown in vero cells with vp-sfm medium ( - , gibco). denv, zikv, hsv and sars-cov- were titrated by a standard plaque formation assay on vero cells (bai et al., ) . iav were titrated using a standard % tissue culture infection dose (tcid ) assay (teferedegne et al., ; varada et al., ) . all experiments involving infectious sars-cov- were performed in a biosafety level (bsl ) containment laboratory. chromobacterium sp. beijing was grown in lb broth at °c for hr at rpm. culture supernatants were obtained by centrifugation and filtering the supernatant through a . µm filter unit (slgp rs, millipore). genscript) before the protein concentration was measured using a bradford assay ( - , bio-rad), and the protein purity was checked with sds-page. vero cells were seeded at ~ × cells per well in -well plates and then incubated at °c overnight before reaching - % confluence. denv, zikv, hsv- and sars-cov- virus stocks were diluted to plaque-forming units (pfu) per ml and incubated untreated or with a serial dilution of the cbaes in five-fold steps at °c for hr before being added onto vero cell monolayers for hr of infection. cell monolayers were washed once with pbs and covered with % agarose overlay dmem with % fbs. after - dpi (denv, zikv and hsv- ) or - dpi (sars-cov- ), vero cell monolayers were fixed and stained with . % crystal violet, and the number of pfu per ml was determined. the concentration of each protein necessary to inhibit virus infection by % (ic ) was calculated by comparison with the untreated cells using the dose-response-inhibition model in graphpad prism . (graphpad software, usa). for pre-infection treatment, vero cells were seeded in -well plates and allowed to form monolayers. ten micrograms/ml cbae- or cbae- was added to vero cell monolayers at °c for hr, and then denv or zikv ( . moi) was added and incubated for another hour at °c. after infection, cell monolayers were washed once with pbs buffer, fresh vp-sfm medium was added, and the cells were incubated at °c for hr before the supernatant was collected for rt-qpcr quantitation of the viral genome. total rna was isolated either from homogenized mosquitoes or infected cell supernatant using supplementary table . the lipase activity of cbae- , cbae- and cbae- -s g was measured with a plate assay as previously described (liu et al., ) . briefly, µg of cbae- , cbae- or cbae- -s g was spot inoculated onto a % agar plate with % egg yolk and incubated for hr at °c. phospholipase activity was indicated by the diameter of the lytic halo around each well. viral rna exposure assay. cbaes or pbs in a total volume of ml for hr at °c. then, the mixtures were treated with µl of rnase a (ge - , transgen) and incubated for hr at °c. viral rna was extracted, and rna degradation was evaluated by rt-qpcr as mentioned above. zikv viral particles were purified as described previously (tan and lok, ) . briefly, virus stocks were pelleted in % w/v peg at , ×g for hr, then purified by % w/v sucrose cushion for hr at , ×g (beckman sw ti rotor) and separated in potassium tartrate-glycerol gradient - % for hrs at , ×g (beckman sw ti rotor). purified viral particles were suspended in µg/ml bsa solution or µg/ml cbae- solution and incubated for hr at room temperature. the samples were then applied to a carbon grid, washed times with water and negatively stained with % w/v uranyl acetate. the images were acquired in a hitachi h- b tem microscope at . kv. the in vitro antiviral efficacy of the cbaes on iav was tested in a cells. briefly, a cells were seeded into a -well plate and incubated at °c for - h. iav ( tcid ) were incubated untreated or with a serial dilution of the cbaes in five-fold steps at °c for hr before being added onto a cell monolayers to allow infection to proceed for hr. then, the virus-protein mixture was removed, and the cells were further cultured with fresh vp-sfm medium. at hr p.i., relative viral rna copy numbers in the infected cells were quantified by rt-qpcr assays with specific primers. the neutralization titre was defined as the concentration of each protein necessary to inhibit the pcr signal by % (i.e., below the threshold of % of the mean value observed in virus control wells). hiv- pseudoviruses were generated by co-transfecting hek- t cells with env expression vectors and the pnl - r-eluciferase viral backbone plasmid, and a neutralization assay was performed as described previously (zhou et al., ) . briefly, pseudovirus titres were measured by luciferase activity in relative light units (rlus) (bright-glo luciferase assay system, promega biosciences, california, usa). neutralization assays were performed by adding tcid (median tissue culture infectious dose) of pseudovirus into serial : dilutions of cbae- or cbae- starting from µg/ml, following incubation at °c for hr and addition of ghostx /r cells. neutralizing activity was measured by the reduction in luciferase activity compared to that in the controls. the fifty percent inhibitory concentration (ic ) was calculated using the dose-response-inhibition model with the -parameter hill slope equation in graphpad prism . (graphpad software, usa). vesicular stomatitis virus g protein (vsv-g) pseudotyped lentiviruses expressing human ace were produced by transient co-transfection of pmd g (addgene # ) and pspax (addgene # ) plasmids and the transfer vector plvx-ace flag-ires-puro with vigofect dna transfection reagent (vigorous) into hek- t cells to generate the hek- t-ace cells for sars-cov- pseudovirus infection. sars-cov- pseudoviruses were purchased from genscript, and neutralization activity was measured using the hek- t-ace cell line with the same procedures as mentioned above. fresh human blood from healthy donors was placed in heparin-coated tubes ( , bd vacutainer) and centrifuged at , ×g and °c for min to separate plasma from blood cells. the plasma was heat-inactivated at °c for min. the separated blood cells were washed three times with pbs to remove the anticoagulant. the blood cells were then resuspended in heat-inactivated plasma. bacterial suspension ( . od) was mixed with viruses and treated blood for mosquito oral feeding via a hemotek system ( w , hemotek). fully engorged female mosquitoes were transferred into new containers and maintained under standard conditions for an additional days. the mosquitoes were subsequently euthanized for further analysis. the mosquitoes used in this experiment were previously antibiotic treated. briefly, mosquitoes were provided with cotton balls moistened with a % sucrose solution including units of penicillin and mg of streptomycin per ml ( - , thermo fisher scientific) for days to remove gut bacteria. the mosquitoes were starved for hr to allow the antibiotics to be metabolized prior to in vitro membrane blood feeding. removal of gut bacteria was confirmed by a colony-forming unit assay. the cytotoxicity of the cbaes was evaluated in vero cells and a cells. cell viability was measured by the mtt [ -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide] (m , solarbio) method. confluent cell monolayers contained in -well plates were exposed to different concentrations of the cbaes for hr at °c. then, a final concentration of . mg/ml mtt was added to each well. after hr of incubation at °c, the supernatant was removed, and µl of dimethyl sulfoxide (dmso) was added to each well to solubilize the formazan crystals. after shaking for min, absorbance was measured at nm. the concentration of each protein necessary to reduce cell viability by % (cc ) was calculated by comparison with the untreated cells using a sigmoidal nonlinear regression function to fit the dose-response curve in graphpad prism . (graphpad software, usa). icr mice were used to test the in vivo safety of the cbaes. a number of icr -week old female mice were divided into groups (n= ) at random. cbae- or cbae- was dissolved in pbs and administered either intravenously once at doses of , , , and µg/kg or intranasally once at doses of , , and µg/kg. pbs and corresponding concentrations of bsa served as negative controls. the general behaviour, signs of toxicity, body weights and mortality of the mice were recorded after the administration of the cbaes. the half-lethal dose (ld ) was calculated using a sigmoidal nonlinear regression function to fit the dose-response curve in graphpad prism . (graphpad software, usa). animals were randomly allocated into different groups. mosquitoes that died before sample collection were excluded from the analysis. the investigators were not blinded to the allocation during the experiments or to the outcome assessment. no statistical methods were used to predetermine the sample size. descriptive statistics are provided in the figure legends. all analyses were performed using graphpad prism statistical software. (d, e) the s g mutant of cbae- fully lost its ability to suppress denv (d) and zikv (e) infection: inhibition curves of cbae- and cbae- -s g against denv (d) or zikv(e). serial concentrations of cbae- or cbae- -s g were mixed with pfu of denv or zikv in vp-sfm medium to perform standard plaque reduction neutralization tests (prnts). (f) representative negative stained transmission electron microscopy images of zikv particles treated with µg/ml bsa (arrow head) and those treated with µg/ml cbae- (empty arrow head); high magnification: , ×, low magnification: , ×. (g, h) rate of denv (g) or zikv (h) replication inhibition following exposure to cbaes before viral infection of vero cell monolayers. the viral genome was quantified by rt-qpcr. (b, c, g, h) significance was determined using unpaired t-tests. data are presented as the mean ± sem. aegypti. a mixture containing human blood ( % v/v), csp_bj bacterial suspension ( % v/v), and supernatant from denv-or zikv-infected vero cells ( % v/v) was used to feed antibiotic-treated a. aegypti rockefeller strain via an in vitro blood feeding system. mosquito infectivity was determined by rt-qpcr at days post blood meal. the final denv or zikv titre was × pfu/ml for oral infection. (a, c) the number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. a nonparametric mann-whitney test was used for the statistical analysis. (b, d) differences in the infectivity ratio were compared using fisher's exact test. (a) conserved domains of cbae- and cbae- protein sequences was analysed using a simple modular architecture research tool (smart) (letunic and bork, ; letunic et al., ) . (b) sequence comparison of cbae- and cbae- was performed using basic local alignment search tool (blast) on ncbi website with the program "needleman-wunsch alignment of two sequences" (altschul et al., ) . gapped blast and psi-blast: a new generation of protein database search programs antiviral peptides targeting the west nile virus envelope protein broad-spectrum agents for flaviviral infections: dengue, zika and beyond a non-live preparation of chromobacterium sp. panama (csp_p) is a highly effective larval mosquito biopesticide lipases: an overview broad-spectrum antiviral agents: secreted phospholipase a targets viral envelope lipid bilayers derived from the endoplasmic reticulum membrane a c-type lectin collaborates with a cd phosphatase homolog to facilitate west nile virus infection of mosquitoes the pandemic in mexico: experience and lessons regarding national preparedness policies for seasonal and epidemic influenza secreted phospholipases a( ), a new class of hiv inhibitors that block virus entry into host cells the a (h n ) influenza virus pandemic: a review structural biology of zika virus and other flaviviruses west nile virus spreads in europe identification of a cd -binding-site antibody to hiv that evolved near-pan neutralization breadth lysis of human immunodeficiency virus type by a specific secreted human phospholipase a years of the smart protein domain annotation resource smart: recent updates, new developments and status in pathogenicity of different isolates of vibrio harveyi in tiger prawn, penaeus monodon infections in engineered human tissues using clinical-grade soluble human ace phospholipase a isolated from the venom of crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope respiratory syncytial virus seasonality: a global overview inter-annual variation in seasonal dengue epidemics driven by multiple interacting factors in guangzhou, china human influenza virus infections chromobacterium csp_p reduces malaria and dengue infection in vector mosquitoes and has entomopathogenic and in vitro anti-pathogen activities the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak chromobacterium spp. mediate their anti-plasmodium activity through secretion of the histone deacetylase inhibitor romidepsin repurposing approved drugs on the pathway to novel therapies west nile virus infection lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis c virus (hcv) through their catalytic activities on hcvassociated lipoproteins zika virus: history, epidemiology, transmission, and clinical presentation respiratory syncytial virus hospitalization and mortality: systematic review and meta-analysis dengue virus purification and sample preparation for cryoelectron microscopy development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription pcr broad-spectrum coronavirus antiviral drug discovery a neutralization assay for respiratory syncytial virus using a quantitative pcr-based endpoint assessment structural and functional basis of sars-cov- entry by using human ace coronavirus disease (covid- ) situation report - epidemic arboviral diseases: priorities for research and public health a gut commensal bacterium promotes mosquito permissiveness to arboviruses sars-cov- : an emerging coronavirus that causes a global threat broadly resistant hiv- against cd -binding site neutralizing antibodies broad-spectrum antiviral agents key: cord- -hgp jwbl authors: carvalho, sandhra m.; mansur, alexandra a.p.; carvalho, isadora c.; costa, Érica a.; guedes, maria isabel m.c.; kroon, erna g.; lobato, zelia i.p.; mansur, herman s. title: fluorescent quantum dots-zika virus hybrid nanoconjugates for biolabeling, bioimaging, and tracking host-cell interactions date: - - journal: mater lett doi: . /j.matlet. . sha: doc_id: cord_uid: hgp jwbl the earliest possible diagnosis and understanding of the infection mechanisms play a crucial role in the outcome of fighting viral diseases. thus, we designed and developed for the first time, novel bioconjugates made of ag-in-s@zns (zais) fluorescent quantum dots coupled with zika virus via covalent amide bond with carboxymethylcellulose (cmc) biopolymer for labeling and bioimaging the virus-host cell interactions mechanisms through confocal laser scanning microscopy. this work offers relevant insights regarding the profile of the zika virus-nanoparticle conjugates interactions with vero cells, which can be applied as a nanoplatform to elucidate the infection mechanisms caused by this viral disease. despite advances in all areas of knowledge, including health and natural sciences and engineering, the current pandemic outbreak of coronavirus (sars-cov- /covid- ) poses one of the greatest challenges in the history of humankind. besides the enormous impact on public health, this outbreak has caused humongous losses to the global economy [ ] . similarly, but on a smaller scale, the zika virus (zikv) epidemic in brazil also had a great impact on public health. the zikv infection rapidly spread across brazil and to more than other american countries. although typical zikv infections are associated with an acute exanthematous disease, it is also associated with microcephaly and fetal abnormalities during pregnancy [ , ] . thus, it is a general consensus that the earliest possible diagnosis of the virus infection amalgamated with the understanding of the virus-host cell interactions is crucial for hampering viral dissemination and the patient outcome. in this scenario, emerging nanotechnologies combined with biology and medicine (termed as nanomedicine) can offer an arsenal of weapons for battling against viral diseases. a new class of hybrid nanostructures comprising inorganic nanomaterials and macromolecules have been developed associated with biomolecules, drugs, and virus for targeted applications in nanomedicine [ , ] . these integrated nanosystems encompass the best characteristics of each component for performing designed multiple functions in biomedical applications, which could not be achieved separately. therefore, fluorescent inorganic semiconductor named as quantum dots (qds) with unique electronic and optical properties have been used in nanomedicine. qds are versatile nanomaterials because their photoluminescence emission band can be tunable from the uv to the ir regions by the proper selection of the chemical composition and size of the nanoparticles. more recently, "cd-free" qds (e.g., agins, cuins, agins/zns) produced by green aqueous colloidal routes have been preferred due to the lower toxicity and facile process aiming at biomedical and environmental applications. additionally, qds synthesized under hydrophilic conditions using biopolymer ligands (e.g., carboxymethylcellulose, chitosan) permits direct chemical conjugation with biomolecules and inactivated pathogens for biolabeling and bioimaging applications via targeting specific cell-receptor sites [ ] [ ] [ ] . herein we report a novel strategy for the synthesis and characterization of hybrid nanostructured materials made of ag-in-s/zns (zais) qds directly stabilized by carboxymethylcellulose as biocompatible polymer ligand and covalently biofunctionalized with zikv virus. they demonstrated to be effective for biolabeling and bioimaging virus-host cell interactions in vitro. these findings deepen our understanding of complex mechanisms of infection of rna viruses and serve as a preliminary resource for developing potential rapid diagnosis and therapeutic approaches. to avoid redundancy, essential information is described in this section, and all of the materials and standard procedures are detailed at electronic supplementary material. dimethylaminopropyl]carbodiimide hydrochloride-mediated (edc) chemistry was chosen for conjugation. the carboxylate groups from cmc and amino groups from viruses (protein-based capsid of the zikv virus) formed amide bonds using edc (sulfo-nhs, nhydroxysulfosuccinimide sodium salt), as a "zero-length" crosslinker, at ph . - . , temperature < ˚c. zais qds were synthesized via an eco-friendly aqueous route using cmc as a stabilizing agent and salts of metals and sulfide precursors. next, the zikv was bioconjugated to the zais qds using edc and sulfo-nhs producing hybrid nanoconjugates (zikv_zais). zais were comprehensively characterized using ultraviolet-visible and photoluminescence spectroscopy (pl), high-resolution transmission electron microscopy (hrtem) coupled with energy-dispersive x-ray spectra (edx), x-ray diffraction (xrd), atomic force microscopy (afm), fourier transformed infrared spectroscopy (ftir), photoelectrons x-ray spectroscopy (xps), zeta potential (zp), and dynamic light scattering (dls). african green monkey kidney cell line (vero) was selected for the biological studies because it is usually employed to research the infective entry of flavivirus in cells [ ] and previous reports that showed that vero cells were susceptible to infection by zika virus [ ] . mtt ( -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide) protocols were performed to evaluate the in vitro cytotoxicity of zais. moreover, the nanoconjugates were evaluated as fluorescent bioprobes for in vitro biolabeling and bioimaging using confocal laser scanning microscopy (clsm). uv-visible spectroscopy of zais qds revealed a broad featureless absorption spectrum, with a long tail extending out into the visible region (fig. a) , which is characteristic of ag-in-s ternary systems. this is mostly associated with the size distribution of nanocrystals and the presence of sub-bandgap transitions arising from the intrinsic point defects (vacancies, interstitials, etc.) [ ] . the surface charge of zais nanocolloids was determined by zeta potential analysis (zp=- . ± . mv). the value is consistent with the predominance of negatively charged surface due to the carboxylic groups (r-coo -) of anionic cmc ligand at physiological ph (pka~ . ) [ ] , which promoted the electrostatic stabilization of the nanosystems. the hydrodynamic sizes (d h ) of the colloidal qds were evaluated by dls analysis. after synthesis, the sum of the contribution of qd inorganic core with the cmc organic shell and its interactions with the surrounding medium resulted in d h = . ± . nm. fig. a (inset) summarizes zp and d h data. tem-edx analyses were performed to access the morphological features, sizes, and elemental composition of the quaternary zais qds nanocolloids stabilized by cmc biopolymer, which demonstrated the formation of fairly monodispersed nanoparticles with a spherical-like shape (fig. b) . the lattice fringes obtained by electron diffraction patterns through hrtem images (inset, fig. b ) evidenced the nanocrystalline characteristics (regular spacing) of zais qds. the histogram of the nanoparticle size distribution (fig. d) indicated an average size of . ± . nm. edx analysis (fig. c) confirmed the presence of the chemical elements zn, ag, in, and s of zais qds in addition to elements from cmc (c, o, and na), grid (cu and c), and edx detector (si). high-resolution xps spectra (fig. s ) showed all of the elements of qds with identified oxidation states zn + , ag + , in + , and s -, proving the formation of quaternary nanoalloys (ag-in-s@zns). ftir-atr spectra of cmc polymer in comparison to zais (fig. s ) indicated changes in the intensities of bands assigned to carboxylic/carboxylate groups due to the formation of the m n+ -coo − complexes as monodentate and bidentate chelates, as well as changes in oh groups/hydrogen bonds associated with coordination with metal ions/stabilization of qds. typical d afm image presented spherical nanoparticles embedded in the "dry" polymer matrix with the zais conjugate size of approximately . nm. xrd pattern of zais exhibited three broad peaks, located in between the diffraction reflections of the agins and zns crystals, which are an indication of the formation of a quaternary alloy [ ] . the average size of the nanocrystal was nm, calculated using scherrer's equation. the dimension of nanoconjugate estimated by afm was relatively higher than that measured from tem because it corresponds to the sum of the inorganic core and polymer shell. conversely, the nanocrystal size (xrd) was smaller than that of the nanoparticle inorganic core (tem), in agreement with the literature [ ] . to evaluate the potential use of zais conjugates as fluorescent biological nanoprobes, d excitation-emission contour curves, quantum yield (qy), and lifetime decay curve were obtained. the d plots ( fig. a) indicated that zais qds behaved as active fluorophores suitable for bioimaging (e.g., cell-virus interactions) with a broad range of excitation wavelengths and emission in the green-red visible window, which are mostly ascribed to intrinsic crystallographic and surface defects [ ] . zais showed qy of %, which has already proven suitable as fluorescent nanoprobes for bioimaging applications [ , ] . the relaxation profile (fig. s ) followed a bi-exponential decay with an average pl lifetime ( av ) of ± ns. the longer lifetimes of qds compared to organic fluorophores favor the tracking of biological processes (continuous, long-term, and enhanced sensitivity) [ , ] . the cytocompatibility in vitro of zais qds for biological applications was assessed using mtt protocol, where the cell viability responses (≥ %) towards vero cells after h of incubation are presented in fig. b . these results proved that the qds nanoconjugates were cytocompatible for biomedical applications. therefore, zais nanoconjugates were tested as fluorescent nanoprobes for vero cells through cell internalization evaluated by clsm, where the results of cellular uptake and mean fluorescence intensity (mfi) are shown in fig. c and d, respectively. the zais nanoconjugates presented a green emission coherent in pl spectroscopy experiments. the results demonstrated the internalization of zais nanoconjugates just after min of incubation, with a significant enhancement of the emission intensities with time, which is vital as they attested their activity as fluorescent bioprobes for tracking cellular events. for biolabeling and bioimaging virus-host cell interactions in vitro, zikv_zais conjugates were incubated with vero cells. fig. a indicates the green fluorescence of zikv_zais in the vero cell line localized predominantly at the cytoplasm. the fluorescence images presented a relative gradual increase of emission with the time of incubation ( to min), which were used to estimate the kinetics profile of interactions of zikv_zais bioconjugates with vero cells using "mean fluorescence intensity" (fig. c) . vero cells treated with "unbound" zikv and zais qds were used as controls (images, fig. b , mfi values, fig. d ) showing less fluorescence than zikv_zais after - min of adsorption. the difference (higher than three times) was associated with the presence of receptors to zikv in vero cells that favored the endocytosis of the nanoparticle-virus conjugates, which can provide a powerful tool for biolabeling and detection of zikv virus as well as tracking virus behavior in situ. zais qds were produced with suitable physicochemical and optical properties to be conjugated with zikv and applied as a cytocompatible biomarker. the qds conjugated with zikv presented higher entry to vero cells in comparison to reference qd (zais), indicating the possibility of labeling and detection of zikv for tracking the viral infection processes including virus entry and transport in cells. new functional quantum dot/polysaccharide/zika-virus nanoconjugates were produced fluorescent ag-in-s/zns-cmc nanocolloids produced via facile green aqueous process -s/zns-cmc nanocolloids biofunctionalized by zika-virus targeted vero cells zais-cmc bioconjugates acted as fluorescent nanoprobes for cell imaging and tracking zais-cmc fluorescent conjugates showed insights of zika virus-host cell interactions this work was supported by the brazilian agencies capes, fapemig, cnpq (process number / - ), finep, decit/ministério da saúde do brasil. the authors thank cenano i/cemucasi/ufmg and the center of microscopy/ufmg for the characterization facilities. no conflict of interest to declare. key: cord- - thdg up authors: payne, kelly; kenny, peter; scovell, jason m.; khodamoradi, kajal; ramasamy, ranjith title: twenty-first century viral pandemics: a literature review of sexual transmission and fertility implications in men date: - - journal: sex med rev doi: . /j.sxmr. . . sha: doc_id: cord_uid: thdg up introduction: the st century has seen a series of viral pandemics that have collectively infected millions of individuals. to understand factors that may contribute to viral spread and address long-term health sequelae for survivors, it is important to review evidence regarding viral presence in semen, sexual transmission potential, and possible effects on fertility. aim: to review the current literature regarding the sexual transmissibility of recent viral pandemics and their effects on semen parameters and fertility. we review evidence for the following viruses: ebola, zika, west nile, pandemic influenza, severe acute respiratory syndrome (sars), and sars-corona virus- (sars-cov- ). methods: a literature search was conducted to identify relevant studies. titles and abstracts were reviewed for relevance. references from identified articles were searched and included, if appropriate. main outcome measures: the main outcome measure of this study was reviewing of peer-reviewed literature. results: both the ebola virus and zika virus are present in semen, but only the zika virus shows consistent evidence of sexual transmission. current evidence does not support the presence of the west nile virus, pandemic influenza, sars, and sars-cov- in semen. the zika virus appears to alter semen parameters in a way that diminishes fertility, but the effect is likely time limited. the west nile virus and sars have been associated with orchitis in a small number of case reports. viruses that cause febrile illness, such as pandemic influenza, sars, and sars-cov- , are associated with decreased sperm count and motility and abnormal morphology. sars and sars-cov- may interact with angiotensin-converting enzyme receptors present in the testes, which could impact spermatogenesis. conclusions: we have reported the presence in semen, sexual transmission potential, and fertility side effects of recent viral pandemics. overall, semen studies and fertility effects are highly understudied in viral pandemics, and rigorous study on these topics should be undertaken as novel pandemics emerge. payne k, kenny p, scovell jm, et al. twenty-first century viral pandemics: a literature review of sexual transmission and fertility implications for men. sex med rev ;xx:xxx–xxx. the st century has seen a series of viral pandemics that have affected the health of millions of individuals and touched every corner of the globe. as one potential means of controlling viral spread, it is necessary to understand what viruses are present in semen, how long they remain detectable, and what their potential is for sexual transmission from male to female partners. in addition, with so many survivors of viral pandemics alive today, knowledge is needed about how these viruses affect the male genital tract and what implications they may have for future fertility. despite the importance of these questions, to our knowledge, these topics have not yet been the subject of systematic review. here, we detail several important foundational principles that affect viral infection in the testes and comment on the differences in immune response seen between men and women. then, we present the state of current research regarding presence in semen, sexual transmission, and fertility effects for the zika virus (zikv), ebola virus (ebov), west nile virus (wnv), pandemic influenza, severe acute respiratory syndrome (sars), and sars-coronavirus- (sars-cov- ) ( table ) . a literature search of pubmed, embase, cochrane, ovid, and clinicaltrials.gov was conducted by independent authors, k.p. and p.k. the literature search was limited to english publications or publications translated into english, and databases were searched from dates of their inception to june . articles with only abstracts available were included. to review the immune status of the testes, searches were conducted using the following keywords: testes, immune privilege, and blood-testes barrier. to review the difference in immune response between men and women, searches were conducted with the following keywords: gender difference in immune response and sex difference in immune response. searches were also conducted for each of the recent viral pandemics with the following key words separated by "or": genitalia, testis, orchitis, prostate, reproductive, semen, sperm, hormones, endocrine, luteinizing hormone, folliclestimulating hormone, gonadotropins, testosterone, fertility, fecundity, sexual transmission, erectile function, erectile dysfunction, sexual function, sexual dysfunction, and libido. case studies, case series, reviews, and meta-analyses were included. both human, animal, and basic science data were included. for each viral pandemic, articles were selected if they met any of the following inclusion criteria: discussion of semen parameters, identification of virus location within the genital tract, discussion of sexual transmission, or discussion of fertility related processes including spermatogenesis, libido, and sexual function. article titles and abstracts were reviewed by k.p. and p.k. to identify relevant studies. full-text articles were then reviewed to determine if they met the stated selection criteria, yielding total articles ( figure ). in any consideration of viral infection of testicular tissue and seminal fluid, it is important to review the function of the bloodtestis barrier (btb). the btb provides protection from autoimmune cell destruction, making the testes an immuneprivileged site. evolving understanding of the btb indicates that it comprises both a physical component and an immune regulatory component. the physical component of the btb comprises a layer of sertoli cells connected by tight junctions, separating undifferentiated spermatogonia from differentiating germ cells in the form of spermatocytes, spermatids, and spermatozoa. local immunosuppression is carried out by constitutive expression of anti-inflammatory factors by testicular cells, most prominently transforming growth factor beta. , testosterone plays an additional role in establishing immune privilege by regulating the numbers of testicular macrophages and lymphocytes and downregulating their expression of proinflammatory transcription factors, cytokines, and adhesion molecules. an in vitro study that treated macrophages with testosterone demonstrated decreased expression of toll-like receptor and reduced sensitivity of toll-like receptor to lipopolysaccharide, a key antigenic molecule for identifying foreign pathogens. furthermore, a study of rats pretreated with estrogen to suppress testosterone production showed a much more rapid rejection of an intratesticular allograft than untreated controls, underscoring testosterone's immune-modulating effects. despite its immune-privileged nature, the testicular environment is still capable of mounting an effective inflammatory response. in fact, the testes are of equal susceptibility to infection as any other tissue type. given the suppression of adaptive immune mechanisms, it is simply more reliant on the innate immune mechanisms of macrophages, natural killer cells, and antimicrobial secretory products such as interferons and defensins. , , nonetheless, there is evidence that the testes can act as a reservoir for pathogens that are no longer detectable systemically. jenabian et al have found that in the testicles of men with hiv, hiv viral rna has remained detectable in the testes even when levels were undetectable in the blood secondary to antiviral therapy (although this is not known to cause increased transmission risk). this becomes especially important in the study of viruses such as the ebola and zika, where asymptomatic individuals may still be capable of sexual transmission of the virus. sex-based differences in immune response present an additional consideration for viruses that affect the reproductive tracts. increasing evidence suggests that the intensity, severity, and mortality of viral disease differs between genders. literature examining this link has generally observed that women mount a stronger immune response to antigens, infections, and vaccines than do men. this effect is most widely observed in the cytokine response to antigens. on inoculation with viruses or direct stimulation of immune toll-like receptors , , , and , men exhibit lower levels of proinflammatory interferon alfa and higher amounts of anti-inflammatory interleukin . helper t cells in men also demonstrate lower cluster of differentiation (cd ), cd , and cd :cd ratio than those in women. the x chromosome contains many important genes for immune function, including tlr , cd ligand (cd l), and forkhead box p (foxp ). notably, it also contains the most micrornas of all chromosomes, which regulate protein-coding genes and modulate cellular activities. the y chromosome, by contrast, uniquely contains none. and, although x inactivation equalizes gene expression for most of the x chromosome, genes within non-recombining regions may be expressed more in women. in addition, other immune-regulatory genes present on both the x and y chromosomes have unequal expression levels, and tissue distribution between the sexes is perhaps due to sex hormones. , the first randomized controlled trial to separate the effect of sex hormones and chromosomes was conducted by palaszynski et al in , using mice in which the sex-determining region y (sry) gene was moved from the y chromosome to an autosome. this allowed separation of sex chromosome complement (xx or xy) from gonadal type (ovaries or testes). the authors reported that among ovariectomized (-sry) mice, those with xy had greater immune response than those with xx, suggesting that the male complement was immune stimulatory. this effect was not detected when comparing intact males (þsry) with xx and xy complements, suggesting that testosterone tempers the effect of the xy complement. supporting this, when testosterone was added to the ovariectomized female (-sry) xy mice, their elevated immune response was suppressed. in , robinson et al similarly found that the increased immune response of female mice to influenza a was eliminated by removal of the gonads. although they did not find any immune response to testosterone or dehydroepiandrosterone in male mice, administration of high-dose estradiol attenuated the induction of tumor necrosis factor alpha and c-c motif chemokine ligand by times and resulted in increased rates of survival compared with no or low-dose estradiol. building on this, administration of b-estradiol to female mice resulted in less secretion of tumor necrosis factor alpha and interferon gamma as well as decreased neutrophil recruitment, improving disease tolerance. taken as a whole, current evidence suggests women exhibit stronger immune response to viral infections than men. this response typically manifests as better long-term outcomes and to subsequent reexposure but also as more severe disease acutely. the size of the effect can be substantial; in the h n pandemic, infected women suffered more than -fold risk of death than men. , further exploration of the mechanisms behind the sexbased differences may allow for modulation of the immune response with hormone treatments. the zikv was first recognized in in a sentinel monkey from zika forest, uganda. the first suspected human case followed in , but the virus did not demonstrate mosquitoborne transmission until . in , the zikv emerged in brazil during preparation for its upcoming international sporting events, creating a large epidemic through the americas. it entered the continental united states in with cases in florida and texas, peaking in . no new cases have been reported since . however, similarities to other flaviviruses such as dengue fever have made the virus difficult to recognize clinically, with several epidemics first noted after rises in associated conditions such as congenital microcephaly and guillain-barre syndrome. the zikv in semen the zikv was first suspected to be in semen following case reports of travel-associated transmission to sexual partners in non-endemic areas. since then, multiple studies have confirmed the presence of zikv rna in semen by reverse transcription polymerase chain reaction (rt-pcr). e a prospective study by mead et al found that semen was positive with zikv in % of men, including % of those tested within days of symptom onset. infectious zikv was detected in % of rna-positive semen samples collected within days of symptom onset, but % of samples obtained after days of disease onset were infectious. evidence from joguet et al although the reported persistence of the zikv varies widely from days to months after the onset of symptoms, it is widely agreed that viral rna persists longer in semen than in other bodily fluids. this reflects a potential reservoir in the male genital tract, perhaps because of the immune privilege of the testes. supporting this, a systemic analysis by counotte et al reported a median duration of zikv rna in semen of days with a maximum of days, vs days in saliva and days in the female genital tract. the persistence of zikv rna in men with vasectomies further indicates that the virus may also replicate in distal genital tract tissues, such as the bulbourethral glands, prostate, and seminal vesicles. several factors are noted to influence persistence of the viral rna in semen. in the analysis from counotte et al, increased persistence was associated with increased age, absence of joint pain, conjunctivitis, and less frequent ejaculation. conversely, men who ejaculated more than times a week cleared viral rna more than days earlier, and for men who reported vasectomies, although the rate of viral rna detection was similar, the viral load was decreased from . to . log rna copies per milliliter. the first suspected case of sexual transmission of the zikv occurred in , when a male patient returned home to the united states from senegal. he developed symptoms of arthralgia, rash, and hematospermia shortly after returning, followed by his wife days later. serologic results confirmed zika infection, although only convalescent phase samples were positive in the wife, and semen samples were not collected. although it is possible that this transmission occurred via other bodily fluids such as saliva, no other family members were affected. after the subsequent zika outbreaks, in a systematic review moreira et al compiled demonstrated cases of sexual transmission from men to women, women to men, and men to men. among the cases examined, sexual transmission was confirmed with positive serum antibodies, zika rna in semen, and semen cultures. in addition, they demonstrated that zikv rna was detected in semen as late as days and infectious virus in semen up to days after symptom onset. notably, transmissions were from entirely asymptomatic patients, who represent the majority of zika cases. further complicating the control of zika transmission, a prospective cohort study by paz-bailey et al demonstrated that the virus exhibits intermittent shedding, making it difficult to determine when an individual has cleared the virus. the number of days between symptom onset and positive samples was up to in serum, in urine, and in semen. taken together, these data represent potential challenges in preventing sexual transmission of the virus, which is of particular concern in nonendemic areas such as the continental united states where mosquito-borne transmission is rare. although mathematical modeling suggests that sexual transmission alone is not likely to drive or sustain a zikv outbreak, serial semen screenings may be effective in preventing the zika-associated congenital microcephaly and guillain-barre syndrome in patients who are or wish to become pregnant. several case reports have described genitourinary symptoms in male zika cases, although their impact on the male reproductive system is as of yet unclear. in a cohort study, paz-bailey et al noted evidence of hematospermia ( . %), painful ejaculation ( . %), and penile discharge ( . %) in patients with the zikv, suggestive of local inflammation and tissue damage. although fever cannot be excluded as a cause, mouse models suggest direct tissue damage may be involved. e examining this effect, huits et al microscopically analyzed semen samples from patients with symptoms of the zikv in cohort studies, demonstrating macrohematospermia and microhematospermia in of patients tested and transient oligospermia in of patients. building on these results, joguet et al monitored the reproductive hormones as well as sperm count, motility, viability, and morphology of patients with the zikv on days , , , , , , and after symptom onset. they detected that total and motile sperm counts were about % lower on day compared with day but recovered by day . the long-term effect on sperm morphology is less clear. in the same study, morphology characteristics recovered to normal by day . results also reflected an early increase of follicle-stimulating hormone and inhibin b, which recovered over time. by contrast, in a cross-sectional study after the epidemic in brazil, avelino-silva et al examined serum hormones and semen samples in patients year after symptoms. in all patients, serum hormones were normal, and semen rt-pcr rna was negative, but impaired motility was seen in all samples tested and low count was noted in of patients. given the recency of the zika outbreaks, the long-term effects of the virus on fertility characteristics should continue to be monitored. history of the ebov ebola virus disease (evd) first emerged in with simultaneous outbreaks in zaire (in a village near the ebola river) and in south sudan, where it is thought to have originated in african fruit bats. recurrent outbreaks took place in in cote d'ivoire and in in kikwit, zaire. most recently, e saw a west african outbreak of unprecedented scale in sierra leone, liberia, and guinea with greater than , cases and , deaths. the large number of evd survivors means that understanding its persistence in semen, its potential for sexual transmission, and its fertility implications is a matter of considerable importance for the control of viral spread. across multiple studies, it has been shown that the crosssectional percentage of ebov survivors of the e west african epidemic with a positive rt-pcr for ebov rna in the semen ranges from . to . %. e with more living ebov survivors from this epidemic than ever before, this number represents a considerable pool of individuals. while disease modeling has shown that % of individuals will clear the virus from semen at days and % will clear it at days, viral ebov rna has been detected in the semen for as long as days after initial infection. , it has also been cultured in the semen up to days after initial infection. as expected, the percentage of individuals with a positive semen rt-pcr for ebov rna has been demonstrated to decrease in a predictable manner over time. deen et al showed in a cohort of male adult survivors of evd in sierra leone that at e months, % of individuals had positive rt-pcr results in semen; at e months, % had positive results; and at e months, % had positive results. these results were echoed by keita et al in a study including evd survivors in guinea where . % of individuals had positive rt-pcr in semen at months, . % at months, . % at months, . % at months, . % at months, and . % at months. interestingly, limited data indicate that older men may be more likely to have a longer length of time in which semen is positive for ebov rna. soka et al showed that men older than years of age were more likely to have a positive semen test than men younger than years of age. animal models have been used to further characterize the presence of filoviruses in the gonadal tissue and have shown viral infiltration of the testicular interstitium and the seminiferous tubules. in situ hybridization (ish) and immunohistochemistry performed on non-human primates (nhps) infected with the marburg virus localized the virus to the interstitium between the seminiferous tubules. in nhps infected with ebov, perry et al similarly showed viral replication in the interstitial tissues of the reproductive tract, including the seminal vesicles, epididymis, prostate, and testis. perry et al also identified the marburg virus in the seminiferous tubules of nhps. indeed, the virus was found to be most prominently localized to sertoli cells, leading to breakdown of the btb and inflammatory cell invasion into the testicular environment. another study in nhps found ebov rna to be localized to the tubular lumen of the epididymis of of twelve rhesus monkey sampled but did not find any ebov localized to the testis. in the lumen of the epididymis, macrophages were found to be the ebov reservoir. overall, it appears that viral persistence may be established in the interstitial tissues of the reproductive tract, where it is transmitted by tissue macrophages across the btb and into the seminal fluid. although the ebov in the semen is clearly demonstrated, it remains far less certain what potential the virus has for sexual transmission. since the first known outbreak of a filovirus (the marburg virus), there have been documented events of suspected human-to-human filovirus sexual transmission. the first case of suspected ebov transmission occurred during the kikwit outbreak, wherein in a -year-old woman was shown to have weakly positive igm antibodies to igm days after exposure from her convalescent partner. the individual was later negative for igg antibodies on a serum sample, making it difficult to confirm whether she was in fact asymptomatically infected. in the west african outbreak, the first confirmed sexual transmission occurred in march . a man transmitted the virus to his sexual partner days after onset of evd, which was confirmed by whole viral genome analysis of his semen compared with an acute blood sample of his female partner where the samples were found to differ by only nucleotide. in october , a suspected sexual transmission event occurred in conakry, guinea. a man presenting with evd was found to have an ebov genome that did not match the country's current strain of transmission but instead differed by only nucleotide submissions from a sample of his brother-in-law's blood collected during prior acute infection. the man's sister was positive for ebov igm at the time of his infection; however, the transmission could not be confirmed as her husband's semen was negative for the ebov at the time a sample was collected. it was thought that the transmission from the male survivor occurred at days after disease onset. the longest recorded convalescent period before sexual transmission occurred days after disease in n'zerekore, guinea in march . the acute blood sample and semen sample from the male survivor differed by mutations from a female sexual partner with acute evd and subsequent cases from the disease cluster. in total, the sexual transmission resulted in a cluster of cases with deaths. sexual transmission from the ebov, while possible, remains a rare event. the world health organization in their guide to clinical care for survivors of evd recommends abstinence or barrier protection during sexual activity for months after the onset of evd. the ebov and fertility given the extremely high mortality associated with evd and the limited number of laboratories with the capability to study such a pathogenic virus, there is much that remains unanswered about the potential reproductive sequelae of evd. in rhesus monkeys, it has been shown that although marburg infection had a deleterious effect on sertoli cells, the overall reproductive function of the sertoli cells was unaffected. spermatogenesis was unaffected, and tissue morphology was normal. further studies are needed to determine if semen parameters or reproductive capacity are altered in male survivors of evd. sexual health complaints are commonly reported among evd survivors. a health clinic for the management of evd survivors in sierra leone reported findings from a group of patients from to . of men, . % reported erectile dysfunction and . % reported loss of sexual desire. an evd survivors' clinic in liberia reported on evd survivors from to . in this cohort, which included men, % reported erectile dysfunction, whereas % reported decreased libido. the causal mechanism for these complaints remains to be determined. although physiologic conditions can play a role, it is also likely that psychosocial factors including residual stress, trauma, stigma, and grief contribute as well. the wnv was first recognized in north america in new york city during the summer of . the virus was previously known to cause small human outbreaks in africa and the middle east but has now established itself as a major worldwide seasonal pandemic. between and , there have been , documented cases of the wnv in the united states, with particularly high-intensity seasonal outbreaks in and . the most concerning sequelae of the wnv is neuroinvasive disease, which occurs in less than % of infected individuals but carries a % fatality rate. there is very little evidence to suggest that wnv is present in semen. while wnv infection in blood, cerebrospinal fluid, and urine has been well studied, to date, there is only study that describes the evaluation of the wnv in semen. gorchakov et al the wnv has been further studied in a cultured line of human sertoli cells. when the wnv was introduced to these cells, it was noted that the sertoli cells supported a robust wnv infection with titers comparable with that of the zikv, a pathogen known to have high infectivity in semen. in a small case series presenting postmortem autopsy results for individuals deceased from neuroinvasive wnv, the wnv was identified by immunofluorescence in the testes and prostate in of men examined. this individual was years of age and on chronic immunosuppression secondary to a kidney transplant, raising the possibility that immunosuppression could contribute to wider systemic dissemination of the disease. owing to the limited nature of currently available evidence, further studies are needed to assess larger sample size populations before any definitive conclusions can be drawn about the presence of the wnv in semen. nevertheless, the studies presented suggest that the wnv in semen is a possibility that bears further investigation. analysis of semen during the period of acute illness is also needed to assess semen infectivity at that time. given the poorly established evidence that the wnv is even present in semen, it follows that the possibility of sexual transmission is even less well characterized. there is only a single case report of male-to-female possible sexual transmission of wnv when a previously healthy middle-aged woman developed viral meningoencephalitis weeks after unprotected intercourse. her husband had serologically confirmed wnv. although no mosquito bite was reported, the women lived in a mosquito endemic area. interestingly, the food and drug administration recommends that all donor semen for assistive reproductive technology be tested for the wnv using a nucleic acid test in any donation made between june and october each year, the season when the virus is most active. further work is needed to determine if this type of testing should be made more routinely available to men with confirmed wnv desiring to father a pregnancy. the only available report of a possible compromise in fertility from the wnv comes from a case report of an autopsy on a year-old man deceased from the wnv. in this case, thickening of the tubular basement membrane was observed with frequent foci of tubular necrosis and absence of spermatogenesis. immunohistochemical staining did not identify wnv antigen in testicular tissue; however, transmission electron microscopy (tem) of formalin-fixed testicular tissue did reveal enveloped particles fitting the size, structure, and location of flavivirus particles, leading the authors to speculate that this was likely wnv causing orchitis. no studies could be identified reporting on semen parameters during or after acute wnv infection, and no studies could be identified detailing possible changes in sexual function. more evidence is required to substantiate a link between the wnv and reduced fertility. the influenza virus is among the most common infectious illnesses worldwide. the virus came into prominence among humans and pigs in the pandemic, shortly after mutations of avian strains enabled transferal to humans and pigs. amplified by the movement and proximity of world war i soldiers, the virus soon infected million people and killed million, more than the military and civilian deaths of world war i combined. since then, type a influenza has produced pandemic outbreaks in , , and most recently in . these events are triggered by reassortment in avian or swine reservoirs of genes for key hemagglutinin (h) and neuraminidase (n) surface glycoproteins, by which the virus is subtyped. the h n and h n strains of type a influenza currently circulate among humans, which along with influenza b cause seasonal flu (who ). notably, in the h n pandemic, young people were particularly at risk, whereas of older than the age of years had antibodies, likely from a historical infection or vaccination with a similar strain. the presence of influenza in semen has not been reported. however, the virus demonstrates extrapulmonary symptoms in many major organ systems, a large number of which have since been found contain to functional influenza receptors. , no studies to date have explored if those receptors are present in the human genital tract, but such tropism has been noted in turkeys. , given the variability of viral tropism among animals, further work is need to explore whether such tropism exists among humans or is reflected by the presence of viral particles and rna in semen. there is a similar dearth of information on sexual transmission of influenza, possibly because of other, more likely routes of infection. evidence suggests that while infection by direct contact is possible, it is not likely to be a significant mechanism compared with transmission via aerosol and respiratory droplets. these airborne routes are both particularly infectious within close contact and should deter high-risk populations from sex during the duration of viral shedding. as a pyrogenic virus, influenza affects sperm quality. macleod noted that medical students who presented with febrile illness had decreased sperm count, motility, and morphology parameters. morphology and motility recovered at weeks, as did sperm count at weeks. buch and havlovec reported a case of a semen donor who suffered a -hour febrile viral illness, who then had decreased sperm count and reduced egg penetration ability and weeks later. other studies have found similar effects from non-febrile heating such as saunas or laptops. , a case study by sergerie et al first detailed the timeline of genital tract effects following febrile influenza. after days of high fever due to the virus ( e c), a -year-old man had decreased total sperm count at days , , and before normalizing at day . motility percentile was similarly decreased but recovered sooner at day . the authors measured the dna fragmentation index and found it to increase from % before the fever to %, %, % and % at , , , and days, respectively. sperm dna fragmentation by terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay increased from % before fever to % at day , but this was not statistically significant (p < . ). evenson and jost found that underlying dna changes included decreased free sh groups and alteration of nuclear protein composition, causing latent effects on sperm chromatin structure and stability. murine models suggest that the viral particulates are also damaging. sharma et al first intraperitoneally inoculated mice with influenza a and recorded a significant increase in numerical and structural alteration of meiotic chromosomes, particularly aneuploidy. subsequent mouse studies noted similar dna damage was induced by both inactivated and purified influenza indicating a possible direct cytotoxicity by the viral particles. therefore, while evidence argues against a local inflammatory response, , the transient sperm changes observed are likely due to a combination of systemic fever and direct dna damage resulting in apoptosis and a transient decrease in fertility. history sars is respiratory disease caused by a novel coronavirus that first appeared in the guangdong province of southern china in . the virus rapidly became an epidemic and spread globally to countries and , people before being contained in . subsequent research uncovered a natural reservoir within horseshoe bats and civets acting as an intermediate host in local meat markets. although no cases have been reported since , the presence of this animal reservoir, the high reported case-fatality rate ( %), and a single reappearance of the virus have driven continued monitoring of the virus near its site of origin. sars in semen investigation into the effect of novel coronaviruses on fertility began after the discovery that angiotensinconverting enzyme (ace- ), which is expressed in the testis, acts as a functional receptor for the sars virus. , zhao et al first reported detection of the virus within testicular epithelial cells and leydig cells through combined ish and tem. however, no other research teams have been able to mirror these results. e ding et al examined all tissues that express ace- in autopsies of patients with sars and were surprised to find that the testes were uniformly negative for the sars rna and signature n protein despite high levels of ace- expression in the testes. similarly, gu et al noted focal testicular atrophy in all autopsies with confirmed sars but found no evidence of sars in parenchymal tissue by ish and tem. therefore, although semen has not been directly examined for sars, indirect evidence suggests that it is unlikely to be a reservoir. similar to other respiratory viruses, sars spreads most readily through respiratory droplets, fomites, and aerosol. to date, no sexual transmissions of sars have been reported. given that most studies do not find sars in genital tissues, any transmission of sars after intercourse is more likely due to direct contact and close proximity rather than through genital secretions. a mechanism for genital injury from sars was furnished in by xu et al. they analyzed the pathologic changes in testicular autopsy specimens from patients with sars compared with those of controls. all patients with sars demonstrated testicular atrophy grossly. the sars samples exhibited extensive germ cell destruction with increased apoptosis on tunel assay. histologically, basement membrane thickening, peritubular fibrosis, leukocytes, and vascular congestion indicative of local inflammation were noted. ish that produced positive readings in the lungs of the patients with sars was negative in the testes. however, extensive deposits of igg were detected within the seminiferous epithelium, interstitium, sertoli cells, and some germ cells of patients with sars compared with controls. this potentially represents an autoimmune orchitis secondary to the immune response to sars. further research is needed to explore this possibility and the persistence of orchitis. in addition, data are needed to ascertain whether the germ cell destruction in sars-associated orchitis is reflected in semen characteristics. history sars-cov- is a novel coronavirus known to cause the disease "coronavirus disease- " or "covid- ". it emerged in late in wuhan, hubei province, china, and likely has its origin in bat populations. on march , , the world health organization declared covid- to be a worldwide pandemic. it has been estimated to cause severe disease in % of cases. older individuals are particularly susceptible to severe illness, with % of deaths occurring in individuals aged years and older. given the recent and novel nature of the covid- outbreak, data remain limited. thus far, there are mixed findings regarding the presence of sars-cov- in semen. pan et al have reported a case series of adult chinese men diagnosed with covid- who showed no sars-cov- in semen at a median follow-up of days. unpublished data from chinese patients in recovery from covid- showed no sars-cov- in rt-pcr of semen. testicular biopsy from deceased individual in this study likewise was negative for sars-cov- viral rna. in addition, paoli et al showed negative rt-pcr in both urine and semen samples of an individual who tested positive for sars-cov- by nasopharyngeal swab. in contrast, li et al reported that of ( . %) patients in a cohort of patients in shangqiu, china, had virus detected in their semen by rt-pcr. of them, were in the acute stage of infection, and were recovering from the virus. in providing analysis and commentary on these findings, paoli et al nevertheless recommended that the small caseload and recency of infection in patients should give readers caution before drawing sharp, fatalistic conclusions. further studies will be essential to confirm these findings. although sars-cov- is not known to be present in semen, precluding the ability for sexual transmission, the intimate nature of physical sexual contact still dictates caution to prevent viral spread. given the presence of sars-cov- in respiratory droplets, saliva, mucus, and fecal matter, as well as current recommendations to maintain feet of distance between individuals to prevent the spread of infection, physical sexual intimacy presents a high-risk scenario for viral transmission, particularly for non-monogamous partners who do not live with one another. , in the chinese case series reported by pan et al, individuals ( % of the cohort) described scrotal discomfort around the time of onset of viral illness, suggesting sars-cov- may induce a viral orchitis. studies on semen quality from covid- survivors have not yet been reported. chen et al have proposed a theoretical link between covid- and reduced fertility based on the virus' strong interaction with angiotensin-converting enzyme (ace ). molecular modeling conducted by the authors showed the virus have a unique structure that allows it to penetrate deep into the hydrophobic pocket of ace . ace is highly expressed in the renal tubular cells, the intestines, leydig cells, and the seminiferous ducts in the testes, raising the possibility that the virus may have a deleterious testicular effect. by contrast, pan et al demonstrate with single-cell transcriptome data that ace rna is sparsely expressed in the testis and argue that this is an unlikely medium of viral entry into target host cells. even so, it is still unknown exactly what function ace serves in the testis. corona et al analyzed these studies and recommended that those of reproductive age consider andrological consultation and evaluation before safely pursuing reproduction. larger studies will be imperative to understand currently conflicting evidence and to deduce what potential fertility sars-cov- may have. in this article, we have reviewed the presence in semen, possibility of sexual transmission, and fertility implications of each of the major recent viral pandemics: zika, ebola, west nile, pandemic influenza, sars, and sars-cov- . the zikv has been reported in semen up to days after disease onset but appears to be present for a median time of days for most individuals. sexual transmission of the zikv has been repeatedly documented, even among entirely asymptomatic individuals, and is of particular concern given its ability to microcephaly in a developing fetus. short-term fertility may be negatively affected by the zikv based on several reports of reduced sperm count, altered morphology, and impaired mobility in semen samples. fortunately, this effect appears time limited. the ebov has also been detected in semen, with studies indicating its presence for an average of days, with a maximum reported duration of days. reports of sexual transmission remain rare, with only suspected cases documented in the history of the virus. semen parameters do not appear altered, although a proportion of survivors decreased sexual function and diminished libido. the wnv has only been reported in semen or to be transmitted sexually in isolated case reports. likewise, only a single autopsy report suggests the possibility of wnv-induced orchitis. influenza, a respiratory pathogen, has not been found in semen or been shown to be sexually transmissible. research demonstrating its impact on fertility has focused on the effects of febrile illness on semen parameters, showing that febrile episodes are linked to transiently decreased sperm count, motility, and morphology. the sars virus has not been shown to be present in semen, but several small case series of autopsy reports have found testicular damage and atrophy in individuals deceased from sars, which may be linked to secondary autoimmune orchitis. sars-cov- , the most recent viral pandemic included in this review, is expected to behave similar to sars virus, but further data are required to validate these assumptions. current evidence from small case series shows that it is not present in semen and thus is unlikely to be sexually transmitted. for both sars and sars-cov- , there is speculation that the viruses' interaction with ace , which is present in the leydig cells and the seminiferous ducts of the testes, could have implications for spermatogenesis. further studies are needed to explore this possibility. we have reported the presence in semen, sexual transmission potential, and fertility side effects of recent viral pandemics. overall, semen studies and fertility effects are highly understudied in viral pandemics, and rigorous study on these topics should be undertaken as novel pandemics emerge. specifically, further semen studies and fertility studies are needed to understand the transmission potential and fertility side effects of the novel sars-cov- . disease control priorities: improving health and reducing poverty expression of interferons-alpha and -gamma in testicular interstitial tissue and spermatogonia of the rat from ancient to emerging infections: the odyssey of viruses in the male genital tract immunoprivileged sites: the testis role of transforming growth factor beta in testicular immunosuppression adeno-associated virus-mediated human il- gene transfer suppresses the development of experimental autoimmune orchitis testosterone reduces macrophage expression in the mouse of toll-like receptor , a trigger for inflammation and innate immunity immune privilege in the testis. ii. evaluation of potential local factors immunological, paracrine and endocrine aspects of testicular immune privilege interferons and interferoninduced antiviral proteins in the testis expression of antimicrobial defensins in the male reproductive tract of rats, mice, and humans puzzling over privilege: how the immune system protects-and fails-the testes immune tolerance properties of the testicular tissue as a viral sanctuary site in art-treated hiv-infected adults sex drives dimorphic immune responses to viral infections sex differences in the response to viral infections: tlr and tlr ligand stimulation induce higher il production in males the x chromosome in immune functions: when a chromosome makes the difference x-chromosome-located micrornas in immunity: might they explain male/female differences? the x chromosome-genomic context may affect x-located mirnas and downstream signaling, thereby contributing to the enhanced immune response of females the azfa gene dby (ddx y) is widely transcribed but the protein is limited to the male germ cells by translation control the rna helicase ddx x is an essential mediator of innate antimicrobial immunity a yin-yang effect between sex chromosome complement and sex hormones on the immune response elevated b-estradiol protects females from influenza a virus pathogenesis by suppressing inflammatory responses critically ill children during the - influenza pandemic in the united states correlates of severe disease in patients with pandemic influenza (h n ) virus infection probable nonevectorborne transmission of zika virus persistence and clinical relevance of zika virus in the male genital tract zika virus shedding in semen of symptomatic infected men effect of acute zika virus infection on sperm and virus clearance in body fluids: a prospective observational study sexual transmission of zika virus and other flaviviruses: a living systematic review persistence of zika virus in body fluids -final report zika virus infects human testicular tissue and germ cells sexually acquired zika virus: a systematic review low risk of a sexuallytransmitted zika virus outbreak the dual role of the immune response in reproductive organs during zika virus infection zika virus causes testicular atrophy zika virus infection damages the testes in mice zika virus in semen: a prospective cohort study of symptomatic travellers returning to belgium potential effect of zika virus infection on human male fertility? ebola (ebola virus disease) ebola virus shedding and transmission: review of current evidence a -month follow-up of ebola virus disease survivors in guinea (postebogui) reveals long-term detection of ebola viral ribonucleic acid in semen and breast milk prevention of sexual transmission of ebola in liberia through a national semen testing and counselling programme for survivors: an analysis of ebola virus rna results and behavioural data resurgence of ebola virus disease in guinea linked to a survivor with virus persistence in seminal fluid for more than days dynamics of ebola rna persistence in semen: a report from the postebogui cohort in guinea ebola virus ribonucleic acid detection in semen more than two years after resolution of acute ebola virus infection persistence and clearance of ebola virus rna from seminal fluid of ebola virus disease survivors: a longitudinal analysis and modelling study persistence and genetic stability of ebola virus during the outbreak in kikwit, democratic republic of the congo ebola rna persistence in semen of ebola virus disease survivors -final report new insights into marburg virus disease pathogenesis in the rhesus macaque model ebola virus localization in the macaque reproductive tract during acute ebola virus disease persistent marburg virus infection in the testes of nonhuman primate survivors identification and pathological characterization of persistent asymptomatic ebola virus infection in rhesus monkeys persistence and sexual transmission of filoviruses molecular evidence of sexual transmission of ebola virus unusual ebola virus chain of transmission clinical care for survivors of ebola virus disease. geneva: world health organization zika virus in semen: lessons from ebola mobile health clinic for the medical management of clinical sequelae experienced by survivors of the - ebola virus disease outbreak in sierra leone, west africa care of ebola survivors and factors associated with clinical sequelae-monrovia west nile virus in the united states -a historical perspective the global ecology and epidemiology of west nile virus west nile virus disease cases reported to cdc by state of residence west nile virus: review of the literature optimizing pcr detection of west nile virus from body fluid specimens to delineate natural history in an infected human cohort zika virus infects human sertoli cells and modulates the integrity of the in vitro blood-testis barrier model systemic distribution of west nile virus infection: postmortem immunohistochemical study of six cases west nile virus meningoencephalitis: possible sexual transmission guidance for industry: use of nucleic acid tests to reduce the risk of transmission of west nile virus from living donors of human cells, tissues, and cellular and tissue-based products (hct/ps) west nile virus encephalitis with myositis and orchitis the threat of pandemic influenza: are we ready? workshop summary influenza (flu)/ pandemic influenza the hidden burden of influenza: a review of the extra-pulmonary complications of influenza infection detection of expression of influenza virus receptors in tissues of balb/c mice by histochemistry susceptibility of turkeys to pandemic-h n virus by reproductive tract insemination interaction of influenza a viruses with oviduct explants of different avian species routes of influenza transmission effect of chickenpox and of pneumonia on semen quality variation in sperm penetration assay related to viral illness the effect of a single sauna exposure on spermatozoa the fundamental reasons why laptop computers should not be used on your lap high risk of temporary alteration of semen parameters after recent acute febrile illness sperm chromatin structure assay is useful for fertility assessment cytogenetic effects of influenza virus infection on male germ cells of mice dominant lethality in mice-a test for mutagenicity of influenza x- virus viral epididymitis and orchitis e myth or reality? center for disease control and prevention. severe acute respiratory syndrome (sars) the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? angiotensin-converting enzyme is a functional receptor for the sars coronavirus quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways multiple organ infection and the pathogenesis of sars orchitis: a complication of severe acute respiratory syndrome (sars) covid- )): national center for immunization and respiratory diseases (ncird), division of viral diseases who declares covid- a pandemic clinical characteristics of coronavirus disease in china no evidence of sars-cov- in semen of males recovering from covid- absence of novel coronavirus in semen and testes of covid- patients study of sars-cov- in semen and urine samples of a volunteer with positive nasopharyngeal swab clinical characteristics and results of semen tests among men with coronavirus disease sars-cov- presence in seminal fluid: myth or reality covid- , or the triumph of monogamy? structure analysis of the receptor binding of -ncov the novel angiotensin-converting enzyme (ace) homolog, ace , is selectively expressed by adult leydig cells of the testis sars-cov- infection, male fertility and sperm cryopreservation: a position statement of the italian society of andrology and sexual medicine (siams) (societa italiana di andrologia e medicina della sessualita) key: cord- -db kb c authors: park, jun-gyu; Ávila-pérez, ginés; madere, ferralita; hilimire, thomas a.; nogales, aitor; almazán, fernando; martínez-sobrido, luis title: potent inhibition of zika virus replication by aurintricarboxylic acid date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: db kb c zika virus (zikv) is one of the recently emerging vector-borne viruses in humans and is responsible for severe congenital abnormalities such as microcephaly in the western hemisphere. currently, only a few vaccine candidates and therapeutic drugs are being developed for the treatment of zikv infections, and as of yet none are commercially available. the polyanionic aromatic compound aurintricarboxylic acid (ata) has been shown to have a broad-spectrum antimicrobial and antiviral activity. in this study, we evaluated ata as a potential antiviral drug against zikv replication. the antiviral activity of ata against zikv replication in vitro showed median inhibitory concentrations (ic( )) of . ± . μm and . ± . μm in vero and a cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (cc( )) > , μm). moreover, ata protected both cell types from zikv-induced cytopathic effect (cpe) and apoptosis in a time- and concentration-dependent manner. in addition, pre-treatment of vero cells with ata for up to h also resulted in effective suppression of zikv replication with similar ic( ). importantly, the inhibitory effect of ata on zikv infection was effective against strains of the african and asian/american lineages, indicating that this inhibitory effect was not strain dependent. overall, these results demonstrate that ata has potent inhibitory activity against zikv replication and may be considered as a potential anti-zikv therapy for future clinical evaluation. zika virus (zikv) belongs to the genus flavivirus within the flaviviridae family. zikv is an enveloped positive sense single-stranded rna virus with a genome size of ∼ . kb that encodes a single polyprotein, which is post-translationally processed by cellular and viral proteases into three structural (capsid, c; pre-membrane, prm; and envelope, e) and seven non-structural (ns , ns a, ns b, ns , ns a, ns b, and ns ) proteins avila-perez et al., ) . zika virus was initially isolated from uganda in and viral infections only occurred sporadically in africa and asia until . zikv appeared explosively as the first large-scale outbreak occurred in the yap island in and french polynesia in (weaver et al., ) . most recently, in , the first local transmission of zikv was found in territories of latin america and the caribbean, resulting in up to . million of zikv infection suspected cases (tang et al., ; tripathi et al., ) . like other members of the flaviviridae family, such as yellow fever virus (yfv), dengue virus (denv), japanese encephalitis virus (jev), and west nile virus (wnv), zikv is commonly transmitted by the bite of infected aedes mosquitos, but it can also be transmitted vertically from mother to child, through sexual contact, and in rare cases from blood transfusions (lessler et al., ; fink et al., ) . upon infection, zikv can be shed in blood, urine, semen, saliva, amniotic fluid, breast milk, and cerebrospinal fluid (nayak et al., ; colt et al., ; nazerai et al., ) . most people ( ∼ %) infected with zikv are asymptomatic or have mild symptoms such as fever, rash, joint pain, and conjunctivitis that can last for several days to a week (fink et al., ) . in rare cases, people with symptoms may have neurological guillain-barré syndrome complications (oehler et al., ; rivera-concepcion et al., ; nazerai et al., ) . in the case of pregnant women, zikv infection can lead to microcephaly and other fetal complications as occurred during the large-scale zikv outbreak in brazil in (lessler et al., ) . because of the significant outbreaks in south, central, and north america, zikv was declared a public health concern by the world health organization (who) in february (lazear and diamond, ; ramos da silva and gao, ; weaver et al., ; tripathi et al., ) . there are several vaccines and antiviral drugs currently under development for the prevention or treatment of zikv infection larocca et al., ; shan et al., ; fink et al., ) . dna-based larocca et al., ) , inactivated larocca et al., ; shan et al., ) , live-attenuated and mrna (richner et al., ) vaccines have been proposed for the prophylactic treatment of zikv infections. on the other hand, arbidol (arb) (fink et al., ; haviernik et al., ) , bortezomib, mycophenolic acid, daptomycin (barrows et al., ) , obatoclax, saliphenylhalamide, gemcitabine (kuivanen et al., ) , emetine (yang et al., ) , and sofosbuvir (bullard-feibelman et al., ) have been proposed for the therapeutic treatment of zikv infection. despite these tremendous efforts, there is currently no food and drug administration (fda)-approved vaccines and/or anti-viral drugs available for the treatment of zikv infection. since vaccination takes at least weeks to several months to show protective effects against zikv infection, vaccination is probably not the most appropriate prophylactic method for those who are traveling to areas where zikv is epidemic, endemic, or have already been infected. moreover, vaccination may cause an important issue, such as antibody-dependent enhancement (ade) priyamvada et al., ) . ade, which has been extensively described in denv (priyamvada et al., ) , is a phenomenon where preexisting antibodies facilitate binding and infection during subsequent exposure to infectious viruses, instead of neutralizing them, resulting in exacerbation of clinical signs priyamvada et al., ) . because of the structural similarities between denv and zikv, denv immunity-linked ade of zikv infection has also been reported priyamvada et al., ) . since vaccination for zikv could lead to denv ade, antivirals could represent a better choice for the control of zikv infection. aurintricarboxylic acid (ata), a polyanionic aromatic compound, has been shown to have inhibitory properties against several bacteria and viruses including, among others, yersinia pestis (liang et al., ) , cryptosporidium parvum (klein et al., ) , human immunodeficient virus (hiv) (mitra et al., ; de clercq, ) , hepatitis c virus (hcv) mukherjee et al., ; shadrick et al., ) , vaccinia virus (myskiw et al., ) , influenza virus (hung et al., ) , enterovirus (hung et al., ) and severe acute respiratory syndrome coronaviruses (sars-cov) (he et al., ) . mechanistic studies have suggested that ata has the ability to modulate various cellular enzymes such as activators of the janus kinase (jak ) and signal transducer and activator of transcription (stat ) families (rui et al., ) , inhibitors of nucleases (shadrick et al., ) , glucose- -phosphate dehydrogenase (bina-stein and tritton, ) , and topoisomerase ii proteins (catchpoole and stewart, ; benchokroun et al., ) as well as the enzymatic activity of the vaccinia virus ah l phosphatase (smee et al., ) . however, to date, the ability of ata to inhibit zikv infection has not been evaluated. herein, we investigated ata as a plausible prophylactic and therapeutic candidate against zikv infection. our results demonstrate that ata has a potent and effective antiviral activity against zikv in pre-and post-infection settings, including broadly antiviral activity against strains of the african and american/asian lineages with no toxicity up to , µm in cultured cells. these data support the feasibility of implementing ata for the treatment of zikv infection. african green monkey kidney epithelial vero (atcc ccl- ) and human adenocarcinoma alveolar basal epithelial a (atcc ccl- ) cells were maintained in dulbecco's modified eagle's medium (dmem; mediatech, inc.) supplemented with % fetal bovine serum (fbs) and % psg ( u/ml penicillin, µg/ml streptomycin, and mm l-glutamine) at • c in a % co atmosphere. paraiba/ zikv isolate was kindly provided by stephen dewhurst (department of microbiology and immunology, university of rochester). uganda/ (mr_ strain, catalog no. nr- ) and nigeria/ (ibh strain, catalog no. nr- ) zikv isolates were obtained from the biodefense and emerging infections research resources repository (bei resources). puerto rico/ (prvabc strain) and french polynesia/ zikv isolates were kindly provided from the centers for disease control and prevention (cdc). virus stocks were propagated in vero cells and titrated by plaque assay as previously described (marquez-jurado et al., ) . aurintricarboxylic acid (catalog no. a ) and arbidol (arb, catalog no. slm ) were purchased from sigma-aldrich, mo, united states. both compounds were prepared at mm stock solution dissolved in dimethyl sulfoxide (dmso) and kept at − • c until experimental use. each drug was diluted into infectious media (dmem % fbs, % psg) for the described experiments, where the maximum dmso concentration was . %. cell viability in vero and a cells was measured using the celltiter non-radioactive cell proliferation assay (promega) following the manufacturer's instructions. briefly, confluent vero or a cells ( -well plate format, × cells/well, triplicates) were treated with µl of dmem containing serially diluted (twofold dilutions, starting concentration of , µm) chemicals or . % dmso (vehicle control). plates were incubated at • c in a % co atmosphere for or h. samples were treated with µl of dye solution and incubated at • c in a % co atmosphere for h. next, cells were treated with µl of solubilization solution/stop mix and absorbance at nm was measured using a vmax kinetic microplate reader (molecular devices, waltham, ma, united states). viability of compound-treated cells was calculated as a percentage relative to values obtained with dmso-treated cells. non-linear regression curves and the median cytotoxic concentration (cc ) were calculated using graphpad prism software version . . confluent monolayers ( -plate format, × cells/well, triplicates) of vero cells were infected with plaque forming units (pfu)/well of paraiba/ , uganda/ , nigeria/ , puerto rico/ , and french polynesia/ at • c in infection media. after h of adsorption, virus inoculum was removed and cells were washed three times with infection media before adding fresh infection media containing % microcrystalline cellulose (avicel, sigma-aldrich) and the indicated concentration of compounds, or . % dmso as vehicle control. in case of pre-treatment experiments, the cell monolayers were treated with the indicated concentration of compound, or . % dmso, for the indicated times before zikv infection. infected cells were incubated at • c for - h, depending on virus strains. for immunostaining, cells were fixed with % paraformaldehyde for h, washed three times with phosphate buffered saline (pbs) and permeabilized with . % triton x- for min at room temperature. then, the plates were blocked with . % bovine serum albumin (bsa) in pbs (blocking solution) for h at room temperature, followed by incubation with µg/ml of the pan-flavivirus envelop (e) protein monoclonal antibody g (atcc, catalog no. vr- ) diluted in blocking solution for h at • c. after incubation with the primary antibody, cells were washed three times with pbs and developed with the vectastain abc kit and the dab peroxidase substrate kit (vector laboratory, inc., ca, united states) according to the manufacturers' instructions. stained plaques were analyzed using the ctl immunospot plate reader and counting software (cellular technology limited, cleveland, oh, united states). virus titers were calculated as pfu/ml (nogales et al., ) . non-linear regression curves and the median inhibitory concentration (ic ) were determined as described above. confluent monolayers ( -well plate format, . × cells/well, triplicates) of vero or a cells were infected (multiplicity of infection, moi, . ) with paraiba/ diluted in infection media for h at room temperature. after viral absorption, cells were incubated with infection media containing the indicated concentrations ( , , . , and µm) of ata. at , , , and h post-infection (h p.i.), tissue culture supernatants were collected and titrated on vero cells by immunostaining as described previously (marquez-jurado et al., ) . levels of apoptosis were measured using the caspase-glo r / assay (promega, wi, united states) following the manufacturer's instruction. briefly, vero and a cells ( -well plate format, . × cells/well, triplicates) were infected with zikv paraiba/ (moi of . ) and, at the indicated times post-infection, cells and tissue culture supernatants were collected and centrifuged. twenty five microliters of supernatants were mixed with µl of caspase- / reagent using a plate shaker, incubated at room temperature for h, and luminescence at nm was measured using a spectramax id (molecular devices, waltham, ma, united states) following the manufacturer's instructions. two-way anova was used to evaluate significant differences. data are expressed as the mean ± standard deviation (sd) of at least three independent experiments in triplicates using microsoft excel software. value were considered statistically significant when * p < . , * * p < . , * * * p < . , * * * * p < . . all data were analyzed with prism software version . (graphpad software, ca, united states). cc and ic were determined using sigmoidal dose response curves (graphpad software, ca, united states). the selective index (si) of each compound was calculated by dividing the cc with the ic . before examining the inhibitory effect of ata (figure ) against zikv infection, we first determined the cc of ata on vero and a cells (figure ) . for this, we treated both cell lines with serial (twofold) dilutions of ata and measured cell viability at and h post-treatment. as an internal control for these studies, we used arb, a drug that has been previously described to have antiviral activity against zikv in vero (haviernik et al., ) and a (fink et al., ) cells. we did not observe any toxicity with ata in vero (figure a ) or a ( figure b ) cells at or h post-treatment, even at the highest concentration tested ( , µm), while arb showed cc values of . ± . or . ± . µm in vero ( figure c ) and . ± . or . ± . µm in a ( figure d ) cells (table ) at or h post-treatment, respectively. to determine the ic of ata, vero and a cells were infected with pfu/well of paraiba/ and after h of viral absorption, virus inoculum was replaced with infection media with twofold serial dilutions (starting concentration of , µm) of ata or arb (figure ) and the ic calculated as described in the section "materials and methods." although the ic of ata ( figure a) and arb ( figure c) in vero cells were similar ( . ± . µm and . ± . µm, respectively), the selective index (si, cc /ic ) of ata (> . ) was significantly higher than that of arb ( . ) ( table ) . likewise, the ic of ata ( figure b ) and arb ( figure d ) in a cells were similar but with clearly different si values (> . for ata and . for arb) ( table ) . notably the cc , ic , and si of arb were similar to those previously described in the literature in these cell lines (fink et al., ; haviernik et al., ) . these data suggest that ata exhibited an effective inhibition of zikv infection with limited toxicity and si values better than those previously described for arb. we also observed that zikv replication was completely inhibited at a concentration of µm of ata in vero ( figure a ) and a ( figure b ) cells while . µm and µm concentrations of ata showed partial viral inhibition in vero and a cells (figures a,b) , respectively, demonstrating a dose-dependent inhibition of viral replication in both cell lines. we next evaluated the ability of ata to protect cells from the cytopathic effect (cpe) induced during zikv infection ( figure ) . to that end, vero and a cells were infected (moi . ) with paraiba/ and, after h of viral absorption, cells were treated with , . , , and µm of ata. at h p.i., cells were observed under a light microscope for evaluation of their morphology and cpe ( figure a ). as expected from our previous results, µm and more clearly µm of ata were able to prevent zikv-induced cpe in both cell lines (figure a) . to quantify the ability of ata to prevent zikv-induced apoptosis, tissue culture supernatants from zikv-infected vero and a cells were harvested at , , and h p.i. to measure the level of apoptotic signal as determined by caspase and activities ( figure b) . zikv-infected cells showed figure | aurintricarboxylic acid inhibition of zikv replication: vero (a) and a (b) cells ( -well plate format, . × cells/well, triplicates) were infected (moi . ) with paraiba/ . tissue culture supernatants were collected at , , and h p.i., and viral titer were calculated by immunostaining (fluorescent forming units, ffu/ml). dotted line indicates the limit of detection ( ffu/ml). data was expressed as mean and standard deviations (sd) from three independent experiments conducted in triplicates. statistical analysis was conducted by two-way anova, * p < . , * * p < . , * * * p < . , * * * * p < . , or no significance (n.s.). figure | aurintricarboxylic acid protects vero and a cells from zikv-induced cell death: vero and a cells ( -well plate format, . × cells/well, triplicates) were infected (moi . ) with paraiba/ . after h viral adsorption, cells were treated with the indicated concentrations ( , , . , and µm) of ata. at h p.i., cells were observed and imaged under an optical microscope. scale bar = µm. (a) caspase / levels were measured in the tissue culture supernatants at , , and h p.i. (b) data of each time point was compared to mock-infected control cells and expressed as mean of relative percentage and sd from three independent experiments conducted in triplicates. statistical analyses were conducted by two-way anova, * p < . , * * p < . , * * * p < . , * * * * p < . , or no significance (n.s.). increased caspase and levels up to eightfolds in vero cells and up to . -folds in a cells compared to mock-infected cells ( figure b ). levels of caspase and activation were dose-dependently reduced by ata with µm of ata showed only . -and . -fold induction as compared to mock-infected vero and a cells, respectively ( figure b ). we next determined whether ata is able to inhibit both ancestor african (uganda/ and nigeria/ ) and contemporary asian/american (puerto rico/ and french polynesia/ ) zikv lineage strains using our microplaque reduction assay (figure ) . we observed similar ic values of ata with uganda/ ( figure a , ic = . ± . µm), nigeria/ ( figure b , ic = . ± . µm), puerto rico/ ( figure c , ic = . ± . µm), and french polynesia/ ( figure d , ic = . ± . µm), compared to those observed with paraiba/ (figure and table ), demonstrating the broad antiviral activity of ata against different zikv strains, regardless of the year and place of isolation. to demonstrate the feasibility of using ata for the prevention of zikv infection, important for travelers to regions where zikv is endemic, we next evaluated whether pre-treatment with ata results in inhibition of zikv replication (figure ) . to that end, vero cells were pre-treated with ata for ( figure a) , ( figure b) , (figure c) , or ( figure d ) h prior to infection (moi . ) with paraiba/ . pre-treatment with ata for - h before zikv infection resulted in similar ic values ( . ± . µm, . ± . µm, . ± . µm, and . ± . µm; respectively) demonstrating that ata is stable and able to prevent zikv infection even when administered days previous to viral infection (figure and table ). the recent outbreak of zikv accompanied with severe pathology, including microcephaly in newborns, prompted many researchers to develop prophylactic vaccines and to identify therapeutic drugs against zikv infection larocca et al., ; lessler et al., ; shan et al., ; fink et al., ) . currently, there are no commercially available vaccines and/or antiviral therapies for the treatment of zikv infection. therefore, there is an urgent medical need for the development of effective counter measurements to control zikv infection. in this study, we demonstrated that ata (figure ) has limited toxicity (figure ) and an effective and dose-dependent antiviral activity against zikv infection (figures , ) in both monkey kidney epithelial vero and human alveolar a cells. notably, ata can prevent zikv-induced cpe and apoptosis in both cell lines ( figure ) and has broad anti-viral activity against representative zikv strains from the african (uganda/ and nigeria/ ) and the asian/american (puerto rico/ and french polynesia/ ) lineages (figure ) . moreover, ata can also prevent zikv infection even when administered days before infection (figure ) . aurintricarboxylic acid is a polyanionic aromatic compound that structurally relates to suramin (balzarini et al., ) and is believed to influence over host and viral enzymes (shadrick et al., ) . although the exact mechanism by which ata inhibits zikv infection was not identified in this study, there are several plausible mechanisms on zikv inhibition mediated by ata, including the targeting of viral and cellular proteins. in terms of inhibiting viral proteins, ata could bind to zikv ns helicase and prevent its binding to either atp or nucleic acids, as previously described for hcv (mukherjee et al., ; shadrick et al., ) . likewise, ata could inhibit the zikv rna-dependent rna polymerase (rdrp) ns protein, as described for hcv mukherjee et al., ; shadrick et al., ) and enterovirus (hung et al., ) . similarly, ata could inhibit the methyltransferase activity of ns involved in mrna capping processes, as previously described for other flaviviruses (denv and yfv) (milani et al., ; garcia et al., ) . because of the structural similarities between denv and zikv ns proteins, it is feasible that, similar to denv, ata binds to ns to inhibit zikv infection . moreover, it is possible that ata targets and has inhibitory activities against one or more of the viral proteins described above. in terms of targeting cellular proteins important for the efficient replication of zikv, it has been previously described that ata has anti-apoptotic properties in a variety of cells (chen et al., ) . it is possible that the anti-apoptotic activity of ata protects against zikv-induced cell death, as demonstrated in this study (figure ) . notably, it has been recently shown that zikv infection induced apoptosis through caspase and in a cells and through caspase in neonatal mice brain (huang et al., ; frumence et al., ) . these results suggest that inhibition of zikv replication results in a decrease in the level of apoptotic cells and that the anti-apoptotic effect of ata affects zikv replication. further research is guaranteed to yield a better understanding of the antiviral activity of ata on zikv infection, and other viruses, before the use of ata as an antiviral drug. during january to february , a total of residents from states in the united states were diagnosed with zikv infection (armstrong et al., ) . out of patients, ( %) traveled to areas of active zikv transmission before the infection and five ( %) did not travel but reported sexual contact with a traveler who had a symptomatic illness (armstrong et al., ) . for these reasons, preventive efforts are required prior to travel to areas of active zikv transmission. in this study, cells pretreated with ata for up to h prior to infection with zikv showed similar ic than those in post-treatment settings, potentially suggesting that ata might target a cellular protein required for zikv replication or that the concentration and stability of ata in pre-treated cells is sufficient to inhibit zikv infection, or both. nevertheless, these results demonstrate the feasibility of using ata for the prophylactic treatment of viral infection, including those traveling to areas where zikv is endemic. moreover, due the broad inhibition effect of ata against others viruses and parasites (liang et al., ; he et al., ; de clercq, ; myskiw et al., ; klein et al., ; chen et al., ; hung et al., hung et al., , mukherjee et al., ; shadrick et al., ) that are present in zikv endemic areas, treatment with ata could be used for the broad prevention of denv, yfv (milani et al., ; shadrick et al., ; garcia et al., ) , hcv mukherjee et al., ; shadrick et al., ) , and parasitic infestation (cryptosporidium parvum) (klein et al., ) for people traveling to these endemic regions. moreover, the broad spectrum antiviral activity of ata against different african and asian/american zikv strains further guarantees the feasibility of implementing ata to prevent zikv infection to travelers around the world. although ata has been amply evaluated in vitro, only few studies have assessed the activity of ata in vivo, including its use as a curative agent against thrombosis (strony et al., ) , apoptosis (roberts-lewis et al., ; heiduschka and thanos, ) , parasite infestations (klein et al., ) , bacterial (y. pestis) (liang et al., ) , and vaccinia virus (smee et al., ) infections. in the case of vaccinia virus, ata did not protect mice from a lethal challenge at a dose of mg/kg/day (smee et al., ) . further studies are needed to evaluate the anti-viral activity of ata in vivo for the treatment of viral infections, including zikv. our studies show limited toxicity, if any, of ata in cultured cells, including human a cells. the lack of knowledge about the use of ata in pregnant women requires future additional safety tests, including studies using validated animal models of zikv infection, before using ata for the treatment of zikv infection during pregnancy. based on the effectiveness of ata against zikv infection (si = . in vero cells and . in a cells) as compared to other previously described drugs, including emetine (si = . in snb- cells and . in env+ cells) (yang et al., ) , obatoclax [si = in human retinal pigment epithelial (rpe) cells] (kuivanen et al., ) , saliphenylhalamide (si > in rpe cells) (kuivanen et al., ) , gemcitabine (si > , in rpe cells) (kuivanen et al., ) , sofosbuvir (si > . in huh- cells and . in jar cells) (bullard-feibelman et al., ) and arb (si = . in vero cells and . in a cells) (fink et al., ; haviernik et al., ) and this study (ata, in vero cells, and . in a cells), it is possible that ata represents one of the most reasonable options of the treatment of zikv infection. protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys travel-associated zika virus disease cases among u.s. residents-united states reverse genetic approaches for the generation of recombinant zika virus aurintricarboxylic acid and evans blue represent two different classes of anionic compounds which selectively inhibit the cytopathogenicity of human t-cell lymphotropic virus type iii/lymphadenopathy-associated virus enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity a screen of fda-approved drugs for inhibitors of zika virus infection aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of dna topoisomerase ii in vitro and in chinese hamster fibrosarcoma cells aurintricarboxylic acid is a nonspecific enzyme inhibitor the fda-approved drug sofosbuvir inhibits zika virus infection inhibition of topoisomerase ii by aurintricarboxylic acid: implications for mechanisms of apoptosis inhibition of cytokine-induced jak-stat signalling pathways by an endonuclease inhibitor aurintricarboxylic acid characterization of aurintricarboxylic acid as a potent hepatitis c virus replicase inhibitor transmission of zika virus through breast milk and other breastfeeding-related bodily-fluids: a systematic review emerging anti-hiv drugs the antiviral drug arbidol inhibits zika virus the south pacific epidemic strain of zika virus replicates efficiently in human epithelial a cells leading to ifn-beta production and apoptosis induction inhibitors compounds of the flavivirus replication process arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses potent and selective inhibition of sars coronavirus replication by aurintricarboxylic acid aurintricarboxylic acid promotes survival and regeneration of axotomised retinal ganglion cells in vivo zika virus infection during the period of maximal brain growth causes microcephaly and corticospinal neuron apoptosis in wild type mice inhibition of enterovirus replication and the viral d polymerase by aurintricarboxylic acid aurintricarboxylic acid inhibits influenza virus neuraminidase in vitro and in vivo activity of aurintricarboxylic acid preparations against cryptosporidium parvum obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism vaccine protection against zika virus from brazil zika virus: new clinical syndromes and its emergence in the western hemisphere assessing the global threat from zika virus aurintricarboxylic acid blocks in vitro and in vivo activity of yoph, an essential virulent factor of yersinia pestis, the agent of plague an alanine-to-valine substitution in the residue of zika virus ns a protein affects viral rna synthesis and attenuates the virus in vivo flaviviral methyltransferase/rna interaction: structural basis for enzyme inhibition hiv- upregulates fas ligand expression in cd + t cells in vitro and in vivo: association with fas-mediated apoptosis and modulation by aurintricarboxylic acid identification and analysis of hepatitis c virus ns helicase inhibitors using nucleic acid binding assays aurintricarboxylic acid inhibits the early stage of vaccinia virus replication by targeting both cellular and viral factors pathogenesis and molecular mechanisms of zika virus a 'furrytale' of zika virus infection: what have we learned from animal models? replication-competent influenza a viruses expressing a red fluorescent protein zika virus infection complicated by guillain-barre syndrome-case report, french polynesia humoral immune responses against zika virus infection and the importance of preexisting flavivirus immunity zika virus: an update on epidemiology, pathology, molecular biology, and animal model modified mrna vaccines protect against zika virus infection the zika virus: an association to guillain-barre syndrome in the united states -a case report aurintricarboxylic acid protects hippocampal neurons from nmda-and ischemia-induced toxicity in vivo activation of the jak -stat signaling pathway in nb lymphoma cells by an anti-apoptotic agent, aurintricarboxylic acid aurintricarboxylic acid modulates the affinity of hepatitis c virus ns helicase for both nucleic acid and atp a live-attenuated zika virus vaccine candidate induces sterilizing immunity in mouse models lack of efficacy of aurintricarboxylic acid and ethacrynic acid against vaccinia virus respiratory infections in mice aurintricarboxylic acid in a canine model of coronary artery thrombosis zika virus infects human cortical neural progenitors and attenuates their growth a novel zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses zika virus: history, emergence, biology, and prospects for control emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry key: cord- -aozxvxxs authors: vermillion, meghan s.; klein, sabra l. title: pregnancy and infection: using disease pathogenesis to inform vaccine strategy date: - - journal: npj vaccines doi: . /s - - - sha: doc_id: cord_uid: aozxvxxs vaccination is the mainstay of preventative medicine for many infectious diseases. pregnant women, unborn fetuses, and neonates represent three at-risk populations that can be simultaneously protected by strategic vaccination protocols. because the pathogenesis of different infectious microbes varies based on tissue tropism, timing of infection, and host susceptibility, the goals of immunization are not uniform across all vaccines. mechanistic understanding of infectious disease pathogenesis and immune responses is therefore essential to inform vaccine design and the implementation of appropriate immunization protocols that optimize protection of pregnant women, fetuses, and neonates. vaccination significantly reduces the health burden of many infectious disease, especially in high-risk populations. pregnant women, unborn fetuses, and neonates represent three populations of high-risk individuals that can all be simultaneously protected from vaccine-preventable infectious disease with strategic maternal immunization protocols. infectious microbes that pose significant health risks during pregnancy can be divided into three broad categories, based on the pathogenesis and disease outcome (fig. ) , with some microbes falling within more than one category. first are maternal infections, which are defined by heightened disease severity in pregnant females, but with rare or inconsequential transmission and disease in the fetus. second are fetal or congenital infections, which are characterized by mild or no disease in pregnant females, but occasional vertical transmission and severe congenital disease in the fetus. third are neonatal and infant infections, which are not considered to pose significant risk to pregnant women or unborn fetuses, but can cause severe, and sometimes fatal disease in neonates and infants that lack protective maternal immunity following birth. the vaccination strategies employed differ for micobes within each of these categories and vary based on the at-risk individual (i.e., mother, fetus, and/or neonate/infant), the timing of the greatest risk of infection (i.e., early pregnancy, late pregnancy, or post-natal), and on the duration of protective immunity following vaccination. in this review, we discuss evidence to suggest that immunization strategies for pregnant women should be tailored to optimize protection for the mother, fetus, neonate, infant, or all individuals. we review vaccine-preventable infections during pregnancy and the current vaccination strategies employed to reduce the burden of infectious diseases, including influenza. further, we examine novel vaccine platforms and consider how their application may provide safe alternatives for enhancing protection of pregnant women. finally, we discuss vaccine development and prevention strategies for combatting emerging infectious diseases, including zika, that pose a threat to pregnant women and their fetuses. owing to physiologic and immunologic changes that support pregnancy and tolerance of a semi-allogenic fetus, pregnant women demonstrate increased susceptibility to certain infectious agents including hepatitis e, varicella zoster, and influenza viruses. infection with these viruses during pregnancy results in severe maternal disease, increased maternal mortality and associated pregnancy complications, which are observed most frequently during the third trimester and peripartum period. for example, the case fatality rate among pregnant women infected with hepatitis e virus is estimated to be - %, , compared with - % in the general population. approximately % of cases of varicella pneumonia in adults reported from - were from pregnant women ; and pregnant women infected with the pandemic h n influenza a virus (iav) were reportedly times more likely to be hospitalized or die than the general population. overall, vertical transmission of these viruses is relatively uncommon, but adverse pregnancy outcomes, including spontaneous abortion and pre-term birth can still occur as an indirect consequence of maternal inflammation. , reports during the h n pandemic in australia and new zealand indicated that among pregnant women who were hospitalized with suspected h n iav infection, % of their infants required intensive care, and % were either stillborn or died shortly after birth; only infants, however, had detectable h n infection. the primary goal of vaccination strategies for protecting against maternal infections is the generation of protective maternal immunity either prior to or during early pregnancy. optimally, vaccination should prevent or reduce disease by inducing sterilizing immunity (i.e., immunity that completely prevents infection). despite reported reductions in antiviral proteins during pregnancy, studies comparing vaccine responses between pregnant and nonpregnant women find no difference in either the magnitude or duration of antibody responses against influenza a viruses. [ ] [ ] [ ] in fact, surveillance data from taiwan reveal that influenza vaccination during pregnancy results in higher levels of seroprotection than does vaccination prior to conception, with no effect of gestational age on vaccineinduced antibody responses. as of , the centers for disease control (cdc) advisory committee on immunization practices (acip) categorizes pregnant women as a target population for receiving the inactivated influenza vaccine and recommends that pregnant women be immunized during any trimester. several studies evaluating adverse vaccine reactions in pregnant women have concluded that there is no link between pregnancy complications or adverse fetal outcomes among women who are vaccinated during pregnancy. [ ] [ ] [ ] [ ] although the live attenuated intranasal influenza vaccine is not recommended for pregnant women, accidental administration during pregnancy was not associated with an increased risk of adverse reactions. , despite the plethora of data that support the benefits and safety of influenza vaccination during pregnancy, coverage remains low, with a less than % maternal vaccination rate during the - influenza season in the united states. misconceptions about the safety and benefits of influenza vaccination represent the largest barriers to vaccine acceptance among pregnant women. varicella zoster virus (vzv) is another vaccine-preventable infection associated with increased severity during pregnancy. vzv is an alpha herpes virus and the causative agent of varicella or chickenpox. in temperate climates, seroprevalence among individuals over years of age is estimated to be %, with almost % of infections occurring prior to years of age. the first modified-live vaccine against varicella zoster virus was licensed in the united states in , and is now recommended for children over months of age. primary vzv infection during pregnancy is therefore uncommon, as most women of childbearing age have been either infected or immunized. in women who have not been previously exposed, however, primary vzv infection between weeks through of gestation is associated with a % risk of congenital transmission and disease in the offspring. because all licensed vzv vaccines contain live-attenuated virus, their use during pregnancy is contraindicated. instead, the cdc recommends that nonpregnant women of childbearing age be vaccinated against vzv at least one month prior to conception. as a herpes virus, infection with vzv is life-long, and reactivation occurs in approximately - % of individuals, which results in a painful skin condition known as shingles or herpes zoster. reactivated vzv, however, is not associated with increased disease severity or congenital infection during pregnancy. acute viral hepatitis caused by hepatitis e virus (hev) is an emerging infectious disease that causes severe disease in pregnant women, with a fatality rate of up to % in endemic regions. in addition to heightened maternal disease severity, hev infection during pregnancy is associated with increased rates of premature birth and prenatal mortality. although vertical hev transmission rates are high, with estimates between - %, the relative contributions of fetal hev infection to adverse perinatal outcomes is unclear. a recombinant hev subunit vaccine has been developed and proven safe and effective following completion of phase ii and iii clinical trials, but commercial use is currently limited to china. furthermore, the vaccine is not approved for use in pregnant women despite being % efficacious in participants receiving all three doses. additional hev vaccine candidates are being tested in preclinical pregnant animal models, and one recombinant hev vaccine has been shown to be safe and highly immunogenic in pregnant mice. additional studies in a susceptible animal model are needed to confirm efficacy following virus challenge. developing fetuses are extremely vulnerable to both infectious and noninfectious insults. certain infectious agents that are often clinically silent in healthy adults can cause severe birth defects if fig. infectious microbes that cause maternal, congenital, or postnatal complications. the infectious microbes are categorized according to the mechanism of transmission and disease, and the population at greatest risk for severe outcome during or after pregnancy. infection with some pathogens (e.g., sars coronavirus, hepatitis e virus, and ebola virus) during pregnancy cause severe disease in pregnant women, but are not transmitted to offspring. other infectious microbes (e.g., toxoplasma gondii, rubella virus, parvovirus b , cytomegalovirus, and zika viruses) infect and cause mild or asymptomatic disease in pregnant females, but can be vertically transmitted to the fetus and congenital complications. another category of microbes (e.g., bordetella pertussis, clostridium tetani, and respiratory syncytial virus) pose the largest risk to neonates after birth. many infectious microbes (e.g., listeria monocytogenes, plasmodium spp., hiv, vzv, influenza viruses, chlamydia trachomatis, gbs, treponema pallidum, and herpes viruses) may cause overlapping syndromes depending on the timing of infection during pregnancy. understanding the pathogenesis of infectious diseases during pregnancy should inform vaccine design and the implementation of appropriate immunization protocols that optimize protection of pregnant women, fetuses and neonates they breach the placental barrier during critical developmental periods during pregnancy. an increasing number of pathogens are being recognized for causing congenital disease, and what was originally designated as the torch complex (toxoplasma gondii, "other," rubella virus, cytomegalovirus, and herpes simplex virus) is now expanded to include other infectious agents including zika virus. development of congenital disease can depend on the timing of infection during gestation, the infectious burden, and the pathogenesis in the fetus. the congenital syndrome for each pathogen is characterized by a variety of different developmental abnormalities, and commonly impact hearing, vision, and central nervous system function. for many congenital infections, the timing of infection during gestation determines the relative risk to the fetus and dictates the spectrum of disease that results. for example, while infection with rubella virus during the first weeks of gestation is associated with an % risk of congenital rubella syndrome (crs), this is reduced to a % risk between - weeks, and minimal risk for infections occurring after weeks of gestation. in contrast, the risk of congenital toxoplasmosis has been demonstrated to be highest during third trimester pregnancy, which is hypothesized to be due to differential expression of placental toll-like receptors, including tlr , within first compared with third trimester trophoblast cells. similarly, maternal infection with listeria monocytogenes is typically associated with adverse pregnancy outcomes during the third trimester, though infection during the first trimester in nonhuman primates also leads to rapid fetal demise. the primary goal of vaccination strategies for protecting against fetal infections is generation of protective maternal immunity prior to pregnancy. because congenital infections can occur in the absence of maternal symptoms, vaccines against congenital agents should ideally provide complete sterilizing immunity. rubella is included in a live-attenuated combination vaccine for measles, mumps, and rubella (mmr), which confers lifelong protective immunity. because of the long duration of protective immunity following rubella vaccination, target populations include children and adolescent girls. however, incomplete vaccination coverage can lead to paradoxical increases in crs due to an increase in the average age of infection, and so it is also recommended that unvaccinated women of childbearing age be counseled to receive the rubella vaccine at least one month prior to conception. the implementation of large-scale rubella vaccination programs has resulted in sufficient population-level immunity, significant reductions in crs, and elimination of rubella virus from several developed countries, including the united states. following successful implementation of mmr vaccination programs, cytomegalovirus (cmv) has emerged as the most common congenital viral infection in the developed world. the incidence of congenital cmv varies based on geographic region and socioeconomic status, but overall birth prevalence is estimated to be . %, which is similar to the incidence of down syndrome and fetal alcohol syndrome. in contrast to rubella virus, however, there is currently no licensed vaccine available for cmv, and with seroprevalence approaching % in some developing countries, vaccine development has been identified as a priority public healthcare goal. , while cmv infection of healthy adults is usually asymptomatic, adaptive immune responses are insufficient to clear the infection, which results in lifelong latent infection of myeloid precursor cells. although latent or reactivated cmv is less likely to cause congenital infection than a primary cmv infection during pregnancy, , preconception immunity does not completely eliminate transplacental transmission and congenital disease. moreover, pregnant women with latent cmv infection are still susceptible to primary infection with different cmv strains, which have been shown to have distinct virulence patterns. , overcoming the challenges associated with latent infections and strain variability are significant hurdles in the development of an effective cmv vaccine, and despite significant advances in our knowledge of cmv pathogenesis, the precise immune targets that constitute fetal protection remain unknown. of the several cmv vaccine candidates that have been tested, none have provided complete protection against infection, and all have failed to protect against reactivation of latent cmv. more research on the pathogenesis cmv infection is needed to define immunological correlates of protection against cmv transmission during pregnancy to inform vaccine development. although not associated with congenital disease, hepatitis b virus (hbv) is another vaccine-preventable infection that can cross the placenta during pregnancy. mother-to-child-transmission remains the most common route of infection in endemic regions, and women with active viral replication have up to a % chance of vertical transmission. of those that are infected perinatally, up to % develop chronic hbv infection. since the initial recommendation of routine hbv vaccination of children in , the rate of new hbv infections has significantly declined in the united states, but chronic hbv remains prevalent in sub-saharan africa and east asia. although combined passive and active immunoprophylaxis of infants has significantly reduced perinatal hbv infection, perinatal transmission occurs in up to % of infected mothers. to augment neonatal prophylactic strategies, the cdc acip recommends that pregnant women who are identified as being at risk for hbv infection be vaccinated with the recombinant hbv vaccine. immunity following receipt of the hbv vaccine is long-lived, with anti-hbv antibodies persisting in most adults for at least years. because of the long-term protection conferred by the hbv vaccine, immunization is not necessary for pregnant women who have already been vaccinated and are at low risk of infection. owing to the limited exposure to foreign antigen and blunted innate immune responses in utero, the neonatal immune system is immature at birth, making neonates (i.e., less than one month of age) particularly susceptible infections. infectious diseases are responsible for over % of child mortality, and over % of these deaths occur within one month of age. during the neonatal period of immune system maturation, protection against pathogens relies primarily on passive immunity from maternal-derived igg antibodies. in humans, most maternal antibodies are transferred into the fetal circulation through the placenta prior to birth, which contrasts with most veterinary species, in which maternal antibody is transferred via colostrum immediately following birth. regardless of species, vaccination during pregnancy increases circulating maternal antibodies and enhances transfer to the fetus/neonate. the goal of vaccination strategies for protecting against neonatal infections is generation of robust maternal antibody responses during pregnancy to enhance placental transfer. further, because neonatal protection is exclusively conferred by maternal-derived antibody, vaccines aimed at protecting infants should prioritize induction of humoral over cellular immune responses, with the induction of igg being most important because this igg isotype is associated with the highest placental transport efficiency in females. moreover, the kinetics of maternal vaccine-induced antibody response, the efficiency of placental antibody transfer, and the half-life of the antibody in the neonate should inform the optimal timing of vaccination during pregnancy. because the peak antibody response is typically observed - weeks following immunization, vaccination during pregnancy as opposed to before conception is likely to result in the greatest benefit to the neonate. further, the efficiency of placental antibody transfer in females increases throughout gestation, with less than % maternal igg transferred to the fetus in the first weeks of gestation, significantly more transferred during the second and third trimesters, and at delivery fetal igg often exceeds maternal levels. vaccination of females during the second and third trimesters of pregnancy is most likely to generate the greatest level of protection in the neonate, but the precise timing for maximum protection is debated. controversy over the timing of pertussis vaccination in pregnancy has been reviewed elsewhere, with some reports claiming peak cord blood antibody concentrations following vaccination in the second trimester, and others reporting peak antibody concentrations following vaccination in the third trimester. antibody avidity also influences the efficiency of placental transfer, with higher avidity antibodies crossing the placenta with greater efficiently than low avidity antibodies. more consideration should be given to the development of high avidity antibodies in the timing of vaccination during pregnancy, as protective immunity in the infant depends on both the concentration and avidity of the maternal-derived antibody. the half-life of maternal antibodies in infants also must be considered in vaccine development and administration. maternalderived igg is reported to have a half-life of approximately days in serum, and depending on serum antibody titers present at birth, this translates into protective immunity for approximately the first - months of life for most infant pathogens. the half-life of the antibody also dictates the vaccination schedules for infants, as the presence of maternalderived antibody interferes with vaccine efficacy, and it is not until maternal-derived antibody has waned below a certain threshold that an infant can mount its own active vaccine response. the goal of the infant vaccine series is to time vaccination to coincide with the time that maternal-derived antibody drops below the threshold at which it can neutralize the vaccine antigen. because the precise timing of these events is unpredictable, infant vaccination schedules are designed so that vaccines are administered in a series that spans the duration of this window, and minimize susceptibility to natural infection. in the united states, infant vaccines are recommended at , and months of age. bordetella pertussis is a vaccine-preventable respiratory pathogen of significant public health importance, and it is a major cause of mortality in infants lacking protective maternal immunity. vaccination of women during pregnancy, however, significantly enhances the transfer of maternal antibody to the fetus, , and these newborns are times more likely to have protective antibody titers at birth compared with those born from women who were not vaccinated during pregnancy. inactivated pertussis antigen is combined with tetanus and diphtheria toxoids in a single vaccine (tdap), which the cdc acip recommends for all pregnant women, regardless of previous vaccine history. in contrast to vaccine formulations that contain killed whole b. pertussis organisms, the tdap vaccine contains only select antigens and confers relatively weak and only transient protective immunity that declines after year. vaccination of women either prior to conception or during early pregnancy does not provide adequate neonatal protection against pertussis. consequently, the cdc considers the third trimester to be the optimal time to administer the tdap vaccine to pregnant women. adverse events reported following tdap vaccination are generally mild, and there are no reported risks of adverse pregnancy outcomes related to tdap vaccination during pregnancy. despite consistent evidence that supports the benefit and safety of tdap vaccination during pregnancy, coverage remains low, with an estimated % of pregnant women receiving the tdap vaccine in the united states in . receipt of the tdap vaccine during pregnancy also confers protection against neonatal tetanus, which is associated with case fatality approaching % in the absence of medical care. disease is caused by the toxin produced by clostridium tetani, and infection occurs most commonly due to contamination of the umbilical stump following delivery. consequently, the incidence of disease is much greater in developing countries, where maternal vaccination is scarce and perinatal hygiene practices are poor. in , the world health assembly called for the elimination of neonatal tetanus, which has inspired an initiative to improve vaccination coverage and birth hygiene in countries with high disease prevalence. as part of this initiative, immunization standards have been expanded and recommend that pregnant women with unknown or inadequate vaccination history receive two doses of the toxoid-containing vaccine, administered one month apart. maternal anti-tetanus antibodies are passively transferred to the fetus, and it is estimated that maternal immunization reduces neonatal tetanus mortality by %. respiratory syncytial virus (rsv) is the most common respiratory viral pathogen of newborns and infants, and accounts for - % of acute bronchiolitis and - % of pneumonia cases in hospitalized children less than years of age. rsv is also reported to cause severe disease and hospitalization in pregnant women when infection occurs during the third trimester, , and therefore dually qualifies as a maternal infection as well. a licensed vaccine against rsv is currently unavailable, but several vaccine candidates have shown promise in various animal models. [ ] [ ] [ ] [ ] given the importance of this pathogen during early life, vaccine development strategies have focused on maternal immunization, with three maternal vaccines currently in clinical trials. maternal vaccination against rsv has direct and indirect benefits to the neonate; neonates are directly protected through passive transfer of maternal antibody through the placenta, and they are indirectly protected because a vaccinated mother is less likely to transmit the infection to her infant. vaccination of pregnant women is controversial, and immunization with live (i.e., replication-competent) viral or bacterial vaccines is generally contraindicated due to the theoretical risk of congenital infection and teratogenic effects from the vaccine strains. however, in a report of over pregnant women who were unknowingly immunized with live attenuated rubella vaccine, there were no cases of vaccine-associated congenital rubella infection, and live virus strains of influenza or yellow fever viruses administered to pregnant women also have no link with pregnancy complications. , vaccination with inactivated vaccines such as influenza and tdap during pregnancy have low uptake, with concerns of safety among both patients and their healthcare providers being a primary barrier. the safety of vaccine adjuvants is debated, and although neither the tdap nor seasonal influenza vaccine recommended during pregnancy contain adjuvants, retrospective studies evaluating safety of the adjuvanted pandemic h n influenza vaccine in pregnant women found no relationship with adverse pregnancy outcomes. the conservative approach to vaccination protocols for pregnant women stems from the lack of controlled safety and efficacy studies for this population. for ethical reasons, pregnant women are exempted from almost all clinical and vaccine trials, and heath care providers are less likely to endorse prophylactic treatments for which safety and efficacy profiles have not been adequately characterized. whereas study in pregnant women is not possible, pre-clinical testing in animal models may provide a useful alternative, and vaccine preclinical trials in pregnant animal models may provide information to inform healthcare policies for pregnant women. although there are some differences in the length of gestation, placental structure, and fetal development between humans and animal models, many structural and functional parallels exist, [ ] [ ] [ ] which serve as tractable platforms for evaluating the safety and efficacy of various therapies during pregnancy. similar to humans, pregnant mice, rats, and rabbits have a hemochorial placenta, and their relatively short gestation and large litters are advantageous for performing high throughput screening of candidate therapeutics for safety and efficacy. preclinical behavioral testing of rodent offspring has proven to be a promising avenue for identifying and predicting adverse effects associated with prenatal drug exposure in children. both rodent and rabbit models have been instrumental in testing teratogenic effects of artemisinin-based combination therapies for treating malaria in pregnant women. these studies concluded that drug-related teratogenic effects are limited to the first trimester, which supports the world health organization (who) recommendation that artemisinin may be administered only during the second or third trimester in pregnant women. , one limitation of mouse and rat models, however, is their inability to recapitulate certain elements of human congenital disease. for instance, because murine cmv is not transmitted vertically as it is in humans, other animal models, including guinea pigs and nonhuman primates, are required for studying this aspect of disease pathogenesis. studies in pregnant nonhuman primates have been instrumental for the identification of cd + t cell responses as critical for early control of cmv infection and transmission during pregnancy, and studies in guinea pigs have demonstrated that a single-cycle infectious cmv vaccine induces immune responses similar to natural infection and protects against congenital infection. guinea pigs are also a useful model of chlamydial genital infection in humans. experimental venereal infection with chlamydophila caviae mimics disease associated with c. trachomatis in humans, including both sexual and perinatal transmission. guinea pigs have therefore served as a useful model for testing candidate vaccines and treatments. rabbits continue to serve as an important model of venereal infection with treponema pallidum, the causative agent of syphilis, which is associated with congenital disease in humans. while natural infection in rabbits is associated with the species-specific t. paraluiscanuculi, rabbits can be experimentally inoculated with human t. pallidum, and have been instrumental in testing the efficacy of candidate vaccines. many mammalian species, including rodents, ruminants, and nonhuman primates, , are susceptible to infection with listeria monocytogenes and demonstrate similar fetal complications when infection occurs during pregnancy. studies in various animal models have uniquely contributed to our understanding of placental listeriosis and serve as a platform for evaluating prevention strategies. , finally, mice, cotton rats, guinea pigs, and sheep are all susceptible to infection with rsv, and vaccination of pregnant animals has facilitated the development and testing of maternal immunization strategies for protecting against neonatal rsv. based on preliminary studies in guinea pigs, an experimental rsv recombinant f nanoparticle vaccine is now being evaluated in third-trimester pregnant women (clintrials.gov, nct ). beyond the direct modeling of human congenital infection in animals, information can also be gained from the study of related veterinary pathogens. for example, bovine viral diarrhea virus (bvdv) is an important reproductive pathogen that infects cattle worldwide, and infection during pregnancy causes congenital infection and disease. persistently infected animals serve as reservoirs within a herd and can have a huge agricultural financial impact. as a result, significant resources have been dedicated to the development and optimization of bvdv vaccines and vaccine protocols, considering variables such as the type and timing of vaccination on immune response and protection against challenge. the information gleaned from these studies may inform vaccine development and optimization protocols for related pathogens in pregnant women, for which similar studies cannot ethically be performed. zika virus (zikv) is a unique flavivirus that causes mild or subclinical disease in pregnant women, , but can have devastating effects for the fetus and neonate. infection during pregnancy is linked with spontaneous abortion and a variety of birth defects, including microcephaly and impaired neurocognitive function. since its initial discovery in african macaques in , zikv has expanded its geographical range and evolved into separate virus lineages, with environmental pressures resulting in the emergence of virulent substrains, raising concerns about vaccine escape mutants once a vaccine is approved. the combination of its unique pathogenesis, diverse modes of transmission, and rapid global spread has increased efforts toward development and licensing of a zikv vaccine. a basic understanding of zikv biology and pathogenesis is essential for development of an effective vaccine against zikv. although we can extrapolate some biological information from related flaviviruses, such as yellow fever, west nile, and dengue viruses, zikv has unique characteristics following in vivo infection, which pose significant challenges to vaccine design. unlike other flaviviruses, zikv has a tropism for reproductive tissues, including the testes, semen, and sperm in males , and the placenta in pregnant females, [ ] [ ] [ ] which is hypothesized to contribute to sexual and vertical modes of transmission, respectively. in addition to unique tissue tropisms, zikv persists in reproductive tissue following clearance of systemic viremia. in males, zikv rna can be detected for months following recovery from symptomatic infection, , and virus persistence in the placenta of pregnant women is hypothesized to contribute to prolonged viremia in this population. evidence of virus persistence suggests that zikv may have evolved mechanisms for evasion of host immune responses when infection occurs in certain immune-privileged tissues. scenarios involving persistent zikv infections should be considered in developing and testing candidate vaccines. considering the potential for viral persistence in the semen, vaccinating men may serve as an additional strategy to reduce transmission to the fetus, as pregnant women may be infected by their sex partners. characterization of the immune response to zikv infection is essential for determining correlates of protection for vaccine efficacy. following infection of both humans and nonhuman animals, zikv induces neutralizing antibodies against the zikv e protein, which prevent fetal infection and demise when administered to pregnant mice. further, mhc class i epitopes have been identified that conferred protection against zikv challenge in immunocompetent mice. to date, candidate zikv vaccines have been developed, and as of february , nine have entered phase i clinical trials. vaccine candidates have been developed using diverse platforms, including dna, mrna, and purified inactivated and live-attenuated virus, many of which have been tested in non-pregnant mouse and nonhuman primate models for their ability to generate immune responses that mimic responses to natural infection and protected against zikv challenge. a candidate dna plasmid vaccine induced robust cellular immunity and neutralizing antibody responses in both nonhuman primates and immunocompetent mice, and conferred complete protection against lethal zikv challenge in type i interferon receptor deficient (ifnar −/−) mice. lnp-mrna vaccines induced similar protective immunity, which was characterized by high neutralizing antibody titers and sterilizing immunity against zikv challenge in non-pregnant mice and nonhuman primates. , whether these candidate vaccines induce protective immunity in pregnant females that is sufficient to prevent fetal and neonatal infections requires further evaluation. also, whether pre-existing immunity to other flaviviruses that co-circulate with zikv, including dengue virus and west nile virus, affects the efficacy of zikv vaccines in pregnant females should be considered in both preclinical animal models and human clinical trials. conventional vaccines are formulated from either live, attenuated pathogen strains or from inactivated pathogens, but there are notable disadvantages to each of these platforms. live, attenuated vaccines are replication-competent with the potential of becoming virulent and causing adverse effects in individuals with weakened immune systems. due to the unknown risk to the developing fetus, live virus vaccines are not recommended for use in pregnant women. inactivated vaccines, on the other hand, are not associated with a risk of reacquisition of virulence, but they tend to induce a weaker host immune response. efforts to balance safety with immunogenicity have led to the development of several novel vaccine technologies, including replicationdeficient nanoparticle-based vaccines and self-assembling recombinant virus-like particles (vlps), replication-competent recombinant viral vectors, and single-cycle infectious viruses that can infect, but not replicate in host cells. nanoparticle delivery platforms, including liposomes and synthetic polymers, can be engineered to enhance selective tissue homing for high potency, targeted delivery of antigen in its native conformation. recombinant vlps combine highly immunogenic surface viral proteins with encapsulated adjuvants that are devoid of infectious nucleic acid, but induce strong cellular and humoral immune responses. both nanoparticle and vlp vaccines are devoid of genetic material and are therefore replication incompetent, enhancing safety in vulnerable individuals. although current production yields and costs associated with these technologies may prohibit large-scale use, these platforms may be well-suited for targeted use in high-risk populations, including pregnant women. since the first nanoparticle-based vaccine was licensed for hepatitis b virus in , the technology has been applied to develop licensed vaccines against human papilloma virus, hepatitis e virus, and malaria. demonstration of safety and efficacy during pregnancy has not yet been documented. recombinant viruses can be engineered to combine the antigenic genes of one virus with the structural genes of another. this targeted manipulation of the virus genome is used to remove virulence genes to enhance safety and alter envelope proteins to change cell tropism. recombinant viruses can be engineered to retain the ability to infect and replicate in the host, while preserving infectious potential and enhancing the generation of innate and adaptive immune responses that mimic natural infection. recombinant virus vaccines, however, warrant careful consideration of the safety of the vector itself, especially in pregnant women. once the safety and efficacy profiles of a viral vector platform have been established, engineering new antigenic targets into the viral genome are relatively simple, and do not require extensive re-validation, as safety and efficacy is most influenced by the vector virus. this can significantly reduce the time to develop and manufacture vaccines against new viral pathogens. viruses from many different families can be used as vectors provided they can infect the host and elicit a productive immune response without causing disease. of note, poxviruses are practical vectors due to ease of growth and manipulation in vitro, wide host range, and robust induction of protective immune responses. although vaccinia virus vectors are contraindicated during pregnancy due to risk of disseminated disease, recent testing of a raccoonpoxvirus-vectored rabies vaccine in pregnant mice proved safe and effective. other novel vaccine platforms are based on genetic engineering and creation of targeted loss-of-function mutations in the viral genome. reverse genetics technology has contributed the identification of the sequences within the viral genomes that are essential for infection and replication in vivo. it is now possible to engineer recombinant virus vaccines with targeted mutations in a cost-and time-efficient manner. in contrast to inactivated vaccines, reverse genetics allows for controlled manipulation of the viral genome that targets a specific process in the virus life cycle. this enables production of a vaccine strain with maximum efficacy and safety, and represents another promising alternative to conventional attenuated or killed virus vaccines. among the safest recombinant vaccine approaches are the single-cycle infectious (sci) viruses, which have been developed from influenza a virus (iav) backbones by deleting or truncating viral proteins necessary for completion of the virus life cycle within the host. such genetic modifications render the virus replication-incompetent, but capable of infecting and inducing an immune response in the host. the infectious capacity of sciiav vaccines results in strong cellular and humoral immune responses, without the risks and adverse effects associated with live-attenuated vaccine strains. studies in nonpregnant mice have demonstrated that a single dose of sciiav vaccine confers protection against heterosubtypic lethal challenge without any adverse effects. [ ] [ ] [ ] [ ] [ ] [ ] similar safety and efficacy profiles of sciiav have been replicated in ferret and pig models, , but studies in pregnant animals have not been performed. compared with the risks associated with live virus vaccination and concerns of efficacy with inactivated vaccines, novel vaccine platforms, such as nanoparticle-based technologies, vlps and replication-deficient viruses have proven benefits. additional safety and efficacy studies in pregnancy models are warranted to validate and expand the use of these vaccine platforms for pregnant women. (table ) conclusions strategic immunization of women, either prior to or during pregnancy, can eliminate or substantially reduce the risk of maternal, fetal, and neonatal infection and disease. the effectiveness of an immunization protocol depends on both the efficacy of the vaccine in inducing protective immune responses and on the timing of vaccine delivery during pregnancy to synchronize the peak vaccine response with the period of greatest susceptibility in the host. optimization of vaccination protocols to achieve this goal requires an understanding of the mechanisms of infection and pathogenesis of disease during pregnancy. successful implementation of vaccine protocols for pregnant women requires consideration of additional challenges, such as the frequency of unplanned pregnancies and access to prenatal health care. human surveillance data provide correlative clues of the character of specific infections, but mechanistic understanding requires additional study in comparative animal model systems. pregnancy and pregnancy-associated hormones alter immune responses and disease pathogenesis incidence and severity of viral hepatitis in pregnancy clinical course and outcome of sporadic acute viral hepatitis in pregnancy hepatitis e virus chickenpox pneumonia: an association with pregnancy h n influenza virus infection during pregnancy in the usa maternal influenza infection causes marked behavioral and pharmacological changes in the offspring maternal influenza infection is likely to alter fetal brain development indirectly: the virus is not detected in the fetus australasian maternity outcomes surveillance, s. critical illness due to a/h n influenza in pregnant and postpartum women: population based cohort study impaired type i and iii interferon response to rhinovirus infection during pregnancy and asthma report of a controlled study during an outbreak of asian influenza antibody response to monovalent a/new jersey/ / influenza vaccine in pregnant women influenza vaccination of pregnant women protects them over two consecutive influenza seasons in a randomized controlled trial seropositivity of influenza a h ni in mothers and infants following maternal vaccination with trivalent seasonal influenza vaccine after the pandemic immunogenicity of trivalent inactivated influenza vaccination received during pregnancy or postpartum prevention and control of influenza. recommendations of the advisory committee on immunization practices (acip) adverse events in pregnant women following administration of trivalent inactivated influenza vaccine and live attenuated influenza vaccine in the vaccine adverse event reporting system trivalent inactivated influenza vaccine and spontaneous abortion monovalent h n influenza vaccine safety in pregnant women, risks for acute adverse events inactivated influenza vaccine during pregnancy and risks for adverse obstetric events maternal outcomes among pregnant women receiving live attenuated influenza vaccine & prevention. influenza vaccination coverage among pregnant women ---united states, - influenza season maternal influenza vaccination: evaluation of a patient-centered pamphlet designed to increase uptake in pregnancy varicella-zoster virus (chickenpox) infection in pregnancy seroprevalence of varicella in the french population centers for disease control and prevention pathogenesis and current approaches to control of varicella-zoster virus infections what does epidemiology tell us about risk factors for herpes zoster? hepatitis e and pregnancy: current state vertical transmission of hepatitis e virus sero-prevalence and mother-to-infant transmission of hepatitis e virus among pregnant women in the united arab emirates fetal and neonatal health consequences of vertically transmitted hepatitis e virus infection efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase trial lessons from hepatitis e vaccine design enhanced humoral response in pregnant mice immunized with liposome encapsulated recombinant neutralizing epitope protein of hepatitis-e virus risks associated with viral infections during pregnancy evaluation of pregnant women exposed to respiratory viruses do the placental barrier, parasite genotype and toll-like receptor polymorphisms contribute to the course of primary infection with various toxoplasma gondii genotypes in pregnant women? listeriosis in human pregnancy: a systematic review acute fetal demise with first trimester maternal infection resulting from listeria monocytogenes in a nonhuman primate model general recommendations on immunization ---recommendations of the advisory committee on immunization practices (acip) impact of birth rate, seasonality and transmission rate on minimum levels of coverage needed for rubella vaccination prevention of measles, rubella, congenital rubella syndrome, and mumps, : summary recommendations of the advisory committee on immunization practices (acip) surveillance for congenital rubella in australia since : cases reported between & prevention. elimination of rubella and congenital rubella syndrome--united states the "silent" global burden of congenital cytomegalovirus review and meta-analysis of the epidemiology of congenital cytomegalovirus (cmv) infection seroprevalence of cytomegalovirus among children to years of age in the united states from the national health and nutrition examination survey of vaccine development to prevent cytomegalovirus disease: report from the national vaccine advisory committee desirability and feasibility of a vaccine against cytomegalovirus latency and reactivation of human cytomegalovirus maternal immunity and prevention of congenital cytomegalovirus infection human cytomegalovirus (hcmv) replication dynamics in hcmv-naive and -experienced immunocompromised hosts strain variation and disease severity in congenital cytomegalovirus infection: in search of a viral marker cytomegalovirus (cmv)-encoded ul (truncated tumor necrosis factor receptor) and outcome of congenital cmv infection prospects of a vaccine for the prevention of congenital cytomegalovirus disease management of hepatitis b virus infection during pregnancy hepatitis b immunisation for newborn infants of hepatitis b surface antigen-positive mothers cesarean section to prevent motherto-child transmission of hepatitis b virus in china: a meta-analysis a comprehensive immunization strategy to eliminate transmission of hepatitis b virus infection in the united states: recommendations of the advisory committee on immunization practices (acip) part ii: immunization of adults persistence of antibodies y after vaccination with a combined hepatitis a and b vaccine role of innate host defenses in susceptibility to early-onset neonatal sepsis global, regional, and national causes of child mortality: an updated systematic analysis for with time trends since evolution of maternofetal transport of immunoglobulins during human pregnancy dynamics of immunoglobulins at the feto-maternal interface placental transfer of immunoglobulin g subclasses placental transfer of antibody and its relationship to vaccination in pregnancy placental transfer favours high avidity igg antibodies half-life of the maternal igg allotype in infants decay of passively acquired maternal antibodies against measles, mumps, and rubella viruses maternal immunization with tetanusdiphtheria-pertussis vaccine: effect on maternal and neonatal serum antibody levels effect of a prepregnancy pertussis booster dose on maternal antibody titers in young infants updated recommendations for use of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (tdap) in pregnant women--advisory committee on immunization practices (acip), . mmwr immunogenicity and reactogenicity of co-administered tetanus-diphtheria-acellular pertussis (tdap) and tetravalent meningococcal conjugate (mcv ) vaccines compared to their separate administration importance of timing of maternal combined tetanus, diphtheria, and acellular pertussis (tdap) immunization and protection of young infants infant outcomes after exposure to tdap vaccine in pregnancy: an observational study coverage with tetanus, diphtheria, and acellular pertussis vaccine and influenza vaccine among pregnant women -minnesota neonatal tetanus: a continuing challenge maternal and neonatal tetanus weekly epidemiological record tetanus toxoid immunization to reduce mortality from neonatal tetanus respiratory syncytial virus and parainfluenza virus maternal effects of respiratory syncytial virus infection during pregnancy clinical presentation and birth outcomes associated with respiratory syncytial virus infection in pregnancy maternal antibodies by passive immunization with formalin inactivated respiratory syncytial virus confer protection without vaccineenhanced disease preclinical assessment of safety of maternal vaccination against respiratory syncytial virus (rsv) in cotton rats maternal immunization with respiratory syncytial virus fusion protein formulated with a novel combination adjuvant provides protection from rsv in newborn lambs modeling maternal fetal rsv f vaccine induced antibody transfer in guinea pigs burden of paediatric respiratory syncytial virus disease and potential effect of different immunisation strategies: a modelling and cost-effectiveness analysis for england strategic priorities for respiratory syncytial virus (rsv) vaccine development pregnancy outcomes following rubella vaccination: a prospective study in the state of rio de janeiro, brazil & campinas group on yellow fever immunization during, p. the effects of yellow fever immunization ( dd) inadvertently used in early pregnancy during a mass campaign in brazil should expectant mothers be vaccinated against flu? a safety review comparative developmental anatomy of the murine and human definitive placentae comparative systems biology of human and mouse as a tool to guide the modeling of human placental pathology animal models to study placental development and function throughout normal and dysfunctional human pregnancy risk mitigation for children exposed to drugs during gestation: a critical role for animal preclinical behavioral testing antimalarial drugs in pregnancy: a review embryotoxicity of the artemisinin antimalarials and potential consequences for use in women in the first trimester maternal cd +t cells protect against severe congenital cytomegalovirus disease in a novel nonhuman primate model of placental cytomegalovirus transmission a novel non-replication-competent cytomegalovirus capsid mutant vaccine strategy is effective in reducing congenital infection the guinea pig as a model of infectious diseases syphilis: using modern approaches to understand an old disease listeriosis in the pregnant guinea pig: a model of vertical transmission experimental infection of pregnant ewes with listeria monocytogenes nonhuman primate model for listeria monocytogenesinduced stillbirths understanding how listeria monocytogenes targets and crosses host barriers conjugated action of two species-specific invasion proteins for fetoplacental listeriosis modelling the spread of bovine viral diarrhea virus (bvdv) in a beef cattle herd and its impact on herd productivity efficacy of bovine viral diarrhea virus vaccination to prevent reproductive disease: a meta-analysis zika virus outbreak on yap island, federated states of micronesia rapid spread of emerging zika virus in the pacific area congenital zika virus syndrome in brazil: a case series of the first livebirths with complete investigation mutational pressure in zika virus: local adar-editing areas associated with pauses in translation and replication zika virus in semen and spermatozoa zika virus infection damages the testes in mice intrauterine zika virus infection of pregnant immunocompetent mice models transplacental transmission and adverse perinatal outcomes zika virus infects human placental macrophages zika virus targets different primary human placental cells, suggesting two routes for vertical transmission presence and persistence of zika virus rna in semen potential sexual transmission of zika virus zika virus rna replication and persistence in brain and placental tissue neutralizing human antibodies prevent zika virus replication and fetal disease in mice analysis of the t cell response to zika virus and identification of a novel cd +t cell epitope in immunocompetent mice world health, o. who vaccine pipeline tracker in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunisation with a prmenv dnavaccine modified mrna vaccines protect against zika virus infection zika virus protection by a single low-dose nucleoside-modified mrna vaccination a dtap-ipv//prp approximately t vaccine (pentaxim): a review of years' clinical experience beyond antigens and adjuvants: formulating future vaccines harnessing nanoparticles for immunomodulation and vaccines novel vaccine strategies against emerging viruses raccoonpoxvirus safety in immunocompromised and pregnant mouse models replication-defective viruses as vaccines and vaccine vectors reverse genetics approaches for the development of influenza vaccines protection against lethal influenza with a viral mimic a replication-incompetent virus possessing an uncleavable hemagglutinin as an influenza vaccine pseudotyped influenza a virus as a vaccine for the induction of heterotypic immunity pb amino acid at position affects replicative efficiency, but not cell tropism, of hong kong h n influenza a viruses in mice a novel bivalent vaccine based on a pb -knockout influenza virus protects mice from pandemic h n and highly pathogenic h n virus challenges a replicationincompetent pb -knockout influenza a virus vaccine vector an eight-segment swine influenza virus harboring h and h hemagglutinins is attenuated and protective against h n and h n subtypes in pigs pertussis antibody transfer to preterm neonates after second-versus third-trimester maternal immunization chlamydia trachomatis infection in pregnancy: the global challenge of preventing adverse pregnancy and infant outcomes in sub-saharan africa and asia perinatal infections and fetal/neonatal brain injury prevention of perinatal group b streptococcal disease. revised guidelines from cdc microbial vertical transmission during human pregnancy current perspectives on prevention of mother-to-child transmission of syphilis syphilis infection during pregnancy: fetal risks and clinical management diagnosing congenital malaria in a high-transmission setting: clinical relevance and usefulness of p. falciparum hrp -based testing treating severe malaria in pregnancy: a review of the evidence toxoplasmosis in pregnancy assessment of laboratory methods used in the diagnosis of congenital toxoplasmosis after maternal treatment with spiramycin in pregnancy neuropathogenesis of congenital cytomegalovirus infection: disease mechanisms and prospects for intervention congenital cytomegalovirus infection following first trimester maternal infection: symptoms at birth and outcome cytomegalovirus neutralization by hyperimmune and standard intravenous immunoglobulin preparations what obstetrician-gynecologists should know about ebola: a perspective from the centers for disease control and prevention ebola viral disease and pregnancy live neonates born to mothers with ebola virus disease: a review of the literature herpes simplex virus and epstein-barr virus infections in pregnancy: consequences of neonatal or intrauterine infection prospects and perspectives for development of a vaccine against herpes simplex virus infections incident hiv during pregnancy and postpartum and risk of mother-to-child hiv transmission: a systematic review and meta-analysis immunology of pediatric hiv infection parvovirus b infection in human pregnancy consequences of confirmed maternal rubella at successive stages of pregnancy severe acute respiratory syndrome (sars) in neonates and children sars during pregnancy, united states microbiology laboratory and the management of mother-child varicella-zoster virus infection initial description of the presumed congenital zika syndrome the authors thank the johns hopkins fisher center discovery program for funding. all authors (m.s.v. and s.l.k.) were involved in the conception and drafting of this review, and approved the manuscript before it was submitted by the corresponding author. competing interests: the authors declare no competing financial interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -nbtkl cx authors: clemente, nuria sanchez; ramond, anna; turchi martelli, celina maria; brickley, elizabeth b. title: geographies of risk: emerging infectious diseases and travel health data date: - - journal: travel med infect dis doi: . /j.tmaid. . sha: doc_id: cord_uid: nbtkl cx nan the battles against these novel threats. not only do travel medicine practitioners provide counsel in the context of the imperfect and evolving information that accompanies epidemics, but also, they are often the first port of call when travellers return to their home countries with medical concerns. reflecting on the findings of petridou and colleagues, describing imported zikv cases to the uk between - , confirmed at the rare and imported pathogens laboratory, we look back to the - zika virus (zikv) pandemic and reflect on some of the opportunities and limitations presented by data obtained from returning travellers in enhancing understanding of emerging infectious diseases. given travellers' well-defined temporal windows of potential exposure, improved recollections of risk behaviors, and access to well-resourced travel clinic laboratories, travel health data are uniquely positioned to provide insights into the pathogenesis of emergent infectious diseases. in the case of zikv, case studies of pregnant travellers provided early evidence that asymptomatic maternal zikv infections can also result in congenital malformations. data from a traveller returning to an ecological setting that does not support zikv vectors, elucidated sexual contact as a new route of transmission. there are, however, noteworthy limitations to using travellers as sentinels for emerging infectious diseases. first, we can assume neither homogenous mixing between travellers and locals nor equivalent risks of exposure to pathogens. considering zikv, travellers' geographic footprint may be limited to tourist sites with enhanced vector control measures limiting the prevalence of aedes spp. mosquitoes. travellers may practice temporarily enhanced preventative behaviours, such as the daily use of insecticides, which may be inaccessible to resident populations. second, travellers and locals may have differing age distributions and underlying risk factors that could modify the clinical severity or complications of the resulting disease. third, travellers may display more health-seeking behaviour traits, be more likely to seek testing for minor symptoms, and have better access to testing laboratories. fourth, travellers and locals may differ with respect to their immunological experiences in ways that could modify testing outcomes. for instance, prior exposure to dengue virus can lead to immunological cross-reactivity that can compromise the specificity of serological testing for zikv. for all these and other reasons, it is therefore important, where possible, for studies utilizing travel data to contextualize and validate their findings with on-the-ground epidemiological investigations led by resident country experts. the sharing of data, knowledge, and expertise between travel medicine specialists and professionals working in areas with active transmission is mutually beneficial and of paramount importance for protecting the public's health in all countries. during outbreaks of emerging infectious diseases, local clinicians and epidemiologists play a critical role in describing the novel features, risk factors, and transmission patterns for emerging infectious diseases. when the epidemic of microcephaly was first identified in northeast brazil, local teams provided the first clinical descriptions of the novel congenital zika syndrome and undertook epidemiological studies that provided robust evidence of zikv as the etiological agent. while travel health data has the opportunity to build on this foundation and provide novel insights about emerging infectious agents, the fastest progress will be made through meaningful bi-directional international partnerships built on respectful collaboration, commitments to capacity building, and cooperative efforts to bolster surveillance. as evidenced by the zikv and covid- pandemics, we are in a new era of emerging infections, rapid research, and potential international partnerships. now more than ever, travel data and databases are becoming invaluable resources in the early stages of outbreak investigations and for on-going support of local surveillance efforts in affected areas. by working together across our shared geographies of risk, we will be best prepared to confront, contain, and mitigate the impact of emerging infectious disease pandemics. i have no competing interests to declare all sources of funding should also be acknowledged and you should declare any involvement of study sponsors in the study design; collection, analysis and interpretation of data; the writing of the manuscript; the decision to submit the manuscript for publication. if the study sponsors had no such involvement, this should be stated. travel medicine and infectious disease requires that all authors sign a declaration of conflicting interests. if you have nothing to declare in any of these categories then this should be stated. a conflicting interest exists when professional judgement concerning a primary interest (such as patient's welfare or the validity of research) may be influenced by a secondary interest (such as financial gain or personal rivalry). it may arise for the authors when they have financial interest that may influence their interpretation of their results or those of others. examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. all sources of funding should also be acknowledged and you should declare any involvement of study sponsors in the study design; collection, analysis and interpretation of data; the writing of the manuscript; the decision to submit the manuscript for publication. if the study sponsors had no such involvement, this should be stated. zika virus infection in travellers returning to the united kingdom during the period of the outbreak in the americas ( - ): a retrospective analysis vital signs: update on zika virus-associated birth defects and evaluation of all u.s. infants with congenital zika virus exposure -u.s. zika pregnancy registry probable non-vector-borne transmission of zika virus clinical infectious diseases : an official publication of the infectious diseases society of sentinel surveillance in travel medicine: years of geosentinel publications ( - ) travel surveillance and genomics uncover a hidden zika outbreak during the waning epidemic travel medicine and infectious disease requires that all authors sign a declaration of conflicting interests. if you have nothing to declare in any of these categories then this should be stated. a conflicting interest exists when professional judgement concerning a primary interest (such as patient's welfare or the validity of research) may be influenced by a secondary interest (such as financial gain or personal rivalry). it may arise for the authors when they have financial interest that may influence their interpretation of their results or those of others. examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. none. all sources of funding should also be acknowledged and you should declare any involvement of study sponsors in the study design; collection, analysis and interpretation of data; the writing of the manuscript; the decision to submit the manuscript for publication. if the study sponsors had no such involvement, this should be stated. key: cord- -h ixhhzz authors: yuan, shuofeng; chu, hin; huang, jingjing; zhao, xiaoyu; ye, zi-wei; lai, pok-man; wen, lei; cai, jian-piao; mo, yufei; cao, jianli; liang, ronghui; poon, vincent kwok-man; sze, kong-hung; zhou, jie; to, kelvin kai-wang; chen, zhiwei; chen, honglin; jin, dong-yan; chan, jasper fuk-woo; yuen, kwok-yung title: viruses harness yxxØ motif to interact with host ap m for replication: a vulnerable broad-spectrum antiviral target date: - - journal: sci adv doi: . /sciadv.aba sha: doc_id: cord_uid: h ixhhzz targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current covid- . here, we found a common yxxØ-motif of multiple viruses that exploits host ap m for intracellular trafficking. a library chemical, n-(p-amylcinnamoyl)anthranilic acid (aca), was identified to interrupt ap m -virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza a viruses (iavs), zika virus (zikv), human immunodeficiency virus, and coronaviruses including mers-cov and sars-cov- . yxxØ mutation, ap m depletion, or disruption by aca causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of iav nucleoprotein and diminished endoplasmic reticulum localization of zikv-ns and enterovirus-a - c proteins, thereby suppressing viral replication. our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target. virus-host interactions drive mutual evolutionary changes which result in the marked diversification of viruses and host antiviral responses ( ) . the emergence of viruses including coronaviruses [severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome-related coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov- )], hiv, zika virus (zikv), avian influenza a (h n ), a (h n ), and pandemic influenza a (pdmh n ) viruses and enteroviruses [enterovirus a (ev-a ) and ev-d ] emphasizes the need to investigate for a conserved and broadly shared mechanism of virus-host interaction with potential therapeutic implications ( ) ( ) ( ) ( ) . viruses live briefly but perpetually. they invade cells and manipulate host machinery to replicate, transmit, and cause disease. host signal transduction in response to virus invasion activates transcription factors that determine the gene expression characteristics and signaling mechanisms of the cell fate. these signaling mechanisms have been well studied in the fields of embryonic development and cancer biology but less well studied in the context of virus infection ( ) . a small set of evolutionarily conserved signaling pathways is associated with the cell fates of apoptosis, differentiation, or virus elimination during virus-host interactions. they include the transforming growth factor- (tgf-)/smad, wnt/-catenin, notch, and phosphatidylinositol -kinase/thymoma viral proto-oncogene (pi k/akt) signaling pathways. here, we show that tgf- signaling is commonly altered by vastly different viruses. modulating the tgf- pathway is primarily mediated by mislocalization of tgf- cytokines, its receptors tgf-r, and smad transcriptional factors, which are largely executed through membrane and intracellular trafficking pathways ( ) . therefore, we hypothesize that one approach to determine the cell fate, to die or to support virus replication, is through virusmanipulated membrane and intracellular trafficking. in this study, we performed integrative chemical and genetic screens that identified host ap m protein as a critical player affecting tgf- signaling and facilitating intracellular trafficking of different viruses. ap m is the mu ( ) subunit of ap adaptor complex, which functions as the major heterotetramer (,  ,  , and  subunits) that orchestrate clathrin-mediated endocytosis (cme) ( ) . the direct binding between tgf-r and ap has been demonstrated in vitro and in vivo ( ) . functionally, ap m recognizes the yxxØ sorting motifs present in the cytosolic tail of different cargo proteins, whereas x refers to any amino acid and Ø indicates hydrophobic residues including l/m/f/i/v ( ) . here, we identify a previously unrecognized role of ap m , which is an intracellular cargo molecule that cotraffics with different internal viral proteins for proper subcellular localizations, in addition to its role in endocytosis. the cotrafficking is mediated through protein-protein interaction (ppi) between host ap m and specific viral proteins harboring a yxxØ motif. our initial pharmacological screening identifies a tool compound, n-(p-amylcinnamoyl)anthranilic acid (aca), that disrupts ap m /yxxØ interaction without affecting the ap m phosphorylation. aca exhibits broad-spectrum antiviral efficacy in cell cultures and mouse models. substitutions made in the influenza a nucleoprotein yxxØ motifs affect viral fitness in vitro and in vitro, indicating a critical role of ap m /yxxØ interaction during virus life cycle. our study reveals ap m /yxxØmediated intracellular trafficking of diverse virus families, which represents a previously unidentified intervention target for a broad spectrum of emerging viral diseases. to determine the virus-induced signaling associated with cell fate pathways, we examined whether virus infection could potentiate host cell signaling by tgf-, wnt, notch, and pi k/akt pathways. reporter gene assays with high multiplicity of infection (moi) and time-course monitoring of luciferase activity were performed in gene-transfected human primary cells including the influenza a pdmh n -infected human primary bronchial/tracheal epithelial cells (hbtecs), zikvinfected human primary fibroblasts hfl and ev-a -infected human neural progenitor cells. tgf- signaling exhibited distinct patterns of marked changes when compared with the marginally changed wnt, notch, and pi k/akt pathways (fig. a) . infection by either pdmh n or zikv triggered tgf- activation, whereas ev-a infection suppressed its signaling, indicating that tgf- signaling is commonly exploited in the cell fate determination pathway by different rna viruses. proper control of tgf-r via membrane and intracellular trafficking is documented to mediate tgf- signaling ( ) . to explore the dependence of viral fitness on these host trafficking genes, we performed a loss-of-function screen to determine the degree of pdmh n replication after individual gene silencing. among the cellular trafficking genes, knockdown of genes reduced virus titers by more than one-log unit ( fig. b and fig. s ). of the influenza a virus (iav) screen hits, had been previously implicated in hiv- assembly, release, and budding: adaptor related protein complex subunit mu (ap m ), caveolin (cav ), ras-related protein rab- a (rab a), and rho-associated protein kinase (rcok ) ( ); depletion of copa or copb profoundly restricted human cytomegalovirus replication ( ) ; copg and ap m were demonstrated to be important for iav production in a genome-wide small interfering rna (sirna) screening ( ) . the results suggested that the selected membrane trafficking genes may serve as proviral factors with broad relevance to a number of virus infections. in view of the vulnerability of intracellular trafficking within virus life cycle, we screened a small-molecule compound library of trafficking inhibitors for finding drug hits with promising antiviral potency, which may also serve as a tool compound enabling the identification and characterization of putative targets within intracellular trafficking pathways. the library consists of inhibitors targeting membrane transporters such as p-glycoprotein, exportin , and ion channels including cystic fibrosis transmembrane conductance regulator, proton pump, calcium pump, etc. multiple rounds of selection were performed using cell protection (supplementary data sheet) and viral load reduction assays, which identified hits that suppressed virus replication for > logs at m and another candidates achieving > logs inhibition at m (table s ). to prioritize these five compounds, we evaluated their antiviral efficacy against other emerging viruses and identified aca as the only inhibitor that exhibited a broad-spectrum antiviral effect against influenza a h n , zikv, hiv- , sars-cov- , ev-a , human adenovirus (adv ), and severe fever with thrombocytopenia syndrome virus (sftsv) (fig. c and fig. s a ). the tool compound aca is a broad-spectrum antiviral in vitro, ex vivo, and in vivo the % cytotoxic concentration of aca ranged from to m in different cell lines, while its half maximal effective concentration (ec ) was at or below micromolar levels ( fig. s a ). aca ( m) potently suppressed sars-cov- replication for > logs in both supernatants and cell lysates in caco cells (ec = . m), indicating a good therapeutic potential for the current covid- pandemic ( fig. s b ). because aca displayed the highest selectivity index of against pdmh n infection, aca was tested against different iav subtypes in the subsequent antiviral evaluations. flow cytometry showed that the percentage of pdmh n -infected madin-darby canine kidney (mdck) cells after m aca treatment decreased by . % at hours post-infection (hpi) ( fig. a ). aca exhibited cross-protection against h n , h n , h n , and h n in a dose-dependent manner (fig. b) . notably, aca treatment reduced supernatant viral titer by > logs hbtecs (fig. c ). using our previously established proximal differentiated threedimensional ( d) human airway organoids (aos) for predicting the infectivity of influenza viruses in humans ( ) , we confirmed that aca reduced virus replication by > logs (fig. d) , with markedly decreased expression of viral nucleoprotein (np) antigen (fig. e) . collectively, aca robustly inhibited iavs replication in vitro and ex vivo. to determine aca's toxicity, we intraperitoneally inoculated the maximal phosphate-buffered saline (pbs)-soluble aca ( . mg/kg) into balb/c mice for days. no body weight loss or decreased activity was observed for a consecutive monitoring of and days, respectively ( fig. s c ). to evaluate the in vivo antiviral protection of aca, we challenged mice with plaque-forming units (pfu) of mouse-adapted h n virus. all mice after a single intranasal (i.n.) dose of aca ( . mg/kg) survived (n = ), whereas all dimethyl sulfoxide (dmso)-treated and zanamivir-treated ( mg/kg) mice died (fig. f ). using the same experimental regimen, aca conferred substantial better survival against avian iav h n ( % versus %; fig. f ), notably less body weight loss (fig. g) , and undetectable lung tissue virus titers at days and after challenge (fig. h ). on days post-infection (dpi), histopathologic examination showed substantially less pulmonary alveolar damage and interstitial inflammatory infiltration in aca-treated mice (fig. i ). together, aca effectively protected mice challenged by two iav subtypes by reducing virus replication and pneumonia. the broad-spectrum antiviral activity of aca in cell cultures warrants further evaluation in other virus disease models (fig. ) . type i interferon receptor-deficient a mice were infected with zikv-pr (a strain of the zika virus originally isolated from a traveler to puerto rico) and treated with either aca ( mg/kg) or dmso by subcutaneous administration. mice receiving one dose of aca therapy showed a remarkably better survival rate ( % versus %) and mean body weight (fig. , a and b) . moreover, zikv titer was undetectable in the brains of aca-treated group, whereas that of dmso group was generally logs higher (fig. c ). less histopathologic changes of meningoencephalitis and zikv-ns antigen expression were observed (fig. d) . furthermore, intranasal aca ( . mg/kg, i.n.) provided good protection against lethal challenge with pfu of mers-cov in human dipeptidyl peptidase (hdpp )transgenic mice, whereas all dmso-treated mice died on or before day after challenge ( % versus %; fig. e ). body weight loss in the dmso-treated mice began since dpi, while that of the aca-treated group continued to increase (fig. f ). about logs lower lung tissue virus titer was detected in the aca group (fig. g) . inflammatory infiltration and mers-cov-np antigen expression in the lung tissues were substantially reduced after aca treatment (fig. h) . thus, aca also exhibited broad-spectrum antiviral efficacy in vivo. small-molecule compound library screening identified aca as a broad-spectrum antiviral in vitro. a membrane transporter/cellular trafficking library was primarily screened in pdmh n -infected madin-darby canine kidney (mdck) cells ( . moi and hpi) through cell protection assays (box , blue dots indicated > % cell viability), followed by secondary screening using viral load reduction assays (box , magenta dots indicated > logs of viral load reduction applying m drug concentration) and tertiary screening (box , yellow dots indicated > logs of viral load reduction using m drug concentration). aca was prioritized due to its broad-spectrum antiviral efficacy. shown in the last panel are the antiviral effects against six different viruses as indicated. shown are the plaque-forming unit (pfu) or % tissue culture infectious dose (tcid ) or od (optical density at nm) value of indicated concentrations relative to controls in the absence of compound (%). aca targets host ap m protein aca is known as a phospholipase a (pla ) inhibitor and transient receptor potential (trp) channel blocker ( ) , but such mechanisms for suppressing viral replication were excluded ( fig. s ). to explore its actual mechanism of antiviral action, we performed time-of-addition assays to investigate how aca interferes with different phases of the viral replication cycle. aca did not inactivate virus particles nor affect virus attachment to host cell surface ( fig. s ). to dissect the postvirus attachment steps, we quantified three types of influenza virus rna [vrna, complementary rna (crna), and mrna] at different time points after virus internalization. distinct dynamics of vrna, crna, and mrna synthesis were observed in the control groups, while all viral rna types in aca groups remained at the baseline level within to hpi of aca addition (fig. a) . the result suggested that aca functions within . hours after internalization before crna synthesis and therefore may inhibit virus endocytosis and nuclear import or blocking viral ribonucleoprotein (vrnps) activity directly. because lysotracker red assay indicated that the acidification of endosomal compartments was not affected, the uncoating of vrna for release into cytoplasm upon virus fusion was unlikely to be blocked by aca ( fig. s c ). hence, we directly tracked the location of the vrna within the incoming vrnps ( fig. b ). at indicated time points, cells were processed and stained for the negative-stranded np vrna of pr using a specific rna probe set (red). in the dmso-treated cells, the dominant nuclear import of vrna was detected by hpi, followed by predominant cytosol location between . and hpi. by comparison, the nuclear vrna signal was rarely observed after aca addition throughout the time course. therefore, aca may inhibit iav replication by blocking vrna nuclear import. to ascertain the target of aca, we developed an unbiased drug target-elucidating platform that integrates click-chemistry, waterlogsy, and protein id techniques using liquid chromatographytandem mass spectrometry (lc-ms/ms), namely, cwid (fig. , c and d). first, click-chemistry was applied to introduce an azido group to aca (azido-aca), without compromising its antiviral efficacy (fig. c, middle) . with an azido-reactive fluorescent dye (dylight ), azido-aca but not aca was visualized in both the cytosol and nucleus (fig. c , right). next, waterlogsy, a ligandbinding determination method through observing the nuclear overhauser effect, was applied for primary nuclear magnetic resonance (nmr) screening of cellular fragments that exhibited high binding affinity to aca. virus-infected mdck cells were fractionated by gel filtration chromatography, followed by waterlogsy to capture the aca-interacting signals, individually (fig. d , middle). iterative rounds of subfractionation and waterlogsy were performed to obtain the maximally separated fraction with detectable aca signals. subsequently, the bound form of fraction-azido-aca mixture was stained by dylight and subject to electrophoresis in a native polyacrylamide gel electrophoresis. a specific band indicating the (c) five mice in each group were euthanized at dpi, and brain tissues were harvested for vial titer determination by plaque assay. the dotted line indicates the lower detection limit of plaque assay (***p < . ; for the purpose of statistical analysis and clarity, a value of to pfu/ml was assigned for any titer below the detection limit). (d) histopathologic and immunohistochemistry (ihc) analyses of the brain samples at dpi indicated less severe meningitis (by h&e, × ) and less virus infected cells as indicated by zikv ns antigen staining (red arrows, by ihc, × magnification) after aca treatment. (e to h) aca protected human dipeptidyl peptidase (hdpp ) transgenic mice from mers-cov infection. the hdpp mice were intranasally inoculated with pfu of mers-cov and intranasally treated by aca or . % dmso for one dose starting hpi. shown are (e) survival rate, (f) mean body weight, (g) lung viral titer at dpi (n = per group), and (h) representative lung tissues stained by h&e and anti-mers-cov-np immunofluorescence. the staining suggested less inflammatory cell filtration (by h&e, × magnification) and less virus infected cell antigens (by immunofluorescence (if) staining, green fluorescence) as detected in aca-treated mouse lungs. results are presented as mean values ± sd. differences in survival rates were compared using log-rank (mantel-cox) tests and viral titer by student's t test. ***p < . , **p < . , *p < . . cells were fixed at the indicated time points and hybridized with rna probes against the iav negative-stranded np vrna (red) and stained for dna (blue), examined by confocal microscopy. images are representative of three independent experiments. scale bars, m. (c and d) click chemistry/waterlogsy/protein id (cwid) platform for identification of drug-binding targets. (c) click-chemistry: chemical structure of azido-aca showing the location of azido group (green circle) on aca. cellular distribution of azido-aca is shown (green), whereas aca was used as a negative control due to the lack of phosphine-reactive azido group. scale bars, m. (d) waterlogsy-guided cellular fractionation was subjected to analysis for aca-featured nmr spectra. "*" and "***" indicate mild and strong binding signals, respectively. the native polyacrylamide gel electrophoresis gel photo shows the selected cell fraction as detected by a fluorescent image analyzer. red arrow indicates the specific azido-aca-binding fragment. (e) mutagenesis analysis of ap m to rescue pdmh n virus replication against aca. full-length ap m (full), longin-like domain (lld), mhd, and mutant ap m were transfected to mdck cells before virus infection and aca treatment. oneway anova. **p < . ; n.s, not significant. (f) partial sequence alignment of human, mouse, and dog ap m is shown. n and k , the key residues for aca binding, are highlighted with a box. the predicted interaction surfaces on ap m (red) are shown, while aca (green) is displayed in stick and mesh representation. protein-compound complex was visualized in the azido-aca group, while it was absent in the aca or cell lysate-only group (fig. d , right). gel plug of the target band was collected for lc-ms/ms, which identified eight candidate proteins that were physically associated with azido-aca. to validate their biological function besides binding, individual open reading frame (orf) clone was overexpressed to overcome the inhibition of virus replication by aca. apparently, only ectopic expression of ap m notably rescued pdmh n growth despite the presence of aca, indicating ap m being one of the most likely targets that accounts for aca's mode of action ( fig. s ). ap m consists of an n-terminal (~ amino acids) longin-like domain (lld) and a c-terminal ( to amino acids) mu homology domain (mhd). functionally, we demonstrated that overexpression of either full-length ap m or mhd enhanced pdmh n replication for about . logs despite adding aca, whereas overexpressed lld did not antagonize aca's antiviral activity (fig. e , left bar charts). thus, mhd harbors sites of aca interaction. amino acid residues of mhd are conserved across the human, dog, and mouse ap m , which is in line with the broad-spectrum antiviral coverage of aca in different species of cells and mouse models. molecular docking predicts that aca interacts mainly with ap m through four amino acids, m , n , k , and k (fig. f ). our mutational experiment showed that substitution in n a or k a failed to antagonize the aca's antiviral activity when compared with that of the m a, k a, or wild-type (wt) ap m (fig. e , right bar charts). collectively, aca targets host ap m by interacting with its n and k residues. the antiviral spectrum of aca spans across enveloped (zikv) and nonenveloped (ev-a ), retro (hiv- ), and nonretro (iav), as well as dna (adv- ) and rna (mers-cov) viruses ( fig. s a ). thus, ap m must be broadly exploited during life cycles of many viruses. first, we excluded the possibility that aca affected phosphorylation of ap m ( fig. s ), which has been approved to be antiviral effective by bekerman et al. ( ) . subsequently, to find the specific virus protein interacting with host ap m , we performed an immunoprecipitation (ip) screening of viral orf clones. previous host-iav interactome suggested m , hemagglutinin (ha), pb , and np as the potential ap m -binding proteins without individual validation ( ) . after coexpression of each viral gene and ha-tagged ap m in human embryonic kidney (hek) t cells, only np could be detected in the ip complex, but adding aca markedly diminished the target np band ( fig. s a ). using the same approach, we identified the zikv-ns , mers-cov-np, and ev-a - c as the interacting partners of ap m ( fig. s , b to d). ap m is known to mediate sorting of cargo proteins harboring yxxØ or dileucine motifs ( ) . using bioinformatic methods, we found that yxxØ but not dileucine motif was consistently present in the implicated ap m -binding viral proteins. the yxxØ motifs are highly conserved in specific proteins across many virus families including the np of orthomyxoviridae, gag of retroviridae, np of bunyaviridae, ns of flaviviridae, np of coronaviridae, c of picornaviridae, and core protein v of adenoviridae by bioinformatics (fig. a ). to determine whether aca blocked the ap m yxxØbinding site, we developed a competitive enzyme-linked immunosorbent assay (elisa). as positive controls, addition of either nonlabeled yxxØ motif peptide dyqrln or the low-affinity mutant d a ap m ( ) resulted in diminished binding signal during binding of biotin-yxxØ substrate to the immobilized his-ap m . aca pretreatment notably reduced ap m /biotin-yxxØ interaction (fig. b ). to explore whether the yxxØ-binding pocket is a druggable target for antiviral therapy, we also tested tyrphostin a , which blocks the tyrosine-binding pocket of ap m ( ) . at nontoxic concentrations, tyrphostin a suppressed a panel of viruses including pdmh n , mers-cov, ev-a , and zikv (fig. c) . last, the effect of ap m gene depletion on viral replication was investigated. ap m knockout led to dramatic viral load reduction in pdmh n -infected t cells, ev-a -infected rhabdomyosarcoma (rd) cells, zikvinfected huh cells, and mers-cov-infected huh cells (fig. d) . together, ap m /yxxØ interaction is a critical step in the viral replication cycle and druggable for antiviral intervention. next, we asked the functional roles of ap m /yxxØ interaction during viral replication cycles using iav, ev-a , and zikv-ns as three representative viruses. nuclear import of iav np through the nuclear pore complex is a prerequisite for efficient vrnp translocation and subsequent genome replication, while our data clearly showed that aca impaired vrna nuclear import and ap m /np binding ( fig. b and fig. s a ). thus, we postulated that ap m facilitated np import from cytoplasm to the nucleus via recruiting the np-yxxØ motif. to quantify the retardation of np import in ap m −/− t cells, cells were infected with moi iav, and cycloheximide (chx) was added to inhibit protein synthesis. crude cell lysate was separated into nuclear (nuc) and cytoplasmic (cyto) fractions at hpi (fig. e ). in wt cells, ap m protein was mainly detected in cytoplasm, whereas considerably more viral nps were found in nucleus. however, in ap m −/− cells, viral np was predominantly found in the cytoplasmic fraction instead. since the only source of np protein was from the incoming vrnps after chx treatment, the finding suggested that ap m facilitates the nuclear import of incoming vrnps. to provide direct evidence for a role of ap m in mediating intracellular np trafficking that beyond endocytosis, we monitored the cotrafficking of np-green fluorescent protein (gfp) with ap m -mcherry using live cell imaging. gfp/mcherry signal was found largely in the host nucleus (movie s ). upon aca treatment, np resides predominantly in the cytosol, and the mobility of the ap m -associated np puncta (yellow) was reduced remarkably, which suggested diminished np trafficking via ap m (fig. f and movie s ). nevertheless, most positive-stranded rna viruses replicate their genomes on the cytoplasmic endoplasmic reticulum (er) membrane without entering the nucleus. for example, ev-a - c and zikv-ns proteins induce the formation of viral rna replication complex by trafficking predominantly to the er membrane ( ) . in the context of synchronized viral infection, we demonstrated by confocal imaging the extensive localization of iav-np and nucleus, so were the ev-a - c and zikv-ns in er, respectively. after aca treatment, however, reduced rates of colocalization were revealed in all three representative viruses ( % versus % for iav-np/nucleus; % versus % for ev-a - c/er; % versus % for zikv-ns /er; fig. g ). in summary, these results confirmed an important role of ap m in trafficking viral proteins beyond endocytosis. to bridge the ap m -mediated trafficking and virus replication, we rescued the recombinant iav with a series of point mutations on two np-yxxØ locations, i.e., y -v (yslv) and y -i (e) ap m −/− and wt t cells were treated with chx before virus infection ( moi). nuclear (nuc) and cytoplasmic (cyto) fractions were separated and detected at hpi by western blotting. (f) a cells transfected with gfp influenza-np and mcherry-ap m were incubated with dmso or aca for hours. live cell imaging was performed, and motile ap m /np puncta were tracked (movies s and s ). shown is the average velocity of trackable puncta within the overall distance traveled. student's t test. (g) ap m facilitates the viral protein localization. synchronized infections were used throughout the experiments. colocalization was quantified using imagej (jacop) colocalization software and manders' colocalization coefficients (mccs). bar charts indicate mean mcc values represented as percent colocalization (the fraction of green intensity that coincides with blue intensity in the case of iav-np/nucleus and the fraction of green intensity that coincides with red intensity in the case of ev-a - c/er and zikv-ns /er) ± sd (error bars, n = to ). scale bars, m. ***p < . by student's t test. (ywai) (fig. s ). mutagenesis includes two y/a substitutions (y a and y a), two Ø/a substitutions (v a and i a), and two Ø/l substitutions (v l and i l). the growth rates of the y a, y a, and Ø a mutant viruses were attenuated for > -log in each time point, while Ø l mutant virus exhibited similar replication kinetics as that of the wt (fig. a) . the results not only indicated the critical role of np-yxxØ in determining the virus fitness but also that yxxØ is functionally interchangeable within homologous Ø signals (i.e., i l substitution). however, we were unable to rescue the recombinant viruses containing Ø l or Ø a mutation for three independent experiments. to validate whether the reduction of iav replication was due to the decreased np nucleus entry, a step which is beyond ap m -mediated endocytosis, the successfully rescued wt and mutant iav were used to infect a cells, followed by measuring np in both the cytosol and nucleus at hpi. apparently, np was not detectable in the host nucleus after infection of iavs carrying y a, y a, and Ø a np substitutions, while Ø l mutant virus exhibited similar amount of np as that of the wt (fig. b ). as expected, influenza polymerase luciferase reporter activity from both Ø/l mutants (Ø l and Ø l) was similar to that of wt, whereas all y/a (y a and y a) and Ø/a (Ø a and Ø a) mutants displayed reduced polymerase activity. moreover, ap m gene depletion reduced the polymerase activity by > % (fig. c) . the results suggested that amino acid residues of np-yxxØ motif determined the iav polymerase activity via modulating the np nuclear import. furthermore, we extended the analysis in vivo and selected the most attenuated y a substitution for a full comparison with the wt. balb/c mice were challenged with two doses of wt-h n -gfp (wt) and y a-h n -gfp (y a) viruses, respectively. survival of y a-challenged ( pfu) group ( % versus % versus %) was obviously better than those of wt-challenged ( pfu) group and wt-infected ( pfu) group (fig. d) . mice in the pfu y a group displayed similar weight loss to those of the pfu wt-infected group but rebounded after dpi, whereas the pfu y a group displayed mild body weight loss (< %) throughout the infection (fig. e ). taking advantage of the gfp reporter feature of the recombinant iav for in vivo dynamic analysis, we examined lung and brain samples from infected mice at different dpi. apparently, spreading of wt virus deeper into the bronchioles and possibly alveoli were detected since dpi, while gfp signal from y a mutant virus was confined to regions around the initial sites of infection around the trachea and bronchi, indicating limited virus spread (fig. f, left) . in line with the reported occurrence of neurological symptoms in highly pathogenic h -infected animals, extensive gfp signals could be visualized in wt virus-infected mouse brain as early as dpi. in contrast, the y a group illustrated reduced gfp intensity throughout the time points (fig. f, right) . these data provided evidence that a single y a substitution in np-yxxØ motif notably restricted iav replication in vivo. together, host ap m /virus yxxØ interaction is critical for intracellular virus trafficking to the sites of replication/ transcription beyond the step of endocytosis, thereby facilitating viral replication (fig. g) . viruses exploit distinct receptors to facilitate cell entry and to evade a hostile extracellular environment that would otherwise abrogate infection. within the intracellular settings, we demonstrated a conserved host ap m /virus yxxØ interaction that is commonly har-nessed by different viruses during intracellular trafficking but beyond the well-defined cme process (fig. g) . using aca as a tool compound, we developed the cwid platform and identified the yxxØbinding pocket of the host ap m as a previously unidentified target for broad-spectrum antiviral development, which is different from the previous antiviral strategy to block ap m phosphorylation ( fig. s ). although the ap m −/− mice is lethal ( ) , the ap m −/− cell line is not susceptible to the infections of iav, ev-a , mers-cov, and zikv (fig. d ). on the virus side, yxxØ motif determines the capacity of np nuclear translocation and therefore affects iav fitness (fig. , a to f) . the outcome of a viral infection with respect to cellular fate is a fundamental aspect of viral biology. we find that tgf- signaling is a cell fate determinant pathway with broad relevance of multiple viruses, and these viruses use ap m as a common intermediate for intracellular trafficking but beyond endocytosis: first, by using live iav infection, vrna was visualized in the perinuclear region but within the cytosol, indicating that the iav entry process has not been affected by aca (fig. b) ; after endocytosis, however, np were predominantly excluded from the nucleus of ap m −/− cells, suggesting that ap m was indispensable for iav-np nuclear localization (fig. e ). the role of ap m associated with hepatitis c virus (hcv) entry and assembly has been defined ( , ) . our study further demonstrated the versatility and broadness of ap m to cotraffic with several other internal viral proteins for the completion of their replication cycles. represented by iav (enveloped and negativestranded), ev-a (nonenveloped), and zikv (enveloped and positivestranded), we demonstrated that recruitment of yxxØ-harboring iav-np, ev-a - c, and zikv-ns proteins by ap m was functionally related in their correct localization as to facilitate viral genome replication. strategically, ap m was harnessed by viral np for efficient nuclear entry, thereby promoting polymerase activity. substitutions introduced in the viral yxxØ motif as y to a or Ø to a greatly diminished virus growth in vitro and in vivo. since the virulence of np y a mutant virus was attenuated, strategic usage of iav strains containing this or other mutants as vaccines might be evaluated in the future. in the case of ev-a and zikv, ap m was required for efficient transportation of c and ns proteins to the er membrane so that their genome replication can occur. furthermore, the essentiality of ap m during virus replication was validated in the context of multiple viruses including pdmh n , ev-a , zikv, and mers-cov infections (fig. d) . together, ap m might be universally exploited by different viruses to complete their replication cycle after cell entry. the architecture of ap has to undergo a large conformational change from a "closed," cargo-inaccessible state to an "open" structure so as to expose the yxxØ binding site, which is regulated by ap associated protein kinase and cyclin g-associated kinase ( ) . although it has been reported that inhibitors of this kinase (e.g., sunitinib and erlotinib) can inhibit rna viruses including dengue, ebola, and hcv, the in vivo antiviral potency of aca ( % survival in iav, zikv, and mers-cov mouse models) is much better than that of sunitinib [ % protection in dengue virus (denv) and % in ebola virus (ebov) mouse models] or erlotinib (conferring no protection in either the denv or ebov mouse model) ( ) . targeting directly the yxxØ-binding pocket instead of the t ap m phosphorylation site, our study not only extends the therapeutic window beyond the conformational change of ap m , which is transient and mediated by kinase activity, but also opens up another synergistic antiviral approach by combining aca with other inhibitors including sunitinib or erlotinib. a major roadblock to translating protein kinase inhibitors into clinical development is the doubt about their poor selectivity, which is largely a consequence of the highly conserved atp-binding site shared by all protein kinases ( ) . disruption of ppis, as exemplified in our study, however, is usually highly specific against the binding interface, which has less concern about cytotoxicity. occupation of ap m yxxØ-binding pocket by using tyrphostin a blocked the replication of multiple viruses (fig. c) . because aca interacts with n and k that lie outside the yxxØ-binding cavity formed by residues f , d , k , and r ( ) , an allosteric activation mechanism of ap may exist (fig. e) . besides ap subunit genes (ap m , ap a , ap m , and ap s ), several syntaxin-relevant genes (stx , stx , and stxbp ) were identified to play a proviral role (fig. b and fig. s ). four pharmacological inhibitors including imperatorin, pyr , zd , and ethosuximide exhibited potent anti-influenza activity with an ec at nanomolar range (fig. c) . further characterization of their underlying mechanisms is warranted. establishment of the cwid platform enables identification of drug-binding target(s) in an unbiased manner, which addresses a common difficulty that challenges all phenotypic forward chemical screening (fig. , c and d) . this platform may be adopted for studies using host-targeting strategies to accelerate the progress of drug target discovery. overall, we demonstrate that the ap m /yxxØ interaction is a druggable target for broad-spectrum antiviral therapy, and virus yxxØ mutant could be tested as attenuated vaccines. these approaches may provide additive and possibly synergistic antiviral effects when aca or its analogs are combined with other antiviral agents for tackling emerging viral infections. the main goal of this study was to identify a next generation of broad-spectrum antiviral with elucidated machinery. first, we comprehensively evaluated the antiviral potency of the selected drug aca in cell cultures, ex vivo and in vivo models. second, we established an integrative platform, named cwid, to identify the host ap m / yxxØ interaction as the aca drug target and in an unbiased manner. third, we investigated the biological importance of ap m /yxxØ interaction in the viral replication cycle using three representative viruses. last, we characterized the effect of virulence of the substitutions in influenza a np yxxØ motif. hbtecs were cultured with airway epithelial cell basal medium according to the manufacturer's protocol. human-induced pluripotent stem cell-derived neural progenitor cells (npcs) were cultured and differentiated according to a previous protocol ( ) . human lung fibroblast hfl was cultured in f- k medium. human t lymphoblast molt- ccr + cells were cultured in rpmi medium supplemented with % heat-inactivated fetal bovine serum (fbs) and g ( mg/ml). human embryonic kidney (hek) t cells, human lung carcinoma (a ) cells, human hepatoma (huh ) cells, human rd cells, human epithelial type cells, mdck cells, and african green monkey kidney (vero) cells were maintained in dulbecco's modified eagle's medium (dmem) medium. all culture medium was supplemented with % heat-inactivated fbs, penicillin ( u/ml), and streptomycin ( g/ml). all cells were confirmed to be free of mycoplasma contamination by the plasmo test (invivogen). the iav strains a/hong kong/ / (h n )pdm , a/anhui/ / (h n ), a/vietnam/ / (h n ), a/netherlands/ / (h n ), and a/hk/ / (h n ) were cultured in embryonated chicken eggs. the sars-cov- hku- a was isolated from the nasopharyngeal aspirate specimen of a laboratoryconfirmed covid- patient in hong kong ( ) . the mers-cov (hcov-emc/ , a gift from r. fouchier) and sars-cov (gz ) were propagated in vero-e cells. the hiv- jr-fl virus (# , national institutes of health aids) was propagated in molt- ccr + cells in the presence of interleukin- ( ng/ml) for days as we previously described ( ) . enterovirus a- (sz/hk - ) was cultured in rd cells. the zikv (puerto rico strain prvabc , a gift from b. russell and b. johnson, cdc, usa) was amplified in vero cells. a clinical isolate of human adv was propagated in a cells. the sftsv hb strain (a gift from m. liang, cdc, china) was propagated in vero cells. all cultured viruses were titrated by pfu assays and/or % tissue culture infectious dose (tcid ) assay and/or quantitative reverse transcription polymerase chain reaction (rt-qpcr) assays and/or p elisa as indicated. all virus stocks were kept at − °c in aliquots. all experiments with live viruses were conducted using biosafety level or facility as we previously described ( ) . wnt signaling reporter containing t cell factor/lymphoid enhancerbinding factor was a gift from r. moon (addgene plasmid # ). tgf- signaling reporter containing four copies of the smad binding site was shared by b. vogelstein (addgene plasmid # ). notch signaling reporter containing centromere-binding protein (cbf )responsive element was obtained from n. gaiano (addgene plasmid # ). pi k/akt signaling reporter containing forkheadresponsive element was a gift from m. greenberg (addgene plasmid # ). all primer sequences used are provided in table s . aca was purchased from cayman chemical (michigan, usa). other chemical inhibitors were obtained from sigma-aldrich (missouri, usa) unless specified. all peptides were synthesized from cellmano biotech (hefei, china) with > % purity. the phosphine-activated fluorescent dye dylight -phosphine (invitrogen) was used for specific labeling and detection of azido-tagged molecules, i.e., azido-aca. the ′, -diamidino- -phenylindole (dapi; sigma-aldrich), calnexin polyclonal ab (abbkine, abp ), and phalloidin-atto n (sigma-aldrich) were used for nuclear, er, and cell membrane staining, respectively. primary antibodies against human ap m (abcam, ab ), anti--actin (abcam, ab ), anti-influenza np (abcam, ab ), anti-zikv ns (genetex, gtx ), anti-ev-a c (genetex, gtx ), anti-his-tag (invitrogen, ma - ), anti-ha-tag (abbkine, a ), anti-flag-tag (sigma-aldrich, f ), anti-lamin a (abcam, ab ), anti-glyceraldehyde- -phosphate dehydrogenase (gapdh; abcam, ab ), and anti-mers-cov np mouse serum (in house) were used in relevant experiments. alexa fluor goat anti-mouse immunoglobulin g (h + l) antibody ( : ; invitrogen, a ) and alexa fluor goat-rabbit ( : ; invitrogen, a ) were used as secondary antibodies for immunofluorescence staining. individual smartpool sirna targeting ap m , trpm , trpm , or pla g a was purchased from dharmacon (lafayette, usa). ne-per nuclear and cytoplasmic extraction reagents (thermo fisher scientific) was used according to the manufacturer's protocol. an ap m human gene knockout kit (origene, kn ) was used to establish the crispr knockout cell lines according to the manufacturer's protocol. immunofluorescence rna was visualized using the viewrna cell plus assay kit (invitrogen, - ) containing a viewrna type probe set designed against the negative stranded rna np genome (vrna) of influenza a h n pr virus (invitrogen, vf - ). to identify host genes essential for h n pdm replication, an rna interference (rnai)-based screen was performed using a commercially available library targeting cellular membrane-trafficking genes (ambion silencer, a ). briefly, . × per well of a cells were seeded in -well plates overnight, followed by sirna transfection once daily for two consecutive days using the lipofectamine rnaimax reagent. at hours after primary sirna transfection, a cells were infected with . moi virus. one hour later, the infectious inoculum was aspirated and replaced with fresh dmem medium containing % bsa and n-tosyl-l-phenylalanine chloromethyl ketone (tpck)-treated trypsin ( g/ml). the cell culture supernatant of individual well was collected after another hours for viral load titrated by the rt-qpcr method. before virus infection, wells exhibited poor cell viability (< %) after gene silencing were excluded. a small-molecule compound library with candidates (medchemexpress), targeting membrane transporters and ion channels, was used for pharmacological screening with antiviral activity. the primary screening of pdmh n inhibitors was cpe inhibition based as we previously established ( ) . viability of mdck cells after . moi virus infection and compound treatment ( m) were determined at hpi using the celltiter-glo luminescent cell viability kit (promega). secondary screening was performed with viral load reduction assay. briefly, the cell culture supernatant at hpi and compound treatment ( and m, respectively) were collected and applied for viral copy quantification by rt-qpcr methods. favipiravir ( g/ml) was used as a positive control throughout the screening process. waterlogsy, a ligand-observed nmr technique, was used to screen for aca-interactive cellular fractions ( ) . to fractionate the pdmh n -infected cell lysate, ml of mdck cells ( cells, moi, hpi) was ultrasonicated three times for s on ice and then centrifuged. the clarified supernatant was applied for fast protein liquid chromatography (Äktaexplorer, ge healthcare) using the -ml hiload / superdex preparative size exclusion chromatography column to harvest each protein fraction with ultraviolet -nm signals. aca waterlogsy experiments were conducted on a -mhz bruker avance spectrometer using a -mm pasei probe. the pulse scheme used for waterlogsy experiment was "ephogsygpno. " with water suppression using excitation sculpting with gradients ( ) . all experiments were conducted at k using -mm-diameter nmr tubes with a sample volume of l and m aca supplemented and recorded using k scans. all samples were dissolved in % h o and % d o with final concentration of m trimethylsilylpropanoic acid as internal standard. control spectrum was recorded under the same conditions without cellular fraction to confirm the absence of self-aggregated aca macromolecules. to identify the aca-binding protein target (protein ids), the cellular fraction was lyophilized before resuspended with l of h o and incubated with m azido-aca for hour. the mixtures were further incubated with m dylight -phosphine for hours to allow linkage of fluorescent dye with the azido group, before loading to a % nondenaturing native gel for electrophoresis. subsequently, a typhoon fla laser scanner using alexafluor filter was used to visualize the fluorescent bands that were associated with azido-aca other than aca. the photomultiplier tube voltage was set as to v and the resolution as m. target bands were excised and subjected to lc-electrospray ionization ms/ ms analysis by q exactive as previously established (shanghai applied protein technology co. ltd.) ( ) . samples with or without aca were also incubated with dylight -phosphine to act as controls. to validate the target protein essential for aca-dependent mode of action, eight protein ids as revealed by the cwid platform were overexpressed individually and screened for their capacity to antagonize the aca's antiviral activity. orf clone of each protein was obtained from mission trc human orf collection (merck). in a -well plate, mdck cells were transfected with ng of each plasmid and incubated for hours before pdmh n infection. cell culture supernatants of the infected cells, with or without aca ( m), were collected for viral titer determination by standard plaque assay. under the protocol approved by the institutional review board (uw - ) of university of hong kong/hospital authority hong kong west cluster, normal human lung tissue from a patient was obtained surgically. informed consent was obtained from the human participant, and the experiments were performed in compliance with the approved standard operating procedures. anti-influenza activity of aca was also evaluated in d human aos, as we previously reported ( ) . briefly, the d aos were sheared mechanically to expose the apical surface to the virus inoculum. the sheared organoids were then incubated with viruses at a moi of . for hours at °c. after washing, the inoculated organoids were re-embedded in matrigel and cultured in the medium containing aca ( m) or dmso ( . %). at the indicated times, aos were harvested for the quantification of intracellular viral load or fixed for immunofluorescence staining. balb/c mice, hdpp transgenic c bl/ mice, and interferon receptor / knockout (ifnar −/− ) a mice were kept in biosafety level or housing and given access to standard pellet feed and water ad libitum, as we previously described ( ) ( ) ( ) . all experimental protocols were approved by the animal ethics committee (culatar - , - , - ) in the university of hong kong and were performed according to the standard operating procedures of the biosafety level or animal facilities. to evaluate the cross-subtype anti-influenza virus efficacy of aca in vivo, balb/c mice ( mice per group) were inoculated intranasally with pfu of influenza a h n virus or pfu of mouse-adapted influenza a h n virus in -l pbs. treatment was performed hour after challenge by intranasal administration. one group of mice was inoculated with l of aca ( . mg/kg). a second group was treated with l of intranasal zanamivir ( mg/kg) ( ) . a third group was given intranasally . % dmso in pbs as an untreated control. animal survival and clinical disease were monitored for days or until death. lung tissues (five mice per group) were collected for viral load detection and hematoxylin and eosin (h&e) histopathologic analyses on days and after challenge, respectively. to evaluate the anti-zikv efficacy of aca in vivo, -to -weekold a mice were randomly divided into two groups to receive aca treatment or sham treatment through the subcutaneous route (n = per group). the mice were inoculated subcutaneously with × pfu (in -l pbs) of zikv-pr under anesthesia. each mouse was then received one dose of subcutaneous-administered aca ( mg/kg) or . % dmso in pbs at hpi. the mice were monitored daily for body weight change and clinical signs of disease. five mice in each group were euthanized at dpi, and brain tissues were harvested for vial titers and histopathologic and immunohistochemistry analyses. the survival of the other mice was monitored until dpi. to examine the anti-mers-cov activity of aca, a total of mice (n = per group) were evaluated. after anesthesia, mice were intranasally inoculated with l of virus suspension containing pfu of mers-cov. intranasal therapeutic treatments were initiated at hpi. one group of mice was inoculated with l of aca ( . mg/kg). the other group was given intranasally . % dmso in pbs as an untreated control. animal survival and clinical disease were monitored for days or until death. five mice in each group were euthanized randomly on dpi, respectively. mouse lungs were collected for virus titration and h&e histopathologic and immunofluorescence staining. the whole-organ lungs and brains of the gfp virus-infected mice were excised at the indicated time after challenge. after fixation in % paraformaldehyde for overnight, the images were acquired in a pe ivis spectrum in vivo imaging system fitted with gfp excitation/ emission filters. live imaging was used to visualize the cotrafficking of ap m and influenza a np according to a previous report with some modifications ( ) . time-lapse images were taken using the nikon ti -e widefield microscope with a × . oil objective in a heated ( °c) chamber. gfp-labeled np protein and mcherry-fused ap m protein were tracked by sequential imaging every s with -and -ms exposures for each channel, respectively. individual colocalized puncta run lengths and transport velocities were calculated using the track points for metamorph analysis software, measuring the distance traveled (in any direction) between frames for each respective puncta. the movies were made using the stack function for metamorph analysis software via accumulating the relative frames in order. the recombinant influenza virus carrying a gfp reporter gene in the ns segment (ns -gfp virus) was rescued using standard reverse genetics techniques ( ) . the -plasmid system, with a/duck/ hubei/wh / (h n ) background, was a gift from j. meilin (huazhong agricultural university, china). np constructs harboring yxxØ mutations, i.e., phwnp-y a, phwnp-v a, phwnp-v l, phwnp-y a, phwnp-i a, and phwnp-i l, were performed by site-directed mutagenesis (stratagene) based on the wt phwnp plasmid. titers of viral stocks were determined by plaque assay on mdck cells. luciferase reporter plasmids reflecting the up-or down-regulation of cell fate determination pathways were used. experimentally, individual reporter plasmid ( ng), together with a transfection efficiency control plasmid (pnl . .tk, promega) construct ( ng), was cotransfected into the indicated cells for hours. subsequently, moi of each virus was used to infect the transfected cells, followed by luminance detection at , , , , or hpi according to the manufacturer's protocol (dual-luciferase reporter assay system, promega). the transfected cell with mock infection was taken as a baseline control for normalization. influenza a virus mini-genome reporter assays were performed as described previously with some modifications ( ) . rnp complexes composed of pa, pb , pb , and np or their mutants were mixed with a luciferase reporter plasmid ( ng each) and pnl . .tk construct ( ng) and then cotransfected into hek t cells. luminescence was determined at hours after transfection. the assay was designed to detect ap m and yxxØ binding. free or biotin-labeled yxxØ motif, with a peptide sequence of dyqrln, was synthesized. to expose the ap m binding site, calyculin a, an inhibitor of ap m dephosphorylation which "locks" ap m in its yxxØ binding active conformation, was added according to a previous report ( ) . hek t cells in six-well plates were transfected with his-ap m or its low binding affinity mutant d a for hours. next, g per well transfected cell lysate containing the overexpressed proteins was incubated with the ni-nta hissorb -well plate (qiagen) for overnight at °c. after washing, drugs were added hour before incubation, followed by input of biotin-yxxØ probe ( g/ml) and detection of binding signal using horseradish peroxidase-conjugated streptavidin (thermo fisher scientific, n ) and trimethylboron substrate (thermo fisher scientific, n ). in this experiment, the unlabeled peptide dyqrln was used as a positive control binding inhibitor, while mock-transfected cell lysate was taken as a background control. the washing and dilution buffer consisting nm calyculin a, pbs, and . % tween- was used throughout the assay. the aca (pubchem cid: ) d structure was downloaded from pubchem database. leadfinder version was used to perform ligand-receptor docking ( ) . extra precision mode (-xp) was applied to search the ligand conformational space more thoroughly. energy grid map was generated according to the binding pose of yxxØ motif in the ap m /yxxØ complex structure (protein data bank code: xa ) ( ). default grid map spacing of . a was set for a good trade-off between accuracy and performance. bond orders were assigned, hydrogens were added, and cap termini were included with the protein preparation wizard module as implemented in maestro. protonation states of side chains were predicted using propka . server. partial charges over all atoms were assigned within the amber force field scheme as implemented in ambertools. the top-ranked pose was visualized by using pymol, while d intermolecular interaction was visualized with ligplot+. data were analyzed using graphpad prism (graphpad software, san diego, ca, usa). the values shown in the graphs are presented as means ± sd of at least three independent experiments. statistical differences between groups were analyzed using a one-way analysis of variance (anova) statistical test with dunnett's multiple comparisons tests or two-tailed unpaired t tests. colocalization rate was quantified using imagej (jacop) colocalization software and manders' colocalization coefficients (mccs) as previously described ( ) . p < . was considered statistically significant. supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/sciadv.aba /dc view/request a protocol for this paper from bio-protocol. evolutionary conflicts between viruses and restriction factors shape immunity middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease zika virus the emergence of influenza a h n in human beings years after influenza a h n : a tale of two cities signaling mechanisms controlling cell fate and embryonic patterning tollip, an intracellular trafficking protein, is a novel modulator of the transforming growth factor- signaling pathway a large-scale conformational change couples membrane recruitment to cargo binding in the ap clathrin adaptor complex transforming growth factor- receptors interact with ap by direct binding to  subunit regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of ap intracellular trafficking of transforming growth factor  receptors an sirna screen of membrane trafficking genes highlights pathways common to hiv- and m-pmv virus assembly and release identification of host factors involved in human cytomegalovirus replication, assembly, and egress using a two-step small interfering rna screen genome-wide rnai screen identifies human host factors crucial for influenza virus replication differentiated human airway organoids to assess infectivity of emerging influenza virus n-(p-amylcinnamoyl)anthranilic acid (aca): a phospholipase a inhibitor and trp channel blocker anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects influenza virus-host interactome screen as a platform for antiviral drug development adaptors for clathrin coats: structure and function inhibition of the receptor-binding function of clathrin adaptor protein ap- by dominant-negative mutant mu subunit and its effects on endocytosis tyrphostin a inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the ap- adaptor complex reticulon binds the c protein of enterovirus and is required for viral replication clathrin adaptor ap- is essential for early embryonal development ap- -associated protein kinase and cyclin g-associated kinase regulate hepatitis c virus entry and are potential drug targets identification and targeting of an interaction between a tyrosine motif within hepatitis c virus core protein and ap m essential for viral assembly flik: a direct-binding assay for the identification and kinetic characterization of stabilizers of inactive kinase conformations molecular architecture and functional model of the endocytic ap complex human induced pluripotent cell-derived sensory neurons for fate commitment of bone marrow-derived schwann cells: implications for remyelination therapy comparative replication and immune activation profiles of sars-cov- and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-   + cd + effector/ effector memory t cells differentiate into productively and latently infected central memory t cells by transforming growth factor  during hiv- infection srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target talaromyces marneffei mp p is a virulence factor that binds and sequesters a key proinflammatory lipid to dampen host innate immune response a tricyclic pyrrolobenzodiazepine produced by klebsiella oxytoca is associated with cytotoxicity in antibiotic-associated hemorrhagic colitis and nanog is essential for oncogenic viral flice-inhibitory protein-induced acetylation of p /rela, nf-b activation, and promotion of cell invasion and angiogenesis human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h n virus the celecoxib derivative kinase inhibitor ar- (osu- ) inhibits zika virus via down-regulation of the pi k/akt pathway and protects zika virus-infected a mice: a host-targeting treatment strategy molecular determinants and dynamics of hepatitis c virus secretion insertion of a gfp reporter gene in influenza virus the k r substitution in viral protein pb enhances the effects of e k on influenza virus replication lead finder: an approach to improve accuracy of protein−ligand docking, binding energy estimation, and virtual screening the hong kong hainan commercial association south china microbiology research fund, the jessie & george ho charitable foundation, and perfect shape medical; and funding from the theme-based research scheme (t - / -r) of the research grants council, hong kong special administrative region; and the high level-hospital program, health commission of guangdong province, china. the sponsors had no role in the design and conduct of the study, in the collection, analysis, and interpretation of data, or in the preparation, review, or approval of the manuscript key: cord- -soffxxqu authors: li, shuang; zhu, anjing; ren, kai; li, shilin; chen, limin title: defa b inhibits zikv replication and retards cell cycle progression through interaction with orc date: - - journal: life sci doi: . /j.lfs. . sha: doc_id: cord_uid: soffxxqu aims: zika virus (zikv) infection causes a public health concern because of its potential association with the development of microcephaly. during viral infections, the host innate immune response is mounted quickly to produce some endogenous functional molecules to limit virus replication and spread. exosomes contain molecules from their cell of origin following virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv-induced exosomes can regulate viral pathogenicity by transferring specific rnas. main methods: in this study, exosomes were isolated from the supernatants of a cells with or without zikv infection. human transcriptome array (hta) was performed to analyze the profiling of rnas wrapped in exosomes. then qpcr, western blotting and elisa were used to determine zikv replication. cck- and flow cytometry were used to test the cell proliferation and cell cycles. co-culture assay was used to analyze the effect of exosomes on the cell cycles of recipient cells. key findings: through human transcriptome array (hta) we found the defensin alpha b (defa b) expression was significantly increased within exosomes isolated from zikv infected a cells. additionally, we found that the extracellular defa b exerts significant anti-zikv activity, mainly before zikv entering host cells. interestingly, up-regulated defa b retards the cell cycle of host cells. further studies demonstrated that defa b interacted with the origin recognition complex (orc ) which is required to initiate dna replication during the cell cycle and increased defa b expression decreased the orc level in the cell nuclei. accordingly, defa b-containing exosomes can be internalized by the recipient cells to retard their cell cycles. significance: together, our results demonstrated that the anti-zikv activity of defa b can be mediated by exosomes, and defa b interacts with orc to retard cell cycles. our study provides a novel concept that defa b not only acts as an antiviral molecule during zikv infection but also may correlate with cell proliferation by retarding the progression of cell cycles. zika virus (zikv) is a re-emerging arbovirus that belongs to the flaviviridae family and is transmitted by infected aedes mosquitoes. zikv is a single positive-stranded rna virus with a genome size of approximately . kilobases [ ] . zikv infection emerged as a public health concern after increasing evidence linking zikv infection with the potential development of microcephaly [ ] . although previous study alluded that zikv could directly infect human neural progenitor cells (hnpcs) to induce cell death and disturb cell cycle progression to affect human brain development [ ] , the detailed underlying molecular mechanism remains elusive. in the battle between pathogens and hosts, the production of endogenous defensins is one of the first lines of protection for the host. various types of human cells can produce defensins that have broad antimicrobial activity to many pathogens, including bacteria, viruses and fungi [ , ] . defensins have been shown to protect against many virus infections, such as human immunodeficiency virus (hiv), influenza a virus (iav), human adenovirus (hadv), severe acute respiratory syndrome coronavirus (sarsc), papillomavirus (hpv), respiratory syncytial virus (rsv), and herpes simplex virus (hsv) [ , ] . currently, no approved vaccines or therapies are available for the prevention and treatment of zikv infection, therefore defensins may be a promising drug target for the treatment of zikv infection. the 'origin recognition complex' (orc), as one of the most important complexes in all eukaryotes, is involved in the initiation of dna replication and has been linked to various diseases [ ] . although orc consists of orc - , orc is the biggest subunit that is necessary for the initiation of dna replication [ ] . notably, several reports linked orc to numerous human diseases [ , ] . of particular interest, mutations in orc have been shown to cause meier-gorlin syndrome, which is characterized by microcephaly and primordial dwarfism [ ] . recent work has pointed out that exosomes function as crucial regulators of cellular cross-talk. exosomes are phospholipid bilayer-bound structures that can transfer packed mrna, mirna, and/or proteins into recipient cells to facilitate intercellular communications and to affect gene expression in recipient cells [ , ] . in addition, emerging evidence indicated that exosomes can mediate the transfer of pathogen-derived antigens and virulence factors [ ] . exosomes play various roles in the pathogenesis of zikv. for example, zikv can be wrapped into a sort of cargo of the placental exosomes and be transmitted through exosomes into trophoblast cells [ ] . furthermore, exosomes have been shown to mediate zikv transmission through smpd neutral sphingomyelinase in cortical neurons [ ] . in order to further decipher the role of exosomes in the pathogenesis of zikv infection, we used human transcriptome array (hta) and bioinformatics analysis to identify candidate molecules in zikv-induced exosomes. we identified defensin alpha b (defa b) is involved in zikv infection and pathogenesis. our data demonstrated that defa b not only inhibits zikv '-gcagagcaacggatgggata- ' and the reverse primer was '-atggtgggagcaaaacggaa- '. plasmids expressing defa b were constructed with routine molecular cloning techniques. the full length human defa b gene was amplified by polymerase chain reaction (pcr) from total rna isolated from a cells and cloned into pcdna . secondary antibodies goat anti-rabbit, goat anti-mouse and goat anti-rat were also obtained from cst (ma, usa). bio-rad image acquisition and analysis software (uvp, upland, ca, usa) were performed to quantify the band density. cell proliferation and viability were evaluated using a cck- assay (transgene biotech, china). for cck , the cells were allowed to grow in a -well plate, at the concentration of cells per well. at each time point, cells were rinsed with / cck- diluted in dmem for . h, and the optical density of the cellular homogenate was measured at nm. each experiment was performed in quintuplicate. the effect of defa b on cell proliferation was evaluated by measuring the distribution of the and protein a&g agarose (santa cruz biotechnology) were used in this co-ip assay. the immunoprecipitates were washed five times and separated by % sds-page gel electrophoresis and blotted using anti-defa b and anti-orc (santa cruz), respectively. pkh green fluorescent cell linker kit (sigma) was used to label exosomes according to the manufacturer's protocol. briefly, exosomes were re-suspended in μl diluent c. in addition, μl pkh was mixed with ml diluent c, followed by mixing with the exosomes suspension and incubated for min. to stop the labeling reaction, an equal volume of % bsa was added into the mixed solution. then, the labeled exosomes were ultracentrifuged at , ×g for h at ℃), washed with × pbs, and ultracentrifuged again. to detect exosomes uptake into recipient cells, hek t cells and sh-sy y cells were grown in -well plates at a density of × cells per well and pkh -labeled exosomes were diluted in whole-medium solution and was added into each well. cells were cultured for h at °c. dapi was used to stain the nucleus, and the cells were observed using fluorescence microscope (olympus ix , germany). student's t-tests were used to analyze the differences between groups. all experiments were repeated at least three times. the p-value < . was considered statistically significant (*p < . ; **p < . ; ***p < . ). . nm, and % of exo-zikv is . nm ( figure c , d, e). western blot revealed that the exosome-specific proteins, cd , cd and tsg , were enriched in all exosomes samples but not in cell lysates-confirming these vesicles are indeed exosomes. calnexin, an endoplasmic reticulum protein, was detectable in whole-cell lysates but not in the exosomes, indicating that the exosomes preparations were not contaminated with other vesicles ( figure f ). together, these results confirmed the isolated vesicles are indeed purified exosomes and the isolation method is reliable. western blotting of exosomes were performed to confirm the presence of exosomal marker protein, cd , cd and tsg . absence the endoplasmic reticulum protein, calnexinan, in exosomes but was detectable in whole cell lysates (f). and a control cells hrs after the plasmids transfection, the mrna level of defa b was significantly up-regulated ( figure a ). elisa data showed the defa b could be secreted into culture supernatants, and the concentration of defa b was significantly increased compared with the control group ( figure b ). furthermore, to examine the anti-viral ability of defa b, we divided the cells into two e. j o u r n a l p r e -p r o o f groups: in one group the supernatants were removed and replaced with fresh medium; the other group the supernatants that contain defa b were added. then, these two groups of cells were infected with zikv at moi= . . our data showed that defa b contained in culture supernatants significantly inhibited zikv replication at mrna level ( figure c ). ns (which is the non-structural protein of zikv) expression level was also significantly decreased in the group with defa b in culture supernatants ( figure d ). to further analyze the anti-zikv effect of extracellular defa b, we added three different concentrations ( . ± . pg/ml, . ± . pg/ml, . ± . pg/ml) of defa b to the supernatants of hek t cells ( figure e ) and infected the cells with zikv. compared with no defa b in supernatants, qpcr data showed that zikv replication was significantly inhibited in a dose-dependent manner ( figure f ). we also verified the same tendency at the viral protein levels using western blot ( figure g ). furthermore, we compared the changes of zikv copies in medium with or without defa b to further validate the anti-zikv effect of extracellular defa b. the qpcr data showed the zikv copies were significantly decreased in medium with defa b when compared with those in normal medium (without defa b) ( figure h ). furthermore, we compared the changes of zikv copies in medium with or without defa b to further validate the anti-zikv effect of extracellular defa b. the qpcr data showed the zikv copies were significantly decreased in medium with defa b when compared with those in normal medium (without defa b) ( figure h ). in order to test whether the intracellular defa b has anti-zikv activity, we transfected different doses ( . ug, . ug and ug per well) of pcdna . -defa b-t a-egfp plasmids into hek t cells. qpcr ( figure i ) and elisa ( figure j ) data showed the defa b was indeed up-regulated in hek t cells after transfection. we then replaced the culture supernatants with fresh medium and infected the cells with zikv (moi= . ). both qpcr ( figure k ) and western blot ( figure l ) data showed no significant changes of zikv replication between untreated hek t cells and cells with up-regulated defa b group. western blot (l) to analysis zikv level. during our research we found that up-regulated defa b expression in hek t reduced the cell growth rate. so we used cck to investigate the proliferation of hek t cells transfected with pcdna . -defa b-t a-egfp or with control vector, and we found that compared with untreated or blank vector control group, the proliferation of cells were obviously decreased at day and day after transfected with pcdna . -defa b-t a-egfp (fig ) . proteins often do not just function as a single substance but rather as team players in a dynamic network. growing evidence shows that protein-protein interactions are crucial in many biological processes in living cells. the database string (https://string-db.org/) is a pre-computed global resource for the exploration and analysis of these associations. we found that defa b was the hub of orc interaction that is the largest subunit of the orc ( fig a) ; therefore, we decided to focus further analysis on orc . to confirm whether defa b specifically interacts with orc , immunoblot analysis of whole cell lysates and anti-defa b affinity immunoprecipitation (ip) derived from t cells transfected with pcdna . -defa b-t a-egfp or blank vector control plasmid were performed. co-ip assay clearly demonstrated that defa b interacts with orc ( fig b) . we further analyzed the expression levels of orc both in the nucleus and cytoplasm. our results demonstrated that following the up-regulated expression of defa b, the levels of orc in whole cell lysis ( fig c) and cytoplasmic protein lysis (fig d) showed no significant changes compared with the control groups. interestingly, although defa b was not expressed in the nucleus, the level of orc in nucleus was significantly decreased in defa b -up-regulated cells (fig e) . we used flow cytometry to analyze the cellular dna content to identify the percentage of g , s and g /m in the cell cycle. our data showed days after transfection, the percentage of g /m in defa b-up-regulated cells was significantly decreased compared with the control groups (fig a-d) . to be able to follow the progression of cell cycle more precisely, we synchronized cultured hek t cells with thymidine which is the most commonly used s-phase blocker and its addition to the culture medium depletes nucleotide pools and inhibits new dna synthesis, causing the slowdown or arrest of s-phase progression [ ] . after released into normal medium, cell populations at distinct cell cycle phase were collected at different time points ( - h) and the overlapped histograms indicated after about - hours some cells finished s+g +m stages and returned to g phase. so we compared the velocity to finish s+g +m stages through analyzing the percentage of g . our data showed with time passing by the cultured cells became more and more asynchronous. hours following releasing, the percentage of g phase was significantly lower in the defa b-up-regulated group, which indicates that defa b retards cell cycle progression (fig e) . the internalization of exosomes is one mechanism of cargo delivery to recipient cells. to examine whether exosomes isolated from zikv infected a cells can be taken up by hek t and j o u r n a l p r e -p r o o f journal pre-proof sh-sy y cells, exo-zikv were labeled with pkh dye (green) and added to cultured hek t cells. data from fluorescence microscope suggested that exo-zikv were internalized into recipient cells after hours co-culture (fig a) . we further determined whether exosomes can transmit the cell cycle retarding effect of defa b to recipient cells, we discriminated the cell cycles of recipient cells using flow cytometry. after co-cultured with exo-zikv for h, the percentage of g /m in hek t cells was decreased compared with cells co-cultured with exo-a (fig b, c) . as expected, exo-zikv was also internalized into sh-sy y cells (fig d) and flow cytometry data showed the percentage of g of undifferentiated human neuroblastoma sh-sy y cells was decreased compared with cells co-cultured with exo-a (fig e, f ). exosomes, as a "carrier" of material and information, play an important role in the interaction between viruses and host cells. loading functional genes into virus-induced exosomes has been demonstrated to modulate viral spread and immune response. in this study, we aimed to understand the role of exosomes in the pathogenesis of zikv infection. we first isolated and characterized the exosomes from zikv infected/uninfected a cells. we showed the exosomes with typical shape,size and protein markers, indicating the vesciles we isolated are indeed exosomes and the isolation method is reliable. next, we used hta to identify the differentially-expressed genes between exosomes isolated from zikv infected a and a control cells and we found the expression level of defa b was significantly increased within exo-zikv compared with exo-a . to further study the role of defa b in zikv replication, j o u r n a l p r e -p r o o f we up-regulated the expression level of defa b in a cells before infected with zikv and the viral replication was assessed by rt-pcr and western blot. we found extracellular but not the intracellular defa b inhibited zikv replication. surprisingly, we also revealed that defa b could retard the progression of cell cycle. to further explore the underlying mechanism, we identified that defa b interacts with orc to arrest orc entering into the nuclei. in addition, exosomes as carriers can transmit the cell cycle progression retarding effect of defa b into recipient cells, such as sh-sy y and t cells (figure ). in contract with the environment [ ] . our data also showed after zikv infection, the defa b increased in host cells as well as in evs, which can protect from proteinase degradation. our data showed defa b could be secreted into culture supernatants. defensins can block viral infection through directly acting on virus particles or indirectly interfering with various stages of the viral life cycle. available data suggest the antiviral activity of defensins occurs predominantly at viral entry steps; however, antiviral effects at other stages of infection have also been reported, particularly affecting viral trafficking within infected cells [ ] . for example, the previous study found that α-defensin- not only had a direct effect on hiv- virions but also blocked hiv- infection at nuclear import and transcription stages [ ] . α-defensins in supernatants also inhibit the infectivity of hsv- and respiratory syncytial virus (rsv) [ ] . our data showed defa b could be secreted into culture supernatants. in addition, the extracellular defa b exerts the anti-zikv effect, and the inhibiting effect mainly before zikv enter host cells. in , physicians in brazil began to report that the number of microcephaly increased among newborns, which was possibly linked to zikv infection during the mothers' pregnancy [ , , , ] . the new emergence of patients with severe nerval system prompted public health emergency of international concerns to explore the suspected association between zikv infection and microcephaly. mouse models showed that zikv could destroy the central nerval system to leave severe pathological changes in mice [ ] . zikv can target human brain cells and zikv may impact their survival and growth by restricting the growth of neurospheres and brain organoids [ ] . previous studies found that meier-gorlin syndrome patients with mutations in orc had increased microcephaly and a significantly proportionally smaller head circumference and brain [ , , ] . some researchers found that a conserved basic amino acid motif of orc (residues - , orc -bp) can specific recognition of thymine residues in the dna replication origin sequence, and decreased orc in the nuclei could impact the cell growth and be developmentally important [ , ] . in our study we identified that defa b can bind with orc in the cytoplasm and decrease the level of orc in the cell nucleus. cell-cycle timings vary markedly during embryo or fetus development, with some stages process rapidly and some stages slow down [ ] . for example, in early neurogenesis, neuronal stem cells have a markedly truncated g phase in which impaired pre-rc assembly could become rate j o u r n a l p r e -p r o o f journal pre-proof limiting. as a result of prolonged cell cycle times, even small changes in the number of cell divisions of progenitor and stem cells could have dramatic effects on eventual tissue and organ size [ ] . our data demonstrated that up-regulated defa b expression could significantly retard the progression of cell cycles, and zikv induced exosomes internalized into nerve cells (sh-sy y cells) and can develivery the detention cell cycle effect. whether the zikv-induced defa b inhibiting cell cycle progression of fetal nerve cells leading to smaller size of brain needs further investigation. during human development, the placental barrier blood-brain barriers contribute to fetus brain protection [ ] . although the zikv rna has been detected in amniotic fluid samples, placental tissues and newborn and fetal brain tissues, how the virus crosses the placental and blood-brain barriers remains unclear [ ] . previous studies indicated that exosomes as small transporters can easily get through placental barrier and blood-brain barrier [ , ] . our data showed the zikv induced exosomes could be internalized into recipient cells and inhibit the cells' dna replication to retard cell cycles. in summary, our innate immune can increase defa b expression during zikv infection, and this can effectively control zikv replication. meanwhile, defa b can retard cell cycles. because there are some correlations between retarded cell cycles and neurodevelopment. so whether the phenomenon we found linked to the formation of microcephaly? if at the early stage of fetal development, can zikv induced exosomes go through the placental barrier and blood-brain barrier to entry fetal brain? can the defa b within these exosomes inhibit cell cycles of fetal nerve cells and consequence lead to small size of brain? more appropriate animal models may be zika virus: history of a newly emerging arbovirus an update on zika virus infection zika virus infects human cortical neural progenitors and attenuates their growth defensins in viral infection and pathogenesis defensins at the mucosal surface: latest insights into defensin-virus interactions dual role of alpha-defensin- in anti-hiv- innate immunity human antimicrobial peptides as therapeutics for viral infections mechanisms and regulation of dna replication initiation in eukaryotes the "orc cycle": a novel pathway for regulating eukaryotic dna replication replication from orip of epstein-barr virus requires human orc and is inhibited by geminin the origin recognition complex in human diseases mutations in the pre-replication complex cause meier-gorlin syndrome meier-gorlin syndrome genotype-phenotype studies: individuals with pre-replication complex gene mutations and without molecular diagnosis communication by extracellular vesicles: where we are and where we j o u r n a l p r e -p r o o f need to go specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication exosomes modulate the viral replication and host immune responses in hbv infection intrauterine zika virus infection of pregnant immunocompetent mice models transplacental transmission and adverse perinatal outcomes exosomes mediate zika virus transmission through smpd neutral sphingomyelinase in cortical neurons isolation and characterization of exosomes from cell culture supernatants and biological fluids cell cycle synchronization and flow cytometry analysis of mammalian cells simple" antimicrobial peptides or broad-spectrum molecules? mammalian defensins in the antimicrobial immune response dual role of alpha-defensin- in anti-hiv- innate immunity -arachidonoyl-glycerol-and arachidonic acid-stimulated neutrophils release antimicrobial effectors against e. coli, s. aureus, hsv- , and current zika virus epidemiology and recent epidemics a new mosquito-borne threat to pregnant women in brazil zika virus: a previously slow pandemic spreads rapidly through the americas the brazilian zika virus strain causes birth defects in experimental models zika virus impairs growth in human neurospheres and brain organoids mutations in orc , encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling meier-gorlin syndrome the bah domain of orc links h k me to dna replication licensing and meier-gorlin syndrome mutations disrupt an orc cdk inhibitory domain and cause centrosome reduplication structure of the origin recognition complex bound to dna replication origin endoreduplication of the mouse genome in the absence of orc the cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall a small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution fetal blood-brain barrier p-glycoprotein contributes to brain protection during human development zika virus damages the human placental barrier and presents marked fetal neurotropism extracellular vesicles and their immunomodulatory functions in pregnancy the transport mechanism of extracellular vesicles at the blood-brain barrier we acknowledge the technical support by dr. wenyu lin and dr. shaobo wu. shuang li conceived the study, analyzed the data, and wrote the paper; anjing zhu, kai ren, shilin li and limin chen provided experimental materials; limin chen revised the paper. all authors have read and agreed to the published version of the manuscript. the authors declare no conflicts of interest. key: cord- - i abjfa authors: schwarz, megan c.; sourisseau, marion; espino, michael m.; gray, essanna s.; chambers, matthew t.; tortorella, domenico; evans, matthew j. title: rescue of the zika virus prototype strain with a cytomegalovirus promoter-driven cdna clone date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: i abjfa the recent zika virus (zikv) outbreak has been linked to severe pathogenesis. here, we report the construction of a plasmid carrying a cytomegalovirus (cmv) promoter-expressed prototype uganda mr zikv cdna that can initiate infection following direct plasmid dna transfection of mammalian cells. incorporation of a synthetic intron in the nonstructural protein (ns ) region of the zikv polyprotein reduced viral cdna-associated toxicity in bacteria. high levels of infectious virus were produced following transfection of the plasmid bearing the wild-type mr zikv genome, but not one with a disruption to the viral nonstructural protein (ns ) polymerase active site. multicycle growth curve and plaque assay experiments indicated that the mr virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published cambodia and brazil cdna-rescued zikv. this zikv infectious clone will be useful for investigating the genetic determinants of zikv infection and pathogenesis and should be amenable to construction of diverse infectious clones expressing reporter proteins and representing a range of zikv isolates. importance the study of zikv, which has become increasingly important with the recent association of this virus with microcephaly and guillain-barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. this work describes a model system to produce infectious virus in cell culture. we created a plasmid carrying the prototype uganda mr zikv genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this dna. furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. this model system will provide a simple and effective means to study how zikv genetics impact viral replication and pathogenesis. the study of zikv, which has become increasingly important with the recent association of this virus with microcephaly and guillain-barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. this work describes a model system to produce infectious virus in cell culture. we created a plasmid carrying the prototype uganda mr zikv genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this dna. furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. this model system will provide a simple and effective means to study how zikv genetics impact viral replication and pathogenesis. ( ) . the approximately , amino acid viral polyprotein is cleaved by host and viral proteases into structural proteins (capsid [c] , premembrane [prm] , and envelope [e]), which form the infectious virion, and nonstructural (ns) proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ). the ns proteins participate in virion assembly, innate immune suppression, and formation of a cytoplasmic replication complex that replicates the viral genome through a negativestrand rna intermediate. zikv was first identified nearly years ago ( ) and has been progressively circulating in various parts of the world, including africa and asia. zikv emergence in south america in the last years marked the first observation of a link between infections and severe forms of pathogenesis, including microcephaly and guillain-barré syndrome ( ) . it is not known if the diseases caused by recent infections are a result of changes to the virus that increase pathogenesis or are influenced by host polymorphisms and immune reactions. the african, asian, and south american zikv isolates are closely related phylogenetically; the original strain, termed mr (accession no. hq ) ( ) , and a recent isolate identified in puerto rico, termed prv (accession no. ku ) ( ), share . % amino acid identity. thus, the characterization of sequence changes between these isolates that impact replication and disease should be reasonably straightforward. such studies would be greatly empowered by the availability of a simple system to assay the functional consequences of altering viral sequences (i.e., a "reverse genetic" system). for most positive-strand rna viruses, where the genome directly encodes the proteins required for its replication, transfection of a permissive cell with viral rna is sufficient to initiate infection that eventually results in the release of viral progeny. these genomes can be cloned into plasmids with elements required for in vitro transcription of the long viral rna for transfection. such an approach has only recently been used to rescue a cambodia zikv isolate ( ) . since flaviviruses have capped rna genomes, the transcription step can be circumvented by transfecting a plasmid carrying the viral genome under the control of an rna polymerase ii promoter, with the correct = end of the viral rna being generated by a hepatitis d virus ribozyme (hdvr) ( , ) . the plasmid dna is required only to initiate transcription of the first round of viral rna, which can serve as both a translation and a replication template to initiate "infection" of transfected cells. indeed, this is the approach that tsetsarkin et al. recently used to rescue a brazil zikv isolate ( ) . flavivirus sequences are notoriously difficult to propagate in bacteria, likely because the presence of cryptic bacterial promoters permits expression of viral proteins that result in bacterial toxicity (reviewed in references and ). numerous strategies have been devised to limit and prevent bacterial death, including the use of bacteria that are more resistant to this toxicity, very-low-copy-number plasmids such that fewer viral translation products are produced, and alternative organisms such as yeast for plasmid propagation. another strategy entails the separation of the viral genome into multiple plasmids, where each piece can be excised by restriction enzyme digestion and ligated into the full-length viral cdna, which can then serve as the template for in vitro transcription. here, we used an alternative strategy to stabilize the mr zikv genome ( ) in bacteria that has been successfully used to stabilize other positive-sense rna viruses, including transmissible gastroenteritis coronavirus and japanese encephalitis virus ( , ) and, more recently, the brazil zikv genome ( ) . we identified the major region of the mr genome that induced toxicity in bacteria and then cloned this sequence with a synthetic intron insertion to interrupt viral translation in bacteria. this rna was spliced in mammalian cells to recreate the authentic viral genome, which efficiently initiated infectious virus production. identification and stabilization of a bacterially toxic zikv cdna region. to generate a plasmid carrying the zikv cdna (fig. b) , we purified rna from mr inoculum and used reverse transcription-pcr (rt-pcr) to amplify overlapping regions of the viral genome, which were progressively cloned into the high-copy-number bacterial plasmid pcdna . (fig. c) . in this plasmid, the authentic = end of the viral sequence was placed at the transcriptional initiation site of the cytomegalovirus (cmv) promoter, the simian virus (sv ) polyadenylation site terminates transcription, and an hdvr trims the viral genome to its authentic = end (fig. b) . the resulting transcript of the zikv genome is identical to that of a genome deposited in a host cell by a virion and is expected to initiate infection. during cloning of the zikv rt-pcr products (scheme illustrated in fig. c ), we were unable to generate plasmids carrying an intact ns through ns coding sequence. our first attempts at cloning this region of the zikv genome resulted in plasmids with large deletions, which we deduced to likely be a result of homologous recombination events by noting short stretches of sequence similarity at either side of the deleted segment. in an attempt to circumvent this issue, we screened a panel of bacterial strains with documented inefficiencies at homologous recombination. indeed, using new england biolabs' turbo competent cells allowed us to propagate a plasmid bearing the entire zikv cdna. however, all such plasmids continued to lack an intact viral open reading frame due to the presence of either nonsense or frameshift mutations within the ns coding sequence. at this stage, we hypothesized that translation of viral polyprotein sequence from this region led to toxicity in bacteria. to test this hypothesis, we inserted a synthetic intron in ns , such that the coding sequence would be disrupted in bacteria but splicing in mammalian cells would restore the viral rna ( fig. b and c) . indeed, this plasmid could be stably propagated in bacteria, thus completing the generation of a plasmid containing the entire zikv cdna. as a negative control for subsequent replication studies, we also generated a version of this plasmid encoding an inactivating gdd to gnn mutation in the viral ns rna-dependent rna polymerase (rdrp) catalytic active site [pol(Ϫ)]. to characterize the ability of the above plasmids to produce zikv proteins in mammalian cells, we transfected t cells with the wild-type and pol(Ϫ) mr zikv plasmids and infected cells in parallel with the parental mr inoculum at an approximate multiplicity of infection (moi) of . % tissue culture infective dose (tcid ) per cell. three days later, the cells were fixed and immunostained with antibodies against the zikv e and ns proteins ( fig. a) . while all transfected and infected cells expressed both viral proteins, cells transfected with the wild-type mr plasmid exhibited larger amounts of antibody staining than those transfected with the pol(Ϫ) plasmid. this difference was confirmed by quantifying e protein immunostaining by fluorescence-activated cell sorting (facs) analysis (fig. b ). to examine viral protein expression over time, cells collected at various time points following transfection or infection were analyzed by facs for e protein antibody staining or by immunoblotting of cell lysates with ns antibodies ( fig. c and d, respectively). while the percentage of e protein-positive cells increased over days following transfection with the wild-type mr plasmid and infection with the parental mr virus, the e protein-positive pol(Ϫ) plasmid-transfected cells remained below % throughout this time course (fig. c ). ns protein levels also increased over time in both wild-type plasmid-transfected and parental virus-infected cell populations and yet remained below the limit of detection in pol(Ϫ) plasmid transfected cells. these results confirmed that although both the wild-type and pol(Ϫ) mr zikv plasmids were able to express viral proteins, protein levels were higher and were detected in a greater percentage of cells transfected with the wild-type plasmid, which may be due to viral rna replication and viral spread. cells transfected with either the wild-type or the pol(Ϫ) mr plasmid, as observed above ( fig. a) , would require splicing of the viral rna transcript. to directly examine this, total rna was harvested days following transfection with either plasmid, or infection with the parental mr virus, and rt-pcr was performed using oligonucleotides flanking the region of the intron-containing ns gene. as shown in fig. a , a single dna product was amplified from wild-type plasmid transfected cells that was similar in size to the parental mr rt-pcr product. this dna fragment likely represented spliced ns rna. however, two distinct rt-pcr products were observed from pol(Ϫ) plasmid transfected cells, likely representing both spliced and unspliced rna [ fig. a , "pol(Ϫ)"]. indeed, sequence analysis of bacterial plasmid clones of these rt-pcrs demonstrated that all products from wild-type plasmid-transfected cell rna lacked the inserted intron (fig. b) . in contrast to parental virus rt-pcr products (fig. b) , these sequences carried a single silent g a mutation that was inserted during intron cloning, which indicated that these rnas were generated from the mr plasmid. while spliced sequences were observed in eight of the bacterial plasmid clones of the pol(Ϫ) rt-pcr products, seven of these clones retained the intron sequence (fig. b) . these results confirmed that splicing can remove the intron from rna transcribed from both the wild-type and pol(Ϫ) mr plasmids. furthermore, the greater abundance of spliced rna in wild-type plasmid-transfected cells may represent amplification due to viral rna replication. to evaluate production of infectious virus, we challenged vero cells with supernatant from t cells transfected with the wild-type or pol(Ϫ) plasmid or infected with the parental mr virus at an approximate moi of . tcid per cell. the vero cells were immunostained with e and ns antibodies days postchallenge (fig. a ). the addition of supernatants from wild-type plasmid-transfected or parental virus-infected t cells resulted in readily detectable levels of viral proteins. conversely, no staining was observed in vero cells challenged with pol(Ϫ) plasmid transfected t cell supernatant. thus, cells transfected with the wild-type mr plasmid produced infectious particles in a manner that was dependent on the capacity of the viral rna to replicate. infectious virus from transfected and infected t cells was quantified by endpoint dilution assay on vero cells (tcid ) by measuring zikv-associated cytopathic effect (cpe). as observed above, t cells transfected with the pol(Ϫ) mr plasmid failed to release detectable levels of infectious virus (data not shown). at h after transfection with the wild-type mr plasmid, t cells produced . ϫ tcid /ml, which increased to . ϫ and . ϫ tcid /ml at and days posttransfection, respectively (fig. b ). parallel infection of t cells with the parent mr virus resulted in supernatants containing between . ϫ and . ϫ tcid /ml. however, it should be noted that the h time point likely contains residual input virus, as we chose to conduct overnight infections and did not wash input virus away prior to collecting the first time point. nevertheless, comparing the kinetics of virus released following plasmid transfection with that following virus infection may not be relevant because rna delivered in a virion can be directly translated and replicated while plasmid-expressed rna requires transcription, splicing, and export from the nucleus. comparison of the growth properties of rescued and parental mr viruses. the previously developed systems for producing infectious cambodia zikv and brazil zikv from plasmids showed that the rescued viruses were attenuated in comparison to the parental inoculum ( , ) . to compare the fitness levels of our parental and rescued mr viruses, we first calculated their vero cell specific infectivities. to ensure that only virion-associated rna was quantified, the rescued virus was initially passaged twice at an moi of . in vero cells. the rescued and parental mr viruses exhibited similar and not significantly different specific infectivity values of and rna molecules per tcid (fig. c ). to directly compare growth properties of the t cell-rescued mr virus to those of the parental inoculum using a strategy similar to that used on previously published systems, we performed plaque assays ( fig. d and e) . when visualized at days postinfection, the rescued and parental mr zikv-induced plaques exhibited a . -fold, but not statistically significant, difference. according to the work of tsetsarkin et al., the rescued brazil zikv isolate made plaques that were . -fold smaller than the parental virus plaques at this time point ( ) . a retrospective analysis of plaque sizes in the publication by shan et al. showed that their rescued cambodia zikv produced plaques that were approximately . -fold smaller than parental virus plaques at days postinfection ( ) . however, we were able to detect slight attenuation of our rescued virus, as we observed statistically significant . -and -fold differences in rescued and parental mr zikv plaque sizes when we stained our plaque assays at and days postinfection, respectively ( fig. d and e) . to further compare the growth properties of the rescued and the parent viruses, multicycle growth curve experiments were performed in which vero cells were infected (moi of . ) for h with either the rescued wild-type virus or the parental virus, followed by several washes to remove input virus. supernatants were collected and filtered daily up to days postinfection, and then titers of the supernatants were determined by vero cell tcid assay. the results of the growth curve revealed that our rescued virus has growth kinetics comparable to those of the parental virus (fig. f) . growth curves initiated at a range of mois also demonstrated comparable growth kinetics between the rescued and parental viruses (data not shown). these results are in stark contrast to previously published multicycle growth curve results with the cambodia and brazil zikv rescued viruses, which exhibited approximately -to 's t test) . -fold slower spread than their respective parental isolates. therefore, although extended plaque assays indicate that our rescued mr virus may exhibit some attenuation in comparison to the parental mr isolate, its replication characteristics are closer to those of its parental isolate than to the cambodia and brazil zikv rescued viruses. in this work, we describe the creation of a plasmid-based rescue system for the prototype uganda mr zikv. other groups have published zikv infectious virus rescue systems. the first such system was a clone of the cambodia zikv isolate cdna ( ) . here, a t rna polymerase promoter directed in vitro transcription of the viral rna for subsequent transfection into permissive host cells for initiation of infection. our approach to express the zikv rna from an rna polymerase ii promoter with a ribozyme positioned at the = end to trim the viral transcript allowed us to avoid the potentially arduous in vitro transcription step and directly transfect the plasmid into host cells. the shan et al. system lessened the toxicity of zikv cdna in bacteria by using a very-low-copy-number bacterial plasmid. in our system, the deleterious sequence within the genome was disrupted by the addition of an intron, which allowed the successful amplification of this sequence in a high-copy-number plasmid (fig. ) . in a more recent publication, tsetsarkin et al. described their derivation of a similar plasmidbased rescue system for a brazil zikv isolate ( ) . they also found that the toxicity of the zikv cdna could be reduced by insertion of introns in the viral open reading frame. a second recently published zikv rescue system avoided bacterial toxicity by transfection of overlapping synthetic dna fragments carrying the mr cdna ( ) . in permissive host cells, these fragments were joined by recombination to launch viral rna transcription from a cmv promoter, analogous to our approach. although not reported, it is likely that the efficiency of recombination limited the initial rescued virus titers. thus, generating high-titer virus stocks in this system required amplification by passage of rescued virus. our plasmid-based system allowed the production of hightiter virus directly from transfected cells, which results in an increased capacity to generate isogenic virus stocks. this would be particularly important when analyzing unstable viral genomes, including genomes that are naturally attenuated in specific host cells or those carrying mutations that limit viral replication and thus may revert during passage. virus produced from the previously published cambodia and brazil zikv isolate rescue systems exhibited slower growth kinetics than the parental viruses, potentially due to sequence diversity within the parental virus populations that provided a fitness advantage ( , ) . conversely, our rescued mr virus grew identically to the parental isolate in vero cell multicycle growth curve experiments and produced similarly sized plaques in standard -day infection plaque assays. thus, even though we observed plaque size differences between our parental and rescued virus in extended infection, and thus likely more sensitive plaque assays, we believe that our mr rescued virus is less attenuated than the rescued cambodia and brazil zikv. it is unlikely that the methods of rescue (transfection of in vitro transcribed rna versus plasmid dna) impacted virus attenuation. instead, we hypothesize that viral genetics influenced postrescue attenuation, and this may be greatly influenced by the passage history of a viral isolate. the mr isolate may not require the same sequence diversity as the cambodia and brazil zikv isolates to reach optimal fitness in vero cells. the mr virus has been subjected to more rounds of amplification in vero cells than these more recent strains, which may have preadapted it to efficient cell culture replication. ultimately, rescued virus attenuation should not be a serious complication to experiments to study viral determinants of replication or pathogenesis. researchers using any zikv rescue system would merely need to monitor the stability of engineered sequences of interest and compare phenotypes between wild-type and mutant viruses rescued in parallel. overall, the plasmid-based rescue system described in this work is a simple and elegant system that paves the way for a variety of future zikv experiments. reporter viruses can be created by cloning fluorescent or bioluminescent proteins into the mr plasmid as an efficient strategy to quantify and monitor infections. this system will also permit the modification of viral sequences to study how genetic determinants impact the viral life cycle and pathogenesis. we will also derive analogous plasmids bearing the cdna sequence of other zikv isolates, including those currently circulating in the americas. such systems will allow the detailed comparison of the molecular determinants for potential strain-specific zikv replication and pathogenesis mechanisms and/or efficiencies. however, all derived clones carried nonsense or frameshift mutations, suggesting that the zikv open reading frame still induced toxicity in these bacteria. we chose a plasmid with a frameshift mutation created by a single g nucleotide insertion after nucleotide position for further cloning. a gblock bearing nucleotides before and after the sali and xhoi sites, respectively, was synthesized with a silent g a mutation in the mr coding sequence followed immediately by the synthetic intron from plasmid phtn (promega, madison, wi) (genbank accession no. jf ), which is based on the human beta-globin intron. in-fusion cloning of this dna into the above plasmid resulted in a plasmid that was stably propagated in bacteria. we termed this plasmid pcdna . atccmr intron hdvr. we also generated a version of this plasmid that carries an inactivating gdd-to-gnn mutation in the viral ns rna-dependent rna polymerase (rdrp). we generated this plasmid by amplifying pcdna . mr intron hdvr with oligonucleotides carrying two nucleotide coding mutations in the ns rdrp active site as well as a silent mutation to create an sphi restriction site, me-o- ( = ctatcat cgattggcttcacaacgcagttatttccactgaccgcc) and me-o- ( = gttgtgaagccaatcgatgata ggtttgcgcatgccctcaggttc). this pcr product was amplified with me-o- ( = gcaagcggccac gcgtctgcaccaaagaagag) and me-o- to clone into the mlui and kpni sites. we termed this plasmid pcdna . mr intron pol(Ϫ) hdvr. t cell transfection and supernatant collection. plasmid-based zikv was produced in t cells by transfections performed in triplicate. one day prior to transfection, ϫ cells/well were seeded in -well polylysine-coated plates. cells were transfected with . sequences of the primers and probe targeting zikv have been modified from previously published sequences ( ) . quantification of zikv rna copies per milliliter of supernatant was performed against a standard curve of in vitro-transcribed mr zikv rna. immunoblot analysis. immunoblot analysis was performed as previously described ( ) with primary antibodies against ns (rabbit polyclonal antiserum , raised against a peptide representing amino acids to of the genbank aav mr sequence) and actin (ac- ; sigma-aldrich, st. louis, mo), horseradish peroxidase (hrp)-conjugated anti-rabbit and anti-mouse secondary antibodies (thermo fisher scientific, waltham, ma), and immobilon chemiluminescent hrp detection reagent (emd millipore, billerica, ma). immunofluorescence. cells were fixed with % paraformaldehyde and stained with the rabbit polyclonal antiserum against zikv ns or a primary antibody targeting the flavivirus e proteins (clone d - g - - ; emd millipore) and a goat anti-rabbit or a goat anti-mouse secondary antibody conjugated to alexa fluor and (thermo fisher scientific, waltham, ma), respectively, using methods previously described ( ) . the nucleus was visualized with hoechst stain (thermo fisher scientific, waltham, ma). images were acquired with the evos microscope (thermo fisher scientific, waltham, ma). flow cytometry. cells were fixed with % paraformaldehyde and stained with the primary g flavivirus e antibody (emd millipore, billerica, ma) and a goat anti-mouse secondary antibody conjugated to alexa fluor (thermo fisher scientific, waltham, ma), using methods previously described ( ) . cells were analyzed using an lsrii flow cytometer (becton, dickinson, franklin lakes, nj). rt-pcr analysis of splicing. rna was extracted from cells h after transfection with the wild-type mr plasmid or pol(Ϫ) plasmid or infection with the parental mr virus or a mock control using the purelink rna minikit (thermo fisher scientific, waltham, ma). the extracted rna was used as a template for random hexamer-primed cdna synthesis using the superscript iii first-strand synthesis system (thermo fisher scientific, waltham, ma). five hundred nanograms of cdna was used for pcr using the expand high-fidelity pcr system (roche life sciences, indianapolis, in) with oligonucleotides flanking the region into which the intron was cloned: me-o- (atgtccgcttgagcacagag) and me-o- (agcgatgttgtcagtgcgtg). pcr products were ligated into pgem-t vector (promega, madison, wi) for subsequent propagation in bacteria and sequencing. growth curves. vero cells ( ϫ ) were seeded in a -cm petri dish, day prior to infection, in triplicate, and then infected at a multiplicity of infection (moi) of . vero cell tcid per cell in dmem with % fbs. virus was removed h later, and cells were washed twice with pbs before ml of dmem was added with % fbs. supernatants were collected and filtered ( . m pore size) daily for the next days. supernatant infectivity was assayed as previously described. plaque assays. one day prior to infection, . ϫ vero cells were seeded in polylysine-coated -well plates in dmem with % fbs. for infection, medium was replaced with l of -fold serial dilutions (with a starting moi of ) of either the parental or rescued virus. after h at °c, . ml . % methylcellulose in dmem with % fbs was added to each well. at to days postinfection, cells were fixed in ml % paraformaldehyde for h at room temperature. cells were washed twice with pbs and stained with l of . % crystal violet solution in ethanol. plates were washed twice with water and air dried. plates were scanned, and plaque sizes were measured using imagej software (national institutes of health, bethesda, md; http://imagej.nih.gov/ij/). statistical analysis. data were analyzed using an unpaired student t test (*, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ) on prism software (graphpad software). values in graphs represent the mean and standard error from experiments performed in triplicate or quadruplicate, with between two and five independent experiments. accession number(s). the sequence of the mr zikv genome with the additional c nucleotide in the = utr was deposited in genbank under accession number kx . the sequence for plasmid pcdna . atccmr intron hdvr has been deposited in genbank under accession number kx . flaviviridae zika virus. i. isolations and serological specificity zika virus in the americas-yet another arbovirus threat local transmission of zika virus-puerto rico an infectious cdna clone of zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors dna vaccine coding for the full-length infectious kunjin virus rna protects mice against the new york strain of west nile virus a rapid and quantitative assay for measuring antibody-mediated neutralization of west nile virus infection a full-length infectious cdna clone of zika virus from the epidemic in brazil as a genetic platform for studies of virus-host interactions and vaccine development flavivirus reverse genetic systems, construction techniques and applications: a historical perspective functional cdna clones of the flaviviridae: strategies and applications stabilization of a full-length infectious cdna clone of transmissible gastroenteritis coronavirus by insertion of an intron a new strategy in design of ϩrna virus infectious clones enabling their stable propagation in e. coli a robust method for the rapid generation of recombinant zika virus expressing the gfp reporter gene temporal analysis of hepatitis c virus cell entry with occludin directed blocking antibodies complete replication of hepatitis c virus in cell culture a simple method of estimating fifty percent end points full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia species-specific regions of occludin required for hepatitis c virus cell entry selection of a hepatitis c virus with altered entry factor requirements reveals a genetic interaction between the e glycoprotein and claudins we thank adolfo garcía-sastre (icahn school of medicine at mount sinai) for the atcc mr zikv isolate, theodore pierson (nih) for the west nile virus replicon plasmid, charles rice (rockefeller university) for t cells, and the dean's flow cytometry core at the icahn school of medicine at mount sinai for facs assistance. key: cord- - zr e lh authors: mohd ropidi, muhammad izzuddin; khazali, ahmad suhail; nor rashid, nurshamimi; yusof, rohana title: endoplasmic reticulum: a focal point of zika virus infection date: - - journal: j biomed sci doi: . /s - - - sha: doc_id: cord_uid: zr e lh zika virus (zikv) belongs to the flavivirus genus of the flaviviridae family. it is an arbovirus that can cause congenital abnormalities and is sexually transmissible. a series of outbreaks accompanied by unexpected severe clinical complications have captured medical attention to further characterize the clinical features of congenital zikv syndrome and its underlying pathophysiological mechanisms. endoplasmic reticulum (er) and er-related proteins are essential in zikv genome replication. this review highlights the subcellular localization of zikv to the er and zikv modulation on the architecture of the er. this review also discusses zikv interaction with er proteins such as signal peptidase complex subunit (spcs ), er membrane complex (emc) subunits, and er translocon for viral replication. furthermore, the review covers several important resulting effects of zikv infection to the er and cellular processes including er stress, reticulophagy, and paraptosis-like death. pharmacological targeting of zikv-affected er-resident proteins and er-associated components demonstrate promising signs of combating zikv infection and rescuing host organisms from severe neurologic sequelae. zika virus (zikv) is a mosquito-borne virus that belongs to the flaviviridae family together with other notable flaviviruses such as dengue virus (denv), west nile virus (wnv), japanese encephalitis virus (jev), and yellow fever virus (yfv). zikv was first isolated from a febrile rhesus monkey in april and was subsequently isolated from aedes africanus mosquitoes months later in zika forest of uganda [ ] {dick, # }. despite its discovery more than a half-century ago, zikv received little attention due to sporadic cases of human infection with mild and self-limiting symptoms [ ] . zikv was put under scrutiny following its first outbreak in the yap island of micronesia in . during this outbreak, suspected cases of zikv infection were reported, and at least % of these patients were either serologically or molecularly confirmed for zikv infection [ ] . the same study estimated that island residents ( %) were infected, of which were symptomatic [ ] . zikv-infected patients typically present mild clinical symptoms such as fever, maculopapular rash, conjunctivitis, and arthralgia [ ] . nevertheless, the second zikv outbreak in french polynesia in provided the first compelling relationship between zikv infection and a neurological complication, where a woman was diagnosed with guillain-barré syndrome (gbs), an autoimmune disease typically affecting motor neuron functions, a week following the onset of zikv-like symptoms [ ] . this epidemic also recorded a -fold increase of gbs incidence, where gbs-diagnosed patients ( %) were serologically positive for zikv [ , ] . the sudden spike of microcephaly cases among newborns in brazil following the - zikv outbreak triggered the brazilian ministry of health and the world health organization to declare zikv as a national and international public health emergency [ , ] . the causative link between zikv and congenital neurological anomalies was established based on several evidence including the detection of zikv rna within the amniotic fluid acquired from zikv-infected mothers with confirmed fetal microcephaly case [ , ] . subsequent clinical and pre-clinical findings further supported the causal relationship between zikv infection and microcephaly in newborns [ ] [ ] [ ] . zikv is believed to infect cells through receptormediated endocytosis. these putative receptors include cluster of differentiation (cd ), tyrosine-protein kinase receptor tyro , and axl, where overexpression of these receptors in zikv-impervious hek t cells rendered the cells susceptible to zikv infection [ ] . in particular, the role of axl in zikv infection has been extensively investigated due to its crucial role in dengue virus infection [ ] . during zikv infection, axl mediates zikv entry indirectly whereby the phosphatidylserine extension on zikv lipid membrane binds to growth arrest-specific (gas ), one of the ligands for axl that serves as a bridge for zikv and axl interaction, resulting in clathrin-mediated virus internalization [ , ] . the acidic microenvironment within the endosome promotes fusion between the virus envelope proteins and the endosomal membrane resulting in the release of zikv genome into the host cell cytosolic space [ , ] . the zikv genome is a positive-sense, single-stranded rna ((+)ssrna) of approximately , bases in length [ ] . zikv genome contains a single open reading frame that encodes three structural and seven non-structural (ns) proteins. these structural proteins consist of capsid (c), pre-membrane (prm), and envelope (env) proteins, which are predominantly involved in viral pathogenesis and virion structure. the seven non-structural proteins, ns , ns a, ns b, ns , ns a, ns b, and ns proteins, largely contribute towards the purposes of viral pathogenesis, replication, and immune evasion [ ] . zikv utilizes host translational machinery to produce a single polyprotein that is further cleaved by viral ns b-ns serine protease and host cell protease into functional viral proteins [ ] . these viral proteins are then distributed to cellular compartments for various functions [ ] . zikv proteins localize to distinct subcellular compartments zikv proteins are primarily distributed within and in close proximity to several endomembrane compartments including the endoplasmic reticulum, golgi apparatus, endosomes, lysosomes, autophagosomes, and nucleus [ ] . molecular cloning and expression of individual zikv proteins reveal distinct and specific organellar localization. zikv capsid localizes to several compartments including the nucleus and the golgi [ ] . in addition to capsid, ns b and ns a also localize to the golgi. three zikv proteins namely env, prm, and ns a are distributed to the er as indicated by their co-localization with calreticulin expression. ns , which contains nuclear localization signals, forms punctate distribution in the nucleus [ ] . however, these data should be interpreted with caution as the subcellular localization of these cloned viral proteins may differ in zikv-infected cells. for example, individual expression of zikv ns localizes to the mitochondria, but ns is instead localized at the er when co-expressed with ns b [ ] . similarly, although ns protein is primarily detected in the nucleus of zikv-infected cells [ ] , zikv ns -rna polymerase has been shown to interact with zikv ns -helicase during viral rna replication in the er [ ] . inhibiting this interaction reduces ns helicase activity. subcellular localization of viral proteins is affected by viral proteins interaction, which is crucial for productive infection. importantly, understanding the mechanisms of viral protein transport and viral protein-protein interaction may provide novel therapeutic targets. the subcellular distribution of certain zikv proteins was corroborated in a separate study where interatomic analyses using proximity-dependent biotin-identification (bioid) labeling and flag-based immunoprecipitation (ip) coupled with mass spectrometry (ms) uncover indepth molecular interactions between zikv proteins with various host organelles and proteins. in general, zikv proteins mainly interact with host proteins that are involved in protein processing, vesicle trafficking, rna processing, and lipid metabolism [ ] . zikv capsid interacts with multiple nucleolar proteins, whereas ns protein targets and disrupts the cajal bodies in the nucleus [ ] . this observation is consistent with the aforementioned experimental study that showed nuclear localization of zikv capsid and ns protein [ ] . the study also identified numerous interactions between viral proteins (prm, env, ns a, ns b, ns a, and ns b) with various er proteins, which highlights the importance of the er in zikv life cycle [ ] . zikv has been reported to be dependent on several er proteins including er-associated signal peptidase complex (spc) proteins, er translocon, and er membrane complex (emc) proteins. spc, especially spc subunit (spcs ), is crucial for zikv pathogenesis as knocking out spcs in t cells significantly reduced zikv infection and drastically impaired the production of infectious zikv particles [ ] . a study using pooled crispr/cas cell survival enrichment assay identified zikv strongly depends on er membrane complex (emc) during early-stage zikv replication [ ] . emc is a highly conserved oligomeric complex residing on the er membrane and is crucial for transmembrane protein folding and lipid trafficking [ ] . the dependency on six emc subunits (emc -emc ) was verified with sirna assays, where depletion of these proteins significantly impaired the replication of several zikv strains [ ] . another study reported that zikv ns b physically interact with emc subunit and depletion of this subunit markedly reduced the level of zikv ns a and ns b protein [ ] , indicating that emc proteins are required for viral protein biogenesis. further investigation using denv ns b identified two marginally hydrophobic domains at the n-terminal of ns b to be crucial for ns b dependence on emc [ ] . since zikv ns b shares moderate sequence identity, high sequence similarity, and similar topology with other flaviviruses [ ] , it is plausible that the two weak hydrophobic domains of zikv ns b are the specific determinants for zikv dependency on emc proteins. in addition to its direct interaction with viral proteins, emc proteins also associate with er sec translocon and oligosaccharyltransferase (ost) complex proteins, both of which are also important for zikv infection [ , ] . sec is a major component of the er translocon that facilitates the entry of nascent polypeptides into the er lumen for protein processing. zikv dependency on sec translocon was validated in a separate study wherein myolactone treatment, a sec α inhibitor, dramatically reduced zikv expression [ ] . zikv replication was restored in cells expressing mutant sec α that conferred resistance against myolactone inhibition [ ] . zikv dependency on ost complex, an integral part of the translocon consisting of eight er-transmembrane protein subunits that catalyzes co-translational n-glycosylation, is based on emc , emc , emc and emc interaction with ost complex subunits namely stt a/b, rpn / , and ddost [ ] . additionally, zikv proteins including ns b have been reported to directly interact with these ost subunits [ ] . zikv dependency on ost complex is corroborated by marceau et al. that reported a significant abrogation of zikv rna replication in stt a knockout cells [ ] . importantly, treatment with ngi- , a small molecule ost complex inhibitor, significantly reduced zikv rna replication with an ec value of . μm [ ] . the inhibitory activity of this non-toxic molecule (cc = . μm) is independent of n-linked glycosylation activity of the ost complex as zikv replication was drastically impaired in hap cells containing wild-type or catalyticinactive stt a [ ] . the specific mechanism of zikv dependency on stt a is unclear, but immunoprecipitation and electron microscopy studies showed physical interactions between denv proteins and stt a/b, forming viral rna replication complexes that reside within the er in close proximity to denv -vesicle packets [ ] . since zikv proteins also directly interact with stt a and zikv infection induces the formation of vesicle packets [ , ] , it is plausible that zikv depends on ost complex in a similar manner. in short, zikv proteins interact with various host proteins and exploit their functions to accommodate the progression of the viral life cycle. er and er-related proteins are especially important in the virus genome replication, which may be useful in developing antiviral therapies. the following sections of this review will discuss the structural and molecular changes in the er following zikv infection. throughout the course of infection, viruses induce various modifications on the host cells' metabolisms and their cytoarchitecture to support the progression of virus life cycle. one of the common targets is the er [ ] . among the important functions of the er include protein processing, synthesis of essential biosynthetic compounds (primarily lipids and proteins), metabolism of steroids and carbohydrates, and storage of calcium ions (ca + ) for intracellular signaling [ ] . structurally, the er is made of a continuous network of membraneenclosed flattened sacs and tubules. the dynamic and fluid nature of these structures enable this organelle to accomplish tubule extension, membrane curvature and shape alteration in response to stress-induced or factordemanding conditions such as cell division and differentiation [ ] . likewise, zikv infection is also capable of exploiting the organelle's plasticity and remodels the er structure for their replicative benefit. recent transmission electron microscopy analyses on zikv-infected cells frequently observed significant expansion of the er [ , , ] . this structural modification typically consisted of proliferating er lamellae and dilated er lumen, which cumulatively increases the overall er size and volume. enlargement of the er is believed to occur in response to virus-induced er stress, due to a supply-demand imbalance that will be elaborated in the next section. zikv infection also forms distinct aggregates of intricate er tubular network that resembles sponge-like matrix called the convoluted membranes (cm) (fig. ) [ , [ ] [ ] [ ] . cm aggregates are frequently seen bordering zikv virions and other zikv-induced structures, with occasional virions also found within the cm itself [ , ] . the function of the cm in zikv infection is unclear; nevertheless, evidence from other flaviviruses studies suggest that the cm possesses roles closely associated to protein synthesis and processing [ ] . interestingly, cm aggregates are not observed in zikv-infected neural progenitor cells, suggesting cell-type-specific factors are required to develop the cm [ ] . no discernible difference in the cm structure is observed between the african and asian zikv strains [ , ] . next, vesicle packets (vps) are also commonly observed in zikv-infected cells ( fig. ) [ , [ ] [ ] [ ] . vps refers to an aggregate of closely packed single-membrane vesicles that are encapsulated within an er cisterna. individually, these vesicles measure between and nm in diameter with a narrow channel of approximately nm that connects the vesicle lumen and the cytosol [ , ] . the radial dimension of these vesicles varies according to the host cell type, thereby hinting possible involvement of cell-type-specific factors in its formation [ ] . modest radial difference was also noted between zikv lineages [ ] ; however, more morphologically pronounced difference between the zikv lineages is observed in the vesicle's shape whereby african strain tend to form ovoid vesicles, whereas the asian strain's vesicles are generally more spherical [ ] . nevertheless, these differences, especially the latter, remain contentious due to limited experimental measurements and warrants additional investigations across various cell types to support these observations. despite these differences, the vesicles' pore maintain a comparable diameter of nm regardless of the virus strain and the host cell type, suggesting that a conserved cellular or viral machinery mediates pore formation [ ] . zikv exploits the er dynamic characteristic and remodels the er structure to generate virus-induced structures, vesicles (ve), vesicle packet (vp), convoluted membrane (cm) and zippered er (zer), for the benefit of virus replication. blue and green box depicts schematic representation of host and viral factors involved in vesicle and virus particle (vi) formation, respectively. zikv ns a utilizes the host reticulon . a (rtn . a) to facilitate membrane curvature during vesicle invagination into the er lumen (blue box). viral genome replication takes place within this vesicle. neosynthesized viral rna genome is released into the cytosol and could undergo either translation, virus particle assembly, or another round of genome replication. virus particle assembly takes place in apposed er leaflet. separate zikv ns a independently recruits viral genome rna, ns b-ns , and unprocessed c-prm-env complexes, and subsequently congregates at the virus particle assembly site through ns a oligomerization (green box). once assembled, ns b-ns proteolytical activity cleaves the recruited c-prm-env complex to generate individual capsid, prm and env protein. cleaved capsid protein interacts with capsid-dense lipid droplets and viral rna to generate nucleocapsid core followed by env and prm proteins encapsulation of the nucleocapsid core invagination of zikv-induced vesicles into the er lumen is mediated through the host reticulon . a (rtn . a) that facilitates er membrane curvature (fig. , blue box) [ ] . silencing of rtn . a impairs ns a protein stability and results in significant reduction of virusinduced vesicles and virus replication [ ] . typically, these vesicles contain the virus' replication complex and therefore carries out the virus rna synthesis or viral replication [ , ] . following viral replication, neosynthesized viral genome rna is released into the cytosol through the vesicle's narrow channel, where it would consequently either undergo another round of genome replication, translation or virion assembly [ , ] . in the latter case, zikv virion assembles and buds into the er lumen directly apposed the narrow pore of the replication vesicle (fig. , green box) [ ] . although detailed mechanistic insights of virion assembly remains uncertain, recent study identified that zikv ns a independently recruits viral (+)-ssrna, ns b-ns , and unprocessed c-prm-env complexes to the virus particle assembly site, possibly through ns a oligomerization [ ] . the same study postulates that virus particle assembly proceeds through ns b-ns protease complex cleaving the recruited c-prm-env complex. cleaved capsid protein form nucleocapsid core with the viral rna and capsid-dense lipid droplet; and subsequently, it is encapsulated by prm and env proteins, which leads to eventual virion budding into the er lumen [ ] . ultrastructural analyses within the dilated er lumen consistently identified virus particles arranged in a two-dimensional paracrystalline array [ , ] . this array is located in close proximity to the replication vesicles and is therefore largely comprised of fully assembled virions and viral-like particles (enveloped virions without viral genome) [ ] . moreover, zikv-infected huh cells also exhibited a unique structural alteration of collapsed er cisterna or more commonly known as zippered er (zer) (fig. ) that reminisce the observation reported in avian infectious bronchitis virus (ibv) infection, a (+)-ssrna virus from the coronaviridae family [ , ] . this finding presents the first identification of such structure among flavivirus-infected cells and has only been observed in zikv-infected huh cells, suggesting that cell-typespecific factors are possibly required to generate this structure [ ] . the role of zer in zikv infection is unclear; however, zer are frequently found adjacent to vps suggestive of functions associated with vesicles formation, which is consistent with observations made in ibvinfected cells [ , ] . nonetheless, this unique finding warrants further research to elucidate the mechanistic details and biological relevance of the zer. in addition to the direct modulation of the er structure, zikv also induces the formation of viroplasm-like structures, which is incidentally the first reported observation of such structure in flavivirus infection [ ] . contrary to other zikv-induced structures, viroplasms are cytoplasmic inclusion of viral replication components and various relevant host factors associated with virus replication that are formed through reorganization of cell membrane or cytoskeletal elements [ ] . these structures are significantly larger than vps and can measure up to nm in diameter [ ] . viroplasms are primarily located in the perinuclear region and in close proximity to the er, mitochondria, and microtubules to facilitate viral genome replication [ ] . the spatial segregation of virus-induced enclosed structures (viroplasms and vesicles) is postulated to create a subcellular microenvironment concentrated with factors required for virus genome replication, and simultaneously provides physical barriers against rna nucleases and the host immune responses [ , ] . the latter, in particular, is consistent with an investigation on wnv that virusinduced structures confer protection, albeit partially, against the host immune mechanism [ ] . in summary, zikv and several zikv proteins localize to the er and eventually leads to the remodeling of the reticular architecture and exploitation of the organelle's unique characteristics. these modifications create an ideal environment for viral genome replication, but simultaneously introduce additional burden and stress on the host cell leading to the activation of several cellular responses, which will be discussed in the following sections. one of the fundamental roles of the er is the folding of secreted and membrane proteins, which depends on a multitude of factors including supply of atps, stable ca + concentration, and a balanced redox environment [ ] . disruption of this specialized environment within the er, whether through glucose deprivation or viral infection, may lead to overwhelming accumulation of misfolded and unfolded proteins-a condition described as er stress [ ] . under normal conditions, glucose regulated protein (grp ), one of the er molecular chaperones, binds to er stress sensor transmembrane proteins: protein kinase rna-activated (pkr)-like er resident kinase (perk), activating transcription factor (atf ), and inositolrequiring enzyme (ire ). in the presence of misfolded or unfolded protein, grp will bind to exposed hydrophobic residues of these proteins to induce proper protein folding [ , ] . accordingly, during er stress, the accumulation of misfolded or unfolded proteins saturates the free pool of grp and titrates the bound grp away from the stress sensors leading to activation of respective sensor's molecular pathways [ ] . these molecular initiatives and their corresponding downstream effects are collectively known as the unfolded protein response (upr). this evolutionarily conserved countermeasure induces pro-survival responses including global arrest of protein synthesis, upregulation of protein degradation factors, and enhanced protein folding capability [ ] . however, under severe er stress conditions, upr will instead trigger apoptosis. following zikv infection, the accumulation of misfolded virus polyproteins in the er lumen overwhelms the er protein-folding capacity leading to er stress and triggers the activation of the upr (fig. ) [ ] . additional evidence of er stress and upr activation are demonstrated in the elevated expression of grp and other chaperones such as calnexin, calreticulin, and protein disulfide isomerase (pdi) in zikv-infected neural cells in vitro and in vivo [ , , ] . increased expression of these er stress markers was accompanied by impaired indirect neurogenesis and microcephalic phenotype in mice [ ] . this finding is consistent with a previous report wherein the induction of upr in cerebral apical progenitor cells tipped neuronal differentiation towards direct neurogenesis at the expense of indirect neurogenesis, leading to depleted intermediate progenitors, reduced overall cortical neuron output, and diminished cerebral volume, which ultimately caused microcephaly in vivo [ ] . in addition to elevating the expression of er chaperone proteins, zikv has been shown to induce upr by activating the stress sensors as summarized below (fig. ) . atf is a type ii er transmembrane protein that contains a transcription activation domain (tad) on its cytosolic-facing n-terminal and two independent golgilocalization signals (gls) on its luminal-facing cterminal [ ] . following the dissociation of grp that unmasks the gls, atf translocate to the golgi apparatus and undergoes sequential proteolytic processing by fig. zikv-induced er stress initiates host cell unfolded protein response (upr). zikv infection induces er stress due to the increased amount of unfolded/misfolded viral (red strand) and host cell (grey strand) protein aggregates in the er lumen. the accumulation of these partially processed proteins leads to the overwhelming demand of correct protein folding. to facilitate protein folding, grp (orange) dissociates from the stress sensors (ire , atf , and perk) and binds to the misfolded/unfolded proteins. expression of other chaperones is also elevated to address the overwhelming protein folding demand during zikv infection. binding of misfolded protein to perk (green) activates the sensor and triggers phosphorylation of eif that in turn stimulates ) global translational block except for selective mrna involved in upr, and ) formation of stress granules. however, zikv bypasses global translation block and inhibits stress granules formation, but the mechanistic details of these viral interference are still unclear. activation of atf (yellow) promotes the protein proteolytical processing in the golgi apparatus into atf n, atf n nuclear translocation, and expression of upr target genes including xbp . activated ire (purple) splices xbp transcript which produces an active transcription factor (xbp s) that stimulates expansion of the er volume, and expression of chop and erad factors such as edem- . alternatively, ire can trigger ask -p mapk pathway that enhances chop apoptotic activities and promotes other apoptotic-related activities under severe er stress condition. blue pointed arrows denote activation, blue blunt-end arrows denote inhibition, and red pointed arrows denote increased expression/activity site- and site- proteases, liberating tad-containing atf n-terminal (atf n) [ ] . subsequently, atf n enters the nucleus and binds to er stress response elements in the promoter region of upr-target genes, including x-box binding protein (xbp ) transcription factor, a downstream effector of ire stress sensor pathway, and grp to autoregulate the upr [ ] [ ] [ ] . zikv infection has been shown to activate the upr via atf , where atf proteolysis and atf n nuclear translocation were induced in vitro and in vivo [ ] . ire is a single-pass transmembrane protein. the protein's er luminal domain serves as a stress sensor, whereas the cytosolic domain is sub-divided into kinase and ribonuclease (rnase) domains. under er stress, grp releases from ire luminal domain and binds onto the misfolded/unfolded proteins. this dissociative event frees the luminal domain and in turn, permits binding of the misfolded/unfolded protein onto the liberated domain [ , ] . consequently, ire oligomerizes at its luminal domain, which brings the kinase domains in close proximity leading to auto-phosphorylation of the kinase domains [ ] . the phosphorylation event at the activation loop is postulated to induce conformational changes of the rnase domain that permit a more efficient binding of rna substrates [ ] . activated ire cleaves off nucleotides from xbp transcripts, resulting in a translational frameshift to produce a potent transcriptional activator (xbp s) that elevates the expression of upr-target genes to promote protein folding, processing, and secretion [ , ] . xbp s also modulates the expression of factors involved in er-associated protein degradation (erad), a cellular process of eliminating aberrant proteins through retro-translocation and proteasomal degradation [ ] . alternatively, ire protein can also mediate er stressinduced apoptosis following chronic stress damage by triggering activation of apoptotic signaling kinase (ask ), which consequently activates p mitogen-activated protein kinase (p mapk) and jun n-terminal kinase (jnk) leading to apoptosis [ ] . jnk mediates apoptosis primarily by inhibiting the anti-apoptotic b-cell lymphoma (bcl- ) and activating the pro-apoptotic bim protein [ ] , whereas p mapk promotes apoptosis by phosphorylating ccaat-enhancer-binding protein (c/ebp) homologous protein (chop) at serine residues and to enhance its transcriptional activity and induce apoptosis [ , ] . chop is expressed downstream of all three er stress sensors, indicating overlapping upr pathways during severe er stress [ ] . chop forms heterodimers with other c/ebp family transcription factors; therefore, the basic domain in chop renders the transcription factors incapable of binding to their respective dna binding sites, which reduces the expression of their target proteins including bcl- [ ] . chop also contains a tad and thus, can bind to a unique sequence to induce the expression of target genes including bim [ , ] . zikv infection has been shown to promote xbp splicing and xbp s translocation into the nucleus, leading to elevated expression of xbp downstream effectors such as er degradation-enhancing α-mannosidase-like (edem- ) and chop, indicating the activation of the ire arm of the upr [ ] . edem- is an enzyme involved in the erad process that recognizes and facilitates retrotranslocation of misfolded proteins to the cytosol for subsequent proteasomal degradation and thus, alleviating stress damage [ ] . instances of aggravated er stress were also demonstrated in zikv-infected cells, which lead to elevated expression of chop protein and initiation of erstress-induced apoptosis [ ] . pharmacological intervention using ire inhibitor, μ c, prevented microcephaly and impaired zikv replication in mouse fetal brains [ ] . modulation of ire -ask pathway during zikv infection is still unclear. previous study reported that ectopic expression of xbp promotes expansion of the er, a visual sign often manifested in response to er stress [ ] . this morphological change is hypothesized to reduce the local concentration of misfolded/unfolded proteins and accommodate the overwhelming demand for protein folding-a significant portion of which is neosynthesized viral polyproteins [ , ] . consistent with increased expression of xbp , ultrastructural characterization of zikv-infected cells reported significant er enlargement extending across the cytoplasm and is related to zikv-induced er stress [ , , , ] . similar to ire , release of grp proteins enable misfolded/unfolded proteins binding onto exposed perk luminal domain leading to oligomerization and autophosphorylation of perk protein to activate its downstream pathway [ , ] . perk phosphorylation, in turn, induces eif α phosphorylation (p-eif α) that mediates three interlinked upr mechanisms: expansion of protein folding capacity, suppression of nascent protein production and induction of apoptosis in the event of severe stress [ ] . phosphorylation of α subunit in the eif complex hinders the assembly of preinitiation complex (pic) by blocking the activity of eif b guanine exchange factor, leading to global suppression of mrna translation [ ] . however, selected mrnas, typically transcripts for upr machinery, can bypass this translational block [ ] . interestingly, viruses have evolved various mechanisms to overcome this translational block, but the mechanism of this process in zikv infection is unclear. recent analyses of zikv infection in vitro and in vivo reported a substantial increase of eif α phosphorylation and elevated expression of several downstream perk effectors such as atf , atf , chac , and chop [ , ] . importantly, intracerebroventricular administration of pharmacological perk inhibitor in zikv-infected mice restored appropriate neurogenesis balance and rescued infected mouse embryos from microcephalic phenotype. however, unlike ire inhibitor, perk inhibitor did not affect zikv replication [ ] . perk inhibitor also prevented microcephaly in placental zikv inoculation model, which mimics natural zikv vertical transmission [ ] . in summary, zikv localizes to the er for viral genome replication. these additional transcriptional and translational processes impart significant burden on the er leading to er stress and upr induction. during the early stage of upr, upr effectors elicit adaptive responses to mitigate er stress. these responses include the upregulation of chaperones and protein processing enzymes to promote and rectify protein folding, induction of erad to degrade misfolded/unfolded proteins, global translational arrest, and activation of autophagy and/or reticulophagy. beside upr, er stress also concomitantly induces several other cellular processes. specifically, stress granules assembly and er autophagy impart fundamental importance in regulating translational arrest and er homeostasis, respectively. zikv have been reported to subvert these processes to allow the progression of viral replication. er stress signal can be transmitted to other cellular components to rescue cell survival. a clear example to illustrate stress signal transmission is the generation of stress granules (sgs) in the cytoplasm to stall initiation of translation [ ] . sgs assembly are generally triggered through two distinct categories of mechanisms: ) eif α-independent and ) eif α-dependent mechanisms [ ] . in particular, the latter category, or more specifically eif α-phosphorylation, is mediated by pkr, perk, general control non-derepressible- (gcn ), and heme-regulated inhibitor kinase (hri) in response to diverse cellular stress signals [ ] . sgs are ribonucleoprotein (rnp) structures primarily composed of non-translating mrnas, stalled translation initiation complexes, and rna-binding proteins [ ] . the formation and components of sgs are reviewed in details here [ ] . sgs assembly during global translational arrest negatively impact viral genome translation as the sgs reduce the accessibility of translational machinery complexes [ ] . following the relief of translation suppression, the sgs are disassembled via several mechanisms, one of which involves eif α dephosphorylation by growth arrest and dna-damage-inducible (gadd ) protein [ ] . this allows the pic to resume protein translation at the surface of the sgs, resulting in sgs shrinkage and disappearance [ ] . viruses have adopted several mechanisms to repress the assembly of stress granules and utilize sg protein components for viral polyprotein synthesis instead [ ] . for example, herpes simplex virus genome encodes γ . protein that functionally mimics the activity of gadd and directs the dephosphorylation of eif α [ ] . alternatively, coronaviruses repress the assembly of stress granules by asserting an inhibitory effect on pkr activation and upregulating the expression of gadd [ ] . likewise, zikv also suppresses the formation of stress granules in favor of virus replication by upregulating the expression of gadd [ ] . consistent with this finding, pharmacological inhibition of gadd -mediated eif α dephosphorylation rescued sgs assembly and decreased zikv particles production [ ] . additionally, another study reported that zikv proteins, namely capsid, ns , ns b-ns , and ns a proteins, suppressed sgs assembly; however, this modulation was observed in an eif α-independent manner [ ] . zikv capsid inhibited sgs assembly by forming stable complexes with sg core proteins, ras gtpase-activating proteinbinding protein (g bp ) and caprin- , but not with t-cell-restricted intracellular antigen related (tiar) protein [ ] . zikv utilized these sg core proteins for viral protein and virion production. beside regulating eif α phosphorylation and hijacking key sg proteins, rna viruses were reportedly capable of interfering sgs assembly through cleaving and redistributing sgs nucleating factors [ ] . it was previously reported that zikv ns b-ns protease could cleave host antiviral factors to impair intrinsic host defense; intriguingly, zikv did not mediate the cleavage of sg factors even though sgs assembly was affected by zikv ns and ns b-ns protease [ ] [ ] [ ] [ ] [ ] . nonetheless, zikv infection was found to facilitate the redistribution of tiar to viral replication sites, which correlates to viral replication output [ , , ] . hence, it is unclear how zikv ns , ns b-ns , and ns a proteins block sgs formation and whether these viral proteins affect eif α phosphorylation. interestingly, a recent study also identified a key sg-interacting protein, human antigen r, exhibits substantial anti-zikv effect potentially by destabilizing the zikv rna [ ] . as previously discussed in section , the induction of er stress initiates several adaptive countermeasures such as upregulation of upr pro-survival factors and er enlargement to repair stress-induced damages and restore normal cellular functions. interestingly, initial studies in mammalian and yeast cells revealed that a fraction of cells also contain autophagosome-like structures following er stress induction [ , ] . these autophagic vacuoles expel selectively excised er membrane containing aberrant protein aggregates [ ] . this process, known as reticulophagy, is an additional mechanism that occurs in parallel with upr to regulate er volume and homeostasis [ ] . autophagy, including reticulophagy, is also a host innate defense mechanism as these autophagosomes have been shown to incorporate viral proteins for degradation [ ] . reticulophagy, also known as er-phagy, is mediated by er-phagy receptors such as family with sequence similarity member b (fam b) and reticulon- (rtn ) proteins that reside on the er membrane. these autophagy receptors sequester er fragments via its lc -interacting-region (lir) domain interaction with autophagosomal-presenting microtubuleassociated protein light chain (map lc ) [ ] . similar to other er-shaping proteins, fam b also possesses a reticulon homology domain (rhd), a motif that promotes membrane curvature [ ] . in zikv-infected cells, depletion of fam b protein renders notable er expansion and significant upregulation of zikv replication activity [ ] . this is due to the proteolytic activity of zikv ns b-ns that cleaves exposed rhd located on fam b protein, which impedes the oligomerization of fam b proteins and consequently, prevents er membrane excision and reticulophagy from taking place [ ] . this efficiently blocks host cell innate defense mechanism from degrading zikv proteins. to summarize, zikv virus bypasses the upr by inhibiting stress granules assembly and reticulophagy to ensure continuous viral protein translation and virion production while simultaneously protecting the virus from host cell defense mechanisms. prolonged er stress exacerbates the stress condition and leads to the activation of another arm of the upr: cell death. countless studies showed that zikv infection causes cell death through several cell death mechanisms such as paraptosis and apoptosis [ , ] apoptotic events in zikv-infected cells occur through both the intrinsic and extrinsic pathways based on the activation of pathway-specific caspase- and caspase- , respectively [ , ] . interestingly, zikv env protein is sufficiently capable to induce pro-apoptotic expression profile that represents intrinsic apoptosis including elevated expression of tumor protein p , which correspondingly mediates the expression and activity of its downstream targets by suppressing anti-apoptotic bcl- and facilitating expression of pro-apoptotic bcl- associated x (bax) [ ] . this modulation consequently reduces mitochondrial membrane permeability; thus, aiding the release of cytochrome c, formation of apoptosome, and initiation of caspase cascade, which eventually leads to cell death [ ] . additionally, increased level of several apoptotic markers, such as tumor necrosis factor alpha and receptor-interacting protein , in zikv-positive microcephalic neural specimens are suggestive of extrinsic apoptotic activation [ ] . as previously mentioned, er stress elevated the expression of chop to initiate apoptosis in zikvinfected cells [ , ] . prolonged er stress could also trigger non-apoptotic cell death as demonstrated by an investigation using time-lapse and electron microscopy that revealed the formation of extensive erderived vacuoles within the cytoplasm of zikvinfected cells, which eventually resulted in paraptosislike death [ ] . this observation is consistent with earlier report that showed cytoplasmic vacuolization in zikv-infected human skin biopsy specimens [ ] . vacuole formation was inhibited with class i pi k/akt inhibitor treatment. more importantly, pan-caspase inhibitor, zvad-fmk, only slightly rescued cell survival but did not affect vacuolization [ ] , implying that certain zikv strains, namely hd , pf and nc , induce cell death primarily through paraptosislike death. the role of pi k/akt pathway in zikvinduced cell death was verified by a recent study that reported treatment with ar- , a celecoxib derivative kinase inhibitor, significantly inhibited the replication of zikv in a mice and improved mice survival mainly through akt down-regulation [ ] . zikv, like other viruses, is an intracellular parasite that largely depends on the host biosynthetic, energetic and structural resources for successful viral replication and propagation. in particular, exploitation of these resources is manifested through the manipulation of the endoplasmic reticulum architecture and the processes that take place within or in proximity to the er as summarized in fig. . briefly, zikv infection leads to structural changes of the er such as er enlargement due to the accumulation of misfolded/unfolded zikv proteins (fig. d ) and the formation of convoluted membranes, vesicle packets, zippered er, and viroplasm-like structures for viral rna replication (fig. e) . the accumulation of misfolded/unfolded proteins induce er stress and triggers the upr (fig. f) . in parallel, er stress also initiate several response mechanisms such as stress granules assembly to impede global protein translation (fig. g ) and reticulophagy to remove damaged er (fig. h ). however, zikv are able to bypass these processes through various strategies, which eventually lead to paraptosis-like cell death (fig. i) . although the number of zikv cases have subsided, zikv remains a significant threat due to the sporadic and unpredictable nature of its outbreak. in addition, zikv shares the same vectors with another widespread flavivirus, denv [ ] , which is projected to increase exponentially in the future [ ] . thus, the potential re-occurrence of outbreaks, coupled with devastating neurological complications, warrants for extensive research to completely understand the virus pathophysiology for antiviral drug development. this is effectively demonstrated by several studies included in this review that employ multiple omics technologies to identify and target er-associated zikv dependency factors and er stress sensors. this strategy could pave the way in developing anti-zikv drugs. other cell receptors such as dc-sign can also serve as zikv entry receptor. b) acidic endosomal microenvironment facilitates endosomal fusion thereby, releasing zikv rna genome that is immediately bound onto cytosolic ribosomes. c) translation of the viral polyprotein (orange) likely initiates in the cytosol and continues with cotranslational insertion into the er membrane via sec translocon complex (blue). post-translational processed zikv proteins induce er structural alterations such as d) enlargement of the er, and e) formation of convoluted membrane (cm), vesicle packets (vps), zippered er (zer) and viroplasms. simultaneously, accumulation of misfolded and unfolded zikv proteins in the er lumen results in f) the induction of er stress and thus, initiates the host unfolded protein response (upr) and other intrinsic defense mechanisms including reticulophagy and stress granule formation. zikv proteins g) impede formation of stress granules and h) inhibit reticulophagy to sustain viral replication. zikv infection also induces i) er-derived cytoplasmic vacuolization, a visual characteristic of paraptosis. blue pointed arrows denote activation, blue blunt-end arrows denote inhibition. ve, virus-induced vesicle; vi, virus particle zika virus. i. isolations and serological specificity zika virus zika virus outbreak on yap island, federated states of micronesia zika virus infection complicated by guillain-barre syndrome -case report guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study pan american health organization / world health organization. epidemiological alert: increase of microcephaly in the northeast of brazil zika virus and microcephaly: why is this situation a pheic? zika virus and birth defects -reviewing the evidence for causality detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study zika virus associated with microcephaly zika virus impairs growth in human neurospheres and brain organoids zika virus disrupts neural progenitor development and leads to microcephaly in mice biology of zika virus infection in human skin cells axl mediates zika virus entry in human glial cells and modulates innate immune responses infection by zika viruses requires the transmembrane protein axl, endocytosis and low ph ph dependence of zika membrane fusion kinetics reveals an off-pathway state zika virus structure, maturation, and receptors role of host cell secretory machinery in zika virus life cycle molecular cloning and characterization of the genes encoding the proteins of zika virus global interactomics uncovers extensive organellar targeting by zika virus zika ns b is a crucial factor recruiting ns to the er and activating its protease activity zika virus targets human stat to inhibit type i interferon signaling zika virus ns is a canonical rna helicase stimulated by ns rna polymerase a crispr screen defines a signal peptide processing pathway required by flaviviruses identification of zika virus and dengue virus dependency factors using functional genomics the er membrane protein complex promotes biogenesis of dengue and zika virus nonstructural multi-pass transmembrane proteins to support infection predicting zika virus structural biology: challenges and opportunities for intervention zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells an orthogonal proteomic survey uncovers novel zika virus host factors genetic dissection of flaviviridae host factors through genome-scale crispr screens a small-molecule oligosaccharyltransferase inhibitor with pan-flaviviral activity ultrastructural characterization of zika virus replication factories endoplasmic reticulum: the favorite intracellular niche for viral replication and assembly the endoplasmic reticulum: structure, function and response to cellular signaling cytoarchitecture of zika virus infection in human neuroblastoma and aedes albopictus cell lines zika virus induced cellular remodelling a novel sheet-like virus particle array is a hallmark of zika virus infection the host protein reticulon . a is utilized by flaviviruses to facilitate membrane remodelling zika virus ns a-mediated virion assembly infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes structural investigation of c / and vero cell cultures infected with a brazilian zika virus virus factories, double membrane vesicles and viroplasm generated in animal cells west nile virus-induced cytoplasmic membrane structures provide partial protection against the interferon-induced antiviral mxa protein protein folding and modification in the mammalian endoplasmic reticulum the role of endoplasmic reticulum stress in human pathology affinity panning of a library of peptides displayed on bacteriophages reveals the binding-specificity of bip crosstalk between endoplasmic reticulum stress and the unfolded protein response during zika virus infection zikv infection activates the ire -xbp and atf pathways of unfolded protein response in neural cells stress-induced unfolded protein response contributes to zika virusassociated microcephaly a dynamic unfolded protein response contributes to the control of cortical neurogenesis er stress regulation of atf localization by dissociation of bip/grp binding and unmasking of golgi localization signals role of the unfolded protein response regulator grp /bip in development, cancer, and neurological disorders xbp mrna is induced by atf and spliced by ire in response to er stress to produce a highly active transcription factor unfolded proteins are ire -activating ligands that directly induce the unfolded protein response endoplasmic reticulum stress sensing in the unfolded protein response structure of the ire autophosphorylation complex and implications for the unfolded protein response phosphoregulation of ire rnase splicing activity from endoplasmic-reticulum stress to the inflammatory response er stress-induced cell death mechanisms stress-induced phosphorylation and activation of the transcription factor chop (gadd ) by p map kinase attenuation of chop-mediated myocardial apoptosis in pressureoverloaded dominant negative p alpha mitogen-activated protein kinase mice the c/ebp homologous protein (chop) transcription factor functions in endoplasmic reticulum stress-induced apoptosis and microbial infection stress-induced binding of the transcriptional factor chop to a novel dna control element er stress triggers apoptosis by activating bh -only protein bim edem recognition and delivery of misfolded proteins to the sel l-containing erad complex xbp , downstream of blimp- , expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response the luminal domain of the er stress sensor protein perk binds misfolded proteins and thereby triggers perk oligomerization translational control in stress and apoptosis eukaryotic stress granules: the ins and outs of translation mechanistic insights into mammalian stress granule dynamics translation inhibition and stress granules in the antiviral immune response growth arrest and dna damageinducible protein gadd targets protein phosphatase alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor viral regulation of rna granules in infected cells the gamma( ) . protein of herpes simplex virus i complexes with protein phosphatase alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor and preclude the shutoff of protein synthesis by double-stranded rna-activated protein kinase inhibition of protein kinase r activation and upregulation of gadd expression play a synergistic role in facilitating coronavirus replication by maintaining de novo protein synthesis in virus-infected cells zika virus inhibits eif alphadependent stress granule assembly zika virus hijacks stress granule proteins and modulates the host stress response species-specific disruption of sting-dependent antiviral cellular defenses by the zika virus ns b protease dengue and zika viruses subvert reticulophagy by ns b -mediated cleavage of fam b zika virus subverts stress granules to promote and restrict viral gene expression autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response differential effects of endoplasmic reticulum stress-induced autophagy on cell survival retention of mutant alpha( )-antitrypsin z in endoplasmic reticulum is associated with an autophagic response er-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery autophagy in immunity and inflammation regulation of endoplasmic reticulum turnover by selective autophagy zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells loss of the tam receptor axl ameliorates severe zika virus pathogenesis and reduces apoptosis in microglia zika virus envelope protein induces g /m cell cycle arrest and apoptosis via an intrinsic cell death signaling pathway in neuroendocrine pc cells correlation between apoptosis and in situ immune response in fatal cases of microcephaly caused by zika virus the celecoxib derivative kinase inhibitor ar- (osu- ) inhibits zika virus via downregulation of the pi k/akt pathway and protects zika virus infected a mice: a host-targeting treatment strategy an overview of mosquito vectors of zika virus the current and future global distribution and population at risk of dengue publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions mimr and ask contributed to the topic conception and writing of the manuscript together. mimr prepared the figures. mimr, ask, nnr and ry edited and revised the manuscript. all authors read and approved the final manuscript. this work was supported by the grant [frgs fp - a] from the ministry of higher education, malaysia.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.received: september accepted: january key: cord- - smnl i authors: chan, jasper f.w.; choi, garnet k.y.; yip, cyril c.y.; cheng, vincent c.c.; yuen, kwok-yung title: zika fever and congenital zika syndrome: an unexpected emerging arboviral disease date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: smnl i unlike its mosquito-borne relatives, such as dengue, west nile, and japanese encephalitis viruses, which can cause severe human diseases, zika virus (zikv) has emerged from obscurity by its association with a suspected “congenital zika syndrome”, while causing asymptomatic or mild exanthematous febrile infections which are dengue- or rubella-like in infected individuals. despite having been discovered in uganda for almost years, < human cases were reported before . the massive epidemics in the pacific islands associated with the zikv asian lineage in and were followed by explosive outbreaks in latin america in . although increased mosquito breeding associated with the el niño effect superimposed on global warming is suspected, genetic changes in its rna virus genome may have led to better adaptation to mosquitoes, other animal reservoirs, and human. we reviewed the epidemiology, clinical manifestation, virology, pathogenesis, laboratory diagnosis, management, and prevention of this emerging infection. laboratory diagnosis can be confounded by cross-reactivity with other circulating flaviviruses. besides mosquito bite and transplacental transmission, the risk of other potential routes of transmission by transfusion, transplantation, sexual activity, breastfeeding, respiratory droplet, and animal bite is discussed. epidemic control requires adequate clearance of mosquito breeding grounds, personal protection against mosquito bite, and hopefully a safe and effective vaccine. globalisation and urbanisation with increasingly frequent and large-scale movements of humans, animals, and commodities by aviation and water transport has led to the spread of previously geographically-restricted microbes and vectors to distant and isolated places. recent examples of emerging viruses that have spilled over to other continents from their original localities via exportation of travelrelated cases include coronaviruses (severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus), influenza viruses, and ebola virus. e moreover, global warming and climate changes have redefined the geographical distributions of important vectors of arthropod-borne viruses (arboviruses), such as the aedes mosquitoes, and facilitated the global spread of these viruses. dengue virus (denv), west nile virus (wnv), and chikungunya virus (chikv), have been introduced (wnv in and chikv in ) and/or spread rapidly in the western hemisphere in the past two decades. zika virus (zikv) is an arbovirus that was little known before it caused a large outbreak on yap island of the federated states of micronesia in . even then, zikv was not considered as an important emerging pathogen because clinical disease was generally mild. the recent report of a possible association between zikv infection and an epidemic of microcephaly among neonates in brazil has attracted global attention. the rapid spread of zikv beyond africa and asia to the americas and europe, and the potentially novel "congenital zika syndrome" outbreak have led the world health organisation (who) to declare the zikv epidemic as a global public health emergency on february . it would therefore be important to review the current knowledge on the epidemiology, virology, clinical manifestations, and laboratory diagnosis of zikv infection, and most importantly, to formulate clinical management options with special reference to perinatal care and control measures based on comparisons made with other mosquito-borne arboviruses. important historical and epidemiological events zikv (strain mr ) was first isolated from the blood of a febrile sentinel rhesus monkey (macaca mulatta), rhesus , during a study on yellow fever virus (yfv) in zika forest of uganda in april (table ) . in , zikv was isolated from aedes africanus mosquitoes caught in zika forest, suggesting that the virus might be mosquito-borne. in , zikv was isolated from the serum of a -year-old nigerian girl who had fever and headache, implying its role as a possible human pathogen. further virological and/or serological evidence of human zikv infection was reported in african (uganda, tanzania, egypt, central african republic, sierra leone, and gabon) and asian (india, malaysia, the philippines, thailand, vietnam, and indonesia) countries. e zikv infection remained relatively restricted geographically with less than sporadic cases reported in these areas in the first years after its discovery. , in , zikv emerged outside africa and asia for the first time and caused a major outbreak on yap island of the federated state of micronesia. over % of the yap residents who were ! years were infected within months. the attack rate of zikv infection in this outbreak was . per residents (range, . e . per residents). subsequently, another major outbreak was reported in french polynesia in october . an estimated , humans (> % of the french polynesian population) were infected by zikv. zikv infection then spread from french polynesia to other pacific islands including new caledonia, cook islands, vanuatu, and solomon islands. e the first cases of human zikv infection in the western hemisphere occurred on easter island, chile, in february , possibly originating from french polynesia during the annual tapati festival. phylogenetic analysis revealed that the ns gene sequence of the chilean strains had ! . % nucleotide and % amino acid identity to the french polynesian strains. the epidemic continued to expand rapidly and autochthonous human cases were reported in many latin american countries in the ensuing years. brazil stands out as the hardest hit latin american country with an estimated , e , , cases of zikv infection since march . based on the close phylogenetic relationship between the south american strains and asian and oceanic strains of zikv, the virus might have been introduced into brazil by asian travellers during the world cup or participants from the oceanic countries of the va'a world sprint championship canoe race in the summer of . , e the climate changes associated with el niño in north and eastern south america in on the background trend of global warming might have facilitated the rapid spread of aedes mosquitoes and zikv. currently, > countries in africa, asia, south america, oceania, and micronesia have reported autochthonous cases of human zikv infection. travel-related cases from endemic and epidemic regions were also reported in europe, north america, australia, and japan. e more worryingly, the brazil health ministry reported the detection of an unusual increase in the number of cases of neonates with microcephaly in northeastern brazil in october , coinciding with the expanding zikv infection epidemic. over suspected cases including some fatal cases were reported during the second half of alone. this represented a > -fold increase in the rate of microcephaly as compared to previous years. on november , the french polynesia health authorities also reported an unusual increase in the number of foetal and neonatal central nervous system malformations in and . like other flaviviruses, zikv is mainly transmitted by mosquitoes. in addition to the sylvatic (enzootic) transmission cycle between the haematophagous mosquito vectors and susceptible primary vertebrate hosts, the recent large-scale epidemics suggest that zikv is also adapting to an urban transmission cycle. , among the various mosquito species, aedes (stegomyia) mosquitoes appear to be the most important vector for zikv transmission, although some anopheles, culex, eretmapodites, and mansonia species have also been proposed as possible vectors (table ) . , , e the animal reservoirs of zikv are unclear. non-human primates including m. mulatta, cercopithecus aethiops, c. ascanius schmidti, c. mona denti, c. albigena johnstoni, chlorocebus sabaeus, colobus abyssinicus, erythrocebus patas, and pongo pygmaeus, and other mammals including zebras, elephants, and rodents, have been suggested as possible vertebrate hosts of zikv in africa and asia, based on virological and/or serological evidence of infection. , , , , e ae. africanus is the first mosquito species from which zikv was isolated, and is likely an important vector in the sylvatic transmission cycle of zikv. , inoculation of unfiltered supernatant of zikv-infected ae. africanus into mice and rhesus macaques led to clinical disease and/or neutralising antibody response. , ae. hensilli is the most commonly found mosquito species on yap island, but no virus isolate was made from field-collected mosquitoes to ascertain its role as a vector for zikv transmission during the outbreak. ae. aegypti and ae. albopictus, which have much wider geographical distributions than other aedes mosquitoes, are considered to be more important vectors in the urban transmission cycle of zikv. these aedes mosquitoes are highly susceptible to zikv infection in vitro with potential for further transmission after an extrinsic incubation period of e days. , they bite both indoors and outdoors, and mostly during daytime. non-vector-borne transmission routes of zikv have been proposed (table ) . like other arboviruses, blood transfusion-related transmission of zikv is possible, especially in endemic regions or where blood products obtained from infected travellers immediately returning from endemic regions are used. zikv rna was detected in the blood of . % of the donors in french polynesia during the epidemic. sexual transmission of zikv appears highly probable, especially in patients presenting with haematospermia with infectious viral particles and rna in semen. , notably, no other arboviruses have been associated with haematospermia or isolated from human semen. this might further complicate the control of the zikv epidemic, since most infected patients are asymptomatic. inadvertent sexual transmission of zikv to the female partner may then lead to virus transmission to the foetus, which may be potentially associated with severe congenital anomalies. besides transplacental transmission, perinatal transmission of zikv may also occur during delivery, via breastfeeding, and/or close contact after birth via exchange of saliva and other bodily fluids. zikv rna could be detected in breast milk and saliva of infected women, although replicative virus particles have not been demonstrated , perinatal transmission of other arboviruses, including denv, chikv, wnv, and yfv, has also been reported. e other suspected routes of transmission of zivk infection are those reported for other flaviviruses. these include mucocutaneous exposure to the virus in infected blood or via monkey bite, haemodialysis, or organ transplantation. e particularly, as zikv may be shed in the urine of infected patients for more than days, the risk to recipients of donated kidneys from donors at or returning from endemic areas has to be considered. , , , , it is unknown whether zikv could be transmitted via respiratory droplets as viral rna could occasionally be detected in nasopharyngeal swab and saliva samples. , , , virology and pathogenesis zikv is an enveloped, positive-sense, single-stranded rna virus belonging to the genus flavivirus in the family flaviviridae. it is closely related to spondweni virus and the viruses represent the only members of their clade within the mosquito-borne cluster of flaviviruses ( fig. ) . , phylogenetic analysis suggests that zikv has likely emerged between and in uganda. the two major lineages of zikv are the african (subdivided into west and east african) and asian lineages, which are responsible for causing the majority of infections in africa and asia (as well as the pacific and americas), respectively. , , the single-stranded rna genome of zikv has a size of , nucleotides encoding amino acids, with flanking untranslated regions ( and utrs) and a single long open reading frame encoding a polyprotein, which is cleaved into capsid (c), precursor of membrane (prm), envelope (e), and non-structural (ns) proteins , reverse transcription-polymerase chain reaction (rt-pcr) using primers targeting the e or ns gene is a key laboratory diagnostic tool for zikv infection in the recent outbreaks. , , the e protein is a major virion surface protein that is involved in receptor binding and membrane fusion. the domain iii of e protein contains a panel of antigenic epitopes that are important targets of serological assays, neutralising antibodies, and vaccines. , loss of the n glycosylation site in the e protein may be associated with adaptation to mosquito vectors and thus facilitate transmission. a single amino acid mutation in the e protein (e -a v) of chikv has been reported to be associated with increased fitness of the virus in ae. albopictus and allows chikv to disseminate in regions lacking the typical ae. aegypti vector. the recent spread of the asian lineage of zikv to oceania and the americas may be associated with significant ns codon usage adaptation to human housekeeping genes, which could facilitate viral replication and increase viral titres. mutations in the e and ns genes should be detected in zikv strains causing the current epidemic. when an infected aedes mosquito bites an infected patient, it ingests a blood meal containing zikv. as in other flaviviruses, zikv likely replicates in the midgut epithelial cells and subsequently the salivary gland cells. after an extrinsic incubation period of e days, zikv can be found in the mosquito's saliva which can then infect human. , moreover, the virus can likely be vertically transmitted transovarially as other flaviviruses. when the mosquito's saliva containing zikv is inoculated into human skin, the virus can infect epidermal keratinocytes, skin fibroblasts in the subcutaneous layer, and the langerhans cells. the keratinocytes and fibroblasts contain axl, tyro , and tim- , which can serve as attachment factors or receptors for zikv. the langerhans cells contain dc-sign, which can also serve as a receptor for virus entry. zikv infection of primary skin fibroblasts is associated with the upregulation with tlr mrna expression, and enhanced transcription of rig-i and mda , which are known innate immune responses to rna virus infection. this is followed by enhanced expression of interferon-alpha and -beta, and their downstream pathways of immune activation. both types i and ii interferons can suppress the viral load of infected cells. moreover, zikv is capable of increasing its replication by the induction of autophagy in host cells. thus, autophagy inhibitors can decrease the viral load of infected cells. infected cells of human skin explant exhibits cytoplasmic vacuolation, pyknotic nuclei, and oedema in the stratum granulosum. after replication in endemic/epidemic areas: + universal nucleic acid testing of blood donors. + temporary discontinuation of blood donation (importation of blood products from blood blank centres in non-endemic regions). non-endemic/epidemic areas: + pre-donation questionnaire to identify donors with recent travel history to endemic/epidemic areas. + deferral of blood donors who have travelled to endemic areas within the preceding ! days. + self-reporting of symptoms after blood donation ( these local tissue cells and the regional lymph nodes, zikv may then disseminate from the lymphatics and bloodstream to reach other organs/tissues, including the central nervous system, the skeletal muscles, myocardium, and perhaps transplacentally to the foetus. zikv was highly neurotropic in infected suckling mice. the brains of infected suckling mice show neuronal degeneration, cellular infiltration, and softening in the brain with virus replication in astroglial cells and neurons on histopathological examination. , , moreover, evidence of inflammation in skeletal muscles and myocardium has also been demonstrated in infected suckling mice. axl and tyro are members of the tam family of receptor tyrosine kinases (rtks). they are also present in neurons and under the influence of gonadotropin releasing hormone (grh), which in turn may affect neuronal survival and migration. furthermore, flaviviruses such as yfv may persist for up to days after intracerebral inoculation in rhesus macaques. the neurotropism and persistence of zikv may therefore partially explain microcephaly and predominantly neurological complications and foetal anomalies in this suspected entity of congenital zikv infection. most patients with zikv infection are asymptomatic. in the outbreak of zikv infection on yap island, only % of cases were estimated to be symptomatic. the incubation period of zikv infection is unclear, but is estimated to be similar to other mosquito-borne flaviviruses ( e days). , the clinical syndromes of symptomatic zikv infection can be broadly divided into zika fever and congenital infection ("congenital zika syndrome") ( table ). zika fever is an acute "dengue fever-like" illness characterized by low-grade fever ( . e . c), rash, retroorbital headache, bilateral non-purulent conjunctivitis, myalgia, and arthritis/arthralgia with periarticular oedema of the small joints of hands and feet. the rash in zika fever is typically described as a generalized, erythematous, maculopapular rash that spreads downward from the face to the limbs. less commonly, some patients may have more prominent systemic symptoms including high-grade fever, chills, rigours, sore throat, hypotension, and cervical, submandibular, axillary, and/or inguinal lymphadenopathies. digestive tract symptoms including nausea, vomiting, diarrhoea, constipation, abdominal pain, and aphthous ulcers may also be present. , , patients with genitourinary symptoms including haematuria, dysuria, perineal pain, and haematospermia often have detectable viral rna or infectious virus particles in urine and/or semen. , haematological and biochemical laboratory parameters are usually normal. however, some patients may have transient and mild leucopenia, neutropenia, lymphopenia or activated lymphocytes, monocytosis, thrombocytopaenia, and elevated serum levels of lactate dehydrogenase, aspartate aminotransferase, g-glutamyl transferase, fibrinogen, ferritin, c-reactive protein, and erythrocyte sedimentation rate during the viraemic phase. associated with restoration of normal number of peripheral immune cells and normal function of antigen-presenting cells. notably, the clinical manifestations of zika fever are non-specific and may mimic those seen in infectious diseases caused by other arthropod-borne pathogens, especially denv and chikv. some suggest that zika fever may be distinguished from dengue fever and chikungunya fever by more prominent oedema of the extremities, less severe headache and malaise, and milder degree of thrombocytopaenia seen in the former. , moreover, haemorrhagic complications seen in dengue fever have not been reported in zika fever, and arthralgia in zika fever is less severe than that in chikungunya fever. however, none of these features are pathognomonic and laboratory confirmation is required to exclude co-infections with these arboviruses and other causes of acute febrile illness in returned travellers from endemic regions, such as malaria. zika fever is usually self-limiting with most clinical manifestations resolving completely within e days. , , no death, hospitalisation, or haemorrhagic complication was reported during the outbreak on yap island. however, some patients may experience more protracted symptoms and other non-haemorrhagic complications. zika fever-related rash usually resolve within the first week, but may last for up to days and may be pruritic. other exanthematous diseases, such as denv, chikv, rubella virus, measles virus, parvovirus b , adenovirus, enterovirus, and rickettsial infection, should be excluded. the median duration of arthralgia is . days, but some patients may develop persistent or recurrent arthralgia for more than a month after symptom onset, mimicking the post-infectious chronic arthritis seen in chikungunya fever and lyme disease. , lymphadenopathies may be present for weeks after symptom onset, and alternative diagnoses such as infectious mononucleosis-like syndrome, streptococcus pyogenes infection, and toxoplasmosis should be considered in refractory cases. a post-infection asthenia appears to be frequent and further investigations may be necessary to determine possible association between zikv infection and chronic fatigue syndrome. , , immune-thrombocytopenic purpura and cardiac complication have also been reported in a few cases. jaundice was observed in patients with virological and/or serological evidence of zikv infection in eastern nigeria in the s who had co-infections (malaria and microfilaraemia) and a patient with sickle cell anaemia. , a possible association between zikv infection and severe neurological complications has been proposed during the recent epidemics in oceania and south america, during which the incidence of guillainebarré syndrome has increased by e times in french polynesia. , / ( . %) patients with suspected zikv infection in the french polynesia outbreak developed neurological syndromes after presenting with a zika fever-like illness. forty-two of these ( . %) patients were diagnosed with guillainebarré syndrome. , , similarly, guil-lainebarré syndrome has been reported among patients with zika fever-like illness in south america. , other neurological complications potentially linked to zikv infection include encephalitis, meningoencephalitis, myelitis, paraesthesia, vertigo, facial paralysis, and , , , , e suspected fatalities due to zikv-related guillainebarré syndrome have been reported. while the neurotropism of zikv may partially explain these neurological manifestations, more details and serial studies on their cerebrospinal fluid and magnetic resonance images by case-control studies are required to ascertain their association. zika fever-related death appears to be extremely rare but a number of probable cases have been reported, especially among immunocompromised patients and neonates with suspected congenital zikv infection. , , a small number of patients with coinfection with denv or hiv did not appear to have more severe disease. , further studies should be conducted to identify patients who are at risk of severe disease or death. microcephaly (head circumference ! standard deviations below the mean for sex and gestational age at birth) is the most prominent and commonly reported clinical feature of suspected congenital zika syndrome. , besides microcephaly, neonates and foetuses with suspected congenital zikv infection also had other malformations (table ). general features included low birth-weight, redundant scalp skin, anasarca, polyhydramnios, and arthrogryposis. neurological abnormalities included cerebral lesions, polymalformative syndromes, brainstem dysfunction, and absence of swallowing. ophthalmological defects included cataract, asymmetrical eye sizes, intraocular calcifications, macular atrophy (well-defined macular neuroretinal atrophy and/or macular pigment mottling and foveal reflex loss), optic nerve hypoplasia, iris coloboma, and lens subluxation. , , notably, other features characteristic of intrauterine infections, such as hepatosplenomegaly, rash, and chorioretinitis have not been reported. ultrasonographic examination revealed cerebral atrophy, intracranial calcifications especially over the white matter of frontal lobes, caudate, lentostriatal vessels, cerebellum, or around the lateral and fourth ventricles, dysgenesis of corpus callosum, vermia, and thalami, enlarged cisterna magna, asymmetrical cerebral hemispheres, severe unilateral ventriculomegaly, displacement of the midline, and thinning of the parenchyma on the dilated side, pons and brainstem. , , , zikv particles and rna may be detected by electron microscopy and rt-pcr, respectively, in autopsied samples. two important questions concerning congenital zikv infection remain unanswered. the first question is whether zikv is indeed the cause of microcephaly and other congenital anomalies in these patients. severe consequences have been reported for materno-foetal transmission of other arboviruses, such as dengue virus (preterm delivery, foetal death, low birth-weight, prematurity, acute foetal distress during labour), wnv (chorioretinitis and focal cerebral destruction), and chikv (encephalopathy and haemorrhagic fever). , , preliminary analysis in the current epidemic of microcephaly has not yet completely excluded other infectious or environmental aetiologies. moreover, there is some virological evidence to support the association between congenital zikv infection and these anomalies. zikv rna has been detected by rt-pcr in the amniotic fluid of pregnant women whose foetuses had ultrasonographic evidence of microcephaly, in the blood and foetal tissues of a neonate with microcephaly and other congenital anomalies who died within the first min of birth, and in the neonatal brain tissues of a few cases of full-term miscarriages and neonates with microcephaly. , , , however, there is still no large-scale prospective cohort or caseecontrol study to demonstrate a causal link between the presence of zikv in the foetus and the congenital anomalies after exclusion of other infectious and toxic causes. some have suggested that the apparent microcephaly surge might be attributable to the intense search for cases due to the heightened awareness of a possible association with the zikv outbreak or the use of larvicide. furthermore, detailed investigations for exclusion of other pathogens associated with congenital malformations have only been reported in a small number of cases. , microcephaly is well reported in congenital cytomegalovirus, rubella virus, and varicella zoster virus infection. chorioretinitis and intracranial calcifications are common in congenital cytomegalovirus infection and toxoplasmosis, but the latter is more commonly associated with hydrocephalus. cataract and cardiac anomalies are characteristic of congenital rubella syndrome, although cataract can also be found in congenital herpes simplex virus infection. thus, the diagnosis of congenital zika syndrome would depend on the exclusion of these "torch" infections in future studies using clinical criteria, histopathological findings, and serological, molecular and conventional cell culture techniques. if zikv is eventually confirmed to be the cause of these congenital anomalies, the second key question would be whether congenital zika syndrome actually comprises a wider spectrum of varying clinical severities than that seen in the reported cases. as with other congenital infections, it is possible that the reported cases of microcephaly represent only the tip the iceberg, focussing on the more severely affected patients, and that the timing of infection is likely to be important in determining the severity and outcome of the affected foetus. early infection during the first or even second trimester may be associated with congenital anomalies or even intrauterine death. , , indeed, preliminary data suggested that the greatest risk of microcephaly or congenital anomalies in the affected neonates appears to be associated with zikv infection in the first trimester of pregnancy. of mothers with infants born with microcephaly, % and % had a rash during the first and second trimester of pregnancy, respectively. besides neurological defects, cardiac and muscular abnormalities should also be excluded, as suckling mice infected with zikv developed evidence of central nervous system infection, myositis and myocarditis. some suspected cases of congenital zika syndrome developed severe arthrogryposis. , , , it is possible that intrauterine zikv infections that occur at a later stage of the pregnancy may present differently, either with less severe manifestations, such as mental retardation, sensorineural deafness, and/or ophthalmological lesions, or as full-term miscarriages. neonates with probable perinatal transmission of zikv infection appear to have mild disease and favourable outcome. further investigations should be conducted to better define the spectrum of manifestations in different gestational stages of congenital zikv infection. definitive diagnosis of zikv infection requires laboratory confirmation as there are no pathognomonic clinical, biochemical, or radiological features that reliably distinguish zika fever from other arboviruses, and congenital zikv infection from other infective, toxic, or genetic causes of congenital anomalies. successful isolation of zikv in viral culture, the gold-standard of laboratory diagnosis of viral infections, mainly depends on the timing of specimen collection and viral loads in the specimens. zikv has been isolated in vero and vero e cells inoculated with infected patients' serum, urine, and/or semen samples (table ) . , , however, infectious virus particles were not recovered by culture in most specimens with low viral loads. a positive serum immunoglobulin (ig) m or -fold rise in the titre of neutralising antibodies in paired serum samples collected approximately weeks apart also establishes the diagnosis of zikv infection. igm may be detected by enzyme-linked immunoassay on as early as day of symptom onset and may last for over months. , igm antibodies to denv and wnv usually persist for months and months, respectively. e the major limitation of these serological tests is possible cross-reactivity with other flaviviruses. neutralising antibodies detected by plaque-reduction neutralisation test may be more specific than igm detection by elisa for primary zikv infection, but may also have indeterminate results for secondary infection, including patients with previous vaccination against or exposed to other flaviviruses. , , , this is especially problematic in areas where there is cocirculation of multiple flaviviruses with the same aedes mosquito vectors. , , , patients with primary zikv infection and past denv infection are more likely to have higher titre (usually ! -fold) of igm and/or neutralising antibodies against zikv than against denv or other flaviviruses. , a positive serum denv ns antigen test without serial increase in igm or the combination of a positive igm response to denv and lack of an igg seroconversion in the convalescent-phase serum sample should prompt the clinician to investigate for another flavivirus such as zikv. moreover, co-infections with other mosquito-borne arboviruses, such as denv, chikv, wnv, and japanese encephalitis virus, are always possible and should be excluded by more extensive laboratory testing if clinically indicated. rapid and accurate diagnosis of zikv infection during the recent epidemics has mainly been achieved by the application of rt-pcr using primers that target the e or ns gene of zikv. , , , , alternatively, rt-pcr sequencing using universal primers that target the conserved regions in the genomes, such as the ns gene, of multiple flaviviruses, may allow simultaneous detection of > different flaviviruses. serum samples should be collected in the early phase of the disease, because viraemia is usually shortlived (usually days, rarely up to days) and may be low-level ( copies/ml). , alternatively, urine and semen samples may have higher viral rna loads table advantages, limitations, and uses of different diagnostic tests and types of specimens for laboratory diagnosis of zikv infection. , , , , , e , , , , , , , e , may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. other tissues brain, liver, spleen, and pooled visceral (kidney, lung, and heart) tissues were positive in a fatal case (an adult male with co-morbidities and immunosuppressive treatment). may be useful to exclude concomitant infections in patients with unusually severe or fatal infection. abbreviations: rt-pcr, reverse transcription-polymerase chain reaction; zikv, zika virus. (> copies/ml) than serum samples, and may be persistently positive for > days and ! days after symptom onset, respectively. , in a few cases, zikv rna has also been detected in saliva and nasopharyngeal swab samples of patients whose serum samples tested negative for zikv. these samples should therefore also be collected in suspected cases of zikv infection. , , , collection of amniotic fluid should be considered in pregnant women with positive zikv test result or if the foetuses show ultrasonographic evidence suggestive of congenital zikv infection. , , cerebrospinal fluid, placental, and/or umbilical cord tissues from neonates with suspected congenital zikv infection should be sent for virological and/or histopathological examinations to establish the diagnosis. , , zikv rna may also be detected in organ tissues in the rare cases of suspected zikv-related deaths. future studies should aim to better stratify the clinical use of these tests and to develop point-of-care tests (eg: antigen tests) that can be widely used in less developed regions without the facilities and expertise for molecular or serological tests. treatment is usually not required for patients with asymptomatic or uncomplicated zika fever. the mainstay of treatment is supportive as there are no specific anti-zikv antiviral agents. acetaminophen may be used to relieve fever and arthralgia. anti-histamines may help to control pruritus. adequate rehydration for fluid loss through sweating, vomiting, and insensible losses should be encouraged. aspirin should be avoided due to the risks of bleeding in those with thrombocytopaenia and developing reye's syndrome in children less than years of age. nonsteroidal antiinflammatory drugs are also contraindicated in cases where denv and chikv infections cannot be confidently excluded in order to avoid haemorrhagic complications. potential neurological complications, especially guillainebarré syndrome, should be diagnosed promptly to allow early use of intravenous immunoglobulins and/or plasmapheresis. the risk of immune enhancement should also be considered if convalescent-phase plasma therapy with neutralising antibodies against zikv is used for treatment of severe cases. virological testing and foetal ultrasound to exclude zikv infection and foetal microcephaly or intracranial calcifications should be offered to pregnant women who develop zika fever-like symptoms during or within weeks of travel to areas with zikv transmission. besides collecting the appropriate specimens for virological tests, serial foetal ultrasound examinations should be performed every e weeks to monitor foetal anatomy and growth in suspected cases of congenital zikv infection. foetal ultrasound and/ or aminocentesis should also be offered to asymptomatic and seropositive pregnant women with history of travel to affected areas. after delivery, serum should be collected either from the umbilical cord or directly from the neonate within days of birth for rt-pcr, igm and/ or neutralising antibodies against zikv. comprehensive physical examination including measurement of the occipitofrontal circumference, length, and weight, evaluation for neurological abnormalities, dysmorphic features, hepatosplenomegaly, rash, ophthalmological lesions, and auditory defects, and laboratory testing for torch screening should be performed. the affected child and the family should be managed and counselled by a multidisciplinary team consisting of paediatric neurologist, clinical geneticist or dysmorphologist, infectious disease specialist, medical social worker, and other relevant specialists. long-term follow-up to monitor physical, intellectual, and functional progress of the child should be offered. both vector control and personal preventive measures are important for interrupting the transmission of zikv. systematic mosquito surveillance and control programs should be established and coordinated by health authorities. mass sanitation campaigns to eliminate mosquito breeding sites in household and high-risk areas such as garbage collection points, construction sites, illegal dumping grounds, and invalid car fields should be organised. mosquitoes should be removed with a radius of at least m around areas with high population densities, such as schools, transport terminals, churches, and healthcare facilities. in areas where autochthonous or imported cases of zikv are detected, the use of adulticide through spraying to remove infected adult mosquitoes should be considered. residents in or travellers to affected areas should stay indoor with air conditioning, window and door screens if possible, wear long sleeves and pants, use permethrintreated clothing and gear, and use insect repellents when outdoor. most environmental protection agency (epa)registered insect repellents, including n,n-diethyl-mtoluamide (deet), should be safe for pregnant and lactating women ( % deet), and children ( % deet) aged > months. individuals returning from affected areas to non-affected regions should continue to use insect repellents for at least an additional days to prevent local non-infected mosquitoes from the acquisition of virus from the asymptomatically infected returned travellers. this will serve to interrupt the mosquito-human-mosquito transmission chain. hospitalised laboratory-confirmed cases should be managed in designated wards to avoid mosquito bites. the effects of other novel mosquito-control measures, such as the wolbachia biological control approach, should be evaluated. other animals such as rodents should also be investigated as potential animal reservoirs and controlled as findings indicate. non-vector-borne transmission of zikv may be prevented by specific measures (table ) . concerning blood transfusion, universal nucleic acid testing of blood donors is recommended. the use of universal primers that can simultaneously detect multiple arboviruses such as denv and zikv should be considered. temporary discontinuation of blood donation should be considered during an outbreak situation. in non-endemic areas, pre-donation questionnaire to identify donors with recent travel history to regions with reported cases of zikv infection and deferral of blood donation from these donors until at least days after returning from affected regions should be implemented. most transfusion-related transmissions of arboviruses are associated with asymptomatic infections, and symptomatic donors who were rt-pcr-positive for zikv usually developed symptoms between and days after blood donation. newer pathogen reduction technologies for blood products should be considered. similarly, donated organs, especially kidneys, from individuals with travel history to affected areas should be tested for zikv as the virus may persist in the genitourinary tract for an undetermined period. , , , , barrier methods should be used to prevent sexual transmission through infected semen. male returned travellers should continue the use of condom with pregnant sex partner throughout the whole duration of pregnancy. future studies should evaluate the duration of virus shedding in semen and the infectiousness of rnapositive semen samples, in order to determine how long barrier methods should be used by men returning to nonendemic regions. some regional authorities have advised women to avoid pregnancy until the epidemic is over. pregnant women or those planning for pregnancy should defer travelling to regions with reported cases of zikv infection. if such travel was unavoidable, they should strictly comply with personal protective measures to avoid mosquito bites. further studies are needed to determine the risk of zikv transmission by breast milk and saliva. other less common transmission routes, including mucocutaneous exposure to infected bodily fluid during laboratory and patient-care procedures, and bites by infected primates should be avoided with strict compliance to infection control measures. in the laboratory setting, zikv can be killed by potassium permanganate, ether, and heat (> c), but it is not effectively neutralised with low concentration ( %) of ethanol. no zikv vaccine is available currently. because the moratorium for pregnancy may be impractical for some people, a safe and effective zikv vaccine is urgently needed. some realistic approaches include liveattenuated or killed vaccine from human cell lines (as in the case of yfv and japanese encephalitis vaccines), attenuated chimeric vaccine (denv vaccine using the yfv vaccine backbone, currently in phase iii clinical trial), dna and recombinant protein vaccine. suitable animal models for evaluation of these potential vaccine candidates should be developed for zikv infection. the role of passive immunisation before and after exposure to zikv should also be assessed in future studies. the zikv epidemic has emerged as an unexpected global health emergency as the rapidly expanding zikv epidemic may turn out to be a major cause of permanent and severe disability in a generation of newborns, which would constitute a huge socioeconomic burden to the affected countries. the future of the zikv epidemic is unpredictable, but the worldwide spread of denv and chikv over the past two decades suggests that zikv has the potential to follow their paths. with more than half of the world's human population living in areas infested with aedes mosquitoes, the ongoing adaptation of zikv to an urban cycle signify the virus' pandemic potential. research preparedness is urgently needed to improve mosquito-control measures, as well as to develop point-of-care laboratory diagnostics, antivirals, and vaccines which are suitable for use in pregnant women and foetuses. interspecies transmission and emergence of novel viruses: lessons from bats and birds severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sars-like pandemic? two years after pandemic influenza a/ /h n : what have we learned? avian influenza a h n virus: a continuous threat to humans from sars coronavirus to novel animal and human coronaviruses the emergence of influenza a h n in human beings years after influenza a h n : a tale of two cities emergence in china of human disease due to avian influenza a(h n ) e cause for concern? ebola virus disease: a highly fatal infectious disease reemerging in west africa zika virus in the americas e yet another arbovirus threat zika virus outbreak on yap island, federated states of micronesia possible association between zika virus infection and microcephaly e brazil who statement on the first meeting of the international health regulations zika virus. i. isolations and serological specificity zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria serological and entomological study on yellow fever in sierra leone zika virus, a cause of fever in central java a serological survey of arboviruses in gabon serological survey for the prevalence of certain arboviruses in the human population of the south-east area of central african republic serological survey for arbovirus antibodies in the human and simian populations of the south-east of gabon surveillance of the circulation of arbovirus of medical interest in the region of eastern senegal a survey for arboviral antibodies in sera of humans and animals in lombok, republic of indonesia potential of selected senegalese aedes spp. mosquitoes (diptera: culicidae) to transmit zika virus epidemiological notes on some viruses isolated in uganda; yellow fever, rift valley fever, bwamba fever notes on zika virus e an emerging pathogen now present in the south pacific concurrent outbreaks of dengue, chikungunya and zika virus infections e an unprecedented epidemic wave of mosquito-borne viruses in the pacific co-infection with zika and dengue viruses in patients zika virus transmission from french polynesia to brazil a report on the outbreak of zika virus on easter island epidemiological update e neurological syndrome, congenital anomalies, and zika virus infection anticipating the international spread of zika virus from brazil zika virus genome from the americas entry routes for zika virus in brazil after world cup: new possibilities zika virus in brazil and the danger of infestation by aedes (stegomyia) mosquitoes. rev soc bras med trop . pii: s e el niño and climate change-contributing factors in the dispersal of zika virus in the americas? zika situation report e neurological syndrome and congenital anomalies zika virus infection acquired during brief travel to indonesia first case of zika virus infection in a returning canadian traveler two cases of zika fever imported from french polynesia to japan first case of laboratory-confirmed zika virus infection imported into europe zika virus infection after travel to tahiti zika virus infection in a traveller returning to europe from brazil zika virus infections imported to italy: clinical, immunological and virological findings, and public health implications cytokine kinetics of zika virus-infected patients from acute to reconvalescent phase zika virus in an american recreational traveler acute zika virus infection after travel to malaysian borneo zika fever imported from thailand to japan, and diagnosed by pcr in the urines zika virus infection in a traveller returning from the maldives epidemiological alert e neurological syndrome, congenital malformations, and zika virus infection. implications for public health in the americas zika virus emergence in mosquitoes in southeastern senegal cross-species transmission and emergence of novel viruses from birds zika virus. ii. pathogenicity and physical properties a simple technique for infection of mosquitoes with viruses; transmission of zika virus isolation of zika virus from aedes aegypti mosquitoes in malaysia investigation surrounding a fatal case of yellow fever in cote d'ivoire in oral susceptibility of singapore aedes (stegomyia) aegypti (linnaeus) to zika virus twelve isolations of zika virus from aedes (stegomyia) africanus (theobald) taken in and above a uganda forest the occurrence of chikungunya virus in uganda. i. isolation from mosquitoes critical review of the vector status of aedes albopictus aedes albopictus, an arbovirus vector: from the darkness to the light aedes (stegomyia) albopictus (skuse): a potential vector of zika virus in singapore zika virus in gabon (central africa) e : a new threat from aedes albopictus yellow fever and zika virus epizootics and enzootics in uganda molecular evolution of zika virus during its emergence in the (th) century aedes hensilli as a potential vector of chikungunya and zika viruses zika virus infections in nigeria: virological and seroepidemiological investigations in oyo state molecular characterization of three zika flaviviruses obtained from sylvatic mosquitoes in the central african republic a sero-epidemiological survey for certain arboviruses (togaviridae) in pakistan sylvatic transmission of arboviruses among bornean orangutans health evaluation of free-ranging and semicaptive orangutans (pongo pygmaeus pygmaeus) in sabah, malaysia a new threat looming over the mediterranean basin: emergence of viral diseases transmitted by aedes albopictus mosquitoes dengue and dengue vectors in the who european region: past, present, and scenarios for the future potential for zika virus transmission through blood transfusion demonstrated during an outbreak in french polynesia probable non-vector-borne transmission of zika virus potential sexual transmission of zika virus evidence of perinatal transmission of zika virus, french polynesia detection of zika virus in saliva dengue infection in pregnancy: prevalence, vertical transmission, and pregnancy outcome maternal dengue and pregnancy outcomes: a systematic review maternal and fetal consequences of dengue fever during pregnancy maternal and perinatal outcomes of dengue in portsudan, eastern sudan breast milk as a possible route of vertical transmission of dengue virus? chikungunya virus infection during pregnancy multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of la reunion west nile virus infection in pregnancy possible west nile virus transmission to an infant through breastfeeding e michigan perinatal transmission of yellow fever, brazil case report: probable transmission of vaccine strain of yellow fever virus to an infant via breast milk transmission of dengue virus without a mosquito vector: nosocomial mucocutaneous transmission and other routes of transmission laboratory-acquired west nile virus infections e united states the dengue and dengue hemorrhagic fever epidemic in puerto rico transmission of west nile virus from an organ donor to four transplant recipients possible dialysis-related west nile virus transmission e georgia zika virus infection in australia following a monkey bite in indonesia detection of zika virus in urine phylogeny of the genus flavivirus a multigene analysis of the phylogenetic relationships among the flaviviruses (family: flaviviridae) and the evolution of vector transmission genetic characterization of zika virus strains: geographic expansion of the asian lineage full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia quantitative real-time pcr detection of zika virus and evaluation with field-caught mosquitoes molecular biology of flaviviruses moraes figueiredo lt. domain iii peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization a single mutation in chikungunya virus affects vector specificity and epidemic potential spread of the pandemic zika virus lineage is associated with ns codon usage adaptation in humans biology of zika virus infection in human skin cells autophagy and viral diseases transmitted by aedes aegypti and aedes albopictus zika virus infection of the central nervous system of mice zika virus: further isolations in the zika area, and some studies on the strains isolated persistence of arboviruses and antiviral antibodies in vertebrate hosts: its occurrence and impacts incubation periods of mosquito-borne viral infections: a systematic review rapid risk assessment: zika virus infection outbreak, french polynesia current zika virus epidemiology and recent epidemics zika virus outbreak zika virus infection in man zika virus infection, cambodia fatal zika virus infection in girl with sickle cell disease, colombia. emerg infect dis zika virus infection complicated by guillain-barre syndrome e case report rapid risk assessment e zika virus epidemic in the americas: potential association with microcephaly and guillain-barré syndrome zika virus in brazil and macular atrophy in a child with microcephaly british broadcasting corporation (bbc) news. colombia links zika to rare nerve disorder deaths first detection of autochthonous zika virus transmission in a hiv-infected patient in rio de janeiro, brazil zika virus associated with microcephaly ophthalmological findings in infants with microcephaly and presumable intra-uterus zika virus infection ocular findings in infants with microcephaly associated with presumed zika virus congenital infection in salvador, brazil zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? zika virus: brazil's surge in small-headed babies questioned by report viral infections during pregnancy levels of igm antibodies against dengue virus in rio de janeiro persistence of virus-reactive serum immunoglobulin m antibody in confirmed west nile virus encephalitis cases development and persistence of west nile virusspecific immunoglobulin m (igm), iga, and igg in viremic blood donors virus and antibody dynamics in acute west nile virus infection one-step rt-pcr for detection of zika virus a diagnostic polymerase chain reaction assay for zika virus universal primers that amplify rna from all three flavivirus subgroups detection of zika virus in semen interim guidelines for pregnant women during a zika virus outbreak e united states interim guidelines for the evaluation and testing of infants with possible congenital zika virus infection e united states zika virus spreads to new areas e region of the americas wolbachia and the biological control of mosquito-borne disease zika virus and the never-ending story of emerging pathogens and transfusion medicine jamaica advises women to avoid pregnancy as zika virus approaches a review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available arthropod-borne viral infections of man in nigeria dengue and other arboviral diseases in south-east asia zika virus, french polynesia, south pacific rapid spread of emerging zika virus in the pacific area first report of autochthonous transmission of zika virus in brazil zika virus spreads across americas as concerns mount over birth defects zika virus-related hypertensive iridocyclitis laboratory infection with zika virus after vaccination against yellow fever the study was partly supported by the consultancy service for enhancing laboratory surveillance of emerging infectious disease of the department of health, the food and health bureau, hong kong special administrative region, china; and by the donations of hui hoy and chow sin lan charity fund limited and mr. larry chi-kin yung. the authors declare no conflict of interest. key: cord- -ji p l j authors: white, sarah k.; mavian, carla; elbadry, maha a.; beau de rochars, valery madsen; paisie, taylor; telisma, taina; salemi, marco; lednicky, john a.; morris, j. glenn title: detection and phylogenetic characterization of arbovirus dual-infections among persons during a chikungunya fever outbreak, haiti date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: ji p l j in the context of recent arbovirus epidemics, questions about the frequency of simultaneous infection of patients with different arbovirus species have been raised. in , a major chikungunya virus (chikv) epidemic impacted the caribbean and south america. as part of ongoing screening of schoolchildren presenting with acute undifferentiated febrile illness in rural haiti, we used rt-pcr to identify chikv infections in of children with this diagnosis during may—august . among these, eight were infected with a second arbovirus: six with zika virus (zikv), one with dengue virus serotype , and one with mayaro virus (mayv). these dual infections were only detected following culture of the specimen, suggesting low viral loads of the co-infecting species. phylogenetic analyses indicated that the zikv and mayv strains differ from those detected later in and , respectively. moreover, chikv and zikv strains from co-infected patients clustered monophyletically in their respective phylogeny, and clock calibration traced back the common ancestor of each clade to an overlapping timeframe of introduction of these arboviruses onto the island. introduction chikungunya virus (chikv) (family togaviridae, genus alphavirus), is the causative agent of chikungunya fever. after first isolation of chikv in in present-day tanzania, outbreaks and epidemics were limited to regions of asia, africa, and the pacific islands. in , chikv emerged for the first time in the americas, with sustained autochthonous transmission and rapid spread through the region [ ] [ ] [ ] . the acute symptoms of chikv infection are similar to those of infection with other arbovirus species, including dengue virus (denv), zika virus (zikv), and mayaro virus (mayv), each presenting with a constellation of symptoms including fever, headache, and myalgias/arthralgias. long-term, chikv infections have been linked with persistent arthralgias in a subset of cases [ ] ; it has also been reported that upwards of % of chikv-infected individuals are symptomatic, in contrast to findings with zikv, where it is estimated that only % of infected persons are symptomatic [ , ] . the similarity of the clinical presentation of acute-phase arbovirus infections is further complicated by the potential for simultaneous infection with multiple arboviruses. in a recent literature review, co-infections with chikv and denv ranged from % to % of patients [ ] . however, virtually no data are available on frequency of co-infection with chikv and arboviruses other than denv. even where good laboratory diagnostic facilities are available, identification of co-infections often does not occur, as there is a tendency to cease investigation once an initial pathogen has been identified, and/or identification of a second pathogen may require facilities for virus isolation, which may not be available. as part of ongoing studies of acute undifferentiated febrile illness in a cohort of school children in rural haiti, we identified children with rt-pcr-confirmed chikv infections during may-august , corresponding with the time period when the caribbean chikv epidemic was moving through haiti. specimens were also simultaneously screened by rt-pcr for denv - , then additionally for zikv. aliquots of the plasma specimens were then inoculated onto cell cultures for the isolation of additional pathogens of potential interest [ ] . we report here results of these studies, focusing on rates of arbovirus co-infection in our patient cohort and potential sources of origin of the co-infecting strains. blood specimens were collected from schoolchildren with acute febrile illness seen at the christianville school clinic in gressier, haiti, beginning in may . this clinic serves as the primary source of healthcare for approximately , children at four school campuses (the main lasalle campus [campus a] and three small satellite elementary school campuses [campuses b, c, and d]) operated by the christianville foundation in the gressier/leogane region; schools are located within a radius of approximately miles. the clinic has facilities for short-term stays of a few hours for sick children, with those requiring hospitalization referred to the local community hospital. as previously described, acute undifferentiated febrile illness was defined as occurrence of fever in a child with no obvious source of infection (i.e., no respiratory symptoms, symptoms of uti, or evidence of malaria or typhoid) [ ] . we have previously reported isolation of zikv [ ] , denv [ ] , human coronavirus nl [ ] , and enterovirus d [ ] from children in this school cohort; however, the cases/outbreaks in these prior publications did not include chikv, or cases within the may-august, , time frame of the current study. clinical features of these cases are reported elsewhere [ ] . whole blood (ca. . - ml) was collected in k edta tubes (bd vacutainer, becton, dickinson and company, franklin lakes, nj). as part of the initial diagnostic evaluation, plasma was screened for chikv viral rna (vrna) by molecular methods [ , ] . all virus isolations and rna extractions on chikv-positive specimens were performed in a bsl- facility at the university of florida (uf) emerging pathogens institute in gainesville, florida. the uf irb and the haitian national irb approved all protocols, and written informed consent was obtained from parents or guardians of all study participants. to assess the sensitivity of rt-pcr tests and for confirmation purposes, chikv isolations from plasma specimens were attempted in epithelioid cells derived from african green monkey kidneys (vero e , atcc crl- ). the vero e cells used for virus isolation were maintained in cell culture medium comprised of admem (advanced dulbecco's modified essential medium) supplemented with % low antibody, heat-inactivated, gamma-irradiated fetal bovine serum (fbs, hyclone, ge healthcare life sciences, pittsburgh, pa), l-alanine and lglutamine supplement (glutamax, invitrogen, carlsbad, ca), and μg/ml penicillin, μg/ ml streptomycin, μg/ml neomycin (psn antibiotics, invitrogen) with incubation at ˚c in % co . confluent cell cultures were split into cm flasks hours prior to inoculation to attain % confluent cell monolayers the following day. prior to inoculation, the culture medium was removed and inoculum containing μl of plasma that had been filtered through a sterile . filter and mixed with μl admem with % fbs, glutamax, and psn antibiotics was added to the monolayer. the inoculated monolayer was incubated at ˚c in % co , and rocked every minutes for hour. a negative control (mock-infected) cell culture was inoculated with ul of dmem without virus or plasma and handled in parallel with the other cultures. after allowing for virus adsorption for hr, the inocula were removed and replaced with ml of admem with % fbs, glutamax, and psn antibiotics, and thereafter incubated at ˚c in % co . cell cultures were refed every days by the replacement of . ml of spent media with admem with % fbs. the cultures were observed for up to days' post-inoculation, or until chikv-induced cytopathic effects (cpe) were observed in the cell monolayers using an inverted microscope. when cpe were observed throughout % of the monolayer, a final collection of ml spent media, and ml of scraped cells in spent media, were cryopreserved at - ˚c for future tests. total rna was extracted from both plasma specimens and spent media, and tested by realtime rt-pcr (rtrt-pcr) following published protocols for chikv vrna [ ] for confirmation of previous tests performed in haiti. rna extracted from plasma specimens were then screened for denv serotypes - [ ] and zikv [ ] vrnas by rtrt-pcr. cycle threshold (ct)-values under were considered positive. viral genomic rna that was extracted from chikv, denv - , and zikv strains that were obtained from the biodefense and emerging infections research resource repository (bei resources, manassas, va) were used as positive control materials for rtrt-pcr. cell cultures were also tested for the presence of denv and zikv vrnas by rtrt-pcr, even if the corresponding plasma specimen tested negative. additionally, spent media from cultures displaying non-chikv cpe, that were denv and zikv negative by rtrt-pcr, were screened with a duplex rt-pcr for the vrnas of other alphaviruses (venezuelan equine encephalitis -, eastern equine encephalitis -, western equine encephalitis -, aura-and mayaro viruses) and flaviviruses (yellow fever -, saint louis encephalitis -, bussaquara -, ilheus -, and rocio viruses) [ ] . the denv- strain from bei and the mayv sample from our laboratory [ ] were used as the flavivirus and alphavirus positive controls, respectively, in the duplex rt-pcr protocol. whole genome sequence data from of the chikv samples ( from children with co-infections, from selected randomly mono-infections) were obtained by sanger sequencing and a primer-walking approach, as previously described [ , ] . similarly, we designed sequencing primers for mayv and zikv that also amplify approximately bp overlapping segments, and used a primer walking method for whole genome sequencing of those viruses [ , ] . for the sequencing of denv, primers described by christenbury et al were utilized [ ] . amplification of each segment was performed using an accuscript high-fidelity first-strand cdna synthesis kit (agilent technologies, santa clara, ca) followed by pcr with phusion polymerase (new england biolabs, ipswich, ma). the ' and ' ends of the viral genomes were obtained using rna-ligase mediated (rlm) systems for ' and ' rapid amplification of cdna ends (race) per the manufacturer's protocols (life technologies, carlsbad, ca). amplicons were purified, sequenced bi-directionally, then the sequences assembled with the use of sequencher dna sequence analysis software v . (gene codes, ann arbor, mi), and subsequently analyzed in comparison to denv, mayv, and zikv sequences available in genbank for nucleotide sequence comparisons. the vrna sequences we obtained differed from those of the corresponding viruses in our repository, confirming the newly analyzed sequences did not arise from laboratory contamination. alignments for each virus-specific dataset (chikv, denv- , mayv, and zikv), including full genome sequences selected to represent the major clades described so far for each virus were obtained using the muscle algorithm implemented in mega (http://www.mega software.net/) [ ] [ ] [ ] . genbank accession numbers (ac), geographical location, and year associated with isolation of each strain are reported in s table. based on previous evidence of recombination reported for mayv, potential presence of recombination was also investigated for the new mayv isolate (ky ) [ ] with algorithms implemented in the rdp [ ] software (http://web.cbio.uct.ac.za/~darren/rdp.html), as previously described [ ] . the phylogenetic signal in each virus-specific data set was evaluated by likelihood mapping using treepuzzle (http://www.tree-puzzle.de/) [ ] , and substitution saturation plots using dambe ((http://dambe.bio.uottawa.ca/dambe/dambe.aspx) [ ] . maximum likelihood (ml) trees were inferred by iq-tree using the best-fitting nucleotide substitution model chosen according to bayesian information criterion (bic) (s fig) [ , ] . statistical robustness for internal branching order in each phylogeny was assessed by ufboot-ultrafast bootstrap (bb) approximation ( , replicates) implemented in iq-tree [ ] , and strong statistical support along the branches was defined as bb> %. ufboot was eployed as it accelerates computing and reduces overestimating branch support [ ] . alignments files are available from the authors upon request. the temporal signal for each dataset was assessed using ml trees with tempest v . (http:// tree.bio.ed.ac.uk/software/tempest/) [ ] . bayesian inference of time-scaled phylogenies were carried out with beast v . . (http://beast.bio.ed.ac.uk/) [ , ] by enforcing either a strict or relaxed molecular clock [ ] and the sdr substitution model with empirical base frequencies and gamma distribution of site-specific rate heterogeneity. two demographic priors were also tested for each analysis: constant population size or non-parametric bayesian skyline plot (bsp). bayesian markov chain monte carlo (mcmc) were run for - million generations (sampled at fixed intervals to obtain posterior distributions with , data points), depending on the data set, in order to assure proper mixing of the mcmc, which was assessed on the basis of the effective sampling size (ess) of each parameter estimate, accepting only ess values > . the best clock/demographic model for each virus-specific data set was chosen by comparing marginal likelihood estimates (mle) [ ] , obtained using path sampling (ps) and stepping-stone sampling (ss) methods [ , ] with bayes factor (bf) (s table) . in practice, the bf natural logarithm was used for comparison with lnbf< indicating no evidence against the null hypothesis (i.e. less parameter-rich model), - -weak evidence, - -strong evidence, and > very strong evidence [ ] . for each data set, the maximum clade credibility (mcc) tree (tree with the largest product of posterior clade probabilities) was selected from the posterior tree distribution of the best fitting clock/demographic model, after appropriate burn-in, using treeannotator v . . implemented in the beast software package. the final trees were manipulated in figtree v . . for display purposes (http://tree.bio.ed.ac.uk/software/figtree/). xml files for the beast runs are available from the authors upon request. with one exception, all rtrt-pcr-confirmed chikv infections in our student cohort occurred in the -week period between may and july , ; the one "outlier" case occurred in mid-august, . among the plasma specimens taken from febrile schoolchildren in the may-august time period, tested positive via rtrt-pcr for chikv, but were negative for denv - and zikv. among the specimens from febrile schoolchildren during this time period that were not positive for chikv, two gave equivocal results in the rtrt-pcr assay and were excluded from the analysis, two were denv type positive, and were negative in all assays. cultures were not performed on these specimens due to insufficient specimen volume, reflecting the large number of assays that had been conducted in the initial screening of what were relatively small blood samples from each child. the mean age of children from whom specimens were collected was . years. ninety-two of the specimens were from students at campus a of the school; were from campus b, and one was from campus c. gender, average age, and school level for the children whose samples were positive for chikv by rtrt-pcr are included in table . of the rtrt-pcr chikv-positive specimens, attempts were made to isolate virus from specimens, with the remainder not inoculated onto cell cultures due to insufficient specimen volume. typical chikv-induced cpe, consisting of cell membrane blebbing, cell lysis, and apoptosis, were observed in cultures from of the samples on average days post inoculation (dpi), with some specimens displaying advanced cpe as early as dpi and others not until dpi (fig ) ; target vrna's were positive by rtrt-pcr for chikv but negative for denv and zikv [ ] [ ] [ ] . all negative control cell cultures lacked cpe and tested negative by rtrt-pcr for the target vrnas of this work. eleven ( %) of the samples cultured did not display any cpe during the period of culture and tested negative for target vrnas in these studies, despite initial positive chikv results by rtrt-pcr. eight cultures displayed cpe inconsistent with those expected for chikv (fig ) . the mixed cpe in these latter cultures was associated with co-infection with chikv together with another virus, as determined by molecular tests of the cultured specimens: co-infecting arboviruses included zikv (n = ), denv type (n = ), and mayv virus (n = ). sex and age of patients were comparable for those with chikv mono-inections and the subset of chikv cases with co-infections. seven of the co-infections were from campus a of the school; one co-infection (chikv and zikv) was in a student from campus b. (fig ) using the best fitting molecular clock/demographic model (s table) , were inferred for each data set. bayesian and ml reconstructions displayed very similar topologies and suggested three independent phylogenetic lineages of chikv in haiti, possibly the result of three separate introductions (figs a and s a) . the chikv strains from the current study were in a well-supported monophyletic clade which was most closely related to a strain from el salvador (fig a) . within this clade, six chikv strains were from patients co-infected with zikv and one with denv- . molecular clock median estimate of the time of the most recent common ancestor (tmrca) for the clade was august , with % highest posterior density ( %hpd) intervals between april and january ( fig a) . as shown in fig , two other chikv isolates, not collected as part of this study but previously reported by lanciotti (kr and kr ) from mono-infected haitian patients, clustered within a second distinct clade related to different central and south american strains, but separate from our gressier strains. one of the sequenced chikv strains from our cohort, from a randomly selected mono-infection in a child from campus b, was in yet a third clade, which clustered most closely with a strain from the dominican republic. the six zikv sequences obtained in june from the chikv co-infected patients were highly similar ( . %) to each other and also cluster within a well-supported monophyletic clade, which, interestingly, includes a divergent strain isolated in from guadaloupe (figs b and s b) . the primary clade of co-infecting zikv strains is closely related to another zikv clade including isolates from the usa florida outbreak [ ] . in contrast, previously reported [ ] haitian strains from this same school cohort in december of (ku ), and haitian strains from early (kx [ ] and kx [ ] ) belonged to distinct phylogenetic lineages, consistent with multiple independent introductions of zikv to haiti. the median origin of the haitian zikv co-infection strains was estimated by molecular clock in april with a %hpd between january and may (fig b) , a timeframe overlapping with the estimates of the corresponding chikv co-infections clade. the specimen from the chikv and denv- co-infection was obtained on june , . the denv- isolate was closely related to a previously reported strain isolated in early (kx ) from a us traveler returning from haiti [ ] and they both cluster within a wellsupported clade including brazilian and peruvian subclades (figs c and s c) . according to the time-scaled phylogeny (fig c) , the tmrca of the denv- isolate from the chikv coinfected patient traced back to february with %hpd between may and october . finally, the mayv strain from a chikv co-infected patient seen on june , is the earliest documented case of mayv in haiti to date. its genome is highly similar ( . %) to a strain from brazil isolated in , and phylogenetic analysis cluster both strains in a well-supported monophyletic clade (figs d and s d) , within genotype l [ , ] , with a tmrca dating back to ( %hpd interval of - ). it should be noted, however, that since we only have one mayv strain clustering with the brazilian strain, the tmrca is unlikely to represent the date of introduction of mayv in haiti. another, previously reported haitian strain (kx ) [ , ] , clusters in a different well-supported clade within genotype l, a scenario once again consistent with multiple independent introductions of the virus in the caribbean. despite increasing recognition of the frequency with which simultaneous co-infection with multiple arbovirus species occurs [ , , [ ] [ ] [ ] , co-infection dynamics are not well understood, and the clinical, pathologic, and immunologic significance of such co-infections remains to be determined. as a starting point in unraveling these dynamics, we were interested in better quantifying the frequency of such infections, and exploring possible origins of co-infecting strains. to optimize our ability to identify co-infections (and following standard practices in our laboratory [ , , , ] ), we continued diagnostic studies even after initial identification of chikv or another pathogen in a patient sample. interestingly, while we identified multiple co-infecting viruses in cell culture, the original patient rtrt-pcr in this study was, in each instance, negative for the second pathogen, suggesting that viral loads for the co-infecting species were low. we also had instances where viral cultures were negative in the setting of a positive rtrt-pcr, possibly reflecting the presence of non-viable virus in the sample, and/or strains that have mutated or which do not grow well in vero cells. if we are to better understand the role of co-infections in disease occurrence, the diagnostic significance of both rtrt-pcr negative/culture positive and rtrt-pcr positive/culture negative samples will require further study. co-infections with chikv and denv are well described, with reported rates of co-infection ranging from % to % [ ] ; the highest reported co-infection rates were from madagascar ( %) and nigeria ( %). another study from the colombian-venezuelan border reports a chikv/denv co-infection prevalence of . % [ ] . in our study, in contrast, the co-infection rate was only %, with only two additional dengue cases (both denv- ) identified among chikv-negative patients; this may reflect a low level of circulating denv in the community during the chikv epidemic, and/or a high existing level of population immunity to the virus. we also identified one chikv/mayv co-infection. we have previously reported a mixed mayv and denv- infection that occurred outside of the time frame of the chikv epidemic (january, ) in the same school cohort [ ] . the high genomic similarity of this haitian mayv isolate with a brazilian strain isolated in corroborates our hypothesis that mayv has been criptically circulating in the human population at a sub-epidemic level, most likely in brazil, for years and that it was introduced just recently onto hispaniola [ ] . what was unexpected in our data set was the relatively high frequency of zikv/chikv coinfections. this occurred in a setting in which we were only screening plasma samples. as we have previously reported [ ] , urine can be positive for zikv for a longer period of time than plasma; if we had also screened urine, there is the possibility that we would have identified additional zikv-positive patients. we, and others, have previously reported cases of zikv/ chikv co-infection [ , , , ] , so finding the two together is not surprising. the zikv case cluster in the current study would appear to have been relatively widespread within the community (i.e., not a point source at the school), as one of the six co-infections was from a child at a different school campus, ca. miles from the main campus. interestingly, our molecular clock analysis indicates that the strains of chikv and zikv found in our patients were both introduced in haiti within the same short time frame, each one giving rise to a well-supported monophyletic clade, one including all chikvs from chikv/zikv co-infected subjects, the other one including all zikvs from the same co-infected subjects. the simultaneous emergence of these two clades in the haitian population is compatible with a simultaneous coinfection scenario or indicate, at the very least, the sequential infection of a patient with two arboviruses within a relatively short period of time. in light of recent reports that mosquito coinfection with zikv and chikv allows simultaneous transmission without affecting vector competence [ ] , it is plausible that the two viruses were co-transmitted by the same mosquito. given that these are among the earliest, if not the earliest, documented zikv infections in the hemisphere, the observation that they appeared together with epidemic chikv raises interesting questions about the possible role of chikv in the initiation of the zikv epidemic in the americas. because our work draws on student populations from four schools located within a radius of about miles, we have some feel for disease prevalence within the immediate study area; however, generalizability of these data to larger regions of haiti may be limited. nonetheless, our findings are consistent with the idea that there were multiple introductions of chikv into haiti between and , with identification of three distinct phylogenetic lineages, each clustering with strains from different parts of the caribbean and/or south america. this was also the case for zikv: the zikv co-infecting strains did not cluster with the zikv strains previously obtained in haiti in and , consistent with multiple introductions or re-introductions of this arbovirus in haiti between and . although for denv- and mayv the data are more limited, the tmrcas for denv- suggested that the introduction in haiti happened between and , a time interval again overlapping with the estimated introduction of both chikv and zikv co-infecting strains circulating in haiti. clustering of both the zikv and denv- strains together with zikv and denv- strains obtained in florida in [ , ] , as well as the multiple independent introductions of chikv, zikv, and mayv to haiti inferred from the phylogenies, demonstrate the potential role of the caribbean as a node for arbovirus infections connecting south, central, and north america. whole-genome sequencing analysis from the chikungunya virus caribbean outbreak reveals novel evolutionary genomic elements emergence of human arboviral diseases in the americas chikungunya virus infection: an overview movement of chikungunya virus into the western hemisphere zika virus: history, emergence, biology, and prospects for control co-distribution and co-infection of chikungunya and dengue viruses spectrum of outpatient illness in a school-based cohort in haiti, with a focus on diarrheal pathogens zika virus outbreak in haiti in : molecular and clinical data complete genomic sequence of dengue virus isolation of coronavirus nl from blood from children in rural haiti: phylogenetic similarities with recent isolates from malaysia isolation of an enterovirus d from blood from a child with pneumonia in rural haiti: close phylogenetic linkage with new york strain clinical and epidemiologic patterns of chikungunya virus infection and coincident arboviral disease in a school cohort in haiti chikungunya virus in us travelers returning from india complete genome sequences of chikungunya viruses isolated from plasma specimens collected from haitians in analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia duplex reverse transcription-pcr followed by nested pcr assays for detection and identification of brazilian alphaviruses and flaviviruses mayaro virus in child with acute febrile illness zika and chikungunya virus coinfection in a traveller returning from colombia, : virus isolation and genetic analysis a method for full genome sequencing of all four serotypes of the dengue virus mega : molecular evolutionary genetics analysis version . for bigger datasets muscle: multiple sequence alignment with high accuracy and high throughput muscle: a multiple sequence alignment method with reduced time and space complexity emergence of recombinant mayaro virus strains from the amazon basin rdp : detection and analysis of recombination patterns in virus genomes tree-puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing dambe: software package for data analysis in molecular biology and evolution iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies w-iq-tree: a fast online phylogenetic tool for maximum likelihood analysis ultrafast approximation for phylogenetic bootstrap exploring the temporal structure of heterochronous sequences using tempest (formerly path-o-gen) bayesian phylogenetics with beauti and the beast . beast: bayesian evolutionary analysis by sampling trees exploring the demographic history of dna sequences using the generalized skyline plot improving marginal likelihood estimation for bayesian phylogenetic model selection improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty zika virus evolution and spread in the americas coinfection with zika and dengue- viruses in a traveler returning from haiti, : clinical presentation and genetic analysis evolutionary and ecological characterization of mayaro virus strains isolated during an outbreak dengue and chikungunya virus coinfection in a german traveller zika, dengue, and chikungunya co-infection in a pregnant woman from colombia co-circulation and simultaneous co-infection of dengue, chikungunya, and zika viruses in patients with febrile syndrome at the colombian-venezuelan border co-infections with chikungunya and dengue viruses coinfections of zika and chikungunya viruses in bahia, brazil, identified by metagenomic next-generation sequencing mosquito co-infection with zika and chikungunya virus allows simultaneous transmission without affecting vector competence of aedes aegypti complete genome sequence of dengue virus type from a resident of north-central florida with locally transmitted dengue fever estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees we thank sonese chavannes and gina anilis of the christianville school clinic, the laboratory staff, and the christianville foundation, for their dedication and support. key: cord- -lgkqwm u authors: yin, yingxian; xu, yi; su, ling; zhu, xun; chen, minxia; zhu, weijin; xia, huimin; huang, xi; gong, sitang title: epidemiologic investigation of a family cluster of imported zikv cases in guangdong, china: probable human-to-human transmission date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: lgkqwm u zika virus (zikv) is an emerging mosquito-borne flavivirus that can potentially threaten south china. a chinese family of four returning from venezuela to china was found to be positive for zikv when the youngest son's fever was first detected at an airport immigration inspection. they were isolated temporarily in a local hospital in enping city, guangdong province, where their clinical data were recorded and urine and saliva were collected to isolate zikv and to obtain viral sequences. all of them except the mother presented mild symptoms of rash and fever. envelope gene sequences from the father, daughter and son were completely identical. phylogenetic analysis demonstrated that this strain is similar to several imported strains reported in recent months, which are all clustered into a group isolated from zika outbreaks in brazil. together with the climatic features in venezuela, new york and guangdong in february, it can be concluded that our subjects are imported cases from venezuela. with the same viral sequence being shared between family members, neither direct human-to-human nor vector transmission can be ruled out in this study, but the former seems more likely. although our subjects had mild illness, epidemiologists and public health officials should be aware of the risk of further expansion of zikv transmission by local competent vectors. although zika virus (zikv) was first identified early in in uganda, africa, outbreaks in french polynesia in significantly accelerated the spread of this virus to other parts of the world. zikv is a reemerging mosquito-borne flavivirus circulating in a wide range of regions including africa, south america, and asia. zikv infection can cause serious damage to the central nervous system, such as infant microcephaly and guillain-barré syndrome. [ ] [ ] [ ] the virus has proven to be neurotropic in animals, and a recent experiment in vitro also showed that it can infect human neural progenitor cells derived from induced pluripotent stem cells. another study showed that human dermal fibroblasts, epidermal keratinocytes and immature dendritic cells are also permissive to the most recent zikv isolates. an animal model of zikv infection has been established in ag mice by foot pad injection. by far, aedes aegypti is considered the principal transmission vector of zikv, although aedes albopictus, which caused several outbreaks of dengue fever in guangdong province of south china in the last two decades, may play a role in the spread of this virus because a. albopictus may be a competent vector. there are over chinese in venezuela, which is one of the regions most heavily affected by zikv infection in south america. with frequent people shuttling between south america and guangdong, there is a potential risk of spreading zikv to south china, where a. albopictus are active in densely populated communities. in this study, a family of four flying from venezuela to guangzhou of guangdong province was found to be zikv positive in their peripheral blood. to gain a better understanding of transmission among communities, the phylogenetic relationship between the isolates from this family and others from diverse regions of the world was analyzed. because this virus may be transmitted directly by body fluids, [ ] [ ] [ ] it was also necessary to explore this possibility in this family. four hospitalized individuals from a family (father, mother, daughter and son) were diagnosed with zikv infection at enping people's hospital. this family had lived in venezuela for more than two months before february ; then they flew to new york on that day and stayed there for~ days. finally, they flew from new york to guangzhou, china on february ( figure ). these four infected individuals were first confirmed by real-time reversetranscription polymerase chain reaction (rt-pcr) in baiyun international airport of guangzhou, where the youngest one (the son) had developed fever, and the family was then isolated by the local department of public health. the infected individuals then lived in a shared ward (without other patients) in the infectious disease department of enping people's hospital. the room had been screened against mosquitoes, and each bed was also covered with a bed net to prevent spreading by local competent vectors. at the time of hospitalization, the subjects' clinical history and results of a general physical examination, blood tests and routine urine tests were documented. the youngest one (a -year-old boy) was the first case whose manifestation was fever and maculopapules. saliva, urine and peripheral blood were collected from the patients during the onset and recovery period and were stored at − °c. all samples were tested for zikv rna by real-time pcr, and some urine was utilized to isolate virus. informed consent was obtained from all patients before sample collection. the study protocols were reviewed and approved by the scientific and ethical committee of guangzhou women and children's medical center. before rna extraction, urine samples were concentrated with amicon ultra- . centrifugal filter units with ultracel- membrane (millipore, shanghai, china). the qiaamp viral rna mini kit (qiagen, hilden, germany) was used to extract viral rna from urine and saliva according to the manufacturer's instructions. rna was eluted in μl of ave buffer and stored at − °c until use. samples positive for zikv were selected to amplify genes encoding envelope protein (e) and nonstructural protein (ns ). complementary dna (cdna) was synthesized from viral rna by using the revertaid first strand cdna synthesis kit (thermo fisher, salt lake city, usa) according to the manufacturer's instructions. four pairs of primers were designed to generate overlapping dna fragments covering the e and ns gene regions by using the software oligo . (http://www.oligo.net/. supplementary table s ). polymerase chain reaction (pcr) was performed with ex taq hs dna polymerase (takara, dalian, china) under conditions of initial heating of °c for min, followed by cycles of °c for s, °c for s and °c for . min. the sequences of the pcr products were identified by using the sanger sequencing method. before phylogenetic analysis, the e and ns coding sequences of four individuals in our study were aligned with other reference sequences using clustal . software (http://www.clustal.org/clustal /). the reference sequences selected were those with highly similar blast (megablast, http://blast.ncbi.nlm.nih.gov/blast.cgi) scores. phylogenetic trees were drawn using the maximum likelihood method in the tamura-nei model with gamma-distributed evolutionary rates in mega . (www.megasoftware.net). an initial tree was made automatically with the nearest-neighbor-interchange method. the gaps/missing data treatment was set as complete deletions. bootstrap analyses with replications were utilized to determine confidence values for groupings within the phylogenetic trees. other parameters were set to default style. the clinical characteristics of four individuals with zikv infection are summarized and compared in table . in this family, whose members were living together for a long period before and after the first onset, the -year-old son was the first patient, and he had both fever and rash . before returning to china, they had no symptoms of any infectious disease, but the son and the daughter both had a history of mosquito bites in venezuela according to the father's memory, although the specific date of biting was not clear. the -year-old daughter was the second one who had symptoms, which included fever and rash, with the rash occurring on the second day after her brother had fever and rash. the -year-old father was the latest patient; the only symptom he had was a rash, and the rash was distributed uniformly on his neck and back ( figure ). his neck rash first occurred on the third day after his son showed fever and rash. the -year-old mother did not have any symptoms during the whole period of observation. a time axis based on rash indicates the sequence, the time interval and the date of positive detection for zikv ( figure ). the four family members had been isolated since february , and they were released on march . the clinical laboratory showed results that the overall symptoms of the four subjects were mild. the daughter and the son were subjected to laboratory blood tests twice and thrice, respectively (table ) . unlike most other viral infectious diseases, low white blood cell counts were not a common feature of our subjects. detection of zikv in saliva and urine from four hospitalized patients. real-time pcr based on taqman probes was used to detect zikv rna. among the four tested urine samples collected on february , patient (father), patient (daughter) and patient (son) were found to be positive for zikv. only patient was positive for zikv in a saliva sample (detected on the night of february , when the samples were collected). the urine and saliva samples from patient (mother) were all negative for zikv detection. nucleic acid sequencing, alignment and phylogenetic analysis pcr products were successfully obtained only from patient , patient and patient . after sequencing and fragment assembly, the e gene sequences of the three patients were found to be exactly the same. the e gene sequence of patient is identical to z (genbank: ku . , submitted by wu et al, guangdong provincial center for disease control and prevention), the viral sequence from the same patient. we found that all our sequences were similar to those of strains circulating in south america, especially brazil ( figure ). to further investigate the origin of the zikv isolated from our subjects, phylogenetic trees were constructed using the maxim likelihood method. zikv infection is becoming a global public health concern since microcephaly cases were linked to zikv infection during pregnancy. so far, the data suggesting that microcephaly cases in brazil might be linked with zikv infection are only epidemiological. , growing evidence has indicated that zikv can damage the fetus, causing intrauterine growth restriction. , a recent report that zikv can be detected in amniotic fluid of fetuses with microcephaly substantiated this point. with the establishment of a prevention and control system for emerging infectious diseases in china after the outbreak of severe acute respiratory syndrome in , imported cases from affected areas with fever will be strictly examined for specific pathogen infections. travelers with fever would usually be intercepted for further quarantine in an international airport when they have a history of being bitten by a mosquito in an area affected by zikv. in our study, when the youngest son had a fever, the whole family needed to be examined. because the four subjects in this family showed zikv positivity in their peripheral blood, isolation of the whole family was obligatory. south american countries closest to the equator have been deeply plagued by zikv infection in recent years. for example, zikv infection has been frequently reported in counties such as brazil, suriname, colombia and venezuela. neighboring caribbean countries including guatemala, haiti, puerto rico and dominica have also been affected. throughout the whole year, these areas have a warm and humid climate suitable for the survival of mosquitoes. the temperature in february in north america, especially northern regions such as new york, is below °c; thus, the family members in our study were unlikely to be bitten by mosquitoes during their -day stay in new york. thus, the zikv isolates carried by this family could only have come from venezuela (figure ). rash and low-grade fever seem to be the main symptoms for most zikv-infected individuals. rash was reported to be the most frequent symptom (presented in . % cases), followed by fever and arthralgia. in general, symptoms of zikv infection (mild flu-like symptoms) are milder than those of dengue virus (denv) infection; the latter often include high-grade fever with myalgia, headache, arthralgia and nausea. leukopenia (o /mm ) has been detected iñ % of dengue fever patients. in our study, one adult and two children had the symptoms of fever, rash and conjunctival congestion, and no one complained of any pain. unexpectedly, the three patients with fever all had normal leukocyte counts ( /mm ). whether the milder symptoms of zikv infection (milder than denv infection) mean a higher leukocyte count is unclear because of the lack of data obtained in past reports on zikv outbreaks. hence, if the mother without any symptoms in our study is considered only a carrier of zikv (not a patient), this proportion of patients in our study is almost consistent with previous reports, in which rashes were presented by almost all patients. information about pruritus, the second most common clinical symptom in the confirmed cases in previous study, was not obtained from the family while inquiring them regarding their case history. perhaps this symptom is transient and unnoticeable at the time of disease onset. rt-pcr based on taqman probes may be the most convenient tool to detect zikv infection in suspected patients , because enzyme-linked immunosorbent assays (elisas) for igm antibody against zikv would cross-react with other flaviviruses, , and the results must be validated by a plaque reduction neutralization test, a labor-intensive and costly method. moreover, compared with blood drawing, urine and saliva collection are more acceptable options for suspected individuals, and the latter have an advantage for viral detection and isolation because virus can be detected at higher titers and for a longer period in urine than in serum. a previous study figure phylogenetic tree based on e gene sequences of zika virus isolates. e gene sequences from the father, daughter and son (genbank: ku ) in our study were used for alignment with other reference sequences. all isolates are indicated with related information (genbank number, country, year and host, some with sample type). phylogenetic trees were drawn using the maximum likelihood method by the tamura-nei model with gamma-distributed evolutionary rates in mega . . an initial tree was made automatically with the nearest-neighbor-interchange (nni) method. gaps/missing data treatment was set as complete deletion. bootstrap analyses with replications were utilized to determine confidence values for groupings within the phylogenetic trees. other parameters were set to default style. first imported familial zikv cases in china y yin et al showed that a patient had prolonged shedding of viral rna in saliva and urine for up to days after symptom onset. nevertheless, positive taqman rt-pcr detection is not usually a guarantee of successful sequencing or isolation of target viruses. urine and saliva usually need to be concentrated before rna extraction or viral inoculation, which was performed in our study. like other flaviviruses, the structure of the single polyprotein encoded by zikv genomic rna is ′-c-prm-e-ns -ns a-ns bns -ns a-ns b-ns - ′, in which the e (envelope) protein is the main antigen recognized by the host immune system, and ns is the principal component of the replication complex of the virus. in previous research, e gene sequences of zikv isolates were usually utilized to construct phylogenetic trees based on experience from molecular study of dengue virus. our phylogenetic tree constructed based on the e protein coding gene is topologically similar to that based on the complete zikv genomic sequence (figure ) , meaning that the phylogenetic tree based on the e gene is strong enough to distinguish all clusters of zikv isolates from all over the world. because complete genome sequencing of an rna virus is a time-consuming procedure, constructing a phylogenetic tree using e protein gene sequences is a simpler method for molecular epidemiologic study, especially in an outbreak. in our phylogenetic tree constructed from e protein gene sequences, zikv can be divided into two lineages, african and asian. , although there is a very short evolutionary distance between our strains and the venezuela imported strain ku . -ve ganxian, the diversity of the strains is apparent, and the latter is nearer to kj . -h/pf/ french polynesian strains. , currently, all cases reported in china seem to be imported from south america and pacific islands, which also indicates there was a great diversity of strains circulating during early in venezuela. our strains are more similar to the strains circulating in south america in late , where there were more strains isolated from patients. a phylogenetic tree based on ns gene sequences cannot efficiently distinguish different subgroups of zikv from various sources of isolates (supplementary figure s ) because the ns gene sequence is more conserved than that of e or even ns , which is a gene sequence used more frequently to build phylogenetic trees. currently, the e gene sequence is the most commonly used sequence in molecular epidemiologic studies of flaviviruses because it has the greatest genetic diversity (supplementary figure s ) . it is worth noting that strains of the african lineage were almost solely isolated from aedes mosquitoes circulating in the african continent according to the phylogenetic tree, probably because zikv did not draw much attention before massive microcephaly cases were reported in brazil. zikv is spread primarily by a. aegypti, a dominant vector for several mosquito-borne viruses in the western hemisphere. zikv was detected in a. albopictus from gabon, a country in central africa. no direct evidence has demonstrated that this virus could be spread by a. albopictus, a local vector for dengue virus in south china, yet the threat is quite real. the fact that zikv can propagate in the c / cell line derived from a. albopictus suggests that this mosquito may be a competent vector and have the potential for spreading the virus, although there have not been any reported isolates from mosquitoes in south china. a previous experiment conducted in laboratory indicated that zikv could be detected in the midgut and salivary glands of a. albopictus ten days after oral infection. outbreaks of denv have proved that south china may be a permissible environment for transmission of emerging viral diseases such as zikv, and imported cases may be sources of subsequent autochthonous cases. , a key difficulty in preventing the spread of infection is that a high proportion of infected individuals have no symptoms. similar to the mother in our study, asymptomatic individuals with viremia may be a source of infection when they are exposed to competent vectors. if the son's fever had not been found at the airport entrance, our subjects would not have been suspected of being infected with zikv, and thus their viremia would not have been confirmed. it is difficult to convince zikv carriers to be examined even if they are returning from an affected region. furthermore, in addition to the mosquito-borne route, other complex modes of transmission such as body fluid transmission will make controlling zikv infection even more difficult than controlling dengue fever. the priorities for control include strengthening the education of travelers from infected areas to non-epidemic regions, identifying virus carriers, preventing local transmission caused by imported cases, and an effective vector management program. in our study, four subjects in one family had lived and traveled together for the past few months, so they had similar opportunities for exposure to mosquito bites. however, because they are a family, the probability of human-to-human transmission by close contact between family members should not be ignored. if their zikv infections were all caused by mosquito-biting, from a time axis showing when symptoms occurred, the incubation periods of three of the patients were all more than five days (figure ), which is consistent with previous reports ( - -day incubation period according to the world health organization website). denv transmission by non-blood routes is almost impossible, so vector transmission is nearly the only way. thus, as for clustered cases in one family, if the vector transmission is considered, zikv can be comparable to denv because they have the same transmission dynamics via vectors. until now, there have been no reports of denv case numbers in one family being more than two, especially when they share the same virus sequence. therefore, because vector transmission of denv between family members is less likely, so is that of zikv. because the zikv isolated from three members is identical and there was a to day interval between initial symptom occurrence, human-to-human transmission may also be a plausible explanation. moreover, viral isolation from urine and saliva samples (collected on february ) of three patients was performed successfully using inoculation of suckling mice (data not shown), indicating that during onset, there was viral shedding in these three patients' body fluids, which was infectious to their family members. regarding direct transmission, sexual contact may be a route, for a relative case had been reported previously but with no direct evidence. because an in vitro experiment demonstrated that human skin cells are permissive to zikv, it might be possible that zikv can be transmitted by other bodily fluids such as saliva and urine. rt-pcr positivity for zikv rna together with successful viral isolation from the three patients' urine samples proved that there were live viruses present in these samples. rt-pcr also indicated the existence of viral rna in the patients' saliva. however, currently there is no reliable evidence demonstrating zikv transmission via these types of bodily fluids. a bibliometric analysis of global zika research zika virus and guillain-barre syndrome: another viral cause to add to the list guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study neurological expertise is essential for zika virus infection zika virus infects human cortical neural progenitors and attenuates their growth biology of zika virus infection in human skin cells characterization of lethal zika virus infection in ag mice zika virus: the latest newcomer zika virus in gabon (central africa)- : a new threat from aedes albopictus the zika outbreak of the st century probable non-vector-borne transmission of zika virus potential sexual transmission of zika virus zika: another sexually transmitted infection? mega : molecular evolutionary genetics analysis version . for bigger datasets zika virus and microcephaly: is the correlation causal or not: applying the bradford hill aspects of evidence to the association between zika virus and microcephaly the brazilian zika virus strain causes birth defects in experimental models western zika virus in human fetal neural progenitors persists long term with partial cytopathic and limited immunogenic effects detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study the clinical spectrum of zika virus in returning travelers the epidemiology, virology and clinical findings of dengue virus infections in a cohort of indonesian adults in western java zika virus outbreak in rio de janeiro, brazil: clinical characterization, epidemiological and virological aspects quantitative real-time pcr detection of zika virus and evaluation with field-caught mosquitoes one-step rt-pcr for detection of zika virus zika virus outbreak on yap island, federated states of micronesia zika virus infections imported to italy: clinical, immunological and virological findings, and public health implications guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses detection of zika virus in urine isolation of infectious zika virus from saliva and prolonged viral rna shedding in a traveller returning from the dominican republic to italy highly divergent dengue virus type genotype sets a new distance record highly diversified zika viruses imported to china molecular evolution of zika virus during its emergence in the (th) century phylogeny of zika virus in western hemisphere zika virus in a traveler returning to china from the convergence of a virus, mosquitoes, and human travel in globalizing the zika epidemic aedes (stegomyia) albopictus (skuse): a potential vector of zika virus in singapore zika virus spreads to new areas -region of the americas first report of autochthonous transmission of zika virus in brazil we thank dr de wu (guangdong provincial center for disease control and prevention, china) for helpful discussions on zika virus isolation. this work was supported by a grant ( y - ) from the science and technology program of guangzhou, china. key: cord- - d eq y authors: nan title: espr date: - - journal: pediatr radiol doi: . /s - - - sha: doc_id: cord_uid: d eq y nan prof. michael riccabona undertook his medical school & university training at the university of innsbruck, tirol, austria, completing his md at karl franzens university in graz, austria. following an internship in neurology, surgery and internal medicine he then specialized in paediatrics at the dept. of paediatrics, university hospital in graz. there he took charge of the paediatric radiology and sonography sections at university hospital in graz, as associate professor of paediatrics. in he additionally started to specialise in radiology, becoming associate professor of radiology in -then taking charge of the subsection of paediatric sonography at the dept. of radiology, university hospital in graz, where in march he was appointed full univ. prof at the medical university graz, austria. he has a distinguished academic career and written over papers, more than chapters and several textbooks, and is a very popular international speaker, delivering numerous lectures at many high profile scientific meetings. he is an active member of several reputable international societies, has been chair of the paediatric ultrasound section of the austrian ultrasound society since , and president of the society of german speaking paediatric radiologists ( ) ( ) ( ) ( ) ( ) ( ) ( ) . he is a constant source of inspiration within his subspecialist areas of interest in ultrasound and abdominal radiology in children. he has been course director at several important meetings and served as president of espr in graz in and as lead of the paediatric subcommittee at ecr in . he has provided inspirational leadership as chair of the espr task force on uroradiology since writing state of the art guidelines and procedural recommendations to facilitate standardised best practice for imaging within paediatric uroradiology. he has been a reviewer for many international journals. he has on-going active roles in postgraduate education for medical colleagues from eastern europe, including basic ultrasound education and refresher courses, and workshops. michael riccabona is very deserving of honorary membership of espr, which reflects his seminal role within paediatric radiology in europe and his tireless dedication working for the good of children. & to discuss how to optimally adapt the various imaging techniques minimising radiation exposure and risks during diagnostic imaging in children. & to consider common restrictions, challenges, and possible solutions in paediatric radiology within the different settings in different countries, regions, continents and clinical scenarios -discussing all these aspects with colleagues, and to mingle with experts from all over the world learning from each other and fostering networking in paediatric radiology to try to grant optimal imaging for all children. an application for the rd annual meeting as well as for the th post graduate course has been submitted to the eaccme® for cme accreditation of this event. the eaccme is an institution of the uems (www.uems.net). the number of cme points will be announced at the espr congress website. each medical specialist should only claim those hours of credit that he/she actually spent in the educational activity. certificates of attendance will be available in the espr myuserarea after the meeting. the answers right away is: yes we can and we constantly have to. it is part of human nature to recognize problems and to find solutions. we define ideals but we also face reality. being aware of the gap in between we are constantly driven to improve. this overview will highlight some milestones and disputes throughout the years of development in the use of plain x-ray imaging. it will hint on scientific literature and sources of information. and it will hint on some swiss contributions as the espr meeting will be held in davos, switzerland. fighting the glow not the fire - years of x-ray imaging development and improvement: in the beginning of the clinical use of x-ray imaging, there was great enthusiasm in its potential without knowing the unfavorable dangers of uncontrolled use of x-ray. the dangers were recognized and the 'beast' was tamed and domesticated. in respect to radiation protection, the most significant achievements took place in the first half of that development. throughout the recent decades, some further considerable steps in dose reduction took place mainly by the improvement of film-screen systems and the recent introduction of computed (cr) and digitized radiography (dr). even if the early computerized x-ray imaging brought a slight increase in patient doses (which was overcompensated later on by direct digital radiography) a whole new world of further advantages launched the digital era in which we live today. as we all know new dangers arose with these techniques as the uncontrolled distribution of images and thereby confidential patient data over hospital departments and across borders throughout. also, the risk of an evitable overexposure in digital radiography is a significant issue. throughout the process of taming the radiation and controlling it, today's doses are attained within the lowest range of the danger scale. this range still is perceived as a black box within which we do not exactly know which concept reflects the potential harm best. the linear no-threshold model [lnt] is acknowledged as the concept which most reliably supports the idea of radiation protection. other concepts partly oppose the linear idea and question the relevance of that dose range because there lie so much greater health benefits in the appropriate use of diagnostic x-rays. several scientists even propagate the idea that very low levels are producing health benefits instead of physical harm [hormesis model]. nevertheless, the lnt model is widely accepted as the most helpful in the context of diagnostic radiology. some recent studies were able to support the idea of potential harm at very low dose levels as they were able to prove the induction of attributable cancers in the pediatric age group. so today we are fighting the glow, not the fire. as trained medical professionals we are fully aware of the fact that there is only little potential harm to the patient by using x-rays in the current state of the art. but on the other hand, we also have to be aware of the fact that our patients and their parents still fear the fire. one of our main tasks, therefore, is to explain the risks and benefits to the patients and their advocates and to educate the public. developed straight from the first radiographic technique's digital radiography today is state-of-the-art in plain d-imaging. throughout the last decade, it has replaced cr and conventional radiography in many institutions. in the united states of america, one of the most developed healthcare systems, healthcare authorities propagate incentives to abolish cr and older imaging systems by making them financially unattractive. the market is fully concentrated on the spread of dr systems. momentarily there are no real milestones but many refinements of existing systems such as tomosynthesis, dual energy subtraction and advanced auto-stitching, fluoroscopy capability, basic angiography applications and -d cone-beam ct images are made available. combinations of these features can be found in some recently designed x-ray machines. grid-less imaging software can reduce patient dose significantly. concerning detectors, there are cr retrofit systems which will support the easy upgrade of existing systems to dr capability. wireless detectors with large internal storage, different sizes and high resolutions of microns are available. dr is becoming a part of the system in the current era of full digitalization of our lives and big data, digital radiology is a cornerstone of our healthcare systems. ris and pacs as part of integrated healthcare (ihs) systems are widely disseminated. at the next step, all accessible data will be used for analyses. the major vendors of imaging systems, as well as pacs suppliers and independent companies, offer readymade software tools for reports and evaluations of all kind. the doses from different x-ray sources can be screened internally and be used for optimization purposes. they also can be sent to remote servers for dose monitoring, comparison and optimization in multi-hospital health care provider settings or to comprehensive databases like the american college of radiology dose index registry, cancer registries, or for central billing. has everything been invented? many technologies have been declared dead before a new transformation appeared. this was the case for example with single slice ct before the invention of spiral ct by willi kalender, germany and peter vock, switzerland in . often the plain x-ray image was meant to be needless or redundant as newer technologies like ct or mri approached. but it still is of value because of different reasons as the low dose, high availability, well known and easy interpretation to name a few. there are some new and sophisticated techniques on the way like the "smart x-ray source" which uses coherent beams of x-rays from an array of micronsized point sources, developed by scientists at the massachusetts institute of technology (mit). the developers promise less radiation, less weight of the equipment and a far better soft tissue resolution. another promising approach is phase contrast x-ray imaging which has the potential to reduce the dose up to / of the actual value. it also has its strengths in additional soft tissue information as recent experimental publications show (paul scherrer institute switzerland) e. g. in functional evaluation of lung fluid (munich, germany). functional imaging of the lungs can also be achieved without any radiation as the development of the known concept of electrical impedance tomography highlights. this functional imaging method usually is not within the modality spectrum of radiologists. dose control and reduction -local -regional -international the most effective measures to achieve significant dose reductions in your own department are still the same strategies which are based on the "eternal rules" as we know them from our teachers: avoiding unnecessary exposures by strictly controlling the appropriateness of a referral. justification is a shared responsibility between radiologists and clinicians. there are many tools available for justification like the appropriateness criteria, guidelines or rules (like wrist or ankle rules) of several national societies and different study groups. the process of optimization is mainly in the hands of the technicians. as many studies show, the proper collimation still has the greatest effect on dose reduction. other important factors are the positioning of the patient and the shielding of radiosensitive organs which are not relevant for image interpretation so that they may be covered by lead shields. the proper use of the grid in bigger children can now partially be replaced by software solutions. in digital radiography, a profound knowledge of postprocessing possibilities is mandatory as well as the active control of the exposure indices. dose limitation procedures should be regularly checked in a team-based approach to avoid overexposure by less experienced staff or "exposure creep". existing standards should be actively used to guarantee a constant satisfactory image quality. in , the image gently campaign released a safety checklist for performing digital radiography examinations on pediatric patients which is easily applicable to every radiology service. organizational improvements: at regional and national level, efforts should be made to check for best practice use in the departments and to compare and discuss imaging strategies. the establishment of national and international dose reference levels helps to keep the overall doses low and to protect the population from unnecessary overexposure. the pidrl project prepared the "european diagnostic reference levels for pediatric imaging" as part of the eurosafe project. momentarily the results of pidrl-workgroup are harmonized with international organizations. the european guidelines on drls for paediatric imaging can be accessed as a preliminary final for workshop drafts on the internet. on a worldwide basis, the world health organization has published a fundamental information brochure concerning radiation risks and the communication of health professionals and patients. health care professionals have a shared responsibility for communicating risks and benefits of imaging procedures to patients, especially in the case of pediatric patients. the document "communicating radiation risks in paediatric imaging-information to support health care discussions about benefit and risk" is intended to serve as a tool for health care providers, to communicate known or potential radiation risks associated with pediatric imaging procedures and to support risk-benefit dialogue in health care settings. as said before we are fighting the glow, not the fire. the paper of the swiss pediatric oncology group stirred a broad discussion. among other issues, there was a question if it shouldn't be a logical consequence to transfer kids from areas with higher background radiation to safer areas. the author's answers were clear: that swiss health authorities better concentrate their efforts more effectively and with greater benefit for more people by supporting prevention "toward modifiable environmental factors leading to larger numbers of deaths from several causes, such as exposure to radon, air pollution, and second-hand tobacco smoke". this leads to the conclusion that we as medical radiological professionals do have the obligation to make every effort to prevent our patients and personnel from harm of the usage or non-usage of radiation. as health specialists, we also should support the fields of prevention with broad mass effects as far as we have the opportunity. and as human beings, we are summoned to do so in respect to other beings, to our environment and to the resources we all share. radiation protection and quality improvement is just a small part of it all, but it is our field -and 'yes we can'. "communicating radiation risks in paediatric imaging. freely available at the who homepage." computed tomography: are we doing enough? e. sorantin; graz/at summary: already in the alara principle was publishedbut the implementation is still far from complete. according to the surveys of the ec tender project "pidrl -european diagnostic reference levels for paediatric imaging" the most frequent computed tomography (ct) examinations in children are, in descending order, head/neck, chest and abdomen thus counting for about % of all pediatric ct's. therefore it makes sense to optimize these examinations first. surveys of the "international atomic energy agency (iaea)" in countries have shown, there is considerable lack of organizationeg in about % of facilities protocols for children were missing, indication based protocols available only in %, ctdi values for head and chest two to five times of those for adults. all of these simple facts indicate we are not doing enough for radiation protection in pediatric ct. actions to lower dose in ct can be categorized in organisational, optimization and alternatives. the interdisciplinary implementation of international guidelines for ct in minor head trauma with trauma surgeons could serve as an example of organisational actions. for dose optimization knowledge about dose relevant factors according the "imaging chain" is mandatory as well as adjusting kv to pediatric needs. dose influence on image quality must be known, by exploiting the fact, that, if all ct parameters are kept constant but hte slice thickness is just halve there must be an increase in noisein particular about two times more. therefore if a standard examination is reconstructed at half slice thickness and image quality is still appropriate the amount of waste radiation is in the range of %. therefore if the next examination will be reduced with eg % mas setting less will be for sure in appropriate quality and the process can be started again. after a couple of examinations the optimal dose will be reached. thus the "half slice thickness" approach is easy to do, does not need special equipment or human resources and will help to find the appropriate dose. the third point is alternatives -ultrasound and mri being the candidates in the first row. new, radiation free, techniques like electrical impedance tomography and others are already developed and can be expected to be release soon. take home points: & we are not doing enough for ct dose savingeven more than years after release of alara principle & dose saving actions can be categorized inthe subtasks organisation, optimization and alternatives & "the half slice thickness approach" is an easy to do technique to elaborate the optimal dose on an particular ct machine. prenatal thoracic mr l. alamo; lausanne/ch the generalization of screening us has considerably increased the detection of congenital anomalies in utero. in the last years, important technological advances and especially, the development of fast heavily t -weighted sequences has led to an increasing use of prenatal mri as additional diagnostic imaging method. mri is increasingly used for evaluation of thoracic pathology, including tumours and vascular malformations as well as anomalies of the diaphragm, the lungs and more recently, even of the foetal heart: -thoracic tumours and vascular malformations: the diagnosis of a congenital tumor during pregnancy involves a tremendous emotional impact for a family. the most frequently observed thoracic tumours are teratoma, myocardial rhabdomyoma and exceptionally, pleuropulmonary blastomas. mri may provide relevant additional information concerning the origin of the lesion and its real anatomical extent. -diaphragmatic pathology: congenital hernia is by far the most commonly reported foetal diaphragmatic anomaly. the large field of view and the multiplanar possibilities of mri may help to clarify the position of the herniated organs and to evaluate the severity of lung hypoplasia, considered the most important parameter for predicting outcome. other rare pathologies include eventration, paralysis and diaphragmatic lung sequestrations. -lung anomalies: congenital lung abnormalities are a heterogeneous group of pathologies consisting of isolated bronchopulmonary or vascular anomalies or a combination of both of them. congenital pulmonary airway malformation, bronchopulmonary sequestration and bronchial atresia are the most often observed pathologies but they present significant overlap imaging findings. mri allows accurate information concerning the location and extension of the lesion and the volume of the normal and abnormal lung. -heart pathology: the evaluation of the foetal heart remains extremely difficult because of its small size and high rate of battements. the unpredictable foetal motions during data acquisition and the absence of a foetal ecg signal to synchronize data acquisition are additional problems. in the last years, different approaches have been made to overcome these challenges. radiologists should know the typical imaging findings of the thoracic pathology most often observed in foetuses. prenatal mri may provide additional relevant information in a wide spectrum of congenital thoracic anomalies, but in general, it should only be performed if it is considered that additional results might influence the management of the pregnancy and/or the therapeutic approach. therefore, it is important to know the right indications for mri and to recognize the limits of the method. interruptions during embryogenesis of the muellerian or wolffian ducts result in various, potentially complex genitourinary abnormalities of a wide spectrum or combinations. multiple imaging modalities are employed to evaluate patients with these abnormalities. ultrasound is the frontline imaging modality. mr imaging is mostly reserved for complex cases and may incorporate an mr urography, too. other imaging modalities are less frequently used or provide only ancillary information. this presentation will demonstrate the utility of ultrasound and mr imaging, in particular, in the routine diagnostic imaging of patients with the wide spectrum of muellerian and wolffian duct abnormalities. & mr imaging is reserved for more complex cases. neonatal hepatic tumors and vascular malformations d. pariente, s. franchi-abella; le kremlin bicêtre/fr neonatal hepatic tumours and vascular malformations are rare but imaging plays a key role in diagnosis and treatment. the most frequent hepatic tumour is haemangioma (fig ) which often is asymptomatic but may be complicated by cardiac failure, coagulopathy or compartment syndrome. the differential diagnosis mainly includes hepatoblastoma, hematoma (fig ), abscess, mesenchymal hamartoma, choriocarcinoma in the solitary form and metastatic neuroblastoma, cirrhosis, neonatal leukemia in the multifocal form. pertinent biological data are alpha-fetoprotein (but level may be normally high in neonate), betahcg, and urinary catechol amines. hepatic vascular malformations are rarer and include intra or extra porto-systemic shunts (pss), arterio-portal fistula or complex mixed forms. intrahepatic pss may be associated with haemangioma and regress in most cases rapidly (fig ). on the contrary the extrahepatic pss which are located below the portal vein, should be urgently closed to avoid occurrence of agenesis of the portal vein. the best imaging modality is us which must be performed with high frequency probes and colour doppler to identify hepatic vessels and assess patency, direction of flow, abnormal communication. mri and ct with contrast injection may also be useful. hepatic mass in a do neonate with increased crp and afp. us showing a hyperechoic mass with thrombosis of the left portal vein (black arrow) and a track (white) extending to the mass: hematoma due to malposition of an umbilical vein catheter. hemangioma of antenatal diagnosis on d . the mass is composed of a large anterior vascular lake corresponding to a porto-systemic shunt and a tissular hyperechoic part. the infant has remained asymptomatic. intrahepatic porto-systemic shunt between the left portal branch of segment (white arrow) and the left hepatic vein (black arrow) in a neonate. at months of age this shunt has completely resolved. haemangioma is the most frequent hepatic tumour in the neonate and is often asymptomatic with spontaneous resolution. levels of alphafetoprotein are physiologically high in the neonate, and can be misleading. hepatic hematoma can be secondary to traumatic delivery, to coagulation disorders or to umbilical vein catheterization. intrahepatic porto-hepatic shunts are the most frequent vascular malformations and regress in most of the cases in the first year of life. us with colour doppler remains the best imaging modality in the neonatal period. imaging in crohn disease: state of the art in diagnosis, prognosis and followup n. colavolpe, a. aschero, b. bourliere-najean, c. roman, f. khachab, h. pico, m. kheiri, g. gorincour, c. desvignes, p. petit; marseille/fr summary: during the past years the inflammatory bowed diseases (ibd) have increase in frequency ( ). less than twenty-five percent of them occur in children of less than years ( ) and crohn's disease (cd) is twice as frequent than ulcerative colitis (uc) in the pediatric age group. specific phenotypic and genotypic subtype of ibd occur in younger children. early onset (eo) pediatric ibd (before years of age) represent % of childhood ibd ( ) . uc and undetermined colitis are more frequent in this age group. eo cd showed a more frequent isolated colonic and upper gastrointestinal involvement than later-onset disease where locations are predominantly colic and terminal ileum later on childhood. some pediatric ibd specificities exist than can interfere with the imaging findings: -cd can be limited to the terminal ileum or to the colon in up to % of children ( ) . isolated jejunal involvement is reported to occur in - % of children. this location is more frequent in the youngest and is more at risk of complicated course of disease ( ) . for auvin et al. ( ) the small bowel is involved in % of cases with less involvement of the terminal ileum than in the adult population. -uc: the classical contiguous alteration of the bowel wall from the rectum to the caecum is inconstant. a macroscopic rectal sparing is reported from et % and the absence of continuous disease from rectum to caecum (caecal patch) described in % of children. transmural inflammation may be present in severe form as well as terminal ileitis without granulomata (backwash ileitis) ( ) . in order to assess these pathologies, and more specifically cd small bowel locations which are difficulty explored by others modalities, small bowel follow-through, barium enema, ultrasound, computed tomography and mr imaging have been widely used. among them, mr-enterography has gained worldwide acceptance due to multiple factors including: a high contrast resolution, a multiplanar ability, an absence of radiation, the possibility to explore in the same exploration the whole bowel and the extra-bowel diseases (perianal fistulae, sacroiliac joint, biliary tract), the ability to compare of side by side consecutive studies in a reproducible manner, a more easily understood exploration by the clinicians than ultrasound, and first of all for its performances. in order to technically harmonize this exploration a recent consensus statements on mre protocol has been published by the esgar and the espr societies ( ) . preparation: -depending on their age children must not have solid oral intake from to hours prior to the examination to reduce bowel wall motility. morning mr appointment is more favorable for this purpose. no gasless fluid restriction is recommended but is reabsorbed too quickly to distend enough the small bowel. none hyperosmolar non absorbable solution is superior to another. its ingestion must start to minutes prior to mre. the recommended volume is ml/kg with a maximum up to ml/kg. explanations long before the mre concerning the importance of such absorption and the use of a refreshed product mixed with aromatized flavors will facilitate the child's participation. -the use of spasmolytic agents is optional. however, there are recommended in adults by multiple societies including esgar ( ) , the society of abdominal radiology ( ) and the acr (www.acr.org/ quality-safety/standards-guidelines). but, mre without antiperistaltic agents result has reached a high diagnostic confidence and excellent agreement with ct enterography for the presence of cd ( ) . if used, they need to be administered immediately prior to motion sensitive sequence (t w dynamic enhanced sequences). if the pictures obtain with these medications are of better quality, there is no evidence that they change the final diagnosis and the children's therapeutic management ( ) . the use of these products increase the length of the exploration and their side effects are frequent (nausea > vomiting) which balance their visual benefice ( ) . if a spasmolytic agent is used, the recommended first line spasmolytic agent is i.v. hyoscine butylbromide ( . mg/kg i.v). the recommended second line agent is i.v. glucagon, . mg (< . kg) and mg (> . kg), given as a slow infusion with i.v. saline at an infusion rate at ml/s. -no rectal enema is needed. -exploration can be performed either at . tesla or testla. more chemical shift and susceptibility artifacts are present with the latter. prone position has been demonstrated to allow better small bowel distension than the supine one with reduction of the peristaltism but without better lesion detections ( , ) . large multi-elements coils are needed to cover with high resolution from the perineum up to the left colonic flexure. sequences: both morphologic steady state free precession gradient echo and d -t -weighted images are needed in the axial and coronal planes. fat saturation in one of this plane is recommended and maximal slice thickness of mm is required. nowadays, non-enhanced then enhanced d t -fat saturation weighted sequences are mandatory. slice thickness does not exceed mm. enhance sequence need to be acquired at the portal phase of injection. however, in recent studies the need for gadolinium has been questioned when dwi is added to the morphologic sequences. dwi sequences have been considered optional ( , ) but we consider their place essential in pediatric practice. they must be done with high b values, from up to in the coronal and axial planes with to mm contiguous cut in free breathing. axial plane is less prone to artefact than the coronal plane. interestingly enough shenoy et al ( ) report in pediatric patients that dwi does not perform as well as standard mre for detection of active crohn disease but the combination of dwi and mre increases imaging accuracy for determining disease activity compared with either technique alone. seo et al ( ) in young adults said that dwi mre was noninferior to contrast-enhanced mre for the evaluation of inflammation in cd. based on the exploration of cd adult and pediatric population, dwi proved to be efficient and would avoid gadolinium injection ( ) . sirin et al. ( ) report in children that dwi revealed lesions that were not detectable with mre done with gadolinium injection. finally, respectively dubron et al. ( ) in children and neubauer et al ( ) in children and young adults demonstrate better performance of dwi than gadolinium enhanced imaging. like the existing mr protocols for suspected appendicitis ( ) it will not be surprising to see fast mr ibd explorations becoming an alternative to emergency us as already proposed ( ) . this fast mr limited to a morphologic t sequence in two planes associated with dwi sequences will allow a positive diagnosis and the ibd work up. apart from bowel obstruction and its spontaneous bowel distension one of the limiting factors will be the need for an oral water agent uptake in a potential surgical patient. however, it has been published in the adult literature than an oral or rectal preparation was not necessary to rule out uc ( ) nor a cd ( ) . the other limiting factor is the length of exploration. mre can be shorten especially if the patient's positioning is easy to do (dorsal decubitus) ( ) and if there is no need for injection, either for spasmolotytic agent and for gadolinium chelates. the suppression of the iv line, the absence of potential side effects (nausea, vomiting) of paralytic agents and the decreased of repeated long apneas with no loss of significant information will be strong progresses toward the holy grail. -positive diagnosis, disease activity, prognosis and follow-up: mre has a better accuracy to detect inflammation for the small bowel than for the colon ( ) . one of its goal is to try to accurately identify features of active inflammation vs fibrotic disease. this is of paramount importance since the former may respond to medical treatment and the latter may need surgical resection. however, inflammation and fibrosis are associated within the same bowel segment and progress in a parallel way making the goal difficult to reach ( ) ( ) ( ) . in their study based on the analyze of children operated for cd strictures, barkmeier et al. ( ) report than strictures demonstrating > cm upstream dilatation with associated feces sign were highly associated with transmural fibrosis. the most severely fibrotic strictures were associated to the greatest amount of inflammation and there was no significant correlation between stricture length, mural thickness, degree of post-contrast enhancement (arterial and delayed venous phases), diffusion-weighted imaging apparent diffusion coefficient, pattern of post-contrast enhancement, or normalized t -weighted signal intensity and histological fibrosis or inflammation scores. however, correlation with histological specimens of cd done on a other series s ( ) (suppl ):s -s pediatr radiol demonstrated that the enhancement ratio of the wall is positively correlated with disease chronicity due to a possible increasing microvessels permeability and inversely correlated to acute disease ( ). on the other hand, several authors have tried to correlate the adc values to cd activity. fibrotic tissue does not restrict diffusion and presents a decrease of signal at high b values and high adc values whether acute inflammation shows decrease adc values. variable thresholds from . x - mm /s to . x - mm /s have been proposed to separate active vs non active disease ( ) . however, others authors have reported low adc value of fibrosis compare to histology ( ). even if promising results have been published with high correlation with the crohn disease endoscopic index of severity ( ) , adc measurements are associated with sever limitation factors including sample size overlap between the bowel wall and its atmosphere, lack of reproducibility between mri-units and mri-vendors, non-standardized sequence b-values parameters ( ) . two mre scores are available to quantify the activity of cd. one is using gadolinium injection ( ) and the other dwi ( ) . due to the complexity of the formula, both are difficult to use in daily practice and have not been evaluated in paediatric practice. interestingly enough if a simplify mre paediatric protocol appears to become a reality, us stays a good imaging challenger and ( ). in a recent meta-analysis, based on adult and pediatric series, calabrese et al ( ) reported that bowel us showed . % sensitivity and . % specificity for the diagnosis of suspected cd, and % sensitivity and . % specificity for initial assessment in established patients with cd. bowel us identified ileal cd with . % sensitivity, . % specificity, and colon cd with . % sensitivity, . % specificity, with lower accuracy for detecting proximal lesions. the absence of abnormal thickness wall would have a negative predictive value, high enough to exclude the need for further exploration, especially when cd is concerned ( , ). concordance between us and mre have been variably reported from excellent ( ) to just correct ( ). rosembaum and al ( ) report that the us findings present in children operated for cd include: bowel wall thickness above . mm (mean, . mm) and an increased frequency of loss of mural stratification and fibrofatty proliferation. others us technologies are used in children to better approach the disease activity. it includes, hydrosonograpy using specific oral agents (mannitol, sorbitol, polyethylene glycol, etc…), contrast-enhanced ultrasound and dynamic contrast-enhanced ultrasound (nowadays, contrast agent is offlabel in children) ( ) and elastography ( ). their enthusiastic results and their efficiency to assess disease activity need to be confirmed ( ). in conclusion, as we suspected years ago ( ), mre has dramatically modified our approach of pediatric ibd especially when considering its orientation toward a less invasive exploration and the increasing importance of dwi imaging. a cost benefice between mre and us remains to be done on this increasing disease. heterotaxy and isomerism c. lapierre; montreal/ca summary: objectives: to review the classification of visceroatrial situs to describe the associated cardiac and non-cardiac anomalies to illustrate typical findings in fetuses, neonates and children to discuss the surgical consideration and the long-term follow-up in these patients abstract: by definition, the type of situs is determined by the relationship between the atria and the adjacent organs. anatomically, the atrial chamber differentiation is based on the morphologic aspect of the atrial appendages, earlike extensions of the atria. three types of situs exist: solitus (normal), inversus (mirror image) and ambiguus. a single type of situs is present in a patient. when the situs is neither solitus nor inversus, it is referred to as situs ambiguus or heterotaxy. heterotaxy may manifest with various abnormal visceroatrial configurations that are associated with cardiac (in - % of cases) and extracardiac anomalies such as splenic abnormalities, biliary atresia and intestinal malrotation. two subsets of situs ambiguus are well-recognized: right isomerism (asplenia) and left isomerism (polysplenia). in heterotaxy, the venoatrial connections are frequently abnormal. left isomerism is usually indicated by bilateral bilobed lungs, interruption of the ivc and multiple spleens. the more likely found cardiac anomalies are: pulmonary or aortic stenosis, isolated atrial and ventricular septal defects, cardiac arrhythmia due to sinus node dysfunction as well as pulmonary veins that drain into both the right and the left atria. in the presence of right isomerism, bilateral trilobed lungs, a large symmetric liver, and absence of the spleen are frequently observed. at the cardiac level, patients are more likely to have a common atrioventricular defect, a double outlet right ventricle and pulmonary stenosis. total anomaly of the pulmonary venous return and absence of coronary sinus will always be present in right isomerism. heterotaxy can be diagnosed with high accuracy by prenatal echography. a diagnosis should be suggested in the presence of congenital heart disease, visceroatrial heterotaxy and interruption of inferior vena cava with azygos continuation for left isomerism or abnormally closed juxtaposition of inferior vena cava and descending aorta in right isomerism. the mortality in fetuses is high in the presence of heart block and hydrops whereas the cardiac anomalies influence the long-term outcome. as discussed in the literature, the clinical outcomes and long-term prognosis in these patients are relatively poor when compared with non-heterotaxy patients. the risk factors are cardiac (underlying anatomy and arrhythmia risk) and non-cardiac. based on the cardiac anatomy, one of the main determinants is left versus right isomerism. with right isomerism, the cardiac malformation is more severe and an univentricular correction is more frequent. another predictor of mortality is pulmonary vein stenosis/obstruction. whatever the severity of cardiac lesions, the postoperative or discharge mortality is higher in patients with heterotaxy. prenatal diagnosis seems not improve the survival. extracardiac anomalies also contribute to the increased morbidity and mortality. three of the more challenging entities are respiratory, immunologic and gastrointestinal. recurrent respiratory infections, failed extubation or chronic respiratory failure are frequently observed in patients with heterotaxy. recent studies revealed an association between heterotaxy and primary ciliary dyskinesia which can explain the increased postoperative respiratory complications. the spleen is important for the bacterial clearance. patients with asplenia or polysplenia are thought to have "functional asplenia". so, they are at risk for sepsis and severe bacterial infection. the incidence of intestinal malrotation is high, approximately % to %. observation versus prophylactic ladd procedure and screening for asymptomatic intestinal malrotation are a growing area of debate. the trend seems to go along conservative management and surveillance of malrotation. bronchopulmonary malformations, such as congenital pulmonary airway malformation (cpam), bronchopulmonary sequestration (bps), and congenital lobar emphysema (currently known as congenital lobar overinflation [clo] ), are common congenital lung diseases. these conditions are detected prenatally, usually in the second trimester, in countries where obstetric sonography is routinely performed. the malformations are seen as hyperechoic images with respect to normal fetal lung parenchyma, with a mass effect and homogenous appearance or with coexisting cysts. the lesions usually decrease in size along gestation. a residual mass is seen on postnatal chest radiography, the first imaging technique performed, in only % of cases. cpam and bps are predominantly located in the posterior lower chest and can be identified postnatally on ultrasound using a small vector probe and a subcostal and subxiphoid approach. potential feeding arteries can be visualized using color or power doppler. based on clinical and sonographic findings, the differential diagnosis between congenital lung malformations and tumors such as neuroblastoma, type i pleuropulmonary blastoma, and myofibroblastic tumor will be discussed. postnatal management and imaging of newborns with congenital lung malformations is controversial, particularly in asymptomatic patients (approximately % of cases). chest radiography is mandatory at birth and chest ultrasound is also recommended to confirm the prenatal diagnosis. computed tomography (ct) or magnetic resonance imaging (mri) using angiographic techniques should be performed some months ( months) after birth in asymptomatic patients. these techniques are also recommended in symptomatic newborns and before surgery to characterize the arterial supply and venous drainage in cpam and bps, as ultrasound is limited in this regard. in premature infants, sonography complements radiography in the study of prematurity-related lung diseases such as respiratory distress syndrome and its pulmonary complications (eg, pneumothorax), in predicting bronchopulmonary dysplasia, and in diagnosing transient tachypnea of the newborn when clinical and radiographic features are inconclusive. the main ultrasound finding in these conditions is visualization of numerous "b-lines", vertical narrow-based hyperechoic bands extending from the pleural surface to the end of the field of view, representing what is currently known as "sonographic interstitial syndrome". b-lines are artifacts originating from variations in the air-fluid relationship of the lung and are better seen using high-frequency linear probes . use of sonography for follow-up of these patients will reduce the number of the chest plain films performed, and therefore, the amount of radiation exposure in this vulnerable population. for proper interpretation of the sonographic findings in these conditions, the radiologist should be familiar with current related terms, such as lines a, lines b, comet tail artifact, interstitial-alveolar syndrome, septal syndrome, and white lung. trauma is the leading cause of mortality and morbidity in children after the first year of life. motor vehicle accidents are the leading cause of death from unintentional injury in children up to the age of . of these cases, the abdomen is the fourth most commonly injured area. in pediatric patients non-operative management of these injuries predominate, hence the importance of early radiologic assessment for appropriate clinical follow-up. anatomically, compared to adults, childrens' abdomens are more square, less muscular and with less intraperitoneal and subcutaneous fat to absorb impact. the diaphragm is more horizontal causing downward displacement of the liver and the spleen outside the protective casement of the ribs. the pelvis is smaller and hence the bladder is displaced upward, also resulting in more vulnerability to this organ. the organ surface area is larger in children and they have a smaller body mass-hence more force applied per-unit of body surface area. the ribs are flexible, and although we see fewer rib fractures, this results in more internal damage. physiologically, children maintain hemodynamic stability longer, often presenting with only mild tachycardia, even when in severe hemodynamic shock. decrease in blood pressure may not be evident before the loss of % blood volume. nevertheless, bleeding is less severe and operative intervention is rarely performed. mechanics of blunt abdominal trauma include organ compression from seat belt injury with the presence of erythema, ecchymossis or abrasion on the abdominal wall increasing the likelihood of internal organ injury ( % likelihood of injury). other common mechanisms include pedestrian-car collisions( % with intra-abdominal injuries), falls ( % with intra-abdominal injuries), or handle bar injuries ( % with intraabdominal injuries). after the child arrives in the hospital, a trauma algorithm is initiated. generally, for the unstable patient, algorithms are similar and require a rapid atls protocol, followed by a fast ultrasound to confirm free fluid prior to operation. in stable patients, institutional algorithms vary greatly between countries and in different centers. some rely solely on mechanism to determine the need for fast vs ct (not complete ultrasound), others will rely on clinical exam (in a conscious patient with reliable exam) and blood work to determine the need for imaging (ct or us) and others may chose to perform an initial us and complete the exam with a constrastenhanced us during work hours. in the literature many management prediction rules exist based on the history, physical examination, mechanism of injury and are supplemented by blood work and/or intial imaging. most are based on retrospective reviews, with only a few controlled clinical trials. however, the validity of these studies is limited because of different populations, institutional policies and variable radiological practices in terms of when imaging is performed, which modalities are most beneficial and which are less valuable, all the while, considering the utilization of the least irradiating techniques. a representative sample of such algorithms will be discussed. routine and extensive initial trauma panels are not required according to a number of studies. abdominal ultrasound and urinalysis together have been found to confirm % of all intra-abdominal injures, in some studies. serial haemoglobins/hematocrit is valuable for determining ongoing s ( ) (suppl ):s -s pediatr radiol blood loss and assists clinical surveillance. electrolyte abnormalities are uncommon in children unless severe shock is present (metabolic acidosis). liver function tests are elevated in most cases of blunt abdominal trauma, hence, are often performed for its high sensitivity, to avoid ct if the liver panel is negative. imaging, however, is needed for grading of the potential liver injury if the liver panel is positive. abdominal xray is not useful in blunt abdominal trauma, and is usually normal. ultrasound has an important role in the pediatric community, as a sensitive and non-irradiating modality. however, this sensitivity is dependent on the type of ultrasound performed (fast vs. complete abdominal ultrasound vs. contrast-enhanced ultrasound) but also on the qualifications and experience of the performing physician. a meta-analysis of fast in pediatrics demonstrates that it has a sensitivity of % (grade i-ii evidence) for identifying hemoperitoneum. a negative fast is not sufficient to rule out intra-abdominal trauma. one prospective observational trial demonstrasted that % of patients without free fluid on fast (performed by formally trained pediatric truama surgeons demonstrated at least grade iii liver or splenic injuries on ct). we know that pediatric ultrasound is operator-dependent, and generally an ultrasound performed by the skillful hand of a pediatric radiologist is more sensitive than that performed by surgeons or by adult radiologists. furthermore, we know that the benefits of contrast-enhanced ultrasound in pediatric trauma exist-highly accurate in visualising lesions, hence avoiding non-contributive ct imaging, however, the feasibility of providing -hr contrast-enhanced ultrasound by a qualified radiologist is resource intesive: both structurally and with respect to personnel. published indications for abdominal ct in stable pediatric patients included suspected mechanism of blunt abdominal trauma, significant fluid resusitation without apparant blood loss, hemoglobin < mg/l without obvious blood loss, multisystem trauma and unreliable abdominal exam. one series with children undergoing ct for blunt abdominal trauma demonstrate postive findings in ( %), of which all solid organ injuries and % of hollow viscus injuries were identified on ct. however, ct has its limitations: it was found to identify gastrointestinal perforation in only % of patients with known perforation, but with findings of free fluid, wall thickening and/or bowel dilatation. it is also less accurate in identifying pancreatic trauma, with normal scans in - % of children with pancreatic trauma. again, findings of pancreatic trauma can be non-specific: free fluid or, less commonly, thickening of the gerota's fascia, presence of mesenteric fluid or of fluid between the pancreas and the superior mesenteric vein. when and where to perform ct depends on the imaging algorithms established by individual centers. generally, unstable patients with very high grade visceral injuries are taken to surgery. the stable patients are treated with non-operative management. the literature on angiographic embolization in pediatric blunt trauma is limited to case series that demonstrate a limited utility in hemodynamically stable patients with ongoing blood loss or for the definitive treatment of traumatic pseudoanevryms. a dialogue with the interventional radiologist is imperative in such cases. common imaging findings and pitfalls will be illustrated with case examples. in conclusion, a child's anatomy and physiology must be taken into account when determing the level of urgency and appropriate imaging work-up in blunt abdominal trauma. imaging of these patients cannot follow a standard algorithm as institutions vary with respect to types of personel, training, frequency of trauma, emergency department trauma protocols and availability of an in-house pediatric radiologist. ultrasound and ct have their advantages and disadvantages with associated pitfalls that the pediatric radiologist must recognize to provide an optimal diagnostic workup with minium irradiation. take home points: a child's anatomy and physiology must be taken into account when determing the level of urgency and appropriate imaging work-up in blunt abdominal trauma. imaging of pediatric abdominal trauma cannot follow a standard algorithm as institutions vary with respect to types of personel, training, frequency of trauma, emergency department trauma protocols and availability of a pediatric radiologist. ultrasound and ct have their advantages and disadvantages with associated pitfalls that the pediatric radiologist must recognize to provide an optimal diagnostic workup with minium irradiation. sport injuries d. jaramillo; miami, fl/us the growing skeleton has unique vulnerabilities to acute and chronic injuries due to sports. the practice of intensive sports during puberty and adolescence has led to a great increase in the incidence of sportsrelated injuries. during the growth spurt of early adolescence, the physis becomes weak, and is the site of fractures and avulsions (particularly in the apophyses) and of physeal widening due to repeated stresses, such as the wrist in gymnasts or the proximal humerus of baseball pitchers. both lesions can result in growth arrest. the chondro-osseous junctions of the ossifying epiphyses and apophyses are also vulnerable to avulsions, and the avulsed fragment may be entirely cartilaginous and not visible radiographically (such as in the patellar sleeve fracture). repeated trauma to epiphyses or round bones can lead to osteonecrosis (panner's disease) but more often to osteochondritis dissecans (ocd). in adolescents, ocd occurs most frequently in the medial femoral condyle, the capitellum of the elbow and the talar dome. juvenile ocd has a better prognosis than the adult form. when the skeleton begins to mature, there are fractures unique to partially closing physes such as the triplane and tillaux fracture. some sturctures have propensity to unique injuries during adolescence. a stress on the anterior cruciate ligament (acl) can lead to a tibial eminence avulsion in puberty ( figure) , an incomplete acl tear in early adolescence or a complete acl tear later. meniscal tears are almost always vertical and often involve large meniscal fragments that can flip. patellar dislocations often result in osteochondral injuries. this review will cover the main types of sports-related injuries and the imaging modalities used to diagnose them. year-old with pain and popping sensation during a fall on a football game. ap radiograph is normal & it is important to take into account the specific sport in order to anticipate subtle injuries that may be difficult to detect. a. c. offiah; sheffield/uk the radiographs obtained when inflicted injury is suspected are collectively termed the "skeletal survey". a full skeletal survey should be performed in all children below years of age in whom abuse is suspected. the investigation is not complete until follow-up skeletal imaging has been performed in the to days following the initial survey. children below one year of age should also receive a ct brain. neurological imaging in older children will depend on the clinical scenario. ct chest/abdomen is indicated when visceral injury is suspected. in terms of imaging in suspected abuse, espr has adopted the rcr guidelines. in the absence of a history of significant trauma, fractures highly specific for abuse in pre-ambulatory children include rib, metaphyseal and diaphyseal fractures. simple linear skull fractures have a relatively low specificity for abuse. the combination of subdural haemorrhage, retinal haemorrhage and diffuse cerebral oedema/encephalopathy (the so-called, "triad") suggests shaking. whereas the presence of a skull fracture implies impact. visceral injury often results from direct blunt trauma and may therefore be accompanied by anterior and/or costochondral rib fractures. the posterior rib arcs are protected by soft tissue and posterior rib fractures result from compressive/squeezing forces rather than direct trauma. the dating of fractures has a subjective element and it is more important to recognise that fractures are in different stages of healing, rather than to assign a definite age/age range to the injuries. the major differential diagnoses are accidental trauma and osteogenesis imperfecta. if rickets is the cause of the fractures, then radiology and/or biochemistry will show evidence of rickets. a low vitamin d level, in the absence of rachitic features, is not the cause of fractures. close liaison between radiologists and paediatricians is vital and any siblings/children in the same household who are below years of age should also receive a skeletal survey. remember that the presence of injury does not always mean abuse and that the absence of injury does not always exclude abuse. scoliosis may be primitive, structural, particularly during adolescence; during this period, careful follow-up is mandatory, because worsening is frequent. clinical examination with evaluation of a hump (gibbosity) with a scoliometer is mandatory, with also neurological assessment. beside radiography, additional tools have been developed to avoid xray exposure: "spinal mouse", back surface topography systems, ultrasound and other computer-assisted systems. but scoliosis can also be secondary, and imaging is important to find a cause and adapt management. among the etiologies, radiologist must recognize spine malformations, dysplastic and neuromuscular scoliosis. in addition, scoliosis may also be in relation with a primitive lesion, tumor-related or not, whether the initial disease could be within the spinal canal, spinal or paravertebral. imaging studies lies first on pa and lateral full spine x-rays, if possible with a low dose device (flat panel, slot-scanning system), keeping in mind that follow-up with repetitive exposures may be necessary. reproducible measures of different curvatures help to assess the overall static spine and the importance of scoliosis with cobb angle. the assessment of axial rotation can be obtained through d simulations, with frontal and axial views (see figure) . morphologic evaluation of the s ( ) (suppl ):s -s pediatr radiol spine is mandatory: if a secondary scoliosis is suspected, the research to etiology needs to perform ct or mri, depending on the clinical signs and the results of plain x rays evaluation. similarly, these explorations are useful in the preoperative assessment when surgical treatment is necessary. girl scoliosis, pa and lateral views with eos®, d simulation, coronal and axial views take home points: clinical evaluation is always the first step in subject with suspected scoliosis radiation burning is quite low with new devices, but repetitive exposures for follow-up need to carefully respect justification for x-rays exposures new tools are available to appreciate d spinal deformation and evaluate prognosis and surgical procedures ct and/or mri are useful in presurgical assessment and to look for etiologies in suspected secondary scoliosis malformations of the spine and spinal cord a. rossi; genoa/it summary: embryology and classification: spinal cord development occurs through three consecutive periods: (i) gastrulation ( nd gestational week): the embryonic disk is converted from a bilaminar into a trilaminar arrangement, with formation of the intervening mesoderm; the notochord is laid down along the midline, identifying the craniocaudal embryonic axis; (ii) primary neurulation ( th - th day): under the induction of the notochord, the midline ectoderm specializes into neural ectoderm. the initially flat neural plate progressively bends and folds until it fuses in the midline to form the neural tube. the primary neural tube produces the uppermost / of spinal cord; (iii) secondary neurulation ( th - th day): a secondary neural tube is laid down caudad to the termination of the primary neural tube. retrogressive differentiation of the secondary neural tube results in the tip of the conus medullaris and filum terminale. defects in one of these three embryological steps produce spinal dysraphisms, characterized by anomalous differentiation and fusion of dorsal midline structures. spinal dysraphisms may be categorized clinically in two subsets: open and closed spinal dysraphisms. in open spinal dysraphisms (osd) the placode (non-neurulated neural tissue) is exposed to the environment through a cutaneous defect along the child's back. osd include myelomeningocele, myelocele, hemimyelomeningocele and hemimyelocele, and are associated with a chiari ii malformation. myelomeningocele is by far the most common of these forms; the placode protrudes through a posterior defect and is elevated above the skin surface due to concurrent dilatation of the subarachnoid spaces. closed spinal dysraphisms (csd) are covered by intact skin, although cutaneous stigmata usually indicate their presence. two subsets may be identified based on whether a subcutaneous mass is present. csd with tumefaction comprise lipomas with dural defect (lipomyelocele and lipomyelomeningocele), meningocele, and myelocystocele. lipomas with dural defect are more common; they are differentiated from one another based on the position of the cord-lipoma interface, that lies within the spinal canal in lipomyelocele, and outside the spinal canal (ie, into a meningocele) in lipomyelomeningocele. csd without tumefaction comprise complex dysraphic states (ranging from complete dorsal enteric fistula to neurenteric cysts, diastematomyelia, dermal sinuses, caudal agenesis, and spinal segmental dysgenesis), bony spina bifida, tight filum terminale, filar and intradural lipomas, and persisting terminal ventricle. the most complicated forms (complex dysraphic states), including diastematomyelia, caudal regression, and segmental spinal dysgenesis) are related to faulty gastrulation. diastematomyelia (literally, split cord) is caused by failure of midline notochordal integration, resulting into two separate hemineural plates. caudal agenesis and segmental spinal dysgenesis are related to defective notochordal formation, characterized by absence or hypoplasia of a segment of the notochord, in turn resulting into absence or hypoplasia of a corresponding segment of the spinal cord. functional neuroimaging of cns is a fast advancing field with frequent new developments in scanner's hardware, protocols, clinical indications, and post-processing techniques. for radiation safety reasons in the case of children, functional neuroimaging is mostly based on mr techniques especially designed to focus on the assessment of functional tissue characteristics, such as neuronal activity (fmri),, metabolism (mrs) and perfusion (dsc perfusion, asl). pediatric coils with multiple elements, multiple slice excitation, d spectroscopy, d asl, reduced fov (zoom) and improved motion compensation techniques are important tools available to meet the permanent challenges of pediatric mr functional imaging: fast motionless acquisitions and increased resolution. functional mri (fmri) reveals brain activation during performance of behavioral tasks, based on the blood oxygen level dependent (bold) mri signal, which is modulated by neural activity via a process of neurovascular coupling. for children, especially of younger age unable to follow a task, resting-state fmri (rfmri) can be performed and correlates brain areas with similar spontaneous fluctuations in the bold signalthereby enabling estimates of 'functional connectivity.' main clinical applications of fmri are the delineation of eloquent cortex near a space-occupying lesion and the determination of the "dominant hemisphere" for language. intense research is conducted in the areas of language organization and development, brain plasticity, and neurobehavioral disorders (e.g. adhd). magnetic resonance spectroscopy (mrs) is a noninvasive mr technique, that detects intracellular metabolites, and may provide neuroimaging biomarkers of normal biological and pathological processes or response to a therapeutic intervention. although the main field of application of mrs is the brain tumors, it has also been of particular ( ) (suppl ):s -s pediatr radiol usefulness in assessing ischemic or traumatic brain injury and neurometabolic disorders. perfusion mr imaging methods detect signal changes that accompany the passage of a tracer through the cerebrovascular system. a less invasive approach is arterial spin labeling (asl) that uses arterial water as an endogenous tracer to measure cbf and thus it is more suitable for pediatric studies. mr perfusion is applied in the evaluation of brain tumors, neurological diseases and developmental disorders. functional neuroimaging clinical applications are expected to expand greatly in the future due to the increasing availability of their techniques, as well as the continuous advancements in the field of pediatric research. good knowledge of these techniques will become more necessary for an effective clinical practice and will enhance the role of radiology in the healthcare system. functional neuroimaging advanced techniques based on mri allow us to study complex cns processes such as cerebral perfusion (dsc, asl), metabolic activity (mrs) and brain activation (fmri). functional neuroimaging techniques already have significant clinical pediatric applications and assisted by recent advances in mr technology are expected to become even more powerful in the near future. kidney: perfusion, excretion, obstruction k. darge; philadelphia/us the functional imaging of the urinary tract entails the evaluation of the renal perfusion and excretion. in this complex process the sites of the main abnormalities could be pre-renal, renal parenchymal, renal pelvicalyceal or post-renal or even a combination at different sites. functional mr urography (fmru) is an advanced tool that not only allows the exquisite morphological depiction of the urinary tract, but also makes it possible to generate comprehensive functional data. these provide information about the function of the kidney as well as the excretion of urine from the renal parenchyma into the pelvicalyces and ureter. the functional results are mainly divided into two groups: . transit timesthese are recorded in minutes and a side comparison gives idea how much time it takes for the contrast to go through the renal parenchymathe longer the more abnormal in general. . differential renal functionsthese can be based on the enhanced renal parenchymal volume or the patalk number generated from this area and provides in percentage the split renal function. this presentation will discuss in detail the functional aspect of mr urography and demonstrate its utility in routine pediatric uroradiologic imaging. in chronic childhood lung disease (e.g. cystic fibrosis) global pulmonary function tests (pft) can be normal although lung damage is already present. moreover, in comparison to imaging, pft is challenging in young children. thus, cross-sectional imaging became more important in the past two decades. regarding morphological evaluation, multidetector computed tomography (mdct) serves as the most sensitive and reproducible modality. for functional evaluation perfusion/ventilation scintigraphy remains the reference standard. although the individual radiation burden by a single chest ct has decreased significantly in the past, radiation doses can cumulate considerably when repeated examinations are performed in a longterm follow-up. pulmonary mri exists as an alternative method, especially for paediatric patients. however, standard h+mr sequences do not demonstrate small airway disease due to inherent limitations of low signal and rapid t * signal decay of lung tissue. for comprehensive diagnosis, functional mri offers the unique possibility to measure regional ventilation and perfusion, and mapping relaxation times and diffusion. focussing on research applications, a variety of methods are available for these purposes. in this context, ventilation imaging using inert fluorinated gas indicates to overcome the limitations of the expensive setting necessary for imaging with hyperpolarized noble gasses. regarding lung perfusion, dynamic contrast-enhanced mri (dce-mri) is the most established method in clinical practice. however, especially in children, techniques that are completely non-invasive and do not require i.v.-contrast agents administration or gas inhalation could be promising to achieve broad acceptance. concerning non-invasive methods, ventilation can be assessed by sequences with ultra-short echo times (ute), perfusion by arterial-spin-labeling (asl) and both by fourier decomposition mri (fd-mri). in conclusion, pulmonary mri offers both, the assessment of morphology and the unique possibility to measure regional ventilation and perfusion, and mapping relaxation times and diffusion. new mr techniques that are completely non-invasive are now available. however, further scientific evaluation is needed. ibd and related arthropaties d. jaramillo; miami, fl/us musculoskeletal diseases affect about % of patients with crohn's disease and are the most frequent extra-intestinal manifestation of inflammatory disease. the articular manifestations of inflammatory bowel disease (ibd) are one of the seronegative arthritides, although they have a lower incidence of hla -b than other seronegative arthritis such as ankylosing spondylitis. there are manifestations in the joints of the extremities, and findings in the pelvis, especially in the sacro-iliac joints, and spine. involvement of the extremities occurs in about % of patients with ibd related arthropathies, are more common with crohn's disease, and can have either manifestations related olygoarticular jia, or can have symmetrical involvement of smaller joints. the axial manifestations include ankylosing spondylitis and sacro-iliitis. sacroilliitis is typically bilateral (figure) and often has radiographic as well as mri abnormalities. enthesitis, tenosynovitis and dactylitis can occur with ibd just as they occur with other arthritides. it is important to differentiate ibd related arthritis from septic arthritis due to extension of an enteric fistula. deceased bone mineral density is a common finding in inflammatory disease. it occurs as a combination of malabsorption of vitamin d due to intestinal involvement and the effects of therapy, particularly corticosteroids. insufficiency fractures of the spine, sacrum and extremities can mimic the symptoms of arthritis. finally, ibd can be associated with chronic non-bacterial osteomyelitis, although this association is relatively rare. this review will illustrate several of the skeletal manifestations of ibd, focusing on the arthropathies. juvenile idiopathic arthritis (jia) can be defined as an arthritis of unknown cause occuring in children younger than years and of at least weeks duration. juvenile spondyloarthritis (jspa) is a subset of jia and is characterized by enthesitis (inflammation at the attachment of tendons, ligaments and the joint capsule), arthritis and an increased risk of axial disease. there is also a strong association with human leukocyte antigen b . jspa accounts for approximately - % of juveniles arthritis cases in europe and is the most common form of juvenile arthritis in asia. the condition is associated with significant long-term morbidity, high health-care costs and poorer outcomes compared with other forms of juvenile arthritis as well as its adult counterpart. up to % of patients continue to be at risk of developing ankylosing spondylitis (as) during the disease course. recognizing spondyloarthritis (spa) in children is challenging, particularly early in the course of disease, as the signs and symptoms at disease onset differ from those seen in adults. jspa typically presents with hip and lower limb arthritis in conjunction with enthesitis. inflammatory back pain as a presenting symptom is less common. as a consequence, jspa may be missed or confused with other juvenile arthritides and patients often experience prolonged delays in diagnosis. currently there is no single diagnostic or classification system that is representative of the jspa population. according to the international league of associations for rheumatology (ilar) classification system, most childhood spa's are classified as enthesitis-related arthritis (era), psoriatic arthritis or undifferentiated arthritis. recent studies indicate that there are two clinical phenotypes of era: those with early axial disease often associated with hip arthritis in addition to peripheral arthritis; and those who follow a more peripheral disease course with arthritis and enthesitis and do not develop axial disease. the ilar classification system places patients with both axial and peripheral involvement into the era subtype, and does not specifically address children who meet the criteria for as. the correct approach to the classification of era is uncertain, and this issue is confusing to both pediatric and adult rheumatologists. unlike other categories of juvenile arthritis, jspa affects boys more often than girls, and peak age of onset is early adolescence. enthesitis is a defining characteristic of jspa. it is more common and affects more sites in the paediatric population compared with the adult one. the most commonly tender entheses are the insertions of the patellar ligament at the inferior patella, plantar fascia at the calcaneus, and the achilles tendon. arthritis in jspa is typically asymmetrical, oligoarticular (< joints) and involves predominantely the weightbearing joints. isolated hip joint arthritis may be the presenting feature and predicts early axial disease. involvement of the small toe joints is common in jspa but rare in other forms of jia. midfoot arthritis and tarsitis (inflammation of the intertarsal bones, overlying tendons, entheses and soft tissue) is highly suggestive of spondyloarthritis. in adults, inflammatory back pain typically heralds the onset of sacroiliitis, whereas children seldom present with symptoms of axial disease. however, according to several studies, sacroilitis can be asymptomatic in jspa and only detectable by imaging. other axial manifestations in jspa are inflammation of the lumbar apophyseal joints and interspinous ligaments, corner lesions of the spine and other sites of axial enthesitis-osteitis including the various ligamentous and muscular attachments of the pelvis. extraarticular manifestations of jspa are highly associated with axial disease and include acute anterior uveitis, bowel inflammation, psoriasis, and cardiac disease. clinical diagnosis of jspa can be difficult and the role of imaging may be more critical than in adult disease. the major goal of imaging in jspa is to identify children with early signs of axial disease, as this group is at the greatest risk for progression to as. the presence of axial disease in spa has also major implications for treatment decisions, since traditional firstline therapies appear to have minimal effectiveness in the management of axial inflammation. in addition, recent studies in adults suggest that earlier initiation of biologic agents (anti-tnfs) may slow radiographic progression. x-rays are not sensitive to acute inflammatory changes and will only show advanced disease in the sacroiliac joints. for these reasons plain radiographs are not useful in children or adolescents. ultrasound is a non-invasive, non-ionizing and relatively inexpensive technique that can be performed in a clinical setting. it is emerging as a valid diagnostic tool in spa and can be used to visualize peripheral synovitis, tendonitis and enthesitis, but the method is heavily operator-dependent and there does not yet exist a clear definition for the diagnosis and grading of enthesitis in children. secondary changes (calcifications, enthesophytes) have been observed much less in children compared with adults. there is a need for better consensus on abnormal ultrasonographic findings that define enthesitis lesions and standardization of methods. magnetic resonance imaging (mri) is a radiation free and sensitive imaging modality for detection of synovitis as well as cartilage and bone destruction. mri of the sacroiliac joints is increasingly obtained for early detection of inflammatory changes, as it shows active inflammatory (bone marrow edema, osteitis, enthesitis and capsulitis) and structural (erosions, subchondral sclerosis, subchondral fatty change and bony ankylosis) lesions of sacroiliitis long before radiographic changes become evident. in adults, mri has become the gold standard imaging modality for detecting arthritis and enthesitis. consensus definitions of lesions indicating pathology on mri are now incorporated into diagnostic criteria for adult with spa. in children and adolescents there is no gold standard mri technique and it is therefore not clearly defined whether changes s ( ) (suppl ):s -s pediatr radiol seen in the sacroiliac joints are pathologic or part of normal maturation in the growing skeleton. the use of contrast enhanced imaging for the detection of active sacroiliitis on mri in jspa is a major controversy. synovial enhancement can be detected without accompanying bone marrow edema in children, and it can be argued that contrast should be administered in order not to miss the diagnosis. some authors argue that contrast administration does not change or add substantially to the mri findings made on non-enhanced scans. certainly, given the risks associated with gadolinium administration, contrast should be used with caution. perhaps the use of contrast agents should be limited to selected cases when high stir signal in the joint is the only finding in order to confirm the presence of synovitis, and when the differential diagnosis includes etiologies such as infection or tumor. the development of new mri techniques has made it possible to perform whole body mri scans (wbmri) that allow assessment of the full range of affected entheses and joints. there is limited data on the utility of wbmri in the pediatric population. it is worth noting that edema-like changes seen in the marrow of healthy children is an important potential pitfall to consider during interpretation and further studies are required in order to establish specific reference standards for mri of the pediatric skeleton. diffusion-weighted imaging (dwi) offers a new approach to detect inflammation. inflammation produces an increase in the apparent diffusion coefficient (adc) of water molecules in affected tissues. several studies in adults and a few recent studies in children have demonstrated that adc is elevated in sacroilitis versus controls and that diffusion scores correlates well with stir images. dwi is promising as a potential biomarker of disease activity in jia and presents a novel approach to contrast-free imaging of synovitis. however, further studies are needed before it can be implemented in clinical practice. jspa is distinct from adult spa and manifests more frequently as peripheral arthritis and enthesitis. symptoms involving the spine and sacroiliac joints often occures later in this population. clinical diagnosis of jspa can be difficult, and imaging therefore plays an important role in the diagnostic workup of disease. identifying early signs of axial disease has major implications for treatment decisions and mri of the sacroiliac joints is increasingly obtained for early detection of inflammatory changes. however, mri criteria for sacroilitis in children are lacking. a major controversy in imaging of sacroilitis in jspa is the use of contrast, as children can have sacroilitis without accompanying bone-marrow edema. dwi presents a novel approach to contrast-free imaging of synovitis but further studies are needed before it can be used in clinical practice.wbmri has been shown to be more sensitive than clinical examination in the assessment of both disease activity and extent, but there is limited data on wbmri in children. normal variants in the growing skeleton may mimic pathologic changes and potentially cause overdiagnosing and -staging of disease. hence, there is an urgent need to establish specific reference standards for mri of the pediatric skeleton and to develop a gold standard mri technique for the axial skeleton in children and adolescents. juvenile idiopathic arthritis o. olsen; london/uk summary: juvenile idiopathic arthritis (jia) is common (about : , children). diagnosis and classification are based on clinical criteria. these criteria are in flux depending on ) contemporaneous knowledge about aetiology and ) available treatment options. radiology has currently no role in establishing the diagnosis. the clinical classification rests on whether the child has few joints affected (oligo jia), many joints (poly jia), has a condition similar to adult spondyloarthritis (entesitis-related arthritis) or other clinical presentations (systemic-onset jia, psoriatic jia, etc). radiology can potentially assess expressions of jia, such as synovitis, tenosynovitis, systemic manifestations and permanent damage caused by inflammation. it is therefore thought to play a part in gauging the disease activity. the clinical care aims at optimising the child's everyday function, reducing acute symptoms (pain, swelling, joint restriction), allowing normal growth, minimising long-term sequelae (joint deformity) and minimising adverse effects of medical treatment. medical treatment in jia is systemic (immuno-modulation) and local (steroid injection to joints and tendon sheaths). both modes of therapy may to some degree be guided by imaging. however, there currently is no evidence that any form of whole-body imaging is efficacious for guiding treatment. this means that, in principle, indication for imaging should be ) specific clinical questions, e.g. uncertainty regarding active inflammation at specific sites, or ) a high pre-test likelihood of inflammation at a site which is difficult to assess clinically and where imaging offers reasonable accuracy. one example of the latter are the temporo-mandibular joints where destruction is frequently seen at an early stage, often without prior symptomatic warning. there is one fundamental challenge for imaging research in jia: what is the reference standard? for lack of anything better, a standardised clinical examination is often used as 'ground truth'. the dilemma is obvious. if clinical examination is reliable and accurate, then why bother with imaging? but we think imaging offers an improvement, then we cannot use an inferior method to set the standard. this problem is not unique to jia. as is often the case, radiology in jia is all about: knowing your clinicians (i.e. the pretest likelihood for disease) being technically eloquent (e.g. using high-resolution us probes, not delaying post-contrast mri acquisitions) knowing what is normal (e.g. normal undulations in the articular surface, focal bone marrow signal variation) not being dogmatic about individual observations or measurements interpreting your findings in a clinical context the lecture will demonstrate similarities and differences among joints and modalities in children with variable-severity jia. the following points will be made: focal areas in the bone marrow with high signal (t ) and corresponding enhancement are often seen in healthy children. in isolation, these do not signify active inflammation. active synovitis in children often is not associated with (much) effusion the combination of synovial thickening with hyperaemia (us)/abnormal contrast enhancement (mri) and surrounding softtissue swelling suggests active inflammation, however there is (yet) no established system for quantifying hyperaemia/enhancement focal pits in the carpal bones do not represent erosions unless there is an associated cartilage defect radiographs are useful for detection of destructive abnormality in mri, scan fairly soon after injecting contrast. gadolinium physiologically leaks into the synovial fluid making it difficult to delineate the synovium a few differential diagnoses to keep in mind when there is mass-like swelling within or adjacent to a joint: vascular and neoplastic lesion, pigmented villonodular synovitis, synovial chondromatosis, lipoma arborescens. synovial inflammation is not always primary. even when there is an established diagnosis of jia, do consider that it may be secondary to biomechanical abnormality (erosion, osteochondral lesion, deformity). focal areas in the bone marrow with high signal (t ) and corresponding enhancement are often seen in healthy children. in isolation, these do not signify active inflammation. active synovitis in children often is not associated with (much) effusion the combination of synovial thickening with hyperaemia (us)/abnormal contrast enhancement (mri) and surrounding soft-tissue swelling suggests active inflammation, however there is (yet) no established system for quantifying hyperaemia/enhancement focal pits in the carpal s ( ) (suppl ):s -s pediatr radiol bones do not represent erosions unless there is an associated cartilage defect radiographs are useful for detection of destructive abnormality in mri, scan fairly soon after injecting contrast. gadolinium physiologically leaks into the synovial fluid making it difficult to delineate the synovium pulmonary manifestation of connective tissue disorders c. m. owens; london/uk summary: connective tissue diseases are an important cause of morbidity and mortality in children with very varied presentations. nomenclature is confusing and a more appropriate descriptive term would be "multisystem inflammatory disorder +/-autoimmunity". it is important for the radiologist to be aware of the protean radiological appearances and clinical manifestations. take home points: different patterns of diffuse lung disease (eg, desquamative interstitial pneumonia, non specific interstitial pneumonia, lymphocytic interstitial pneumonia, organising pneumonia, diffuse alveolar damage) may be present in several forms of collagen vascular disease, (and indeed other rheumatological conditions such as jia) including scleroderma, systemic lupus erythematosis, juvenile dermato and polymyositis, sjogren's syndrome and mixed connective tissue disease. these will be discussed in detail with illustrations for thin section high resolution ct with histopathological correlation. the clinical presentation, prognosis and response to therapy vary depending on the histological pattern of diffuse lung disease, as well as on the underlying collagen vascular disease. whole body imaging in children: sonography, ct, mri, nuclear medicine -what and when? r. a. nievelstein; utrecht/nl there are several (benign and malignant) disease processes in children that frequently involve more than one organ system or body region. diagnostic imaging of children with such multifocal or multisystem diseases has been quite challenging, often requiring a combination of different imaging techniques for a whole body coverage. the recent technical developments in computed tomography (ct), magnetic resonance imaging (mri) and nuclear medicine (nm) have changed the role of imaging in these children revolutionary. in the past, imaging techniques have been mainly used as a tool to detect the cause of illness and to assess the extent of disease spread before, during and after therapy (i.e. structural imaging). but nowadays, it has also become possible to use imaging techniques to gain information on the biological behavior of diseases before and during therapy (i.e. functional imaging). plain radiography, ultrasonography (us) and computed tomography (ct) have been the structural imaging techniques of choice for many decades, more recently supplemented by functional imaging techniques like single-photon emission tomography (spect) and positron emission tomography (pet). a major disadvantage of most of these techniques is the use of ionizing radiation, which may be associated with induction of second cancers later during life. this small but not negligible health risk is of particular concern in children as their tissues are more radiosensitive than adults and they have more years ahead in which cancerous changes might occur. that is why there is an increasing interest in the use of alternative imaging techniques that do not use ionizing radiation. with mri it is nowadays possible to acquire images with a high spatial resolution and excellent soft tissue contrast throughout the body, which makes it an ideal radiation-free tool for the detection of pathology, especially in soft tissue, parenchymal and bone marrow locations. moreover, recent technological advances have resulted in fast diagnostic sequences for whole-body mr imaging (wb-mri), including functional techniques such as diffusion weighted imaging (dwi). as a result, wb-mri has become a clinically feasible imaging modality for diagnosis and follow-up of multifocal and multisystem diseases in children. in this scope, the recent development of integrated pet/mri systems is very interesting, combining the superior structural imaging of mri with the functional (molecular) information of both imaging techniques while decreasing the radiation dose. traditionally, whole body imaging techniques have been mainly used for oncological indications, such as staging of malignancies, and monitoring of the effectiveness of therapy. however, whole-body imaging techniques are increasingly used for the diagnostic imaging of other benign multisystem diseases and indications, including chronic recurrent multifocal osteomyelitis (crmo), rheumatological diseases, neuromuscular diseases, neurofibromatosis type , generalized vascular malformations, multifocal osteonecrosis after intensive chemotherapy, fever of unknown origin, and post-mortem imaging. finally, these imaging techniques may be used for the screening of children with a cancer predisposition syndrome. during this lecture, imaging protocols and indications of the different whole body imaging techniques will be discussed with a focus on their clinical application in children with benign and malignant multifocal or multisystem diseases. ( ) (suppl ):s -s pediatr radiol appearances is important for any radiologist involved in child imaging, because we have an important role in characterizing the lesions and guiding purposeful and minimally invasive but successful diagnostic procedures. most head and neck masses in children are benign and have an inflammatory, infective, vascular or congenital cause (cf. special presentation on vascular malformations). malignant lesions are less common, however, early diagnosis is paramount as many of these cancers are readily treatable and often curable. differential diagnosis is guided by patient age, clinical presentation, tumour localisation, and imaging characteristics. while some masses such as (epi-)dermoids, fibromatosis colli and swollen lymph nodes including atypical mycobacterial infections (mott) may be readily diagnosed by clinical inspection und ultrasound, others present special diagnostic challenges. fibromatosis, for example, is a benign lesion with an often complex and potentially destructive local spread. some malignant lesions tend to be localised such as the embryonal rhabdomyosarcoma, while others may be part of a systemic disease such as lymphoma and langerhans cell histiocytosis (lchc). in case of a suspected malignancy, patients should be referred to a specialized centre which will be able to provide the full spectrum of multidisciplinary evaluation and treatment according to the guidelines of an international oncology study group. this is also important for image guided or surgical biopsies as long term outcome and survival of many of the young patients are directly associated with these initial diagnostic and therapeutic strategies. with its excellent spatial resolution in the near field, ultrasound is the method of choice for all superficial masses. an experienced paediatric radiologist will be able to identify most of the benign lesions and in other cases will be able to guide further diagnostic decisions. tumours in the midline require thorough workup to exclude an encephalocele or a dermal sinus with connection to the intracranial space. high resolution mri is required if such an extension cannot be ruled out by ultrasound or if a tumour is larger than the transducer's scan area. soft tissue tumours in the deeper parts of face and neck as well as tumours of osseous origin are also best delineated by mri. in lesions adjacent to the skull base contrast enhanced and fat saturated mr images with high spatial resolution are of utmost importance to completely depict the tumour's extension through the foramina and along the meninges (fig. ) . ct can provide additional information on the involvement of osseous structures. embryonal rhabdomyosarcoma. high resolution mri with fat saturation after contrast injection depicts the tumour's extension through the foramen ovale (long arrow) and along the meninges (short arrows). skull base and face lesions are less frequent in children than in adults. symptoms may be subtle or unspecific. depending on their localization, clinical findings may be common (nasal obstruction, otitis…) or more disturbing (cranial nerves palsies, exophthalmos, vision loss …). clinical history and physical examination findings are important to reduce the spectrum of differential diagnosis, but imaging data are the key features to determine the nature of these lesions. ct and mri play an important role in diagnosis, treatment survey and surgery planning of skull base and face lesions. skull base and face bone lesions are either intrinsic lesions of the bone or secondary to soft-tissue tumors or pseudo tumors invasion. this lecture will focus on bone intrinsic lesions, and include soft-tissue and pseudo tumors only as differential diagnoses. computed tomography plays the role for skull base and face of plain radiograph for long bones. therefore, the same semiology may be used to determine if the lesion is slowly or rapidly growing, aggressive or looks benign. helical ct allows reconstructions with both soft-tissue and bone algorithms as well as multiplanar reformations. it gives a good visualization of the anatomy of the skull base and allows a good depiction of the bone architecture. ct is first used for the initial work up of the disease but also for surgery and therapeutic planning (endoscopic sinus surgery with navigation). however, ct analysis may be challenging in children due to growth changes: normal process of pneumatization according to age, sutures not yet fused has to be recognized. some variations in pneumatization must not be mistaken for pathology: asymmetrical pneumatization of the petrous apex and arrested pneumatization of the sphenoid mimicking intraosseous lesion are the most common. both ct and mr imaging are complementary: most preferably, contrast-enhanced mr is associated with non-contrast high resolution ct. mri allows a good delineation of bone involvement of skull base lesion due to bone marrow changes, whether ct can fail to detect subtle extension within the bone. in addition to t and t weighted sequences, the use of specific sequences and/or techniques such as fat-saturation, diffusion, dynamic-contrast-enhanced sequences, and mr angiography helps to characterize the lesions. t spin echo sequence is mandatory to appreciate bone marrow infiltration in adults and older children. but when red bone marrow has not yet be replaced by fatty bone marrow, in young children, this can be challenging. it is useful to know the bone marrow fatty conversion of the skull base chronology. cranial mr can also be associated to whole body mr to look for multifocal or metastatic disease. epidemiologic data concerning bone tumors of the skull base are scarce due the rarity of these lesions. they can be classified according to their location within anterior, middle or posterior cranial fossa or classified according to their origin: osteogenic (osteoblastoma, osteoma, osteosarcoma...), chondrogenic (chondroma, chondrosarcoma), fibrous ( fibrous dysplasia, fibro-s ( ) (suppl ):s -s pediatr radiol osseous lesions..), notochord (chordoma), hematopoietic (leukemia, histiocytosis ), vascular (hemangioma), neuro ectodermic ( ewing sarcoma) or unknown origin (aneurysmal cyst, giant cell tumor). the aim of this presentation is to draw attention to skull base growth changes that can mimic pathology and to describe the imaging specificities of the most common bone tumors of the skull base and face in children. because conflicted nomenclature can cause confusion, accurate diagnosis and classification of these anomalies is important for proper clinical evaluation and management. many of these patients require multidisciplinary care, consequently the usage of a correct nomenclature across all disciplines is a sine qua non. the international society for the study of vascular anomalies (issva) classification, updated in , offers a comprehensive classification accepted by many subspecialities. this approach/ classification has facilitated correct communication for all medical subspecialties involved in the care of these complex vascular anomalies. pediatric radiologists play a critical role in evaluating these patients since the majority present during childhood. in this presentation, we present a state of the art mri imaging protocol with exemplary cases of the most common types of vascular anomalies in the pediatric trunk and extremities, using the current issva classification. in addition, we discuss the common syndromes associated with vascular anomalies such as klippel-trenaunay and lumbar syndrome. genetic skeletal disorders (gsd's) are a heterogeneous group of syndromes characterized by an intrinsic abnormality in growth and (re-)modeling of cartilage and bone. a large sub-group of gsd's may have additional involvement of other structures/organs beside the skeleton, such as the central nervous system (cns). cns abnormalities have an important role in long-term prognosis of children with gsd's and should consequently not be missed. sensitive and specific identification of cns lesions while evaluating a child with a gsd requires a detailed knowledge of the possible associated cns abnormalities. here, we will present and discuss a pattern-recognition approach for identifying relevant neuroimaging findings in gsd's guided by the obvious skeletal manifestations of gsd. in particular, we will discuss which cns findings should be ruled out for the various gsd. to facilitate this diagnostic approach the multiple gsd are classified based on the pattern of skeletal involvement ( . abnormal metaphysis or epiphysis, ) abnormal size/number of bones, ) abnormal shape of bones and joints, and ) abnormal dynamic or structural changes). skeletal involvement is defined in accordance with online mendelian inheritance in man. the spectrum of co-existing cns involvement is extracted from an extensive literature search. selected examples will be shown based on prevalence of the diseases and significance of the cns involvement. cns involvement is common in gsd's. a wide spectrum of morphological abnormalities is associated with gsd's. early diagnosis of cns involvement is important in the management of children with gsd's. this pattern-recognition approach aims to assist and guide physicians in the diagnostic work-up of cns involvement in children with gsd's and their management. not infrequently the correct radiological differentiation of skeletal and/or central nervous system findings secondary to non-accidental injury versus inherited genetic and/or metabolic disorders may be challenging. imaging findings may be non-specific, can result in incorrect diagnosis and subsequently inadequate patient management or initiation of faulty treatment. the diagnostic work-up of children suspected of non-accidental injury or genetic/metabolic disorders requires a multi-disciplinary approach involving many key players including physicans of various disciplines, nurses, psychologists, social workers and many more. a proper and detailed medical history and physical examination of the patient, collection of the relevant family history, a metabolic and genetic work up, a detailed interview of care givers, friends and family are essential for the correct and comprehensive evaluation of imaging findings. in the current session, various exemplary and possibly confusing cases will be interactively discussed with the audience by a panel of experts (susan blaser, thierry a.g.m. huisman and andrea superti-furga). goal is to offer a case based approach to challenging patients with discussion of the best diagnostic approach including differential considerations. the zikv is transmitted mainly by the bite of female aedes aegypti and aedes albopictus mosquitoes. other forms of transmission, including through sexual intercourse, blood transfusion, and neonatal, are currently under evaluation, although more elements are still needed to assess the real importance of these transmission routes . the course of the zikv infection is self-limited. so far, no specific symptoms have been attributed to the disease, and a wide variety of manifestations ranging from absent to mild symptoms (in % of cases) have been described. when symptoms are present, they may lead to a misdiagnosis of other bacterial and viral infections, especially other arboviroses in endemic areas. the most frequently reported symptoms are mild fever, cutaneous rash, fatigue, arthralgia/myalgia, and conjunctivitis. dizziness, malaise, edema of the extremities, anorexia, retro orbital pain, photophobia, gastrointestinal disorders, sore throat, cough, sweating, and lymphadenopathy have also been reported. infection by the zikv in adults may be associated with autoimmune complications such as guillain-barré syndrome . the laboratory diagnosis of zikv infection is based on the demonstration of the virus in the urine and blood using real-time reverse transcription polymerase chain reaction (rt-pcr). the main limitation of this diagnostic method is a false-negative result after the viremia is resolved. the serological diagnosis of the disease is limited due to cross-reactivity of the zikv with other viruses of the flavivirus genus, especially those causing dengue and chikungunya. physicians should be aware of this fact when the diagnosis of zikv infection relies solely on serological results. the diagnosis is also possible by igm measurement in serum, urine, or cerebrospinal fluid using enzyme-linked immunosorbent assay (elisa) . the prevention against zikv infection is similar to that of other arboviroses, including vector control and mosquito bite prevention. the first major zikv epidemics were reported in the french polynesia in and . at that time, some neurological changes were observed in neonates of infected pregnant women but were not associated with a maternal-fetal transmission of the virus. the growing increase in the number of cases and the severity of the infection specific to this subpopulation then led to the evidence of a congenital disease . in brazil, the situation became alarming with the report of a high number of infected individuals in the second half of , . the brazilian ministry of health attributed to congenital zikv infection the -fold increase in cases of neonatal microcephaly in the northeastern part of the country, particularly in the state of pernambuco. this led the world health organization (who) to declare the zikv infection a "public health emergency of international concern" in february . the main challenge for radiologists practicing in regions of endemic zikv infection is to become familiarized with findings of congenital zikv infection in perinatal imaging studies; this is particularly important for the prenatal screening of pregnant women , . the diagnosis of zikv infection in the fetus by neuroimaging is based on prenatal ultrasound (us), especially in the third trimester, and complemented with magnetic resonance imaging (mri). postnatal imaging was obtained by transfontanellar us, ct or mri. the main imaging findings on ct are microcephaly, an exuberant external occipital protuberance, rectification of the frontonasal angle, and a redundant scalp skin. three-dimensional ( d) reconstruction of al skull permits a better evaluation of these findings and enhances the parents' understanding of the disease. moreover, ct scan data may yield a d virtual physical model that can maybe obtained from ct scan data and printed onto using thermoplastic acrylonitrile butadiene styrene . the aim of this study was to describe the perinatal imaging findings in cases of congenital zikv infection. we studied mothers diagnosed with zikv infection from october to november . they had all presented a maculopapular rash and fever during the first or second trimester of pregnancy, and their neonates presented neurological defects that were attributed to intrauterine transmission of the zikv. the maternal diagnosis of zikv infection was confirmed by serology (n= ) or rt-pcr (n= ). all patients were torch (toxoplasma, rubella, cytomegalovirus, herpes simplex) negative. prenatal us was performed every weeks after the first imaging findings, and fetal mri was obtained in all cases. microcephaly was considered present when the infant's head circumference was two standard deviations below the mean value for age and sex or below the second percentile. postnatal imaging follow-up was obtained in all cases by transfontanellar us, ct or mri. we found several cns malformations, including lissencephaly, pachygyria and/or polymicrogyria, cerebral atrophy (panel ), enlarged cisterna magna with abnormalities of the corpus callosum, ventriculomegaly, brainstem hypoplasia, malformation of the cortical development, and cortical and/or periventricular calcifications mainly in the junction between the cortical and subcortical white matter (panel ). the skull of the infants had a collapsed appearance, with overlapping sutures and redundant skinfolds (panel ). craniofacial disproportion was easily identifiable, and arthrogryposis was identified in one case. similar neurological findings were observed in the infected patients and seemed to differ from findings of other infectious diseases. the finding of microcephaly in neonates with congenital zikv infection seems to be only the tip of the iceberg, as several cns malformations have been identified in connection with the disease. in brazil, a spectrum of imaging findings associated with congenital zikv infection has been observed. such findings are useful in helping radiologists to identify suspected cases of the disease. panel : prenatal ultrasound ( weeks) shows calcifications (arrows) and microcephaly. axial and sagittal t shows relative smoothness of the brain surface (arrows) and assymmetric colpocephaly. panel :ax t -wi multiple cortical-subcortical fronto-parietal hyperintense foci (arrows) and markedly hypointense on swi. sagittal t : dysgenesis of the corpus callosum, with dilation of the posterior horns of the lateral ventricles (colpocephaly). pre-and postnatal imaging in zika virus: where are we? early insights into zika's microcephaly physiopathology, from the epicenter of the outbreak: a case for teratogenic apoptosis of central nervous system. p. jungmann; recife/br early insights into zika's microcephaly physiopathology, from the epicenter of the outbreak: a case for teratogenic apoptosis of central nervous system. in mid-october , intense interaction among surgical pathology and fetal medicine specialists from university of pernambuco was only focused on the dramatic and non explained ultrasonographic (us) findings and hopelessness due to lack of explanations on the odd us discoveries on the first gestational cases of zika's microcephaly. this is the field of our history of a physiopathological hypothesis on zika virus (zikv) related microcephaly when it first struck pernambuco state (pe), northeast brazil, the place that has been at the front line of the global response to the microcephaly and responsible for a large amount of data from affected children. the outbreak onset came with a sudden increase in microcephalic newborns being reported in pe state from august (panel, fig. ) . zikv was previously thought to cause a relatively mild disease, but was recently accepted to lead to severe and diverse neurologic conditions in s ( ) (suppl ):s -s pediatr radiol some children born from infected mothers and in adults . the scientific community is actively trying to uncover the extent of these disorders but little has been reported on the early days of the outbreak when doctors were approaching the unknown. while evidence that zikv is related to microcephaly in newborns is accumulating, the mechanisms of how the virus affects the fetus is still uncertain. in the outbreak onset we had to face daunting challenges to search the cause of microcephaly and the emotional toll on the families. we took a very early approach from microcephalic fetuses on gestation and microcephalic babies on clinical follow-up from different pe areas, evaluated between october and december in oswaldo cruz hospital, to propose the early physiopathologic hypothesis that, a viral-related brain developmental disruption could be the basic neuropathogenesis in zikv babies instead of a direct injurious process due to viral insult followed by active inflammation. the eight pregnant women were all in the rd gestational trimester and had had normal us follow-ups till week th . crucially, we were facing a temporal-geographic association of cases presenting an unanticipated pattern of us alterations. because of their late alarming findings they were re-examined and the us scans revealed sudden encephalic alterations after th gestational week. such devastating us clustering images were not seen here before, but are now considered as part of the "congenital zika syndrome". we observed late appearing severe dysmorphic encephalic changes in out of fetuses, including small skull, small brain, sub arachnoidal space enlargement, ventricular dilation, brain calcifications of varied shape and distribution, inclined frontal bone, progressive decline of head growth potential, early fontanels closure and redundant scalp (panel, figs. a, b). we had no clues on the causes and mechanisms responsible for this phenotype of severe alterations. thus, we had no explanation to offer to patients, in particular, or to the medical community. both as physicians and human beings, we were committed straightaway to continue the study of these victims of an unknown medical tragedy, engaging our expertise in fetal imaging and immunopathology. from beginning october, the first microcephalic babies were referred to the upe pediatric infectology service for initial investigation. strikingly, the newborns exhibited "healthy" appearance, excluded the microcephaly itself and motor sequels. we then looked for csf analysis of the microcephalic babies. for that, we obtained from dr. patricia travassos, a csf specialist at upe, a cohort of csf samples that have been studied for signs of meningitis or encephalitis. about % ( cases) of the csf analyzed looked normal for any signs of central nervous system ongoing inflammatory responses (panel, fig. ). the babies had been examined by outpatient clinic dr angela rocha, from the upe hospital infectology reference center that have stated that although small, the babies were near to full term gestation ( - weeks gestation), had good apgar scores and variable degrees of microcephaly and neurologic impairments, i.e. contractures, spasms, irritability and in some retinal macular atrophy. during the follow-up, the babies were cared at home, breastfeeding, gaining weight and having routine vaccines. none of them expressed signs of ongoing inflammatory reaction in the cns (panel, fig. ) or alterations on peripheral blood count and other routine laboratory tests up to months of age. despite the striking neurological phenotype, % of the babies were negative for torch agents, no deaths were recorded. furthermore only in january , the first evidence associating zikv to microcephaly from rt-pcr test on amniotic fluid was reported . astonishingly, a particular kind of physiopathological process linked to fetal brain development was arising without clinical manifestation of inflammatory reactions or necrotic processes in these babies. unfortunately, no necroscopic samples of affected brain tissues were available to us to monitor the presence of putative neural dysgenesis and the very nature of brain calcifications background offering histological support for our hypothesis. nevertheless, with this restricted dataset we hypothesized that whatever the etiologic agent involved in these cases, its physiopathologic mechanism must trigger the cellular death programthe apoptotic process -at a particular development window on the cns, assuming clinically that the agent was not encephaalitogenic but silently tertogenic. if not, the clinical outcome of affected babies would not be so mild as far as signs of inflammation on cns was concerned. consequently, the inflammation-free clinical status of patients suggested us that a massive enhanced apoptotic cell death during the window of telencephalic expansion was the most probable physiopathologic process operating this microcephaly phenotype, with no direct direct lytic brain lesion or significant necrosis due to usual injury. furthermore, knowles and penn stated that this window is very active to select the "fittest" neural cells by a constitutive apoptotic pathway. we so hypothetized that during this developmental time window, the "fit or not fit" status of the rapid, transient amplifying neural progenitors cells facing zikv, would heavily shift the selective process toward the self-elimination of virusbearing cells through apoptotic pathways. thus, zikv-enhanced constitutive apoptotic mechanisms would lead a massive loss of developing telencephalic neuronal precursors and, consequently, provoking losses of dividing cells and the arrest of further brain development. this could be particularly inferred by the absence of the characteristic morphology of late stages structures of neocortex, according with our us images of zikv microcephalics in gestation. similar processo could also be inferred to neurocrest derivatives as deformities in the viscercrany always accompany the cephalic malformation. our initial understandings based on clinical examination on the field, when no specific laboratory test, necroscopic data or experimental evidence on the disease causality were available, conducted our physiopathological approach to the "apoptosis hypothesis" for zika microcephaly that is now gaining strong support. in february, mlarkar et al showed clear connection between zikv and microcephaly, presenting cns histopathologic analyses, revealing remnants of neural germinative matrix, intense gliosis, alterations in cortical ribbon, calcifications in gray and white matters without associated necrosis, encephalitis or meningitis and the presence of the virus, further supporting neurodevelopmental arrest. similar results were showed by driggers et al . the ct scans from microcephalic babies from hazin at al , have added details of brain development arrest with no radiological signs of brain destruction or active inflammation. finally, experimental models have provided a body of evidence f or neuroprogenitors permissiveness to zikv and viralinduced apoptotic process. tang et al demonstrated by icq that the zikv infection of cortical neural progenitors attenuates their growth and increases caspase- activation, calling for an apoptotic process. this finding was corroborated by the up regulations of caspase- genes by rna sequencing. nowakowsky et al demonstrated that zikv may hijack axl protein as an entryway to infection. interestingly, axl is highly abundant on the surface of neural stem cells but not on differentiated neurons in the developing brain. recently, cugola et al demonstrated that zikv was able to cause cns congenital brain dysgenesis upon vertical transmission in mice. in parallel, human brain organoids infected by zikv show a reduction of proliferative zones and disrupted cortical layers, so targeting cortical progenitors and inducing apoptotic cell death with impaired development. for babies born with zikv-related microcephaly, the many expected consequences besides the evolving congenital neurosequels, are the unanticipated pattern of persistence of zikv in cns host cells, unsafe maintenance of neuron genome stability on remaining arrested populations, implying risks for brain tumors, risks for impaired adult type neuron wiring and neuron survival in an affected neuronal circuitry. in brief, evolve life with a wide vulnerable brain. the outbreak of zikv in the americas will eventually decline as herd immunity increases, but the world remains at risk of further waves of infection in affected countries and spread into new territories . while experimental studies will be carried out to fully understand the pathophysiology of zikv infection in the developing fetus, our findings provide a coherent and testable physiopathological hypothesis for cns teratogenic phenotype linked to zikv congenital infection, which may be critical for the clinical care of pregnant mothers and their babies before and after birth. take home points: fetal dysmorphisms detected by ultrassonographic and mri images in congenital zika syndrome are late findings, usually after the th gestational week and requires acurate analyses. clinically, zika's virus microcephaly is an infectiuos congenital condition that is not encephalitogenic but primarily teratogenic on the nervous system. the most important process leading to zika's virus microcephaly is pathologically induced apoptosis in telencephalic neuroprecursosrs cells and neurocrest precurssors cells. viral induced autophagy and low antiviral responses during the fetal period are linked to zika virus persistence in the central nervous system of affected new borns and babies a. vossough; philadelphia/us summary: susceptibility-weighted imaging (swi) has proven to be a valuable mr imaging sequence in a variety of applications. pediatric imaging has also immensely benefitted from this technique. in this presentation we will review pediatric neuroimaging applications in trauma, arterial and venous vascular disorders, hypoxic-anoxic injury, congenital malformations, congenital heart disorders, neoplasms, and pediatric degenerative disease. use of swi in pediatrics other than demonstrating hemorrhage and calcification will be reviewed. challenges in the clinical use of swi in pediatrics, interpretive pitfalls, and sources of clinical misinterpretation of swi will also be explored. we will also briefly present ongoing research and clinical use of swi in pediatrics and potentials for future collaborative investigations. & swi is highly sensitive in detection of susceptibility effects on mri. & in many cases, but not all, swi processing can differentiate between calclium and blood products. & quantitation information can also be obtained from swi with further processing. state of the art imaging of the single ventricle d.m. biko; philadelphia/us there are many congenital heart defects that result in a functional single ventricle. this may be functional or anatomical as a result of a dysfunctional valve or absent or ineffective pumping chamber. the repair of single ventricle physiology most often involves a staged reconstruction due to changing physiology ultimately resulting in a total cavopulomonary connection or fontan procedure. to appropriately image the single ventricle throughout its stages of palliation, familiarity with the physiology of the various steps in surgical palliation of the single ventricle is essential although echocardiography is a mainstay of cardiac imaging, cross sectional imaging has a vital role in the evaluation of the single ventricle. the role of ct angiography is mostly for anatomic evaluation. although it is fast and has high spatial resolution for evaluation of vasculature, ct has lower temporal resolution than mri and is unable to quantify flow. ventricular performance along with quantification of flow can be performed with mri. systemic to pulmonary collateral flow, which has been shown to result in adverse outcomes after fontan, can be quantified. valvular insufficiency and myocardial scarring can also be assessed. additionally, high anatomic vascular detail can be obtained with mri, particularly with the recent investigational use of the blood pool agent ferumoxytol. mri also has the ability to assess the lymphatics either through non-contrast t weighted imaging and/or dynamic contrast mr lymphangiography as lymphatic pathology may play a role in postsurgical hemodynamics in single ventricle patients. this lecture will focus on the use of ct and mri in the evaluation of the single ventricle particularly concentrating on the developing use of mri for anatomic and physiologic assessment. take home points: in single ventricle physiology, there is only one effective pumping chamber. familiarity with the physiology of the various steps in surgical palliation of the single ventricle is essential in imaging this disorder ct angiography provides high anatomic detail but limited in its assessment of physiology since it cannot quantify flow and has lower temporal resolution than mri. mri can evaluate ventricular performance, quantify flow and valvular insufficiency, and assess myocardial scarring. high anatomic vascular detail can also be obtained with mri particularly with the emerging investigational use of ferumoxytol. with non-contrast t weighted mri and/or dynamic contrast mr lymphangiography, lymphatic evaluation can be performed which may play a role in post-surgical hemodynamics in single ventricle patients. neuroimaging in head trauma m. argyropoulou, g. alexiou; ioannina/gr summary: objective: head trauma in children is one of the most common reasons for visiting emergency department. however, only a small portion of patients will have a traumatic brain injury. patients with moderate or severe head trauma should undergo ct scan, however, a debate exists for the indication and yield of neuroimaging for minor head trauma. we performed a systematic literature review on the accuracy of symptoms and signs in children with minor head trauma in order to identify those with severe intracranial injuries. materials: a systematic literature search of medline ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was performed to identify studies assessing the diagnosis of intracranial injuries in children. the authors independently performed critical appraisal and data extraction. results: we identified studies that evaluated the performance of findings for detecting intracranial injury using the reference standard of neuroimaging or follow-up examination. mechanism of injury, multiple vomiting episodes and decline in gcs score were more commonly associated with severe intracranial injury on ct. normal variations in the amount of joint fluid, ganglion cysts, bone marrow edema, and bony depressions that resemble erosions are frequent in the wrists of children. the results of a follow-up of a healthy cohort aged - will be presented. the cohort was examined twice with mr of the wrist, and the second time also with a cartilage sequence for better visualization of the bony depressions. knowledge of these normal variations is important because they can resemble disease. bone marrow edema, joint fluid more than mm, and bony depressions that can resemble erosion are frequent findings in the normal wrist. take home points: bone marrow edema, joint fluid more than mm, and bony depressions that can resemble erosion are frequent findings in the normal wrist. these findings can not be attributed to dissease without additional findings of synovitis. a cartilage sequence can be of use in the differentiation between true erosions and bony depressions. mri scoring of the wrist in patients with jia-current status and future perspectives c. nusman; amsterdam/nl the wrist is a frequently affected joint in patients with juvenile idiopathic arthritis. due to recent improvements in treatment strategies, permanent damage is not that common anymore. also, imaging has been playing a key role in monitoring the disease activity in the wrist of jia patients. the past years lots of efforts have been made to improve the assessment of acute and permanent changes of the jia wrist. requisites and recommendations for the mri protocol to use for of the jia wrist are available in literature. currently, the features of scoring the jia wrist are synovitis, tenosynovitis, bone marrow edema and bone erosions. the repeatability of the above-mentioned scoring features proved to be acceptable. recent studies showed that the appearance of the wrist in healthy children can mimic pathology. therefore, construct validity of the scoring features needs to be assessed by comparing wrists of healthy children with the wrists of jia patients. & construct validity of the scoring features needs to be assessed by comparing wrists of healthy children with the wrists of jia patients a novel radiographic scoring system for permanent hip involvement l. tanturri de horatio , p.l. di paolo , s.c. shelmerdine , p. toma , k. rosendahl ; rome/it, london/uk, approximately - % of children with jia, particularly those with systemic onset disease, will have hip-involvement within - years after disease onset. as scoring systems for radiographic changes in children with hip involvement are lacking, we aimed to examine the reliability of potential markers and suggest a novel scoring system. a set of hip-radiographs from children with jia and clinical hipinvolvement: seen at the outpatient clinic at great ormond street hospital (gosh), london, and seen at ospedale pediatrico bambino gesù, rome, was used. all hip radiographs were scored in a blinded fashion, once by an experienced paediatric radiologist and a paediatric radiologist with minor experience in musculo-skeletal imaging in rome, and twice by an experienced radiologist and a research fellow in bergen/ london. radiographic findings suggestive of ) destructive change (bone erosion, flattening of the femoral head, squaring of the femoral head contour, presence of sclerosis, joint space height, and ) growth abnormality (length and width of the femoral neck, varus/valgus deformity, the ccd angle and the trochanteric-femoral head height) were assessed. assessment of erosions of the femoral head, femoral neck and the acetabulum showed moderate to good agreement for the same reader. the inter-reader agreement was, however lower. there was a high to moderate ( ) (suppl ):s -s pediatr radiol agreement for the assessment of femoral head flattening using the mose' circle. the measurements of femoral neck length and width, the ccd and trochanteric-femoral head lengths were precise, with % limits of agreements within - % of the mean. we have identified a set of relative robust radiographic findings suggestive of growth abnormalities and destructive change in children with hip-jia, and suggested a novel scoring system. x-ray of a years-old jia patient with severe chronic hip involvement. x-ray of a years-old boy with growth abnormalities on hips (bilateral coxa magna). in jia hip involvement is often a predictor of a severe disease course. radiographic findings vary according to mode of onset and age: in younger children the initial findings may be developmental rather than destructive while children with later onset jia may have destruction/narrowed joint space as the first feature. several of the commonly used radiographic findings for chronic hipchange are inaccurate. we have identified a set of relative robust radiographic findings suggestive of growth abnormalities and destructive changes in children with hip-jia, and suggested a novel scoring system. bone age assessment -statement from the msk task force k. rosendahl; bergen/no summary: age assessment is an important, yet complex and challenging issue that authorities may need to perform to determine whether an individual is an adult or a child in circumstances where their age is unknown. there is currently no method which can identify the exact age of an individual and there are concerns about the invasiveness and accuracy of the methods in use, namely analysis of documentary evidence, interviews, physical or other form of medical examination such as imaging. the main imaging methods include carpal, collar bone and dental examinations. whilst many countries make use of these methods they do not apply them in the same way and often use different combinations and/or order. one of the main reasons for this is the fact that age assessment procedures remain to a large extent determined by national legislation, with procedures evolving through national jurisprudence (ref.: european asylum support office (easo age assessment practice in europe)). the ethical and legal aspects of using bone age to determine age will be addressed in a statement from the msk task force. the ethical and legal aspects of using bone age to determine age will be addressed in a statement from the msk task force. & the application of drls should be the responsibility of all providers of x-ray imaging. this means that drls should also be applied to imaging performed outside the radiology department. & the physical quantity used to establish drls should be an easily measurable quantity, usually directly obtainable from the x-ray equipment console, obtained either by manual recording or preferably by automatic recording and analysis. organ doses and effective dose are not considered feasible as a drl quantity because these cannot be easily determined. the ultimate mission of eurosafe imaging is to support and strengthen medical radiation protection across europe following a holistic, inclusive approach. most common imaging procedures in children and their contribution to collective dose e. sorantin , c. granata ; graz/at, genova/it summary: several countries have released "diagnostic reference levels (drl)" for imaging procedures using ionizing radiation. unfortunately those drl differ in types of procedures and granularity as well as information about the proportion of pediatric patients within the different examinations are sparse. therefore an more evidence based approach seems to be feasable -meaning releasing drl first for frequent and radiation burdened examinations. therefore a survey within europe was conducted and a questionnaire was sent to key persons of the european society of pediatric radiology (www.espr.org) as well to members of a large academic, interdisciplinary, international network within the ceepus programme (central european exchange programm for university studieswww.ceepus.info). alltogether centers were contacted and an response was received from ( . .%). from one center only frequencies for interventional radiology was sent. plain films: most frequent procedures are extremities ( . %), followed by chest films ( . %) -both account together for more than ¾. flouroscopy: voding cysto urethrography (vcu) . %, followed by upper gastro intestinal (gi) series with . % -again representing / of those examinations. computed tomography: head & neck . %, chest . %, abdomen . % -together almost %. interventional radiology and cardiac interventions: only limited data available and procedures quite hardly standardize and comparable. it seems adviseable, that only a few procedures are suitable for drl like peripheral insertion of vascular lines, occlusion of ductus arteriosus botalli or stent implantation for coarctation. in order to estimate the contribution to the relative collective dose all values were normalized to a chest xrays ( . ) and the following numbers could be calculated: abdominal plain film . , skull . , ct head . , ct chest . , ct abdomen . . the most frequent imaging procedures using on ionizing radiation are: in plain films extremities and chest xrays in flouroscopy vcu and upper gi series in ct head, chest and abdomen therefore eu wide drl should be released for those examinations. as it could be expected chest ct is the main contributor to the collective dose. since the espr abdominal (gi and gu) imaging task force has changed its name and agenda, extending from initially only genitourinary queries to also other abdominal imaging topics, new projects have been added such as for example imaging in anorectal and cloacal malformations, imaging in paediatric inflammatory bowel disease (ibd, a joint project with esgar), or paediatric abdominal ceus applications. results of these new projects will be presented in the upcoming talkshoping that again (as the last procedural recommendations and proposed imaging algorithms) our proposals and recommendations will help to standardise paediatric imaging, to reduce radiation burden, and to facilitate comparable imaging data for future research. other topics in this session are a proposal for a more standardised approach to gastrointestinal ultrasonography, and considerations on gadolinium applications in children in the light of new observations (i.e., gadolinium deposit in tissue even in children with normal renal function). the work goes ononly achievable with active participation of interested and competent members. many interesting topics for either recommendations or joint research are on the list such as addressing late decompensating pujo or specific imaging needs in ibd in early childhood; other new ones may be proposed by any task force member. thus all espr members are invited to join the group, work with us and share their expertise. ( ) (suppl ):s -s pediatr radiol contrast enhanced us in childhood -applications in children: literature review and results from the questionnaire c. bruno; verona/it in adults, following the characterization of focal liver lesions, several applications of contrast-enhanced ultrasound (ceus) have emerged in the last two decades, since second-generation contrast agents have been introduced and approved for use in most european countries. from many points of view, children represent an ideal population for ceus, because of the absence of radiation exposure and of need of sedation. moreover, due to the small body size many anatomical targets in children can be adequately explored with high-frequency ultrasound, obtaining images with higher spatial resolution than in adults. however, to date comparatively few data on pediatric ceus are available. although very rare and usually mild, possible adverse effects of contrast agents probably limit their use in many centers. in addition, the intravenous administration of ultrasound contrast agents in children is still off-label in europe, which makes informed consent necessary in every case. finally, for unclear reasons information on this topic does not flow easily. & from the comparison between the data available, similar or better results are likely to be obtained with ceus in children than in adults, and some specific pediatric indications might be proposed. imaging in ibd-joint recommendation statement with esgar f.e. avni , m. napolitano , p. petit ; brussels/be, milan/it, marseille/fr the first joint esgar/espr consensus statement on the technical performance of cross-sectional small bowel and colonic imaging ( ) objective: to develop guidelines describing a standardized approach to patient preparation and acquisition protocols for magnetic resonance imaging (mri), computed tomography (ct) and ultrasound (us) of the small bowel and colon, with an emphasis on imaging inflammatory bowel disease. methods: an expert consensus committee of members from the european society of gastrointestinal and abdominal radiology (esgar) and european society of paediatric radiology (espr) undertook a six-stage modified delphi process, including a detailed literature review, to create a series of consensus statements concerning patient preparation, imaging hardware and image acquisition protocols in pediatric and adult patients. the delphi process is constructed as follow: step questionnaire construction to includes all contents relevant to the guideline and set up of working groups; step questionnaire completed by all committee member, step literature search; step draft consensus produced by each wg based on the literature review and questionnaire responses; step committee members indicate agreement or otherwise for each individual draft consensus; step acceptance of agreed statements (more than % of members), face to face meeting to modify statements without agreement. committee members indicate agreement or otherwise for each modified consensus statement and final consensus statements. the questionnaire was split into four broad topics, each of them treated by a subgroup including in each of them a pediatric radiologist: ( ) patient preparation for mre/mr enteroclysis/cte/ct enteroclysis, ( ) mre/ mr enteroclysis technique and sequence selection, ( ) cte/ct enteroclysis technique, and ( ) enteric us patient preparation and technique. after an extensive literature research each member were instructed to always base their statements on the retrieved literature wherever possible, and to this end graded the strength of retrieved relevant publications from i (high) to v (low) using the criteria of the oxford centre for evidence based medicine ( ) during their review process. if no relevant literature was available for a particular item, members used expert opinion to construct the consensus statements. the pediatric guidelines were based on the opinion of pediatric radiologists and adult radiologists who have experience in pediatric practice. & it is recommended that children aged - should not eat any solid & it is recommended that the use of a spasmolytic agent is optional. unlike adult practice, the use of spasmolytic prior to mre is considered optional in paediatric patients and use is likely dependent on the age of the patient, with older children more likely to tolerate spasmolytic injection. there are data supporting the benefits of glucagon on image quality, at the expense of prolonged imaging time and precipitation of nausea in just under half of paediatric patients ( , ) . however, high diagnostic accuracy can also be achieved without spasmolytic ( ) . & it is recommended that children aged over years should be nil by mouth for carbonated and milk beverages for - h. ingestion of still water or non-carbonated fruit juice is recommended. & it is recommended that for dedicated colonic evaluation, a standard protocol without specific modification is used. & use of a spasmolytic agent is not recommended. & the use of i.v. us contrast is not recommended. & it is recommended that scan coverage should include an abdominal and pelvic examination, including the liver. there are no specific recommendations as to the use of hydro us in the paediatric patient as practice is not well developed. if oral contrast is given prior to us, it would seem sensible to follow the recommendations for mre in the paediatric population & it is recommended that if ct scanning is used in the paediatric population, no specific preparation is usually required although administration of positive oral contrast could be considered; for example, prior to percutaneous drainage of abscesses. limitations: there is little evidence in the literature to ascertain all these proposals. the recommendations were mainly based on expert opinion. no recommendations have been proposed for children before years of age. especially the benefice of mre under sedation ( ) in the younger compare to us doppler need to be explored. contrast media application is essential for a number of mri studies in children. there is some evidence that gadolinium-based contrast agents (gbca) are well tolerated in infants and children. the risk of adverse reaction is no higher in children than in adults. there are only few data available about pharamakokinetics in children, especially for the use of gbca in neonates. age-adapted reference values of the glomerular filtration rate (gfr) have to be used to identify children with a potential risk. in the past few years there was some attention toward the potential cellular toxicity of gadolinium and its role in the development of nephrogenic systemic fibrosis (nsf). there were only few children identified with proof of nsf. but, particulary renal insufficiency, poor hydration, acidosis and inflammation increase the risk for nsf. because the cases of nsf have been observed with linear componds the guidelines ( ) (suppl ):s -s pediatr radiol from the esur and the espr and others propose to avoid linear compounds and to prefer macrocyclic gbca. in the past year several studies have described observations about possible gadolinium retention in the brain; hyperintense brain structures in native t weighted sequences were verified -globus pallidum and dentate nucleaus -also in children undergoing multiple mri examinations with gbca application. so, repeated mr investigations within a short time should be avoided -the cumulative dose of gbca should be recorded. consider all these points, the benefit of a contrast-enhanced study should be weighted against the potential risks before administering a gbca for each child separately. but, never deny a child an indicated cemri study. use single dose application ( . - . ml/kg body weight), improve renal function and hydration, balance acidosis -and ask your pediatric nephrologist íf necessary. gadolinium-based contrast agents are safe. macrocyclic compounds should be used in children. avoid contrast media in neonates and be careful in infants. identify risk factors. avoid repetivite application. procedural recommendation: how to perform pediatric gastrointestinal us m.l. lobo , m. riccabona ; lisbon/pt, graz/at summary: ultrasound (us) is the first imaging modality applied in the investigation of abdominal complaints in children, and an increasingly valuable imaging tool in the assessment of the gastrointestinal (gi) tract in neonates, infants and children. a comprehensive us examination is a critical first-step to optimize the potential of us diagnostic yield in many paediatric gi conditions. using proper high resolution transducers and graded compression technique is an essential part of gi us examination. a methodical and systematic analysis is crucial to facilitate a thorough evaluation of the bowel segments as complete as possible: follow bowel in a cross section, complete by longitudinal and oblique views. for some bowel sections filling is essentialsuch as stomach for gastroesophageal reflux and pyloric function, and distensibility and size of the colon by enema (e.g. for query microcolon). modern us methods are valuable, but not a pre-requisite. proper documentation of abnormal size of the gi tract segments, their luminal content, peristalsis, bowel wall characteristics and its surroundings, as well as local tenderness should be noted. a proposal for recommendation on how to perform paediatric gastrointestinal us will be presented for public discussion. & careful and dedicated us examination is crucial to obtain maximum anatomic and functional information in many gastrointestinal disorders in children. & systematic and methodical analysis helps to assess the bowel as complete as possible. & satndardization of us technique is essential to optimize us diagnostic capabilities and to allow for comparable examinations wich is essential to improve future evidence-based knowledge. hominid evo-devo: reconstructing the evolution of human development c. zollikofer; zurich/ch from an evolutionary biologist's perspective, modern humans represent the only surviving species of a group of highly specialized "bipedal great apes". they evolved more than seven million years ago in africa and managed to spread over the entire globe. in this talk, i will trace the history of our species with an emphasis on key developmental innovations that underlie major evolutionary innovations. why are we born with brains that have the size of adult great ape brains? and why do we grow up so slowly and get so old? i will highlight how advanced biomedical imaging methods help addressing these questions, and show how combined fossil, clinical and great ape data yield surprising insights into the evolution of our development. to present our experience with innovative imaging in pediatric interventional radiology. imaging technologies presented will include: . use of bubble contrast (lumeson) for indicatons including; complex pleural effusion and abdominal collection assessment pre and post therapy, primary g tube placement, renal perfusion pre and post rena artery angioplasty, vascular patency during central venous line placement, vascular malformation therapy and biliary tube assessment. . intravascular us (ivus) in arterial intervention pre and post angioplasty and venous thrombolysis intervention. . optical coherence tomography pilot study assesssment for renal artery intervention -validation in normal subjects. currently this imagng which uses laser light technology to assess vascular mural detail at the micron level, is only validated in coronary artery intervention in adults. . mr overlay -a technology that fuses mr imaging with low dose fluoroscopy and can faciltate biopsy of mr positive/ct negative lesions in the ir suite. focus will be on bone lesion biopsy and vascular malformation therapy. critical structures to be avoided can be outlined on the mr and transposed onto the fluoroscopic image during biopsy. in our experience this technology has promise in the pediatric setting with significant dose reduction when compared to ct. . mr fusion and i guide fusion technology enables an mr positive/ct negative lesion that would require ct guided imaging to be biopsed, using low dose c -arm ct, with fusion of the ct and mri images performed using landmarks, facilitating fluoroscopically guided biopsy in the ir suite. critical landmarks/structures to be avoided can be outlined on the ct or mr and transposed onto the fluoroscopic image during biopsy path planning and orchestration. focus will be on bone lesion biopsy. . color parametric flow related imaging in vascular interventionthis software enables time to peak opacification of arterial or venous contrast to be color coded in time and can provide adjunctive information for assessent of perfusion change during vascular intervention such as renal artery angioplasty, dialysis access intervention and cerebral embolization. . mr guided intervention -this focus will be on the initial development of an mr interventional program and our initial experience with mr arthrography. discussion will also involve the use of this modality for vascular malformation sclerotherapy and other msk interventions such as biopsy and nerve injections. . high frequency us imaging-focus will be on the use of a mhz us probe in the ir suite for various indications including visualization of smaller targets such as neonatal central venous access, superficial vascular malformation therapy and thyroid fine needle biopsy. . participants will become more familiar with exisitng and emerging innovative imaging technologies for pediatric intervention. participants will learn about the various indications and limitations of these technologies. . participants will gain insight into the process of introducing new imaging modalities into their pediatric interventional practice. increasing evidence supports the notion that autism spectrum disorder is associated with anomalies of brain function and connectivity. it is also evident that there are atypicalities in development/maturation of brain systems. particular promise arises from findings of atypical electrophysiology -indexing brain neuronal activity in real time. in particular, this talk will address a characteristic electrophsyiologic signature of delayed auditory evoked response latency (at~ ms). this, and related timing anomalies, have been proposed as biomarkers for asd -with candidate use for diagnosis, prognosis, stratification and therapy monitoring. progress along each of these axes will be discussed. however, to justify the term "biomarker", we demonstrate converging evidence from spectrally-edited (megapress) mrs and diffusion-mri. mrs offers insights into neurotransmitter levels, especially gaba and glutamate, imbalance of which may be associated with anomalous electrophysiologic oscillations in the gamma band. diffusion offers insights into the white matter of the brain (auditory pathway will be illustrated) and an interpretation of diffusion parameters as an index of central conduction velocity will be offered. combining these mechanistic measures with the spectrospatio-temporal capabilities of magnetoencephalography (meg), this talk will present a state of the art review of multimodal biomarker development in asd. take home points: meg captures brain activity in space and time as well as showing sensitivity to activity at different frequencies (where, when and what) delays in cortical neuronal response latency are evidence in asd atypical coupling between diffusion evidence of conduction velocity and timing of cortical responses in shown in asd oscillatory activity is atypical in asd (elevated "noise", decreased "synchrony") diminished inhibitory neurotransmitter (gaba) levels are shown in asd disturbance of teh typical coupling between gaba and gamma-band oscillations in development leads to anomalous adult oscillatory activity (taken to index local circuit function). multimodal and longitudinal approaches may be required to tackjle the complex and heterogeneous landscape of asd the paediatric radiologist can play an important role in establishing vascular access in paediatric patients ranging from neonates to teenagers. a breadth of knowledge and skills are needed to deal with changing body morphology and varied pathology in this age range. some of the skills particular to performing and managing vascular access in children will be discussed. different devices which can be placed, their indications, advantages and disadvantages will be reviewed. choice of access vessel is important in children, because there are known long term complication such as central venous stenosis and thrombosis, which can have a huge impact for future venous procedures or potential creation of an arteriovenous fistula of the arm for dialysis. preserving venous access sites is a ( ) (suppl ):s -s pediatr radiol key responsibility especially in children with complex medical and surgical co-morbidities. because vascular access in children has associated morbidity it's important to manage and maintain devices that are placed. the risk of infection when repairing or exchanging a broken line will be highlighted. image guided biopsy is a very frequent procedure in pediatric patients. they range from random organ parenchyma for the diagnosis of medical disease up to tumor biopsies for histopathology analysis. different imaging modalities can be used for guidance as well as different biopsy devices and needles. ultrasound guidance is the most common modality used for this purpose in the pediatric population. the success of this procedure depends on multiple factors: from pain control up to choosing the correct device and area to sample. the radiologist performing the procedure also needs to be familiar with the potential complications of the intervention, how to prevent them and how to manage them. the intention is to perform the safer procedure as possible, obtaining the best quality of sample. the goal of this lecture is to present in a didactic way technical tips to perform safe and effective image guided pediatric biopsies, which may be applicable to different groups of operators, ranging from general pediatric radiologists performing occasional biopsies up to pediatric interventional radiologists. the objectives will be: to identify the safest approach to different types of biopsies; to describe ways to obtain the better quality of sample as possible; to demonstrate the use of different approaches in challenging clinical scenarios; to illustrate new devices currently used in specific applications; to discuss potential complications and its management and to show imaging modality integration applied to biopsy planning an performance. image guided biopsy is a frequent procedure in pediatric patients. a pre-procedure planning is fundamental in the success of the intervention. the operator must be aware of the aims of the biopsy and based on this choose the best approach, device and site for sampling. preparation and competency to manage complications is mandatory. pediatric interventional oncology: big cases in little people m. heran; vancouver/ca summary: the pediatric patient presents unique challenges in diagnosis and management of oncologic disorders. interventional radiology (ir) has a prominent role in the care of these children, with improvements in imaging and equipment offering better and safer options to traditional diagnostic and therapeutic procedures. as cancer can involve any organ system, consultations to the ir service can involve any part of the body, and can be non-vascular and vascular, simple and complex. the most common ir procedures in the pediatric oncology patient are enteric tube placement/change, vascular access, and percutaneous image-guided tissue/organ biopsy. however, with the explosion of interventional oncology in the adult setting, the variety and complexity of ir in pediatric oncology has begun to increase as well. ir techniques, such as thermal ablation, transarterial pharmacotherapy, and preoperative embolization, are now increasingly discussed in multi-disciplinary conferences as complementary or primary modes of treatment of oncologic disorders or related diseases/complications. however, although the principles of these diagnostic and therapeutic ir procedures remain essentially the same in their translation from adults to children, well recognized differences in pediatric physiology and metabolism, as well as the range in weight, size, and age of children, result in a practical question of "how do we do this?" the aim of this presentation is to provide an overview of the role of ir in the pediatric oncology patient, and to highlight areas of research and innovation. vascular anomalies encompass a spectrum of disorders including vascular tumours and vascular malformations. incorrect nomenclature and misdiagnoses resulting in inappropriate treatment are commonly experienced by patients with vascular anomalies. the currently accepted method for classification of vascular anomalies is straightforward and clinically relevant. vascular malformations can be divided into high flow lesions such as arteriovenous malformation or low flow lesions such as venous or lymphatic malformations. in children, a diagnosis can often be made with the history, examination and ultrasound. the classification of vascular anomalies will be briefly reviewed with examples of commonly encountered pathologies. a multidisciplinary team approach to the management of these conditions is vital. paediatric radiologists can play a key role not only in diagnosis but also in management, principally by injection sclerotherapy of low flow lesions and embolization of the much rarer arteriovenous malformation. many sclerotherapy agents are available with sodium tetradecyl sulphate the most commonly used for venous malformations and doxycycline for lymphatic malformations. different sclerotherapy agents have different characteristics and uses which will be covered. symptomatic relief is often achieved with treatment but multiple treatment episodes may be needed to achieve the desired outcome. ensuring the child and family understand this is vital to ensure they are satisfied with the management of the condition. contrast media is commonly used during imaging in children whatever their age and whatever the pathologic conditions. still, youngest patients are vulnerable and unstable. therefore, in neonates and infants the use of s ( ) (suppl ):s -s pediatr radiol contrast media should be carefully evaluated and customized putting in balance the risk versus the benefit of its use. when using contrast media in neonates and infants, several features should be highlighted: -prematures and neonates have rather immature kidneys and some contrast media might be difficult harmful -the thyroid gland in prematures may be (transitorily) depressed by iodinated contrast media -the use of high osmolar contrast may induce a fluid shift and dehydration especially in premature and neonates -most contrast media are used off label; almost none has obtained the authorization to be used in neonates. -there are very few studies evaluating the short and long term adverse reactions in neonates and infants below the age of two. fortunately these reactions seem very rare in these age groups. -using contrast extends the duration of the examination and the need for sedation different types of techniques will potentially need ingestion, instillation or injection of contrast media: ) opacification of the entire gi tract pre-and post-operatively ) retrograde uretro-cystography ) contrast enhanced ct ) contrast enhanced mr imaging ) contrast enhanced us ) angiography furthermore, different types of contrast media can be used to achieve these purposes ) barium (sulfate) ) iodinated water-soluble contrast media (hyper-, iso-or hypoosmolar) remarks regarding opacification of the upper gi tract: -the upper or lower gi tract should be opacified using water soluble contrast in the immediate postoperative period or whenever a bowel perforation is suspected. -air can be used to confirm esophageal atresia and duodenal atresia -barium should be preferred in case of t-e fistula -either barium or water soluble iodinate contrast can be used in order to opacify (sub)obstructed upper gi tract remarks regarding the opacification of the lower gi tract -iodinated iso/hypo osmolar contrast should be used to opacify the colon in case of obstruction -a higher osmolarity iodinated contrast can be used in case of suspected meconium ileus or plug; still this contrast should be used diluted and under close clinical surveillance and adequate hydration. -in some more specific cases, for instance whenever hirschprung disease or a stenosis post nectotizing enterocolitis are suspected, barium enema can be used remarks regarding ct scan -contrast enhancement may help for the global assessment of various pathologies especially in case of cardio-vascular malformations or for the evaluation of abdominal masses. any iodinate contrast among those available is acceptable in neonates. higher osmolality contrast allows to inject a lower volume -injected volumes of . ml/kg seem adequate using - gauge needles -power injectors are acceptable as long as adequate catheters can be used -allergic or side effects are very rare and should be managed similarly to adults. remarks regarding mr imaging -the use of gd chelates in neonates remains controversial as there is no data available on the long term effects of gd injected so early in life -gd should be used only when enhancement may provide additional information compared to the non-enhanced study (cns infections, tumors, cardiovascular imaging, abdominal tumors, uro-mr imaging...) -only gd with low nsf risk should be used -gd should not be used in children with renal failure remarks regarding contrast enhanced us -little is known about the use of ce-us in neonates -indications seem equal to older children -there are very few side or allergic effects -doses suggested are . ml/year of age children present varied histological types of brain tumours. it's now possible to combine different information and image techniques to improve the diagnosis of paediatric brain tumours. the multimodal approach has increased the diagnostic specificity and permits, in most cases, the pre-operative differentiation between low and grade tumours. children with low grade lesions, and in particular the less accessible tumours, would benefit the most from avoiding biopsy. in addition, preoperative spinal mri evaluation to rule out drop metastases should be performed in patients with suspected high grade tumours. in general paediatric brain tumours are less necrotic, i.e. aggressive tumours in paediatric patients tend to be more hypercellular and homogeneous. because of its ready availability and speed, computed tomography ( ) (suppl ):s -s pediatr radiol (ct) is the first investigation generally performed for a suspected brain tumour. ct can rule out haemorrhage or calcifications, but can also be used to evaluate tumour cellularity. a hyperdense tumour on ct reflects hypercellularity and is very often high grade. medulloblastoma are, for example, typically hyperdense on ct scans and paediatric low-grade astrocytomas are almost always hypodense. mri plays a major role in the evaluation of brain tumours. in conventional mri, the "general aspect" is the single most important parameter in predicting high-grade tumours in children. the same does not hold true for low-grade tumours, of which only % can be predicted using the general aspect. in our previous study, hyperintensity on t -w and the lack of diffusion changes were the most important single parameters with % positive prediction. embryonic tumours, such as medulloblastoma or pnet have high tumour cellularity with consequent very low adc and hypo/isointense t compared to the cortex. adc values derived from dwi have been shown to be decrease in highly cellular tumours. adc values cannot reliably be used in individual cases due to the substantial overlap between tumour types previously described in the literature. nevertheless, adc has a higher predictive value in children and increases the accuracy of preoperative differentiation between low grade and high grade paediatric tumours. the cut-off values for differentiating between low and high grade paediatric brain tumours are . x mm /s and . x mm /s for minimum adc and average adc values, respectively. perfusion with relative cerebral blood volume (rcbv) is considered a marker of angiogenesis and is helpful in distinguishing high and low grade tumours. however, perfusion can be difficult to perform in small children; small catheters with manual injection are therefore used in such cases (or, as an alternative, arterial labelling). it should however be taken into account that choroid plexus tumours can have high rcbvs resulting from highly leaky capillaries. mr spectroscopy (mrs) shows the metabolic profile of the tumour. high grade tumours show elevated choline (cho) -reflecting increase in cell membrane turnover -and decreased n-acetylaspartate (naa), which represents a neuronal marker. the absolute values of the mrs peaks are not used by us; we favour to normalize the signal intensities of metabolites to their values in contralateral brain tissue. mrs is helpful not only as guidance for stereotactic biopsy (cho hot spot) but also for determining whether the tumour is high or low grade. as a rule of thumb, a % increase of cho when compared to the contralateral brain tissue is highly suggestive of a high-grade tumour. however, in children, increased cho levels can also be found in pilocytic astrocytoma; in this case the typical aspect with cystic component and location can suggest the diagnosis, despite the mrs result. therefore, in children, high cho levels do not necessarily imply the presence of a malignant tumour. task based functional mri (fmri) can be used for pre-operative localization of the eloquent cortex together with the identification of the language and somatomotor function. in the future, small children who are unable to cooperate will probably profit from resting-state fmri. pet mri has the advantage of integrating structural mr imaging with physiologic pet. take home points: take home points although the histology of paediatric brain tumours is diverse, their general morphological aspect on mri has a very high diagnostic reliability. unlike adult grade iv brain tumours, malignant paediatric brain tumours are less necrotic, but are highly cellular with high nuclear-to-cytoplasmic ratios. adding information on signal intensities on t w and dwi further increases the diagnostic accuracy of conventional mri. the solid areas of high-grade tumours are iso-or hypointense on t w and hyperintense on dwi, whereas low-grade tumours show inverse signal characteristics. advanced mr techniques (perfusion and spectroscopy) provide important biological information which can be used to correctly identify grading (high vs. low) and to guide biopsy. in children high cho levels, although suggestive, do not necessarily mean a malignant tumour. experience with central review of paediatric renal tumours g. khanna; st louis/us summary: central imaging review of pediatric renal tumors has been performed in children's oncology group since . to date, more than cases of pediatric renal tumors have been centrally reviewed real time by the study radiologists. the mean time for central review was < days. discrepancies between local and central risk stratification were identified for detection of bilateral disease and pulmonary metastasis. in addition, central archiving of images has created a rich repository of cases for future research. the role of imaging in detection of key diagnostic features in pediatric renal tumors will be reviewed. the diagnostic performance of imaging for staging, detection of vascular invasion and tumor rupture will be discussed. real time central review of imaging is feasible in pediatric oncology wilms tumor remains the most common pediatric renal malignancy, followed by renal cell carcinoma cystic nephroma typically presents as a bosniak lesion, and has high association with dicer- mutations is there a role for dwi in nephroblastoma? a.s. littooij; utrecht/nl wilms tumour or nephroblastoma is the most common malignant renal tumour in children. ultrasound is usually the first line investigation. mri of the abdomen is often performed to further delineate the tumor and its surroundings. the addition of diffusion-weighted imaging (dwi) to the standard mri protocol may enable subtype characterisation and allows assessing treatment response beyond necrosis and volume change. overall, the survival rate in patients with nephroblastoma is relatively good and the current focus is on finding biomarkers to further improve outcomes while reducing therapy-related side effects in these children. therefore, identifying low-or high-risk type nephroblastoma might be relevant for treatment planning. diffuse anaplastic nephroblastoma and extensive blastema in residual tumour after preoperative chemotherapy may require more intensive treatment. the limited available literature suggest a linear relation between adc values and subtypes nephroblastoma at histopathology. furthermore, the addition of dwi to the standard mri protocol may detect lesions (e.g. nephrogenic rests of nephroblastomatosis) that remain undetected at post contrast t -weighted images. unfortunately, there is a considerable heterogeneity in acquisition techniques and methods of adc measurements. nephroblastoma often contains areas of necrosis and/or hemorrhage that can demonstrate very low adc values and consequently mimic highly cellular portions of tumours. therefore these areas should be excluded from further analysis. this lecture will highlight the potential additional benefit and limitations of dwi in children presenting with renal tumour. significantly lower radiation exposure even in comparison to low-dose pet/ct, (b) the higher diagnostic accuracy as compared to pet/ct even when using diagnostic contrast-enhanced ct, (c) the unique possibility to combine distinct mr-inherent contrasts (e.g. dwi) with specific pettracers (e.g. cu-labeled antibody imaging) for the evaluation of novel targeted therapies, and (d) the opportunity to stage local and systemic tumour burden within a single and highly resolved examination. on the other hand, many circumstances are challenging the extensive use of pet/mri in children. in general, the availability of pet/mri systems is low, particularly for children. thus, only a few sites in europe have experience with this technique in children, and therefore the generated scientific evidence is limited. moreover, whole-body-mri is still not a broadly adopted method for the combined assessment of local disease extent and whole-body staging, potentially replacing other whole-body modalities like the bone scan. in this context, especially the detection of pulmonary metastases is biased also against pet/mri. finally harmonized sequence protocols and specific recommendations for trace dosage are not available for pet/mri. in conclusion, further efforts are needed to keep the promises of pet-mri in the daily practice. common artefacts in paediatric mri-how to recognise, avoid or take advantage of them c. kellenberger; zurich/ch summary: while mri is a robust and radiation free imaging technique for assessing anatomy and pathology of most tissues and organs throughout the body, it is inherently prone to artefacts as no other imaging modality is. mri artefacts may impair image quality potentially leading to difficulties or errors in interpretation, but in some instances can contribute diagnostic information. main sources of image degradation are motion, disturbances of the local magnetic field and other factors inherent to image acquisition. strategies to reduce effects from various kinds of motion and adjustment of sequence parameters for eliminating artefacts will be discussed. & understanding the origin and effects of artefacts encountered in paediatric mri is essential for modification of mri protocols, so that artefacts and associated errors can be avoided. & for safely and successfully imaging children with implants and devices, the composition, location and functionality of the foreign body needs to be known. injuries to the central nervous system in abusive head trauma are responsible for the primary cause of morbidity and mortality in infants. neuroradiology has an important role in diagnosis but also in depicting injury and extent of brain damage of poor outcome. computerized tomography (ct) and magnetic resonance imaging (mri) are the primary imaging techniques. ct is usually performed in the acute phase while mri is performed the following days after injury. some injuries are better identified on mri such as diffuse axonal injury and cerebral edema with susceptibility and diffusion weighted images. abusive head trauma (aht) is the primary cause of morbidity and mortality in infancy, especially during the first year of life. aht is clinically characterized by a triad consisting of subdural hematoma, retinal hemorrhage and encephalopathy caused by brain swelling ( ). the most common mechanism responsible for brain damage is thought to be caused by whiplash shaking injury explaining that abusive head trauma is also referred as shaken baby syndrome. impaction, compression and penetrating injury are also possible mechanisms as well as strangulation. however because of the variability of types and severity of injury, clinical symptoms vary from subtle to severe such as alteration of consciousness or coma ( ) . the most common symptoms include vomiting, seizure, lethargy, poor feeding and apnea of which vomiting and respiratory pauses are non-specific ( ). poor feeding, irritability or lethargy is also nonspecific signs. however apnea and/or retinal hemorrhages seen in children with brain injury are strongly associated with inflicted trauma ( ) . in contrast to acute injury some children may manifest with increased head circumference related to chronic subdural hematomas. neuroimaging is therefore playing a crucial role to assess infants and children with a suspicion of abusive head trauma. computerized tomography (ct) and magnetic resonance imaging (mri) are the primary imaging techniques. ct is performed for the initial evaluation in cases with acute symptoms to look for hemorrhagic intracranial injury as subdural hematoma. mri is more often performed in the following days to further evaluate brain injury and to look for spine and spinal cord damage ( , ) or in the presence of normal or equivocal ct findings ( ) . however brain mri may be the first option in children presenting with increased head circumference. recently the study from flom et al showed the high sensitivity of mri for intracranial hemorrhage in well appearing infants at risk for abusive head trauma suggesting mri as a screening tool with pulse sequences (axial t , axial gradient recalled echo and coronal t weighted inversion recovery) ( ) . ct is generally performed without intravenous contrast injection with d volume rendering (vr) reconstructions for identification of fractures. in some cases postcontrast images are also obtained specially to rule out deep venous thrombosis especially when children present with nonspecific clinical symptoms. mri protocol should include axial t , t * or susceptibility weighted images, coronal t images, diffusion or diffusion tensor images, and postcontrast dt images including mip reconstructions to evaluate the venous structures. mr venography can also be performed. susceptibility-weighted images are usually preferred because they allow the depiction of smaller hemorrhagic dai lesions and greater number of lesions compared to gre t ( ) . it was also reported by colbert et al ( ) that the presence of micro-hemorrhages alone was useful for outcome prediction in abusive head trauma with significant poor long-term outcome. the sensitivity and specificity of microhemorrhages was also higher than the other clinical (such as retinal hemorrhages and glasgow coma scale score) and other imaging findings for prediction of outcome. diffusion tensor imaging (dti) measurements were reported in abusive head trauma by imagawa et al: decreased axial diffusivity related to axonal injury with consequent reduced mean diffusivity did correlate with poor outcomes ( ) . magnetic resonance spectroscopy (mrs) is usually not part of the standard protocol. however aaen et al ( ) showed that n-acetylaspartate/creatine and/or nacetylaspartate/choline ratios were decreased significantly in the corpus callosum, frontal white matter, parieto-occipital white matter, and parietooccipital gray matter in children with poor outcomes. this study mentioned above also reported that the prediction of outcome was accurate in % of patients by using a logistic regression model that include age, initial glasgow coma scale score, presence of retinal hemorrhage, lactate on mrs, and mean total n-acetylaspartate/creatine. functional mri, ( ) (suppl ):s -s pediatr radiol volumetry may be performed in long-term follow up of victims of child abuse. physical abuse is associated with altered emotion with greater activation in the salience network in response to negative stimuli, that includes amygdala, thalamus, putamen and anterior insula ( ) . increased responsiveness of the right amygdala to fearful and angry faces (negative stimuli) and structural changes as reduced hippocampal volume, are reported by dannlowski et al ( ) . impaired attention was also reported in patients with childhood abuse ( ) with reduced activation during attention tasks in the left hemispheric ventral and dorsolateral prefrontal regions. intracranial injuries include extracerebral hemorrhages and parenchymal damage as brain swelling and ischemia, venous infarction, diffuse axonal injury, contusions and intraparenchymal hematomas ( , ) . extracerebral hemorrhages subdural hematoma is a characteristic finding of inflicted traumatic brain injury, is generally multifocal and most commonly seen along the posterior interhemispheric scissure, over de convexities at the vertex level and/ or in the posterior fossa ( ) ( ) ( ) . subdural hematomas are most likely bilateral but may be unilateral. all locations are related to disruption of bridging veins. the identification of bridging vein rupture allows the diagnosis of traumatism in relation to acceleration/deceleration, rotational and shearing forces due to violent shaking ( ) . a mixed density appearance of subdural hematomas is frequent but is also seen in accidental traumatic brain injury ( ) ( ) ( ) . indeed this feature is often present in the very early hours following trauma and is thought to be secondary to early sedimentation of blood clots and supernatant serum. tubular high density is often seen on non-contrast ct over the convexities in abusive head trauma. this ct feature is related to a clot secondary to venous disruption ( , ) that can end up in thrombophlebitis. this tubular high density was reported more recently as tadpole sign ( ) and lollipop sign ( ) respectively seen in and % of abusive head trauma. this appearance is strongly associated to inflicted trauma and much less frequent in accidental trauma ( out of cases ( , %) of accidental trauma in our experience). associated venous infarction is reported in % of cases of abusive head trauma ( ) and often located in the parieto-occipital region, unilaterally at the site of venous disruption of bridging veins. subdural hemorrhages, when multiple, in the convexity and interhemispheric, or in the posterior fossa were found significantly associated with abusive head trauma in the meta-analysis reported by kemp et al ( ). in addition subdural hematoma, cerebral ischemia, skull fracture, retinal hemorrhage and intracranial injury were significantly associated with abusive head trauma in the review from piteau et al ( ). subarachnoid hemorrhages (sah) and epidural hematomas are also found in inflicted trauma and are not considered discriminant-imaging features. however epidural hemorrhages, isolated skull fracture and scalp swelling were reported as significantly associated with accidental traumatic brain injury ( ). sah in shaking injury is usually caused by tears of the vessels within the pia and arachnoid predominantly in the interhemispheric fissure and high convexity ( ). parenchymal injury parenchymal injury include brain swelling and ischemia, venous infarction (discussed above), diffuse axonal injury related to rotationallyinduced shear-strain injury with different inertia for grey and white matter due to their different specific gravities, contusions seen in deceleration trauma with friction between the skull and brain, and in blunt trauma and intraparenchymal hematomas related to lacerated vessels. brain swelling/ischemia may be related to increased blood volume (congestive swelling), increased presence of water in the nervous tissue, and the combination of both. increased water in the nervous tissue may manifest as vasogenic edema located in the white matter due to extravasation of plasma like fluid related to incompetent blood-brain-barrier and as cytotoxic edema located in the grey matter, related to ionic imbalance. cerebral edema can be recognize on ct within the hours following injury as loss of gray-white matter differentiation and decreased attenuation of grey and white matter. cytotoxic and vasogenic edema are better characterized on mri with diffusion-weighted imaging. brain swelling and edema occur early after trauma with consequent underestimation of subdural hematoma. therefore imaging should be repeated (ct or mri) especially when neurologic symptoms change rapidly. brain swelling/ edema may also involve the posterior fossa and is better identified on brain mri. two frequent patterns have been reported in abusive head trauma ( ). diffuse supratentorial brain swelling (infarction) involving the cortex and white matter was reported in % of cases and is considered as severe hypoxic-ischemic injury with poor outcome ( ). watershed infarction was reported in % of cases and considered a less severe form of hypoxia-ischemia. apparent diffusion coefficient (adc) values are strongly associated with poor neurodevelopmental outcomes in the acute phase (within days) especially basal ganglia, thalamus, brainstem, cerebral cortex, cerebellar vermis, cerebellar cortex and mean total brain ( ). during the early phase up to month adc values in fewer regions (basal ganglia, thalamus, brainstem and corpus callosum) were associated with poor outcome. when patients with and without parenchymal lesions are compared, the detection of diffuse lesions during the first months as well as beyond months is significantly associated with severe developmental outcome ( ). late mri (beyond months after injury) also showed that recovery depends on the extent of brain damage. patients with diffuse lesions show more severe motor and intellectual impairments and are more likely to have blindness and epilepsy than patients with focal or hemispheric lesions ( ). diffuse axonal injury (dai) is related to shear-strain injury of small medullary veins and was reported in % of cases of abusive head trauma ( ). it is encountered in trauma with sudden acceleration-deceleration associated with rotational angular forces and in shaking-impact trauma. the lesions may be hemorrhagic or non hemorrhagic (related to axonal swelling). dai is most often located in the subcortical white matter at the gray-white matter junction, corpus callosum, basal ganglia, brainstem and internal capsule. if the lesions are large enough and hemorrhagic dai may be seen on ct. however dai is usually better identified on mri with susceptibility and diffusion weighted images. the detection of changes in the basal ganglia or brainstem during the first days as well as during the first month after injury is significantly associated with poor long-term outcome in survivors ( ). the presence of intraparenchymal brain micro-haemorrhages detected on swi in children with abusive head trauma correlates with significantly poor long-term neurologic outcome ( ) contusion is also reported in abusive head trauma and is seen in blunt trauma with impact with or without contrecoup contusion. contusions are located at the surface of the brain (crest of gyri) and may be pial and haemorrhagic (disruption of cortical arteries). they are also found in the frontal and temporal regions related to impact of the brain on the roof of the orbit, middle cranial fossa and sphenoid wing. white matter tears are also seen in the frontal and temporal area related to the vulnerability of unmyelinated and soft white matter in infants. skull fractures are seen in blunt impact and are less frequent than long bones and rib fractures in non-accidental trauma. the most common site is the parietal bone (because of bulging of parietal bones below year of age). the fracture may be linear as in accidental trauma. radiologic features significant for inflicted trauma are multiple fractures, bilateral fractures and fractures that cross suture lines ( , ). focal underlying brain damage can be seen such as subdural hematoma and hemorrhagic contusion. hypoxic-ischemic encephalopathy is seen in strangulation injury with involvement of the territories of the internal carotid artery related to their anatomic vulnerability. neuroradiology (ct and mr) is crucial for the diagnosis of trauma, to predict outcome when showing edema and hypoxic-ischemic injury. this presentation will present an update on post mortem mri (pmmr) with relevance to clinical developments over the last years. in particular, reference will be made to diagnostic accuracy of pmmr across different body parts, the current limitations of post mortem mr, and protocol development at different field strengths. imaging correlates of post mortem interval are also being investigated. maceration (autolysis within intrauterine fluid) and perimortem hypoxic brain changes caused difficulties in image interpretation, which more advanced and quantitative techniques may be able to address. jawad take home points: below g, . -t pmmr shows a significant reduction in diagnostic yield, compared with conventional autopsy, and therefore its clinical usefulness in this setting will depend on individual circumstances. t pmmr performs better than . t particularly < weeks gestation, and particularly for the chest, heart and abdomen. diffusion characteristics in different fetal brain areas are multifactorial, with maceration the strongest predictor in most areas. international pm ct protocols c.y. gerrard , o.j. arthurs ; albuquerque, nm/us, london/uk the european society of pediatric radiology (espr) taskforce and the international society of forensic radiology and imaging (isfri) pediatric working group have combined efforts to establish best practice standards for performing perinatal and pediatric post mortem computed tomography (pmct) examinations. use of pmct in the investigation of pediatric death has increased significantly in the past decade. due to quick acquisition times and the ability to acquire thin slice, high detailed images of the whole body, ( ) (suppl ):s -s pediatr radiol many hospitals and forensic institutes have implemented pmct into daily practice. however, there lack an overall standardization of how cases are triaged and the acquisition methods when comparing institutes using pmct. in an effort to address inconsistencies in acquisition parameters, post processing, and case selection, pmct protocols were compiled from international institutes and centres currently performing pediatric imaging. this paper will describe both the uniform and divergent elements of image acquisition and procedural uses identified among the participating centres. the outcome is to provide a single source of information that can guide already established and new centres on the best practice standards for implementing pediatric pmct. take home points: describe how pediatric post mortem computed tomography (pmct) has increased in utility over the past decade. identify the differences in acquisition methods for clinical computed tomography versus post mortem computed tomography. discuss the overall consensus of case triage and scan acquisitions when comparing institutes in aggregate. provide comprehensive statement of best practice standards for pediatric pmct. post mortem imaging research: updates and future proposals o.j. arthurs; london/uk paediatric and perinatal post mortem imaging is a new and rapidly growing field, and the post mortem imaging taskforce was founded in graz at espr . the pmi taskforce aims to help reach consensus and guidance regarding imaging protocols and the potential yield of post mortem ultrasound, ct and mr. the key priorities are the themes of collaboration, image acquisition, best practice guidelines, training and education, raising awareness and access to imaging. this presentation will give updates on the latest developments in perinatal and paediatric imaging, with particular focus on where the pmi taskforce can help. in particular, protocol development is underway, and the espr meeting acts as an opportunity for collaborative working and network development, to facilitate best clinical practice and welcome new members. arthurs oj et al., espr post mortem imaging task force: where we begin. pediatr radiol ( ) ; : - take home points: post mortem imaging is an exciting sub-specialty which requires a combination of in depth fetal medicine, perinatal autopsy and pediatric imaging knowledge to help shape and grow the clinical and research arena. dedicated personnel have an opportunity to create the evidence-based behind a growing clinical service, with clear benefits to patients, families and referring clinicians. abstracts appear as submitted to the online submission system and have not been checked for correctness and completeness. sequences, are an emerging tool for evaluating intracranial vessel disease. improved survival due to emended treatment protocols results in an increasing number of long-term medulloblastoma survivors who experience delayed treatment effects. microbleedings, developement of cavernomas, vasculitis and atherosclerotic lesions are cerebrovascular structures affecting sequelae of the applied radiochemotherapy. this study evaluates radiation-induced intracranial vascular changes. twenty-two long-term pediatric medulloblastoma survivors (mean age . years, range - years; mean years after primary radiochemotherapy . years, range - years) underwent mri. the scan protocol included precontrast -dimensional time of flight (tof)magnetic resonance angiography (mra), precontrast d t -and d t -vwisequences and postcontrast d t -vwi-sequences of the medium and large intracranial arteries. vessel wall thickening, contrast enhancement and luminal narrowing were analyzed. additionally precontrast t -, t -swi and t -weighted images of the supra-and infratentorial brain were acquired. results: vwi-sequences: vessel wall changes could be found in ( %) and patients ( %) of the right and left ica, respectively. for the ba ( %) patients revealed vessel wall changes; for the left and right va ( %) patients were detected with vessel wall changes, respectively. in the tof angiography no alteration of the ica, ba or vas could be identified. in total vessel wall changes for the vertebrobasilar system and the icas could be found in ( %) patients. swi-sequences: all patients ( %) revealed swi lesions, the smallest lesion measuring less than mm, the biggest up to mm. sixteen patients ( %) were presented with lesions > mm, suspicious for cavernomas. to ensure quality of life in long term childhood medulloblastoma survivors, monitoring of long-term effects, like vascular changes after rct is gaining in importance. high resolution mri, including swi and vwisequences could be used here for. this study images, asymptomatic vessel wall alterations in former childhood medulloblastoma patients through vwi sequences and micro bleedings through swi sequences. vessel wall alterations, revealing rct induced arteriosclerosis, can lead to symptomatic intracranial stenosis which is associated with ischemia, furthermore micro bleedings and cavernomas can lead to intracranial hemorrhage. however further studies are needed to standardize mri sequence protocols to ensure a high standard follow up protocol, detecting clinically still asymptomatic vascular changes. fast "black-bone" mr imaging in evaluation of craniofacial abnormalities: comparison with high resolution ct z. habib, a. talib, c. parks, s. avula, l.j. abernethy; liverpool/uk to evaluate the feasibility and diagnostic value of a fast field echo, "black bone" mri sequence in children with craniofacial abnormalities. a fast "black bone" mri sequence has been used in addition to standard brain mri in children (mean age months, age range months to years and months) referred to the supra-regional craniofacial surgery unit at alder hey children's hospital, liverpool, uk. a subgroup of of these patients with complex craniofacial abnormalities additionally had high resolution volume ct performed at the same visit. "black bone" mr imaging was performed on philips ingenia t and . t scanners, using a d fast field echo sequence (tr= . ms, te= . ms, flip angle ). this sequence can be performed with an acquisition time of less than minutes. the "black bone" sequences were assessed for accuracy in evaluating the patency of the sagittal, coronal and lambdoid sutures, and, where applicable, were compared with high resolution ct. the fast "black bone" mri sequence was shown to be technically feasible in all cases. the resultant images successfully demonstrated both patent sutures, which were confidently seen, and prematurely fused sutures which were confidently not seen. visualisation of patent sutures was found to be further enhanced by the use of minimum intensity projection. in the subgroup of patients with complex craniofacial abnormalities, comparison with high resolution volume ct confirmed good sensitivity for patency of cranial sutures. there was complete agreement in out of sutures assessed. the "black bone" mr images were also found to produce good-quality surface-rendered images and were also suitable for -d printing of models for pre-operative planning. fast "black-bone" mri has proven to be technically feasible and to demonstrate cranial suture patency with good agreement with high resolution ct. additionally "black-bone" mri can be used to produce good quality surface-rendered images and -d printed models for surgical planning. main symptom of mucopolysaccharidosis type iva (mps iva) is progressive systemic skeletal dysplasia. this is routinely monitored by cerebral and spinal mri. the vascular system is generally not in the primary focus of interest. in our population of mps iva patients we observed vessel shape alterations of the vertebrobasilar arteries, which has not been described before materials: mri-datasets of patients with mps iva acquired between and were eligible for retrospective analysis of the vertebrobasilar arteries. the vessel length and angle of the basilar artery (ba) and both vertebral arteries (va) were analyzed. a deflection angle between °and °in the vessel course was defined as tortuosity, less than °as kinking. the results were compared to an matched control group of patients not suffering from mps. the deflection angle [°] of the va and ba was significantly decreased in the majority ( %) of mps iva patients (fig. ) mps iva is associated with significantly increased tortuosity of vertebrobasilar arteries. therefore the vascular system of mps iva patients should be monitored on routinely basis, as vessel shape alterations had been associated with dissections, leading to a higher risk of cerebrovascular events. in the pediatric population, intraspinal cysts (arachnoid or neurenteric cysts) are rare lesions mainly located in the thoracic region, whose acute onset is not well described in the literature. ( ) (suppl ):s -s pediatr radiol we present a series of four children seen in the last two years as spinal cord emergencies and discuss the clinical aspects, imaging diagnosis, and management approaches, particularly in the emergency setting. a comparison of our cases with those reported in the literature is also provided. as in other types of spinal cord lesions, mr imaging is the diagnostic procedure of choice, because of its potential to demonstrate the exact location and extent of the cyst and its relationship to the spinal cord, valuable information for planning surgical treatment. this is a retrospective review of cases of pediatric intraspinal cyst occurring in boys and girl, aged to years, treated at our institution between and . onset was sudden in all cases and mimicked transverse myelitis or infarction. all our affected patients had no preceding history of trauma and presented with signs of spinal cord compression-back pain and less commonly abdominal pain-followed by weakness. all patients underwent emergent mr imaging, including t , t , t *, d ciss, diffusion imaging and enhanced t sequences, mainly in the sagittal and axial planes. in each sequence, mr imaging showed a well-defined cystic lesion with signal intensity similar to cerebrospinal fluid, and secondary spinal cord compression that was severe in most cases. blood remnants were not visualized within or around the arachnoid cyst in any patient, which correlated with the absence of trauma antecedents. three of the four cysts were located in an anterior position relative to the spinal cord, and only one was located posteriorly; this latter had an associated subdural effusion. none of our patients had an associated neural tube defect. all patients were urgently treated with cyst wall fenestration or resection. the symptoms improved in all except one patient, whose symptoms did not abate, but ceased to progress. a prompt emergent diagnosis with mr imaging is important, as the symptoms can resolve if surgical treatment is performed before the spinal cord becomes irreversibly damaged. urgent surgery is essential in these cases, particularly if progressive neurological dysfunction develops over the course of spinal cord compression. the outcome following surgical fenestration or excision is excellent in most cases. nevertheless, a long-term imaging follow-up is recommended to detect possible recurrence. the objective of this study was to evaluate the usefulness of multiparametric quantitative mri model for myelination quantification in children. twenty-two children (age range: - , days) were scanned with multiparametric quantitative mri. total volume of myelin water fraction (mwf) (msum), the percentage of msum within the whole brain parenchyma (mbpv), and the percentage of msum within intracranial volume (micv) were obtained. mwf values of brain regions were acquired by drawing regions of interests. the values were fitted to representative models of myelin maturation. spatiotemporal pattern of mwf mapping was visually assessed. values of msum, mbpv, and micv well fitted to a developmental model of myelination. mwf of brain regions well fitted to a developmental model with high r values: pons (r = . ), middle cerebeller peduncle (r = . ), genu of corpus callosum (r = . ), splenium of corpus callosum (r = . ), thalamus (r = . ), frontal white matter (wm) (r = . ), parietal wm (r = . ), temporal wm (r = . ), occipital wm (r = . ), and centrum semiovale (r = . ). mwf mapping followed the known spatiotemporal pattern of myelination. multiparametric quantitative mri is a useful tool for mwf quantification in children. retinoblastoma is the most common intraocular tumour of childhood. it is a highly malignant. retinoblastoma is curable. if detected while still confined to the globe and if there are no metastatic risk factors, the child will nearly always survive following appropriate treatment. our aim is to assess diagnostic accuracy of preoperatively performed magnetic resonance (mr) imaging for detection of tumor extent in patients with histopathologically proved retinoblastoma. local ethics committee approval and informed consent were required for reviewing of patients' images and records. fifty-eight eyes in girls and boys with retinoblastoma (mean age at diagnosis was months ± . ) were reviewed on unenhanced t wi, t wi, and gadolinium-enhanced t -weighted mri with and without fat suppression. mri parameters such as anterior chamber hyperintensity, involvement of choroid, ciliary body, optic nerve, sclera, orbital fat, and pineal gland were determined. maximum tumor diameter was measured and correlated to metastatic risk factors. imaging and pathologic findings were compared. choroidal invasion was suspected with mr imaging in / eyes; findings were false-positive in eyes and false-negative in two (accuracy, . %; sensitivity, . %; specificity, %). mr imaging findings were true-positive in of eyes with proved prelaminar optic nerve invasion ( % sensitivity) and false-positive in ( . % specificity, . % accuracy). postlaminar optic nerve invasion was correctly detected in eyes; eyes were false positive, in other eyes, this metastatic risk factor was missed (accuracy, . %; sensitivity, . %; specificity, %). of nine eyes with histologically proven scleral invasion, eyes were true positive . in the other eyes, scleral involvement was missed on mri (accuracy, %; sensitivity, . %; specificity, %).extraocular fat invasion was suspected on mri in / eyes. of these, findings were truly positive in eyes ( %) and in eye ( %) was incorrect (false positive) (accuracy, . %; sensitivity, %; specificity, %).anterior chamber hyperintensity on t -weighted mr images obtained after contrast agent administration correlated well with main mri and histolopathology findings. tumor size (assessed in our study by the maximum diameter in mm) was statistically associated with postlaminar optic nerve invasion (ρ=. ) and choroidal invasion (ρ=. ). mr imaging shows promising role for tumor staging and detection of metastatic risk factors. tumor diameter, measured with mr imaging, is associated with postlaminar optic nerve and choroidal involvement. patterns of the cortical watershed continuum of term gestation hypoxic ischaemic injurythe "wish-bone sign" a. chacko , s. andronikou , s. vedajallam , j. thai ; east london/za, bristol/uk objective: background partial-prolonged term hypoxic ischaemic injury (hii) involves the cortical and subcortical watershed zones of the brain, which are visually difficult to conceive. new innovative methods of demonstrating watershed cortical atrophy using flattened maps of the brain surface gives added insight into distribution of the watershed zone by demonstrating the entire brain surface. aim determining and validating patterns of hii sustained at birth in term infants using cross-sectional mri and the innovative mercator and scroll map views of cortical surface anatomy, to define the distribution of the watershed zones in children with partial-prolonged injury. one hundred paediatric mri brain scans with an mri and clinical diagnosis of chronic term hypoxic injury were read by radiologists independently. all sites of abnormality were recorded and patterns classified. ( ) (suppl ):s -s pediatr radiol patients with partial-prolonged and combined patterns were evaluated using mercator and scroll map reconstructions, generating schematics of the watershed zone. predominant patterns of disease were partial-prolonged and acuteprofound types. the watershed zone was demonstrated, on the derived maps, representing a continuum of involvement in the shape of a 'wish-bone' extending bilateral from frontal lobes to posterior parietal lobes in band-like fashion along the para-falcine cortex and intersected by another band of atrophy in the peri-rolandic regions extending along peri-sylvian cortices. this is defined in schematics as a visual aid. predominant patterns of injury in term hypoxic ischaemic injury are described and quantified, with the 'wish-bone sign' introduced to describe the typical distribution pattern of partial-prolonged hii in the watershed zone. correlation of brain edema degree and biochemical parameters in pediatric posterior reversible encephalopathy syndrome with hematologic/oncologic diseases t. akbas , s. ulus , b. karagun , t. arpaci , c. kalayci , b. antmen ; adana/tr, istanbul/tr posterior reversible encephalopathy syndrome (pres) often associated with hypertension is characterized by typical transient parietooccipital predominantly brain edema on magnetic resonance imaging (mri) with neurological symptoms such as seizures, headache and visual disturbances. even if endothelial dysfunction, increased blood-brain barrier permeability and hyper-hypoperfusion remain as controversial mechanisms to explain, the pathophysiology of pres is unremain. the aim of our study was to investigate the correlation between brain edema degree on mri and serum biochemical parameters such as lactate dehydrogenase (ldh), albumin (alb), creatinine, uric acid (ua) and urea. a total of pediatric hematology and oncology patients ( male, female, aged - , mean age: years months) diagnosed with pres during treatment and after hematopoietic stem cell transplantation (hsct) were included in this retrospective study. underlying diseases were beta thalassemia (n: ), aplastic anemia (n: ), acute lymphoblastic leukemia (n: ), acute myeloid leukemia (n: ), lymphoid leukemia (n: ) and burkitt's lymphoma (n: ). pres was seen after undergoing hsct in patients. the brain edema degree according to specified anatomical regions on fluid attenuation inversion recovery (flair) mri sequence was scored by two radiologists blinded to patients' records. the levels of serum biochemical parameters at onset of symptoms were correlated with score of brain edema degree on mri. serum ldh concentration was statistically correlated with the score of brain edema degree (spearman's rho correlation, r= . , p= . ). no relationship was found between other biochemical parameters and the score of brain edema degree. our results suggest that increased serum ldh as a marker of endothelial dysfunction is the main biomarker for development of brain edema in pediatric pres patients under treatment and after hsct with underlying hematologic and oncologic diseases. objective: gadolinium based contrast agents (gbcas) have been associated with increasing signal intensities in deep brain nuclei on unenhanced t -weighted brain imaging. until now, most studies have been performed in adults, while results on pediatric patients are sparse. therefore, the aim of this study was to evaluate if there is any difference between signs of gadolinium retention in pediatric and adult patients. in this irb-approved, single center retrospective study, we extracted all patients with at least contrast-enhanced mris archived on pacs between - . all patients with gadobenate dimeglumine only enhanced mris were reviewed. seventy-six pediatric patients with the most injections and adult patients with the most injections were included in the final evaluation. therapies were documented. t signal intensity measurements for the initial and last unenhanced brain mris were performed for dentate nucleus, pons, globus pallidus and thalamus. signal intensity ratios for dentate-to-pons (dnp) and globus pallidus-to-thalamus (gpt) were calculated and correlated with number of injections and time interval as well as therapy. differences between adults and pediatrics were assessed. mean age for the pediatric group was . years compared to . years in the adults. no significant difference was found for gender distribution ( vs. % females) and follow up time ( . vs. years). there was no difference concerning the signal intensities on first and last mri in children and adults (p= . / . , respectively). for each additional year of follow-up the change in ratio increases by . for adults but only . for peds (p= . ). comparing therapies, in children a statistically significant difference between patients with and without former radiation was found (p< . ) while there was no difference in adult patients with and without therapy (p= . ). children and adults show a similar increase in t signal in deep brain nuclei ascribed to gadolinium deposition. in children, radiation and chemotherapy) seem to have a higher influence on gadolinium deposition. this correlation cannot be found in our adult cohort, indicating therapies have no (additional) influence. kearns-sayre syndrome (kss) is a rare mitochondrial dna-deletion syndrome characterized by early onset (< years), progressive external ophthalmoplegia and pigmentary retinopathy, often associated with cerebellar ataxia, muscle weakness, bilateral sensorineural hearing loss and cardiomyopathy. pyramidal symptoms may be present in kss, but they are poorly reported in the literature. through this case series, we aim to evaluate the concordance with the imaging patterns proposed by literature, correlating them with clinical and laboratory data, and to investigate possible microstructural damage with diffusion tensor imaging (dti) and magnetic resonance spectroscopy (mrs). we evaluated eight patients ( - years of age) with genetically confirmed diagnosis of kss. all pts. were studied with t/ . t mri. in / pts. the study was completed by mrs and in / by dti imaging with reconstruction of cortico-spinal tracts (cst) using a -rois approach. a t-test comparative study between mean fractional anisotropy (fa) of cst in the kss patients with dti and a group of healthy controls was performed. cst reconstruction in a patient suffering from kss (images a-c), compared to an healthy control (images d-f). the dti study showed significantly reduced fa values, pointing out a possible microstructural damage. the disease showed an mr pattern of mixed white and gray matter signal abnormality, with periventricular and/or subcortical white matter hyperintense lesions, which in / patient presented a "tigroid pattern" (fig. ) three patients displayed a disease extension to the cervical spinal cord. (fig. ) dwi images demonstrated restricted diffusivity in almost all lesions (fig. ) , with persistence of low adc values. mrs study documented a high lactate peak in / pts. and a naa reduction in / pts; an increment of gsh was noted in one patient (fig. ) . the t-test comparative study of cst showed a significant reduction of mean fa value in kss patients compared to healthy controls (p= , ). involvement of the spinal cord (a-c, yellow arrows). comorbidity was suspected in "a" (myelitis). below (d-f): pale nuclei (d, green arrows) and subcortical white matter (e) alterations. right image displays the "tigroid pattern" (purple arrow). mrs showing the presence of a gsh peak, which may suggest an augmented antioxidative activity within the encephalic tissue. below: dwi hyperintensity in many regions of the brain in patients suffering from kss, due to diffusion resctriction. the integration of neuroimaging with clinical data can implement the diagnosis of mitochondrial diseases such as kss. according to our experience, comorbidities can delay the achievement of a correct diagnosis. the finding of an altered signal in the spinal cord of / pts. may suggest a new possible localization of the disease, while in one patient was referable to myelitis (fig. , a) the evidence of a "tigroid patter" in should be taken in count in the differential diagnosis with lysosomal disorders. the presence of a prominent gsh peak may represent an augmented antioxidant activity, which may correlate with a more favorable outcome. an involvment of cst can be speculated even if pyramidal symptoms are poorly represented in kss. remotely distractible, magnetically controlled growing rod (mcgr, fig. ) system has been developed to allow for gradual lengthening on an outpatient basis. this allows for safe spinal lengthening with continuous neurologic monitoring and real-time feedback by the patient. this study aims to evaluate retrospectively our ultrasound (us) geometric method and his accuracy compared with the plain radiograph (gold standard) for assessing mcgr distractions. this is a retrospective study that includes patients with early-onset scoliosis undergoing multiple consecutive distractions after mcgr implant. the rods length was measured for with us, for each distraction ( -months interval), and compared with plain radiograph follow-up ( -year interval). all patients included were treated with dual-rod systems. distraction length was monitored by a senior radiologist with us at each visit, one rod at a ( ) (suppl ):s -s pediatr radiol time, before and after magnetic lengthening, with our geometric measurement method (fig. ) . low-dose upright two-projections radiograph were taken immediately after surgery and at -year intervals and measured by two radiologists ( and years of experience respectfully) (fig. ) . we compared measurements with the wilcoxon signed-rank test. from january to october , a total of patients ( females and male), which diagnoses included mitochondrial encephalopathy syndrome (n= ), spina bifida (n= ), ataxia of unknown cause (n= ), juvenile idiopathic scoliosis (n= ) and trisomy (n= ), with a mean of distractions per patient (standard deviation [sd] ± , ), were recruited. fifty distractions for each system ( measurements in total) were performed, targeting different lengths of distraction (from - . mm to + . mm) on each occasion. a total of sets of plain radiographs were taken. from these, sets of data points were used for correlation analysis. the mean distracted length per year on plain radiographs was , mm (sd ± , mm) and the mean distracted length on us per -months interval was , mm (sd ± , mm). excellent correlation was observed between radiographic and ultrasound measurements. in particular, correlation between rx measurements and ultrasound was excellent both for junior ( . <p< . , w= ) and senior radiologist (p< . , w= , ). there were no rod's breaches identified neither in ultrasound or radiographs in this series. the measured changes in rod length between the two imaging modalities were highly correlated. thus, ultrasonography is at least as accurate as radiographs in measuring changes in rod length. our geometric measurement method adds reproducibility to an effective, non-ionizing and lowcost procedure for monitoring distraction of mcgr in children population. refuting child abuse denialistshealing rickets is clearly different from child abuse metaphyseal fracture a.e. oestreich; cincinnati/us objective: child abuse denialists, who testify against the diagnosis of child abuse, often claim that radiographic findings generally recognized by pediatric radiologists as likely due to abusive trauma are instead a consequence of rickets, including healing rickets. in particular, bucket handle transverse bony density at metaphyses are declared by some denialists to be identical to the pattern of healing rickets. i demonstrate by reviewing the details of these different patterns that this is an incorrect declaration. additionally, concave distal ulna margins alone are not a sign of rickets. rickets is radiologically distinct from classic metaphyseal fractures. i reviewed medicolegal documents and published articles by several well known physicians who claim that certain children suspected of having suffered child abuse instead demonstrate rachitic bone disease. their statements and images in support of their opinions are critically reviewed and contrasted with known examples of rickets, including healing rickets. at the physeal region of long bones in rickets, the zone of provisional calcification (zpc) is not calcified and the normal metaphyseal collar shaftward from that zone is not maintained. in metaphyseal fractures from abuse, the zpc and collar are maintained. in early healing rickets, the zone of provisional calcification calcifies first, giving a transverse calcification separated from the already ossified metaphysis. this calcification is different from a bucket handle fracture pattern without signs of rickets. moreover, concave distal margin of the ulna without other signs of rickets is seen in about % of normal infants. repeatedly, denialists have testified against the diagnosis of child abuse, claiming rickets in the absence of radiographic evidence -i critique one well known child abuse is a serious diagnosisclaiming by denialists of an alternate diagnosis, such as rickets, when radiographically no rickets is present, should be refuted. a clear understanding of the differences between abuse fractures and rickets is obligatory. when no rickets is seen at other growth sites, including seeing the presence of zones of provisional calcification, rickets is not present. simultaneous multi-slice turbo spin echo: a -fold time-saving alternative to conventional tse in mr imaging of the pediatric knee s. benali, a. gholipour, p.r. johnston, s.d. bixby; boston/us objective: simultaneous multi-slice (sms) is a novel iteration of parallel imaging in mri that reduces acquisition time at least two-fold over conventional turbo spin echo (tse) acquisitions. the aim of this study is to determine whether -dimensional ( d) sms tse can replace conventional d tse in imaging of the knee in pediatric patients without compromising diagnostic quality. this was an irb-approved retrospective study. subjects included all pediatric patients referred for routine -tesla mri of the knee between october through december . mri examination consisted of institutional basic protocol with both traditional t tse and sms t tse in axial and sagittal planes with fat suppression. two anonymized study folders were created for each subject, one containing all basic sequences (basic exam), and one in which t tse sequences were substituted with t sms sequences (sms exam). two pediatric radiologists independently and blindly reviewed each study and noted presence or absence of pre-determined abnormalities (table ) . discrepancies between radiologists were settled by consensus. qualitative assessment was performed using a likert scale to evaluate edge blurring, contrast resolution, image noise, and flow artifact. thirty-five subjects comprised the study population (mean age . years, range - , female, male). diagnostic performance was assessed by comparing findings (table ) from basic exam to sms exam based on absolute agreement and cohen's kappa statistic. for all findings, the proportion of absolute agreements between basic and sms exam was > . for reader , > . for reader , and . for consensus between readers. kappas for consensus reads were . on all structures (p< . , lower % confidence limit > . ). for reader , kappas were . for / structures (p< . ) and . for pcl. for reader , kappas were . for / structures (p< . ) and . for cartilage defects. paired t-test was used to compare mean likert scores for image quality characteristics. for both readers, sms was preferred for flow artifacts whereas tse was preferred for the three remaining image quality characteristics (p< . ). our primary assessment suggests that sms t tse is comparable to standard tse in terms of diagnostic performance in the evaluation of the pediatric knee despite modest decrease in overall image quality. the -fold decreased acquisition time of sms is a significant advantage which is felt to offset the mild decrease in image quality, particularly as it increases the likelihood that children will tolerate the examination without motion. mri for sacroiliitis in children: panel findings and inter-observer evaluation using standardised reporting k.e. orr , m.j. bramham , s. andronikou ; plymouth/uk, bristol/uk there is little evidence regarding mri for diagnosing sacroiliitis in children with juvenile idiopathic arthritis (jia). the limited literature presents varied opinions but no published recommendations for standardisation of reporting. axial disease in jia responds poorly to conventional first-line treatments but identifying these children using history and examination findings is unreliable. standardised mri reporting ( ) (suppl ):s -s pediatr radiol may improve diagnosis and selection of patients in whom newer biologic treatments are indicated. the aim was to use a standardised reporting proforma based on published definitions for recording mri findings in suspected sacroiliitis to evaluate inter-observer agreement and determine the reliability of findings according to specific sequences. ninety-nine sacroiliac joint mris ( joints) were included, were initial examinations and were follow-up mris. the age range was between . and . years (mean age . years). three readers retrospectively reported all mris using the standardised proforma. 'reader ' was the study group panel while readers and were specialist paediatric radiology consultants working in the united kingdom. readers were blinded to additional clinical information and other imaging. inter-reader variation was evaluated for the presence of bone marrow oedema, erosions, effusions, ankylosis, sclerosis and enhancement, as well as the presence or absence of sacroiliitis. the quality of mri examinations was evaluated, including presence and adequacy of sequences performed and alignment of the coronal/oblique studies. mri findings were correlated with clinical details and final diagnosis. there is significant variability in sacroiliac joint mri protocols. refinement of these to include only necessary sequences based on inter-reader reliability and reinforcement of good positioning will improve reporting and result in universal standardisation. there is inconsistency in current reporting practice of sacroiliac joint mri in children but increasingly, clinicians rely on imaging to select patients with sacroiliitis and guide appropriate treatment. using a standardised reporting proforma may improve the quality and consistency of reporting. ultrasound-guided steroid tendon sheath injections in juvenile idiopathic arthritis s. peters, d.a. parra; toronto/ca objective: juvenile idiopathic arthritis (jia) is the most common chronic rheumatic disease in childhood. tenosynovitis is one of the manifestations of jia, which can explain the absence of response to treatment when adjacent joints are injected. steroid injection is one of the treatment options for tenosynovitis and it has been shown to be effective in the literature. utilizing ultrasound (us) guidance for injections into tendon sheaths has shown clinical advantage to conventional blind injections in the adult rheumatoid arthritis population. the aims of this study are to: (a) identify tendon sheaths most commonly treated in our patient population with jia referred for steroid injections; (b) describe technical aspects of the procedure; (c) characterize sonographic appearance of tenosynovitis in jia; (d) assess agreement between clinical request and sites injected. this was a year single-center retrospective study ( may -april in which we recruited patients with jia referred by rheumatology for us-guided tendon sheath injections. we collected patient demographics, clinical assessment information, sonographic appearance of the tendons and technical aspects of the intervention from the procedure records. we collected data from visits of patients ( % female, mean age years months) with a total of injections. the ankle region was most commonly injected ( %), specifically the tendon sheaths of tibialis posterior ( %), peroneus longus ( %) and brevis ( %). % of the procedures were performed under general anesthesia and triamcinolone hexacetonide was used in % of the injections. an "out of plane" approach was used in % of the interventions and the mhz "hockey stick" us probe was preferred for guidance ( %). we found minor intra-procedure complications without sequelae. the majority of treated sites ( %) showed peritendinous fluid and sheath thickening on us. other findings were increased color-doppler signal and echogenic peritendinous fluid. a strong agreement between clinical request and sites injected was observed and most patients required one visit ( %). us-guided tendon sheath injections are used frequently to treat patients with jia. it is a safe intervention with a high technical success rate. the ankle region, specifically the medial compartment, is the area most commonly injected in this cohort of patients. the most common sonographic finding is peritendinous fluid and sheath thickening. these findings might assist radiologists and rheumatologists to characterize and more effectively manage tenosynovitis in patients with jia. to evaluate the accuracy of the software for automatic bone age (ba) estimation based on deep learning technique, and to validate the feasibility of this system in clinical practice. the software for automatic ba estimation was developed based on deep learning technique using , left hand radiographs and estimated ba of each radiograph based on greulich-pyle method. ba estimation was done for left hand radiographs of consecutive patients ( months - years; boys and girls) in three methods: ( ) ai bone age (assessed by the software), ( ) ai-assisted ba (assessed by two radiologists with the assistance of the software), ( ) gp atlas-assisted ba (assessed by two radiologists with only gp atlas but the software). the reference ba was determined by two radiologists by consensus. the accuracy of the estimated ba by each method was assessed using concordance rate (%), pearson's correlation analysis, the root mean square error (rmse), and bland-altman plot. reading time for ba estimation by each method was evaluated. ai bone age showed % of concordance rate, and a significant correlation with reference ba (r = . , p< . ). the bland-altman plot of agreement between the reference ba and ai bone age showed the mean difference of - . years ( % limit of agreement, ± . years). rmse was . years. in reviewer , concordance rates were same between both gp atlasassisted ba and ai-assisted ba ( %), and rmse of ai-assisted ba ( . ) was slightly lower than that of gp atlas-assisted ba s ( ) (suppl ):s -s pediatr radiol ( . ). in reviewer , concordance rate was slightly higher in aiassisted ba ( %) than gp atlas-assisted ba ( %), and rmse was almost the same ( . in ai-assisted ba, . in gp atlasassisted ba). the reading time was reduced . % in reviewer and . % in reviewer . the software for automatic ba estimation based on deep learning technique showed high accuracy and may enhance work efficiency in ba estimation by allowing radiologists to save reading time and to improve accuracy. temporomandibular joint mri findings in adolescents with primary disk displacement in comparison to those in juvenile idiopathic arthritis j. bucheli, d. ettlin, c. kellenberger; zurich/ch to investigate potential differences of morphology and degree of inflammation in temporomandibular joints (tmjs) affected by primary anterior disk displacement (add) and juvenile idiopathic arthritis (jia). in adolescents ( female, age ± y), contrast enhanced magnetic resonance images (fig. a) of tmjs with add were retrospectively compared to those of age-and gender-matched controls with jia. morphology of articular disk and bony structures were described. osseous deformity and inflammation were qualitatively scored with progressive -grade scales and compared between groups with mann-whitney-u test. mandibular ramus length, measured on gradient echo minimum intensity projection images (fig. b) , was compared between groups and to normal values with independent samples t-test. in the add-group, / disks were dislocated anteriorly and showed thickening of the posterior band ( / ). in contrast, tmj disks of jia patients were mainly flattened (n= ) and/or centrally perforated (n= ) and rarely dislocated (n= ). tmjs with add showed similar overall grades of inflammation (p= . ) and osseous deformation (p= . ) as tmjs in the jia group. while erosions were frequent in both groups (add / ; jia / , p= . ), the mandibular condyle (p< . ) and glenoid fossa (p< . ) were less flattened in tmjs with add. in add tmjs, bone marrow oedema was less frequent (p= . ) and grades of joint enhancement slightly lower (p= . ), but presence of synovial thickening (p= . ) and degree of effusion (p= . ) were not significantly different between groups. mandibular ramus length was not significantly different (p= . ) between groups, but in both groups clearly decreased compared to mean normal values (p< . ). articular disks in tmjs affected by jia are rarely dislocated. surprisingly, tmjs with primary add show considerable inflammatory change including condylar erosions. still, chronic systemic inflammation in jia joints results in considerable higher deformity of the mandibular condyle and the temporal joint surface. observation of the mostly preserved normal shape of the temporal bone may help differentiating primary add from jia. retrospective magnetic resonance imaging (mri) study of consecutive jia patients ( female, median age y) with at least two consecutive tmj mri examinations ≥ y apart and no csi. degree of tmj inflammation was determined on t -weighted and contrast-enhanced t weighted fast spin echo images (fig. a) , and degree of osseous deformity on gradient echo images (fig. b) by progressive -grade scales ( - ). change of respective grades was assessed with wilcoxon test. mandibular growth was determined by ramus length change and compared to normal values. over a median period of . y (interquartile range, . - . y), degree of tmj inflammation improved (p< . ) with decrease in frequency of grade ( . % to %) and grade ( . % to . %). inflammatory grades improved both in patients with (n= , p= . ) and without (n= , p= . ) systemic disease modifying medication. the degree of osseous deformation slightly improved (p= . ), with decrease in frequency of grade ( . % to . %) and grade ( . % to . %), and increase of grade ( % to . %). overall growth rates of mandibular ramus (median, . mm/y) were not significantly different from normal growth rates (p= . ) (fig. c) . growth rates of tmjs from patients only receiving non-steroidal anti-inflammatory drugs (median, . mm/y) were not significantly different (p= . ) compared to patients treated with systemic disease modifying drugs (median, . mm/y). in patients with systemic treatment of jia, both the degree of tmj inflammation and osseous deformity as seen on mri improved at midterm follow-up. normal growth of the mandibular ramus was maintained. these results are in contrast to those from an earlier cohort treated with csi, in which on average deformities deteriorated and growth was impaired. objective: pediatric ileocolic intussusception, ici, is a common abdominal condition for which pediatric radiologists are asked to attempt emergency pneumatic reduction. because of the high success and low complication rates of pneumatic reductions, radiologists are able to make several attempts at reduction in stable patients if the initial enema attempt is unsuccessful. we have observed patients with successful reductions with rather long periods between initial symptoms of ici and performance of the air enema. we hypothesize that successful pneumatic reduction rates are independent of length of symptoms and in stable patients, repeated reduction attempts can be performed with the expectation of successful reduction. we performed an irb-approved retrospective review of all ici with a pneumatic reduction attempt between - at xxx. clinical, imaging and surgical data was reviewed. time to enema was defined as the time from first symptom to first air enema attempt. linear and second order polynomial statistical analysis was performed to assess the relationship between time to enema and enema outcome. results: ici were identified in patients. air enema was successful in ici, %. the mean time to enema was . hours, range - hours with sd of . hours for successfully reduced ici and . hours, range - hours with sd of . hours for unsuccessfully reduced ici. surgical resection was required in patients with ischemic bowel including one with an irreducible meckel's diverticulum as lead point. there was no correlation between time to enema and successful reduction, fig . no patient with a successful pneumatic reduction of a ici required subsequent bowel resection. conclusions: air enema for ici can be safely performed despite prolonged time to enema with the anticipation of a successful reduction. the lack of correlation of pneumatic reducibility and time to enema suggests that in surgically cleared patients with ici, the pneumatic reduction attempt may not be a true emergency and that repeated attempts at reduction are safe. additionally, though our numbers are small, they suggest that an ici is reducible or not from the beginning and do not "become irreducible" with prolongation of the time to enema. evaluation of splenic stiffness measurements for the diagnosis and the follow-up of portal stenosis after paediatric liver transplantation c. escalard , a. dabadie , s. chapeliere , d. pariente , c. adamsbaum , s. franchi ; le kremlin-bicêtre, paris/fr, la timone, marseille/fr to report our preliminary findings about the role of splenic and hepatic supersonic shear-wave elastography (sswe) in the diagnosis and followup after treatment of portal stenosis in paediatric liver graft recipients. all paediatric liver recipients with portal stenosis treated by the interventional radiology procedure, and who underwent splenic and hepatic sswe pre and post interventional procedures, were retrospectively reviewed. demographics, data about the portal stenosis (delay post transplantation, clinical presentation, initial radiological findings, hemoglobin and platelet counts), ir procedure performed, clinical and ultrasonographic follow-up and spleen stiffness pre and post ir procedure were collected. four patients were included, median age , years (range , months to years) and median delay post transplantation , years (range month to . years). two patients presented with anemia, associated in one case with progressive splenomegaly. one patient had liver test abnormalities, and one had decreased portal flow found on systematic doppler followup. spleen stiffness was elevated pre-procedure in all patients, from to kpa (normal < kpa), and liver stiffness was normal or mildly elevated in all. portal stenosis was successfully treated by ir in patients. spleen stiffness decreased rapidly, ranging from to % (figure ) . however, the size of the spleen remained unchanged. in the last patient, angioplasty of the portal stenosis failed leading to portal thrombosis. spleen stiffness increased on the subsequent ultrasound ( figure ). mr elastography (mre) is a novel imaging technique that provides a non-invasive evaluation of liver fibrosis. the standard sequence used for this purpose on a siemens scanner has been gradient echo (gre). we also implemented echo planar imaging (epi) available as a work-in-progress (wip). our aim is to compare the liver elastogram values between gre and epi in children. after consent from both research and referred clinical subjects, a dedicated mre of the liver was performed on a t mr scanner (magnetom® skyra, siemens) with a pediatric mechanical driver over the right upper quadrant. an axial t blade with fat saturation, coronal t vibe dixon and axial diffusion weighted imaging (dwi) were obtained. elastograms were obtained using both standard gre and epi, in the axial plane. for the gre sequence, different slices were selected and each scanned sequentially. the epi sequence incorporated different slices in just one series. images were post-processed placing regions-of-interest (roi) and measuring the stiffness in kilopascals (kpa). for each sequence and each slice the mean stiffness and then the average of the means was calculated. a spleen elastogram was simultaneously generated, without changing the mechanical driver location, and the mean stiffness was also calculated. increased stiffness was defined as > . kpa in the liver and > . kpa in the spleen. we focused on a technical comparison between the sequences without clinical or histological correlation of findings. we included subjects that had elastogram measurements of liver and of them spleen stiffness on both gre and epi sequences. mean liver stiffness on gre was . (sd+/- . ) and on epi was . (sd+/- . ), with a pearson's correlation of r= . (p< . ). increased liver stiffness was found in / ( . %) of the cases in gre and / ( %) of the cases in epi. mean spleen stiffness on gre was . (sd+/- . ) and on epi was . (sd +/- . ) with a pearson's correlation of r= . (p= . ). epi reported consistently higher values than gre in both liver and spleen stiffness. our preliminary data shows a moderate to high correlation between gre and epi sequences; however, the epi values were higher in both liver and spleen. in the future, larger studies are needed to validate these thresholds and patterns among different sequences. were also reviewed if done. patient's medical & surgical treatment, and clinical progress were also reviewed. active telephone follow-up days after cevus was performed. results: patients giving a total of pelviureteric units were referred for vus study during the study period, with age ranging from month to years old. no contrast-related complication was encountered. except cases with failed catheterization, were investigations of urinary tract infection (uti), antenatal hydronephrosis and congenital anomalies etc., and remaining were follow up studies of known reflux. of all cases of uti, refluxing units were picked up by vus, ranging from grade i to v. of the refluxing units diagnosed by cevus, were missed on mcu, among which were high grade refluxes (grade iii to v) requiring treatment; whereas cevus only missed one grade i refluxing unit detected by mcu. besides, one grade iv refluxing unit identified on vus was under graded by mcu to grade i. regarding patient outcomes, one patient with mcu-missed refluxing unit presented with breakthrough uti on follow up. two refluxing units that were missed on mcu but detected on cevus demonstrated scarring on dmsa. conclusion: cevus is shown to be more sensitive in detecting vesicoureteric reflux than mcu. the fact that mcu-missed refluxes detected by cevus were associated with breakthrough urinary tract infection and scarring on dmsa indicated that the extra sensitivity brought by cevus did translate to clinical significance. difficulty in visualizing low-grade reflux is a potential limitation of this technique. with favourable diagnostic performance and safety profile, cevus can be further applied in this community in the era of radiation reduction. percutaneous transbiliary needle or forceps biopsy in hepatic masses with biliary dilatation a. dabadie , s. franchi , d. pariente ; la timone, marseille/fr, le kremlin-bicêtre, paris/fr hepatic masses with biliary dilatation are rare in children and mainly include rhabdomyosarcoma of the biliary ducts, but also other masses or pseudo-masses compressing the hepatic hilum. in these patients histological diagnosis of the lesion as well as temporary biliary drainage are warranted. the objective of this study is to report our experience in percutaneous transbiliary biopsy performed simultaneously and using the same access as the percutaneous biliary drainage in children with hepatic mass obstructing the biliary ducts. children presenting with a hepatic mass causing biliary obstruction, with need for biliary drainage, were considered candidates for percutaneous transbiliary biopsy of the lesion performed at the same time. the biopsy was performed under ultrasound guidance, through a sheath introduced in the dilated biliary system, using a semi-automatic gauge needle or the transluminal biliary biopsy forceps set (cook medical, bloomington, usa). between and , four patients were included, three females and one male, median age . years (range . - . ). all presented with jaundice and were diagnosed with a hepatic mass with secondary biliary obstruction. percutaneous transbiliary biopsy was performed in all patients using the gauge needle. in one patient, the biopsy did not demonstrate any tumoral cells and a second biopsy was performed using the forceps device through the same biliary access. the samples deemed adequate for analysis by the pathology department in all patients, however the samples were larger when using the needle. a retrospective -prospective study included patients of both sexes ( , +/- , y), in a two-year span. patients were divided into two groups according to the used diagnostic method (positivegroup a on us and a on mri, with intestine mural thickness above mm, and negativegroup b on us and b on mri, with mural thickness below mm). overall sensitivity and specificity of us and mri in diagnosing ibd was calculated in comparison to pathohistological (ph) findings. us examination showed an average intestinal mural thickness of . ± . mm and . + . mm in group a ( patients) and group b ( patients) respectively. mri examination showed an average intestinal mural thickness of . ± . mm and , + , mm in group a ( patients) and group b ( patients) respectively. out of patients from group a, ( %) had irregular mural architecture, contrary to group b in which mural architecture irregularities have not been observed. in groups a and b ( . %) and ( . %) patients had irregular mural architecture respectively. average length of affected intestinal segment on us and mri was mm and mm respectively. five patients from group a and four from group a had signs of fibrosis. color doppler showed hyperemia in and patients of group a and a respectively. transmural signs of inflammation were found in % of patients on us, and . % of patients on mri. average longer diameter of mesentery lymph nodes measured by us and mri was . ± . mm and . ± . mm, respectively. overall sensitivity of us and mri was . % and . % respectively. both us and mri showed a specificity of %. us and mri are reliable and compatible methods in diagnosing ibd, with mri being slightly more accurate. us is an extremely valuable and widely available imaging modality in every-day clinical work, both in diagnosing and follow-up of therapy effects in children with ibd. findings in percutaneous transhepatic cholecysto-cholangiography in neonates and young infants presenting with conjugated hyperbilirubinemia d.a. parra, s. peters, j. amaral; toronto/ca objective: conjugated hyperbilirubinemia is a concerning finding in neonates and young infants, biliary atresia (ba) being one of the main diagnostic considerations. ba is a rare disease characterized by fibrosis of the biliary tree. the obliteration of the biliary system leads to cholestasis and ultimately liver parenchymal injury, cirrhosis and death. an early diagnosis of ba along with a kasai portoenterostomy operation significantly improves the long-term prognosis. percutaneous transhepatic cholecysto-cholangiography (ptcc) is one of the options described in the diagnostic algorithm of ba. the aims of this study are to: (a) describe ptcc findings in patients with conjugated hyperbilirubinemia; (b) identify the abnormal patterns encountered that justify further investigations; (c) analyze technical aspects of the procedure. this is a year single-center retrospective study ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in which we recruited patients with the diagnosis of cholestasis (less than months old) referred for ptcc. we collected patient demographics, clinical information, findings in ptcc, post-procedure management and long term clinical outcome. eigthy-nine patients were referred for ptcc in the study period. the procedure was technically feasible and successfully performed in patients ( % male, mean age . months). forty-one had a pre-procedure hida scan suggestive of ba. fifty-nine patients had an ultrasound-guided biopsy in conjunction with the ptcc and in all of them the cholangiography was performed through a needle placed using ultrasound guidance in the gallbladder. % ( ) of the patients had a normal ptcc. abnormal patterns encountered were: ) variable degrees of hypoplastic bile ducts seen in %; ) atretic gallbladder without demonstration of communication with bile ducts seen in %; and ) gallbladder communication with a cystic structure not communicated with the biliary ducts (cystic biliary atresia) seen in %. the most common diagnosis in the abnormal group was ba ( %). alagille's syndrome, alpha- antitrypsin deficiency and progressive familial intrahepatic cholestasis were other diagnoses in this group. no complications related to the procedure were observed. ptcc is a safe and effective option in the diagnostic algorithm of patients presenting with cholestasis early in life. visualization of the gallbladder is fundamental to perform the procedure. the majority of studies were normal in our patient population preventing further invasive investigations. three types of abnormal ptcc patters were encountered, with ba being the most common diagnosis in this group of patients. to evaluate the additive role of shear wave elastography in the sonographic distinction of biliary atresia from other causes of neonatal/ infantile cholestatic liver disease. neonates and infants with clinical and biochemical diagnosis of cholestatic jaundice were enrolled in our study after obtaining informed written consent from the parents. grey scale, doppler and shear wave elastographic findings were recorded after hours of fasting using aixplorer® ultrasound system (supersonic imagine, aix en provence, france). sedation was not needed during the study. for obtaining elastographic values, linear transducer ( - hz) was used and after image stabilization a q-box measuring mm was placed in the most homogenous vessel free area. the mean of three elastographic values were recorded. hida scan, liver biopsy, intra-operative cholangiogram and histopathological evaluation of resected specimens was done wherever feasible and clinically indicated. the prospectively obtained elastographic values were retrospectively evaluated. eleven of patients included in our study were proven to be biliary atresia (ba) by intra operative cholangiogram and histopathological reports. the diagnosis in the remaining patients included other causes of infantile cholestatic jaundice like infantile choledochal cyst, neonatal idiopathic hepatitis, progressive familial intrahepatic cholestasis, abernathy malformation, cmv hepatitis etc. the elastographic values of ba and non-ba patients were compared. six of infants were younger than days which included four patients with ba and their elastographic values ( . ± . kpa) were significantly different from that of non-biliary atresia ( ± kpa) in the same age group (p value < . ). similarly, for patients aged > days also we had a significant difference (p value < . ) in elastographic stiffness between ba ( . ± kpa; n= ) and non-ba ( . ± . kpa; n= ) groups. the mean echogenic area anterior to right portal vein (earpv) was . ± . mm in ba and . ± . mm in non-ba group (p value < . ). the mean gall bladder (gb) length was . ± . mm in biliary atresia group in contrast to . ± . mm in the rest (p value < . ). the roc plot for earpv and gb length gave a youden index cut off value of > . mm (sensitivity . & specificity . %) and < . cm (sensitivity & specificity . %) respectively. infants with biliary atresia have a significantly higher elastographic value when compared to age matched patients with other causes of neonatal cholestasis. we expect to validate the findings in our ongoing study with a larger sample size. to retrospectively define in a large pediatric population the association between testicular microlithiasis and testicular neoplasia. retrospective multicenter study of scrotal ultrasounds performed between january and april in subjects < years of age. all unique subject scrotal ultrasound reports from each institution were reviewed for mention of microlithiasis. for subjects with serial exams, the most recent exam performed was included in the analysis. all exams mentioning microlithiasis were reviewed by site-specific investigators to confirm the presence of ≥ punctate calcifications in the testicle on a single image. the presence of testicular germ cell and stromal tumors were determined for subjects with and without microlithiasis through review of institutional pathology and imaging databases. the risk of testicular neoplasia in the context of microlithiasis was expressed in terms of odds ratios with (a-or) and without adjustment (u-or) for fixed study site (institution) effects by logistic regression. the study population included , unique subjects with confirmed microlithiasis in , ( . %). mean subject age was . ± . years for subjects with microlithiasis and . ± . years for subjects without (p< . ). one hundred thirty-nine subjects ( . this large, multicenter study confirms that there is a significant, strong association between testicular microlithiasis and testicular neoplasia, particularly malignant germ cell tumors. children with microlithiasis have approximately x greater odds of having a malignant germ cell tumor than children without microlithiasis. this reinforces the need for a large prospective study assessing the risk of developing testicular neoplasia in children with incidentally identified diffuse microlithiasis. do adc-values reflect renal function or obstruction in children with uretero-pelvic-junction obstruction? p. grehten, a.c. eichenberger, c. kellenberger; zurich/ch the use of diffusion weighted imaging (dwi) in renal mri is increasing. in adults as well as in infants a positive linear correlation between adcvalues and glomerular filtration rate has been demonstrated. the aim of our study was to assess whether renal dwi can provide information on the grade of urinary tract obstruction or renal function in children with uretero-pelvic-junction (upj)-obstruction. retrospective analysis of children (age . +/- . y) with unilateral upj-obstruction who underwent pre-and postoperative mri at . t and normal controls (age . +/- . y). functional mr-urography and multiple b-value dwi were part of the mr-protocol. renal adc-values were correlated to measures of obstruction and function, and compared between obstructed and non-obstructed kidneys and between pre-and postoperative studies. no correlation was found between mean parenchymal, cortical or medullary adc-values and calyceal transit time (ctt), renal transit time (rtt) and measures of differential renal function (%parenchymal s ( ) (suppl ):s -s pediatr radiol volume, vdrf, pdrf). there was moderate correlation with absolute parenchymal volume and total kidney volume, and low correlation with pelvic volume. adc-values showed high correlation with age and patient's weight. adc-values normalized for age or weight showed low correlation with rtt and ctt, but no correlation with functional measures. adc-values were not significantly different between obstructed and contralateral normal kidneys (p= . - . ) or between pre-and postoperative studies (p= . - ). renal adc is dependent on age and weight in young children and does not correlate with differential renal function. for assessing urinary tract obstruction with adc normative values need to be established. to determine the level of knowledge and awareness of medical staff, medical students and parents concerning possible risks associated with ionizing radiation. a prospective study has been conducted at children's hospital, center for adult's radiology, and medical faculty, by filling out two anonymous questionnaires (questionnaire medical staff and medical students, questionnaire parents of the children exposed to x-ray based procedures), and it included participants. statistical analysis was performed using the spss . . the majority of examinees assessed their knowledge about ionizing radiation as moderate. knowledge level was statistically significantly higher only in the group of medical students who passed the course of radiology, in comparison to the group of those who have not attended the course yet. only % of radiologists and up to . % of pediatricians, pediatric surgeons and anesthesiologists are informed about "image gently" campaign. up to % of radiologists, and up to % of clinicians, both specialists and residents, are aware of alara principle. over % of medical doctors think that diagnostic radiology procedures are very often performed unnecessarily among children, while only . % of parents share this opinion. most of the radiologists and clinicians consider it necessary to inform parents about potentially harmful effects of ionizing radiation, but even though - % of clinicians claim they do inform parents in every-day clinical practice, over % of parents affirm that they had never been informed about effects of ionizing radiation before diagnostic procedures were performed on their children. only % of pediatric surgeons and pediatricians, but . % of radiologist and % of anesthesiologists are concerned that informing parents about ionizing radiation would cause problems in every-day work. nearly % of parents claimed that they would not refuse to expose their child to x-ray based diagnostic procedure, after the given information about potential harmful effects. over % of radiologists and less than % of pediatric surgeons and pediatricians support the initiative to calculate the total effective dose child was exposed to during hospitalization, and place it on the discharge list. between % and % of pediatricians and pediatric surgeons greatly underestimated the effective doses in ct and fluoroscopy procedures. there are - % of clinicians who are aware that ct increases the risk of carcinoma development. this study showed that general knowledge about ionizing radiation, potential risks and effective doses in pediatric population is poor, and that organized education is required. fluoroscopy in pediatric radiology -how important is an individual impact to radiation exposure of children? j. lovrenski, i. varga; novi sad/rs to determine whether there are differences between different pediatric radiologists and radiology residents in exposure of pediatric population to ionizing radiation during fluoroscopy procedures. a retrospective study has been conducted at the regional children's hospital, and included all the diagnostic fluoroscopy examinations performed within a one-year period. the fluoroscopic data along with the names of pediatric radiologists/radiology residents performing these examinations were retrieved from the evidentiary notebooks, and included: dose-area product (dap), skin dose, and fluoroscopy time. there were radiologists (r -r ), and radiology residents (r -r ) involved in fluoroscopic examinations. we found all the fluoroscopic findings in the hospital's data base, which enabled a differentiation between positive and negative findings. statistical analysis was performed using the spss . . a p-value less than . was considered statistically significant. a total of fluoroscopy procedures in children (mean age , y, males and females) have been performed within a one-year period, most of which were voiding cystourethrograms (vcug) - , and an upper gastrointestinal (gi) series - examinations. radiology residents and radiologists carried out and examinations respectively. duration of fluoroscopy procedures performed by residents (av. . s) was statistically significantly shorter in comparison with duration of fluoroscopy examinations performed by radiologists (av. s). dap and skin dose did not show statistically significant difference between these two groups, as well as the number of positive and negative fluoroscopic findings in groups of examinations performed by radiologists and radiology residents. mean dap value ranged from . μgym (r ) to . μgym (r ) when performing vcugs, and from . μgym (r ) to . μgym (r ) for upper gi series. mean skin dose ranged from . mgy (r ) to . mgy (r ) for vcugs, and from . mgy (r ) to . mgy (r ) for upper gi series. mean fluoroscopy time ranged from . s (r ) to . s (r ) for vcug, and from . s (r ) to . s (r ) for upper gi series. statistically significant difference was shown only between radiologists r and r for dap and skin dose values in performing vcug, and for fluoroscopy time in performing an upper gi series. for all examinations dap and skin dose were statistically significantly higher in the group of positive fluoroscopic findings. this study has shown that exposure of children to ionizing radiation during fluoroscopy procedures significantly depends on radiologist/ radiology resident and the nature of fluoroscopic finding. to evaluate image quality and radiation exposure of non-contrast pediatric chest ct with automated tube voltage selection (atvs), in combination with automated tube current modulation (atcm). non-contrast chest ct scans of children ( male and female; mean age, . ± . years) were analysed retrospectively with regard to radiation exposure and image quality before and after the implementation of an automated tube voltage selection. correlations of volume ct dose index (ctdi vol ) and the effective diameter (edm), before and after the implementation of atvs were compared, and confidence intervals related to the change in correlations with and without atvs were determined using fisher's z-transformation. image quality was assessed by mean signal-difference-tonoise ratios (snrs) in the aorta and in the left principal bronchus with the independent samples t-test. subjective image quality was rated by two pediatric radiologists and a general radiologist on a point scale. agreement between the readers was assessed using weighted kappa coefficients. a p< . were considered significant. automated tube voltage selection, in combination with an automated tube current modulation, resulted in optimization of scan protocols, homogeneity of image quality, and reduction of radiation exposure for pediatric patients. advantages and disadvantages of cone beam ct for pediatric interventions l. dance, r.b. towbin, d. aria, c. schaefer, r. kaye; phoenix/us objective: illustrate the advantages and disadvantages of cone beam ct (cbct) as an alternative to conventional ct guidance and an adjunct to angiography. there is a steep learning curve to optimize utilization of cbct. we found that cbct reliably identifies high-contrast lesions. however, the lower dose and decreased penetration of cbct resulted in poorer visualization of low-contrast lesions. also cbct can be degraded by streak artifact from hardware or dense contrast. the relatively narrow field of view can be restrictive for peripherally located lesions in larger patients. however, the anatomic display is adequate for guidance in most instances. these findings are illustrated in a series of cbct-guided cases including pulmonary nodule localization, osteoid osteoma ablation, abc sclerotherapy, renal av fistula embolization, and liver lesion biopsy. the advent of cbct as an adjunct modality in the ir suite has significantly decreased the use of conventional ct guidance and significantly decreased the radiation dose in children. we have found cbct to be a practice changer. the aim of this study is to review our local drl in pediatric fluoroscopy and to compare them to values proposed by pidrl guidelines and recent international litterature. data were prospectively collected on consecutive procedures ( total) performed from january to december on different fluoroscopy units (siemens iconos r , luminos drf). of each procedure patients data (name, weight and birth date), examination-data (kind of procedure, date, dap [cgy*cm ], total fluoroscopy time, number of images) were recorded. data from micturating-cystourethrography(mcu), barium meal/swallow(bs) and most commonly performed procedures were divided into weight-groups (< kg, - kg, - kg, - kg) and of each one th-percentile was calculated. data were compared to europeandrl and recent literature data (by age:newborn, -, -, years old). weight-groups are considered a representative sample if at least -patients per procedure-type and per patient-group are included. our local-drl for mcu are (< kg), ( - kg), ( - kg) and ( - kg). they results to be lower than pidrls values ( , , , ) but higher if compared to a previous local survey of ( , , , ) . bs data are (< kg), ( - kg), ( - kg); these data are lower than that of a previous local survey of ( , , ) . the update of local-drl is helpful in daily practice to identify (and solve) critical issues such as incorrect technique or poor practice with new flat-panel equipment. pidrl guidelines: a review of local drl for pediatric head, thorax and abdomen ct in a italian referral center a. magistrelli, v. cannatà, e. genovese, m. cirillo, r. lombardi, p. toma; rome/it the aim of this study is to review our local drl in pediatric ct and to compare them to values proposed by pidrl guidelines and recent international litterature. data were prospectively collected on consecutive procedures ( total) performed from january to june on a somatom definition flash siemens. of each procedure patients data (name, weight and birth date), examination-data (kind of procedure, clincal question, date, ctdivol / and dlp / ) were recorded. ctdivo/dlp from head ct were divided into age-groups (< weeks, weeks- y, - y,≥ y) and of each one th-percentile was calculated. ctdivol/dlp from thorax (chest, cardiovascular ct angiography) and abdomen+pelvis ct examination were divided into weightgroups (< kg, - kg, - kg, - kg,> kg) and of each one th-percentile was calculated. data were compared to europeandrl and recent literature data. weight-groups are considered a representative sample if at least patients per procedure-type and per patient-group are included. our local drl are substantially lower than that proposed by pidrl guidelines. specifically ctdivol/dlp for chest ct are / (< kg), , / ( - kg), , / ( - kg), , / ( - kg), , / (> kg) respectively. for cardiovascular ct angiography are , / (< kg), , / ( - kg), , / ( - kg), , / ( - kg), , / (> kg). while for abdomen+pelvis ct are , / (< kg), , / ( - kg), , / ( - kg), , / ( - kg), , / (> kg). data for trunk sere not collected. for head ct local drl are higher in age-group and but lower in other age-group if compared to routine head ct pidrl ones. the update of local-drl allowed us to identify (and solve) some critical issues such as incorrect technique. drl-curve in optimization of pediatric body ct r. seuri , p. laarne , a. nikkola-sihto , k. nygaard bolstad , m.s. perhomaa , a. thilander klang , k. rosendahl , j. ruohonen , e. tyrvainen ; helsinki/fi, tampere/fi, seinäjoki/fi, bergen/no, oulu/fi, gothenburg/se, kuopio/fi objective: diagnostic reference levels (drls) in medical imaging represent valuable tools to study dose optimization in clinical practice. this is particularly important in pediatric computed tomography (ct) as the number of the examinations in many institutions is low. drls are typically given as a percentile point, usually as % or rd quartile of the observed distribution of patient dose. in pediatric practice drls are often given for each age-or weight group separately. we present continuous drl-curve as a feasible way to compare dose levels in pediatric body ct. during - a selected group of nordic hospitals collected dose values (ct dose index by volume, ctdi vol , and dose-length product, dlp) from pediatric body ct examinations on children aged - years. the dose values were imported into a dynamic excel table, previously established by the radiation and nuclear safety authority in finland, stuk (fig ) . the stuk-table includes a graphic presentation of a continuous drl-curve presented as a function of body weight, and the program automatically calculates a dose curve and compares it to the established reference level (fig ) . the dose values were easily exported to the excel tables, and the graphic presentation and comparison with an established drl-curve was clear and readily understandable for both radiologists and radiographers. in some of the institutions included in the present study, the weight of the patient was not recorded routinely. this represents a challenge for the use of the drl-curves provided by stuk. the drl-curves provided by stuk were feasible for clinical practice. the automatic calculation of the dose curve and graphic presentation were helpful to interpret the results. the drl-curve also allows relevant comparison even with a smaller number of patients. fifty randomly selected ct chest studies performed over years to assess diffuse lung disease were included in the study sample ( females, males; mean age . years + . years), comprising disorders. two pediatric radiologists and a pediatric radiology fellow blinded to the results of the cts evaluated four subsets of complete chest cts ( slices, every third slice, every other slice, and all images below the thyroid) and compared the subsets with the entire chest ct, interpreted as the control. accuracy of evaluating the primary diagnosis and determination if significant diagnoses were missed in the reduced slice ct subsets were rendered. we assume linear distribution of dose across the anatomy to estimate dose reduction on reduced slice subsets. most significant findings were present on all reduced slice ct subsets. all relevant findings were present in % of subthyroid, % of every other slice, % of every rd slice, and % of regional slice subsets respectively. excluded findings included small foci of ground glass opacity, consolidation, focal mosaic attenuation, and linear parenchymal bands; peribronchial thickening, dextrocardia vs dextropositioning, tree-in-bud opacities, extent of mild bronchiectasis. with the exception of consolidation in of the studies, these findings were not thought to inhibit diagnostic assessment. the underlying diagnosis was correctly identified in most of the subsets: % subthyroid and every other slice, % every rd slice, and % of regional slice subsets. dose is significantly decreased by using any of these methods. while some findings are excluded with increasing gaps between slices, equivalent diagnostic information can be provided on reduced slice ct and can serve as a viable strategy to reduce lifetime radiation dose to children and young adults with diffuse lung disease imaged for routine follow-up. as findings are missed with larger gaps, this strategy should be used with caution in patients presenting with acute symptoms . to extrapolate the significance of early diagnosis which will compliment to treatment planning and management. case presentation: types a and b niemann-pick disease are lysosomal storage disorders that result from deficient acid sphingomyelinase activity and lead to the accumulation of sphingomyelin, primarily in tissues of the reticuloendothelial system. type b niemann-pick disease manifestations are hepatosplenomegaly, excess bleeding and bruising, growth retardation, and recurrent respiratory infections. features of hrct include thickened peribronchovascular and interlobular septal thickening, ground-glass opacities. the intermixed regions could be characterized as showing crazy paving, although this is not the predominant pattern. type b niemann-pick disease should be added to the list of clinical entities that can demonstrate crazy paving. our patient is a sevenyear old girl, presented with dry cough and fever. physical examination revealed hepato splenomegaly. radiological work up included abdominal ultrasound examination, which showed mild hepatosplenomegaly. chest radiography revealed diffuse reticulonodular infiltration in both lungs. chest hrct was done for more comprehensive evaluation which showed multilobar bilateral peribronchovascular interstitial thickening and interlobular septal thickening with ground-glass opacities and crazy paving appearance. no honeycomb pattern was seen. no sizable pulmonary nodule or sizable mediastinal lymphadenopathy was seen. no pleural effusion was seen. finding were indicating extensive pulmonary intestitial disease. a corroborative analysis along with lab tests and genetic studies revealed the diagnosis of type b niemann pick disease. . the spectra of hrct features including crazy paving pattern may be encountered; though not frequent. hence should be included in the differential diagnosis of crazy paving pattern. blast from the past: lemierre's syndrome in adolescents with sore throat o. kvist; stockholm/se a minor ailment such as a sore throat could prove to be a severe disorder known as lemierre's syndrome. this syndrome mostly affects previously healthy adolescents and young adults and in its classical form should meet four diagnostic criteria; primary infection of the oropharynx, septicemia, clinical-or radiographic evidence of thrombosis of the internal jugular vein (ijv) plus secondary metastatic abscesses. the infection is caused by fusobacterium necrophorum, a species of obligate anaerobe bacteria forming part of the normal human flora. the syndrome should be suspected in any patient with pharyngitis, cervicalgia and pulmonary symptoms. the incidence of lemierre's syndrome decreased dramatically after the introduction of antibiotics but has, of unknown reasons, increased over the past years. we will present four patients diagnosed with lemierre's syndrome in our department during the last years. the purpose of this case report is to raise awareness of this "forgotten disease". of the four patients diagnosed with lemierre's syndrome two fulfilled all criteria while two fulfilled out of . (table ). the first two presented at the emergency department with one week's history of a sore throat, left sided cervical lymphadenopathy, erythematous tonsils, leukocytosis and elevated crp. in both cases the clinical condition deteriorated and they were referred to the icu. one developed ards and required initiation of ecmo. in both patients, chest ct revealed multiple pulmonary consolidations with cavitations, findings consistent with septic emboli (image , and ). incidentally ct-neck revealed thrombosis in the left ejvand ijv (image ). ultrasound of the neck veins confirmed the finding (image and ). blood cultures taken on admission later proved positive to f. necrophorum. the third and fourth case, with similar clinical histories but with a less aggressive development, had positive blood cultures but no thrombosis and vice versa. (table and all four patients recovered and could be discharged with oral antibiotics and anticoagulants. unique teaching points: in conclusion, lemierre's syndrome is less common today thanks to antibiotics but may still occur in previously healthy adolescents and may lead to a fatal outcome. the pediatric radiologist should be aware of typical findings like septic emboli in the lungs and thrombosis in the ijv. unicameral bone cyst associated with secondary aneurysmal bone cyst of clavicle i. dasic, g.j. djuricic, s. ducic; belgrade/rs objective: aneurysmal bone cyst (abc) accounts for , % of all bone tumors. they are benign but locally destructive lesion of the bone characterized by presence of spongy or multiloculated cystic tissue filled with blood. abcs are metaphyseal, excentric, bulging, fluid-filled and multicameral, and may develop in all bones of the skeleton. most common locations include the proximal humerus, distal femur, proximal tibia, and spine. clavicle is a very rare site for aneurysmal bone cyst with only few cases reported in literature. a -year-old boy reported to the university children's hospital for detailed examination of swelling of right shoulder. - days before admission parents noticed tumefaction of right shoulder. there was no history of trauma or fever. physical examination revealed tumefaction of the right shoulder, in projection of acromial end of clavicle, measuring approximately x cm, which was tender and fixed. the swelling was not hot to the touch, and there was no skin discoloration over that area. regional lymph nodes were not palpable. (fig. a) x-ray revealed osteolytic, expansible lesion in the lateral end of clavicle and there was no pathological fracture. (fig. b) laboratory analyzes were within normal limits. blood cultures remained sterile. chest x ray and abdominal ultrasound were normal. computed tomography (ct) revealed a thinwalled multiloculate lesion in lateral end of right clavicle. (fig. a) there was no extension in the soft tissues on magnetic resonance imaging (mri). mri shows the multiloculate cavities and fluid levels. (fig. b) . the open biopsy was done. histopathological examination confirmed the secondary aneurysmal bone cyst on the field of simple bone cyst of clavicle. the clavicle is an uncommon site for bone tumors. review of literature shows clavicle accounts for less than % of all bone tumors. the patient with an aneurysmal bone cyst generally presents with pain and swelling, which may vary in duration from weeks to several years. up to % of bone tumors occur in less than years of age with peak incidence in second decade. radiologically, lesion is lytic and may have a soap-bubble appearance with ballooned distension of the periosteum. the differential diagnosis for aneurysmal bone cyst include giant cell tumor, chondromyxoid fibroma and telangiectatic osteosarcoma. distinction from telangiectatic osteosarcoma is difficult because the conditions have overlapping clinical and radiologic features. the differentiation is made from the histologic features. imaging of glomus tumor of liver in a child (case report) n. tewattanarat, j. srinakarind, j. wongwiwatchai, p. komvilaisak, s. areemit, p. ungarereevittaya, p. intarawichian; khonkaen/th objective: glomus tumors occur preferentially in subcutaneous tissue of fingers and toes, but extremely rare in visceral organs. most cases of the tumors are diagnosed in adults. several cases of glomus tumors in liver have been reported in adults. a literature review, no case of glomus tumor in liver in children was published. therefore, we present clinical, imaging findings of the first case of pediatric patient with glomus tumor in liver and also histopathological features. a previously healthy year-old-girl was admitted with a twoweek history of progressive dyspnea on exertion and vomiting. family history was unremarkable. physical examination revealed hypertension and smooth and firm mass at epigastrium. systolic apical murmur on heart examination was noted. liver function test ( ) (suppl ):s -s pediatr radiol showed elevated cholesterol ( mg/dl). other laboratory tests (complete blood count, blood chemistry, renal and liver function test, coagulation test, hepatitis profiles and alpha-fetoprotein) were within normal limits. echocardiogram found mitral and tricuspid regurgitation and poor left ventricular systolic dysfunction. abdominal mri demonstrated a -cm well-defined exophytic hypervascular mass with intratumoral hemorrhage at segment / b of the liver. there were no other suspicious lesions in other organs. the biopsy was done and revealed glomus tumor. patient underwent preoperative embolization and the liver mass revealed decreased size to -cm after -month follow up with ultrasound. after that, exploratory laparotomy with left lateral segmentectomy was performed. the pathological results showed dilated vascular channels surrounded by uniform neoplastic cells, uniform with round nuclei, fine chromatin, inconspicuous nucleoli, and pale eosinophilic cytoplasm, and well-defined cytoplasmic border. no mitotic figures and necrosis are identified. immunohistochemical (ihc) staining of tumor was positive for cd , smooth muscle actin (sma) and h-caldesmon. others ihc including ae /ae , heppar , cd , desmin and myogenin were negative. from these findings, the tumor was finally diagnosed as glomus tumor of uncertain malignant potential due to deep location and large size. primary glomus tumor is a rare entity of liver tumor diagnosed in children. however, it should be considered in the differential diagnosis of a hypervascular liver mass. most of these tumors are benign, however tumor in liver have malignant potential due to deep seated position. therefore, tumor removal with pre-operative embolization should be considered. brain mri in a pediatric patient with linear scleroderma en coup de sabre m. mortilla, a. rosati, e. canale, c. filippi; florence/it objective: linear scleroderma "en coup de sabre" (ecds) is a rare subset of localized scleroderma. affected individuals typically have a characteristic atrophic skin lesion involving the fronto-parietal scalp. the disease usually has a benign course but rare neurologic symptoms can be seen associated: the most common described is epilepsy. intracranial mri findings described in the literature include: focal brain atrophy, calcifications and t -hyperintense white matter lesions that may demonstrate contrast enhancement. white matter lesions and calcifications are found in the cerebral hemisphere ipsilateral to the skin abnormality. in the literature only a few pediatric cases have been described. a yrs. old girl was hospitalized at our institution for evaluation of a lesion of the frontal skin associated to a history of febrile seizures and mri alterations. she presented febrile seizures at the age of on april . on january parents noted a frontal cutaneous lesion that was defined as "linear scleroderma, port-wine stain type". on november she performed an mri at another institution showing a diffuse white matter alteration in the left emisphere with focal lesions with high susceptibility and mild contrast enhancement. she was addressed to immunosuppresive therapy with steroids and methotrexate, with steroids stopped after months. a clinical cutaneous improvement was noted. on july a second mri showed a worsening of the findings. we describe a case of a little girl with ecds with no neurologic deficits or symptoms that shows extensive and progressive neuroradiologic alterations. only a few pediatric cases have been described, but it has to be known that also in absence of symptoms, patients with linear scleroderma should be screened with mri to look for cns involvement in this immune disease. brain mri can also be used to monitor the progression of the disease and the response to therapy. mals is a vascular compression syndrome which symptoms can overlap chronic functional abdominal pain. in mals the proximal part of the celiac artery is compressed by the too low located median arcuate ligament during expiration resulting in hemodynamically significant symptoms. we report two cases with mals diagnosed primarily by ultrasonography. case -year-old girl was admitted to tartu university children's clinic (tucc) due to recurrent acute epigastric pain episodes with nausea and loss of appetite during years. previous analyses were normal, abdominal uss and gastroscopy did not show any abnormalities. she was referred to paediatric radiology department for doppler us (dus) which showed narrowed proximal celiac artery (ca) with turbulent flow, increased peak-systolic and end-diastolic velocities on deep inspiration and expiration, and positive ca deflexion angle on expiration. superior mesenteric artery (sma) was markedly widened, indicating possible collateral blood-supply due to severe ca stenosis. according us findings mals was suspected. abdominal mra showed proximal ca kinking, stenosis and poststenotic dilatation and confirmed diagnosis. during dsa collateral blood-supply from sma via pancreaticoduodenale arcade (pda) was seen. laparoscopic release of mal resulted in relief of patient's symptoms, she has been pain-free for two years. case -year-old girl applied to tucc due to recurrent abdominal pain episodes for - years. usually, pain occurred - times per week about minutes after the start of intense cycling training or competitions, and passed about minutes resting in squat position. mild mid-epigastric bruit was audible at physical examination. dus showed two-fold increase in expiratory peak-systolic and enddiastolic blood flow velocities compared to inspiratory velocities which indicated to the hemodynamically significant worsening of ca compression by mal during expiration. mra showed proximal ca compression, upward angulation and poststenotic dilatation. preoperative ct-angiography depicted collateral supply via pda. during laparoscopic surgery ca was released by transecting mal and surrounding fibrous tissue. after surgery the girl has been pain-free for one year except single pain episode during intense competition. the diagnosis of median arcuate ligament syndrome should be considered in patients with postprandial abdominal pain that does not have other clearly established etiology. colour doppler us should be the first choice imaging method. to confirm diagnosis in pediatric patients abdominal mra is preferred in our institution, but as mra may still have a tendency to movement artifacts and inadequate spatial resolution for smaller blood vessels, in these two cases mra was followed by cta or dsa. understand the unique predilection of infantile malignancies to metastasize and present as skin-based masses, most commonly lymphoma/leukemia. case presentation: an otherwise healthy day old male presented to dermatology with a pedunculated, friable red glabellar mass (centered between the eyes). first noticed as a flat, bluish lesion at days, its subsequent rapid growth led to an emergency department visit where dermatology diagnosed a hemangioma and initiated propranolol treatment. despite this, the mass continued to grow rapidly, encroaching upon the patient's right eye. the patient was admitted for further workup. an elevated beta hcg, anemia ( . mg/dl), and thrombocytopenia ( , ) suggested an alternate diagnosis. an mri and ultrasound led to a percutaneous biopsy; pathology was consistent with choriocarcinoma. pet ct found fdg-avid glabellar, liver and lung lesions. maternal and placental testing was negative for choriocarcinoma. ultrasound demonstrates a hypoechoic hypervascular mass. mri brain demonstrates cutaneous confinement of the solid avidly enhancing glabellar mass. ct shows a peripherally enhancing liver mass with a masslike area of consolidation in the right lung. initial pet/ct demonstrated fdg avid liver and lung metastases with a small focus of residual activity at the glabella consistent with incomplete resection. follow-up pet/ct showed astoundingly rapid re-growth of the glabellar mass and enlargement of the hepatic and pulmonary masses just days later demonstrating the extremely aggressive nature of this cancer. month follow-up pet/ct showed significantly decreased size and activity of the metastases consistent with a treatment response. in a series of infants with cutaneous metastases, the following diseases presented with cutaneous involvement (ordered most to least common): leukemia, langerhans cell histiocytosis, neuroblastoma, rhabdoid tumor, rhabdomyosarcoma, primitive neuroectodermal tumor, choriocarcinoma, and adrenocortical carcinoma. pathology slides ( ) (suppl ):s -s pediatr radiol unique teaching points: considered one of the fastest growing tumors, infantile choriocarcinoma classically presents with hepatomegaly, anemia, failure to thrive, and precocious puberty between days and months of life. left untreated, the disease is usually fatal within weeks of presentation. a solitary cutaneous metastasis can be mistaken for infantile hemangioma both clinically and radiographically. atypical mri appearance is one important clue that can suggest an alternative diagnosis. pet/ct may be useful for staging and follow-up. a rare case of ovarian juvenile granulosa cell tumor associated with ollier's disease -generalised mesodermal dysplasia p. joshi; pune/in to demonstrate a rare case of mesodermal dysplasia -association of ovarian granulosa cell tumour with enchondromatosis case presentation: two year month old girl presented with precocious puberty i.e thelarche. left hand radiograph showed the radiological age corresponding to chronological age, suggestive of peripheral precious puberty. the patient subsequently underwent a sonography which revealed a pelvic mass probably arising from the right ovary ? sex cord stromal tumour. a mri of the abdomen and pelvis confirmed the pelvic mass and revealed multiple bone lesions in the right hemipelvis -on the side of the tumour she was later operated. hpe of pelvis mass revealed juvenile granulosa cell tumour. ultrasound pelvis images reveal a solid pelvic mass, probably ovarian in etiology mri pelvis also reveals multiple bone lesions unique teaching points: the aim of the poster is to create awareness about this association. the bone lesions should not be mistaken for metastasis juvenile granulosa cell tumour of the ovary (jgct) is a well-known sexcord stromal ovarian neoplasm. ollier's disease is a rare, non hereditary mesosermal dysplasia consisting of multiple enchondromas. the association of granulosa call tumour with asymmetric ipsilateral hemiskeletal distribution may indicate generalised mesodermal dysplasia as there is also association of jgct with maffucci's syndrome, other dysplastic conditions such as microcephaly, facial asymmetry,' and potter's syndrome. review of literature showed previous cases of juvenile granulosa cell tumor associated with enchondromatosis, three associated with maffucci's syndrome, and the rest with ollier's disease goldbloom's syndrome is a paediatric idiopathic disease characterized by transient bone marrow oedema with recurrent crisis of bone pain, periosteal hyperostosis, fever, increased inflammatory markers and dysproteinaemia. a case series of wbmr studies in goldbloom's syndrome is reported and differential diagnosis discussed. case presentation: a -year-old female girl was admitted to our paediatric department because of daily crisis of bone pain of the lower limbs, associated with fever spikes, limping and nocturnal awakenings. no history of trauma was reported. laboratory tests showed mild anaemia (hb . g/dl), thrombocytosis (plt /mmc), increased inflammatory markers (ers mm/h, crp mg/dl), high streptolysine o and dnase-b antibody levels (aso iu/ml and adn-b ui/ml, respectively). throat swab was positive for group a β-haemolytic streptococcus (gas). unusual dysproteinaemia, characterized by hypoalbuminemia ( . g/dl) with increased a , a and g globulinaemia, was noted. x-ray examinations of both legs resulted normal. wbmri showed markedly delineated, high and homogeneous hyper-hypointensity respectively in stir/t of the distal tibialperoneal meta-diaphysis of both legs (fig a,b). distal metaphysis of femur, humerus, radius-ulna and proximal tibia were also homogeneously mildly hyperintense on stir sequences bilaterally (fig a). bone biopsy revealed signs of chronic inflammation. infectious and neoplastic diseases were ruled out and the diagnosis of gs with dysproteinaemia seemed conceivable. steroid treatment was started in association with indomethacin, leading to a prompt resolution of the clinical picture within a few days. the follow-up stir total body mri, performed after months, showed the complete resolution of bone oedema. (fig a,b) the sock sign is a pathognomonic whole-body magnetic resonance imaging (wbmri) feature of goldbloom's syndrome (gs).it is a well marked, symmetric, homogeneous and high bone marrow hyperintensity, localized both at the distal tibial and peroneal meta-diaphysis, which looks like a pair of socks. objective: left ventricle hypoplasia is generally thought as a part of hypoplastic left ventricle syndrome or aortic hypoplasia. it is estimated that about - ml/m left ventricle volume is needed in order to support systemic circulation. less than that volume generally precludes biventricular repair. however conditions associated with severe preload decrease such as total anomolous pulmonary venous return (tapvr) should be considered in the differential diagnosis. tapvr presenting as hypoplastic left ventricle syndrome is presented in this study. six month old female patient admitted to emergency service with symptoms of fever, dyspnea and coughing. emergency staff started intravenous antibiotic theraphy and from medical records learned that she has been followed for partial anomolous pulmonary venous return (papvr) and atrial septal defect (asd). lung x-rays revealed pulmonary edema. echocardiography was performed and revealed very small left ventricle, papvr and mm wide asd. ecg gated cardiac ct was requested with the prediagnosis of hypoplastic left ventricle syndrome. ct images revealed dilated right cavities, very small left ventricle, pulmonary edema, tapvd and peritoneal fluid plus hepatomegaly. we then retrospectively searched our archive and found she was diagnosed as papvr when she was days old. all the cavities that time, were normal sized. according to these we confirmed our diagnosis as tapvr and hypoplastic appearing cavities due to reduced preload and right chamber dilatation due pulmonary overcirculation. surgical team decided to perform corrective operation and they confirmed our diagnosis unique teaching points: small left ventricle cavity in an infant need not to be due to intrinsic hypoplasia. whenever we experience such a situtation we should search for other reasons of pseudohypoplasia in order to give a chance for corrective surgery instead of palliative procesures. we present a case report of kimura disease, a rare benign chronic inflammatory disease that involves the deep subcutaneous tissues and lymph nodes of the head and neck. we report the case of a thirteen year old male who presented with a right sided facial mass which had been present for two years but had enlarged rapidly in the preceding three months. us and mr were interpreted locally as an arteriovenous malformation. review of these examinations and catheter angiography performed at this quaternary referral centre favoured a vascular tumour. subsequent percutaneous biopsy demonstrated angiolymphoid hyperplasia with eosinophilia and blood tests showed a serum eosinophilia, consistent with kimura disease. us shows a mass consisting of scattered heterogenous foci within the fat with multiple large feeding vessels. contrast enhanced mri demonstrated a solid, homogenously enhancing, mass with multiple vascular flow voids from the right external carotid artery branches. catheter angiography showed tumour blood supply from branches of the right transverse facial artery and distal right ima. the dominant supply arose superficially from the transverse facial artery. kimura disease is a rare chronic inflammatory disorder of unknown aetiology that involves the deep subcutaneous tissues and lymph nodes of the head and neck region, most common in asian men in the third decade and sporadic in the non-asian population. the histopathological and biochemical characteristics are eosinophilic lymphfolliculoid granuloma, increased eosinophils in the peripheral blood and increased ige levels. whilst ultrasound and mri are effective imaging modalities, imaging alone does not allow confident differentiation from malignant lesions and biopsy is necessitated. kimura disease has a benign indolent course with an excellent prognosis following surgical excision although local recurrence has been reported. increased naa: is it surely canavan disease? e. varga, p. barsi, g. rudas; budapest/hu leukodystrophies are a group of rare genetic, metabolic diseases that affect the central nervous system, mainly the brain. each type of them is caused by a specific gene abnormality that leads to abnormal development or destruction of the white matter of the brain. the differential diagnosis are made on the basis of clinical and neuroradiological signs. there are some diseases which show typical changes on mr spectroscopy. we present a case of a year-old boy, who has been investigated due to somatomental retardation and muscle dystrophy since his six months of age. his perinatal period was normal except of a nystagmus visible from his birth. the child has muscle dystrophy, spastic quadriparesis, contractures, scoliosis, truncal hypotonia and ataxia and mental retardation. we started examinations to find out the background pathology of his idiopathic encephalo-myopathy. the brain mri showed a bilateral, symmetrical white matter signal alteration, which referred to some kind of metabolic ( ) (suppl ):s -s pediatr radiol disease. the mr spectroscopy revealed decreased cholin and increased naa levels, which are typical of canavan disease. despite of this, the clinical aspects and the location of the involved brain areas were more typical of pelizaeus-merzbacher disease (pmd). the pmd is a genetic disorder, which is originated of the mutation of the proteolipid protein gene (plp ) located on long arm of x-chromosome (xq - ) . this gene has an impact on growth of the myelin sheath. various types of mutations (deletion, duplication, point mutation, insertion) of plp gene lead to various severity of clinical picture. all form of mutations show decreased naa level on spectroscopy, except the duplication of plp gene. in connection with our case, we present briefly the clinical and neuroradiological differences between the two entities. magnetic resonance imaging findings in medium-chain acyl-coenzyme a dehydrogenase (mcad) deficiency l. talamanca, d. narese, m.c. rossi espagnet, l. pasquini, d. longo; rome/it we report serial brain magnetic resonance (mri) in a patient with medium-chain acyl-coenzyme a dehydrogenase (mcad) deficiency who developed acute encephalopathy. a -months-old girl was admitted in the emergency department of our hospital with sudden onset of acute encephalopathy with drowsiness. baseline laboratory investigations revealed severe hypoglycemia, hyperammonemia, hyperchloremic metabolic acidosis and hyperuricemia. the patient was treated with glucose solution infusion that resulted in a gradual resolution of symptoms. the first brain mri, performed within hours of onset of symptoms showed bilateral symmetric restricted diffusion on diffusion-weighted imaging (dwi) in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter; the adc map showed reduced diffusivity (fig ) . the second mri, at hours after the onset, revealed bilateral and symmetric hyperintensity on t -weighted images in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter. dwi showed restricted diffusion in both globus pallidus (fig ) . a single voxel h-mrs study performed by placing a roi in the right nucleus lenticularis revealed increased values of gaba and glutamine (fig ) . a further mri was performed weeks after the first neuroimaging and indicated widespread atrophy and the appearance of a hyperintense signal in t -wi in both globus pallidus while dwi did not reveal any remarkable signal abnormality. single-voxel mrs of the same region showed a normalization of gaba and glutamine values. brain mri showed bilateral symmetric restricted diffusion on diffusionweighted imaging (dwi) in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter; the adc map showed reduced diffusivity the second mri, at hours after the onset, revealed bilateral symmetric restricted diffusion on diffusion-weighted imaging (dwi) in both globus pallidus. a single voxel h-mrs study performed by placing a roi in the right nucleus lenticularis revealed increased values of gaba and glutamine. mcad is an enzyme of the mitochondrial b-oxidation of fatty acids, an essential source of energy for cells during stress. mcad deficiency is the most common genetic disorder of fatty acid oxidation. the clinical manifestation of the disorder is typically precipitated by stress due to fasting, vomiting, fever or muscular exertion and occurs in the majority of cases before the age of with the onset of acute hypoketotic hypoglycemia. clinical features of this decompensated state include seizures and lethargy proceeding to coma and death in the absence of prompt treatment with intravenous dextrose infusion. mcad deficiency usually appears in an acute form and has high morbidity and mortality rates; early diagnosis is therefore extremely important in order to promptly begin treatment and obtain a complete recovery from symptoms. mr can play a significant role in the early diagnosis of the decompensated state of the disease; in our case dwi revealed the presence of lesions with a bilateral symmetric topographic distribution that strongly suggested a metabolic disease leading to acute encephalopathy. a full-term male neonate ( days old) with external perineal anomalies was referred to our hospital. the physical perineal examination revealed a bifid scrotum containing palpable testis and a normal configured penis located at the bottom of the bifid scrotum. two soft masses of and cm respectively, divided from a cutaneous notch, were located below the bifid scrotum and on the right of the midline. the rear biggest mass was normal epithelized, instead the other one was a rugged pigmented mass, which resembled the scrotum (figure ). there were no additional abnormalities of the external genitalia. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content. a mild hydrocele in the right scrotum and a sliding testis on the left side were also revealed. us examination showed a hyperechoic solid tissue, corresponding to the rear biggest perineal mass. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content (figure ) . a mild hydrocele in the right scrotum and a sliding testis on the left side were also revealed. mri also confirmed two perineal peduncolar masses: the biggest and posterior one, was made up by homogeneous fatty matter without contrast-enhancement after intravenous gadolinium injection (figure ). the patient underwent excision of perineal masses and no complications occurred in the surgery. the histopathological examination of the perineal masses revealed two areas with different histological features: the first one was characterized by the presence of smooth muscle bundles dispersed in the dermal collagen, instead the other contiguous area showed abundant mature adipose tissue in the deep dermis and hypodermis ( figure ). at last the rugged swelling mass was definitively diagnosed as as without testis tissue inside, and the rear mass was diagnosed as lipoma. the physical perineal examination revealed a bifid scrotum containing palpable testis. two soft masses of and cm respectively, divided from a cutaneous notch, were located below the bifid scrotum. us examination showed a hyperechoic solid tissue, corresponding to the rear biggest perineal mass. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content mri confirmed the presence of two perineal peduncolar masses: the biggest and posterior one, was made up by homogeneous fatty matter without contrast-enhancement after intravenous gadolinium injection. neonates presenting with perineal masses are uncommon. these anomalies can occur isolated or more rarely in combination with other abnormalities such as uro-genital or ano-rectal anomalies or with contiguous subcutaneous tumors. when perineal masses are found, with prenatal diagnosis or during a newborn physical examination, it is important to look for any associated congenital anomalies or subcutaneous tumors by using imaging. to describe and emphasize the significance of the "half-moon" sign in pelvic mri. a -year-old adolescent, karate athlete, was submitted with left hip pain, decreased range of movement and asymmetry in thigh circumference. markers for infection or inflammation were negative. frog-leg radiograph was negative for hip effusion, slipped epiphysis and equivocal for a left trochanteric abnormality. mri demonstrated a half-moon pattern of bone marrow edema at the left intertrochanteric area and at the major trochanter, surrounding an apophyseal low-intensity lesion. ap radiograph and limited ct confirmed the presence of a lytic lesion with sclerotic margins, containing calcified chondroid matrix. chondroblastoma was histologically confirmed following excision. mri, coronal stir sequence, demonstrates semilunar-shaped hyperintense area abutting the growth plate and the cortex of the femoral neck, consistent with the half-moon sign. note edema surrounding an apophyseal low-intensity lesion and soft-tissue edema. ct confirms a typical apophyseal lesion with sclerotic margins containing chondroid matrix. unique teaching points: "half-moon" sign refers to a semilunar shape of bone marrow edema at the intertrochanteric area of the hip with its base located at the cortex of the femoral neck. this distribution differs from the distribution of edema in metaphyseal and metaphyseal-equivalent osteomyelitis. "half-moon" sign has been described in patients with stress fractures and osteoid osteomas. to our knowledge, this is the first case of chondroblastoma exhibiting this sign. whenever the "half-moon" pattern of edema is identified at pelvic mri scans, a thorough search for an occult fracture line or a nidus corresponding to an osteoid osteoma or a chondroblastoma is mandatory. mr elastography (mre) is a noninvasive imaging technique that quantitatively measures liver stiffness and provides an estimate of the degree of fibrosis. our aim was to evaluate the feasibility of performing mre using both gradient echo (gre) and echo planar (epi) sequences on siemens scanners. a dedicated mre of the liver was performed on a t mr scanner (magnetom® skyra, siemens) with a pediatric mechanical ( ) (suppl ):s -s pediatr radiol driver (courtesy of mayo clinic) over the right upper quadrant. an axial t blade with fat saturation, a coronal t vibe dixon and axial diffusion weighted imaging (dwi) were obtained. elastograms were obtained using both an axial standard gre and a works in-progress (wip) epi sequence. for the gre sequence, different slices were selected and each scanned sequentially. the epi sequence incorporated different slices in just one series. images were post-processed placing regions-of-interest (roi) and measuring the stiffness in kilopascals (kpa). for each sequence and each slice the stiffness mean was measured and then the average of the means was obtained. a spleen elastogram was simultaneously generated, without changing the mechanical driver location, and mean stiffness was also calculated. based on cutoffs in the literature, values were considered abnormal if liver stiffness > . kpa and spleen stiffness > . kpa. our initial experience shows that mre is feasible on siemens scanners using both gre and epi sequences. epi sequences are a promising addition to standard gre. prone versus supine ultrasound positioning for evaluation of urinary tract dilation (utd) in children c. maya , y. gorfu , e. dunn , k. darge , s. back ; philadelphia/us, addis ababa/et objective: ultrasound (us) is used in the initial evaluation and surveillance of utd in children. utd classification systems, including the multidisciplinary consensus, assess anterior-posterior renal pelvic diameter (aprpd) and calyceal dilation. there is currently no consensus regarding optimal patient positioning-prone versus supine-during us assessment of utd. this study was performed to determine if there is a significant difference in the measurement of the aprpd, presence of calyceal dilation, or resulting utd consensus score obtained between supine and prone positions. two raters retrospectively reviewed renal bladder ultrasounds of patients with utd of one or both kidneys. technically adequate ultrasound examinations of orthotopic kidneys that were imaged in both supine and prone positions were included. those with renal anomalies or prior surgery were excluded. aprpd measurements, as well as central and peripheral calyceal dilation, were documented in both prone and supine positions. a postnatal utd consensus score was assigned to each kidney based only on these features. kidneys ( left) in subjects had utd in either the supine or prone position. mean age was . years (range: . - . y). female to male ratio was : ( / ). the interclass correlation (icc) of the aprpd between raters was . and . in the supine and prone positions respectively (ps< . ). central calyceal dilation was found in / supine kidneys and / prone kidneys by rater and / supine and / prone kidneys by rater (kappa . ). peripheral calyceal dilation was found in / supine kidneys and / prone kidneys by rater and / supine kidneys and / prone kidneys by rater (kappa . ). as such the results are presented as one. the aprpd tended to be greater when prone with a strong correlation between prone and supine measurements ( . , p< . ). the mean difference between supine and prone aprpd was . mm (p< . ). in kidneys, calyceal dilation was seen in the prone position and not supine while kidney had central calyceal dilation only when supine. the utd score differed between supine and prone in / kidneys, with all but one higher when prone. in other kidneys, the aprpd differed between positions however concurrent calyceal dilation resulted in no change in utd class. as a screening tool, performing ultrasounds in the prone position may help identify more kidneys with utd. further research is needed to determine if these differences are clinically significant. during the evaluation of magnetic resonance enterography (mre), diffusion restriction (dr) has been utilized as a marker for bowel inflammation, but in our practice we commonly see dr in otherwise normal segments of jejunum. the purpose of this article is to assess the dr in normal loops of jejunum on mre and to determine if there is a correlation between dr and luminal distention, age, magnet field strength, and bowel segment location. a retrospective analysis of subjects with a normal mre and normal clinical work up (based on available clinical history, endoscopy reports, serum white blood cell count and inflammatory markers, and stool samples) was performed. the abdomen was divided into quadrants. if available, loops of jejunum were randomly chosen in each quadrant. two radiologists independently evaluated these same loops of jejunum for the following: luminal distension, wall thickness, and enhancement pattern. additionally, the loops were then evaluated for the presence or absence of dr. inter-rater reliability was determined. disagreement was resolved by consensus. presence or absence of dr was correlated with luminal distension, age, magnet field strength ( . versus tesla), and abdominal quadrant. one hundred ninety-seven loops of jejunum were evaluated in patients. not all subjects had jejunal loops in all quadrants. sixteen subjects ( %) had jejunal loops with dr for a total of loops. one loop had increased wall thickness and another increased enhancement but both did not demonstrate dr. no other loops demonstrate increased enhancement or wall thickening. for the presence or absence of dr, inter-rater reliability was fair (kappa= . ). there was no correlation between the presence/ absence of dr in relation to luminal distension, age, magnet field strength, or quadrant location. of the subjects who had a single loop with dr, a nd loop with dr was found in %. year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. a. coronal haste demonstrates the craniocaudal position of the axial diffusion sequence for reference (line). year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. b. axial diffusion weighted seqeunce (b= ) shows diffusion restriction within loops of jejunum (arrow) within the anterior abdomen. year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. c. corresponding adc map demonstrates low signal within the jejunal wall consistent with diffusion restriction (arrow). diffusion restriction in normal loops of jejunum on mre was present in % of patients. if dr is seen in an otherwise normal segment of jejunum, this can be considered non-pathologic. a patient with a loop of jejunum with dr is likely to have an additional loop of jejunum demonstrating dr. there is no correlation with dr of normal jejunum with luminal distension, magnet field strength, or patient age. our data may help reduce overestimation of disease burden when clinically applied. imaging findings in the newborn with meconium peritonitis that require surgery p. caro dominguez , a. zani , a. daneman ; cordoba/es, toronto/ca objective: meconium peritonitis is a rare condition caused by an in-utero bowel perforation resulting in spillage of meconium into the peritoneal cavity and subsequent calcification. the role of prenatal and postnatal imaging is to identify infants who require surgery. the aim of this study was to evaluate the role of postnatal imaging in meconium peritonitis and to correlate the radiologic and sonographic patterns with the need for surgery. imaging studies in infants with meconium peritonitis performed between and at our institution were reviewed separately by a pediatric radiologist, a pediatric radiology fellow and a pediatric surgeon. patients were divided in a surgical and a non-surgical group. clinical, surgical and pathology reports were reviewed to validate the diagnosis. statistical analysis: comparisons between sonographic and radiographic findings and patterns in the surgical and non-surgical groups were performed using unpaired t-test and chi-square. during the study period, there were infants with meconium peritonitis managed at our institution. in the ( %) who needed surgery, the most frequent surgical findings were idiopathic perforation, jejunal and ileal atresia. ultrasound identified more cases with hepatic calcifications, meconium pseudocyst, ascites and pneumoperitoneum than radiography and radiography more cases of small bowel obstruction. ascites identified with ultrasound (p= . ) [fig ] and bowel obstruction [fig ] diagnosed either with ultrasound (p= . ) or radiography (p= . ) were associated with the need for surgical intervention. one third of children with meconium pseudocysts ( / ) [fig ] , did not require surgery. diffuse peritoneal or hepatic calcifications as an isolated postnatal finding were not associated with the need for surgery. both radiography and ultrasonography give valuable information to the surgeon to take the decision for surgery. dilatation of bowel loops and ascites detected postnatally with radiography and/or ultrasound require surgical intervention in children with meconium peritonitis. interestingly, a large proportion of infants with meconium peritonitis can be managed conservatively. . - . ) . those included had complete fmru analysis, dti (b= and b= , directions), and upjo configuration in at least kidney. cases with motion artifact (n= ), post-pyeloplasty (n= ) or duplex collecting systems (n= ) were excluded. pelvicalyceal dilation grade (pcd), corticomedullary differentiation (cmd), and functional parameters were included. pyeloplasty following fmru was recorded. dti tractography was reconstructed using a fractional anisotropy (fa) and an angle threshold of . and °, respectively (figure ) . user-defined regions-of-interest (roi) of the renal parenchyma, excluding the collecting system, were drawn to quantify dti parameters: mean fa, apparent diffusion coefficient (adc), tract length and tract volume. the relationships between dti quantitative parameters and fmru parameters were analyzed. age and adc (roi) (p< . , r = . ), tract volume (p< . , r = . ) and tract length (p< . , r = . ) were positively correlated. age and fa (roi) (p< . , r = . ) were negatively correlated. there was a correlation between fmru parenchymal volume and tract volume (p< . , r = . ), but median volumes were higher on dti (tractography= . cm vs. fmru= . cm ; p< . ). of the children, had pyeloplasty, had nephrectomy, were managed conservatively and was lost to follow-up. fa was significantly lower in kidneys that went on to have pyeloplasty in comparison to those without pyeloplasty, but the %ci and the iqr overlapped (table ) . the adc, tract length and tract volume were similar between these groups (table ) . there was no difference between the adc of fa values in kidneys with and without pcd or cmd (p> . ). linear hierarchical regressions controlling the age did not show a significant relation between adc and cortical or renal transit times (p> . ), but lower fa values were related to a higher renal transit time (p< . , r = . ). table . quantitative dti parameters between kidneys with and without pyeloplasty following fmru. renal adc, fa, tract volume and tract length change with age but tractography overestimates renal parenchymal volume. there was a tendency towards a lower fa in kidneys that went on to pyeloplasty. otherwise, none of the quantitative parameters evaluated in this study differentiated degrees of upjo. echo-enhanced voiding urosonography (eevus) has become an important imaging tool in urodiagnostics; however, it has been observed that during eevus the premature destruction of ultrasound contrast agent microbubbles might occur. the purpose of this study was to evaluate the possible causes of contrast vanishing during investigations and propose the protocol to avoid false negative results. eevus was performed in children from april to december . sonovue mixed with saline solution in a plastic bottle is applied by continuous flow through the urine catheter. the collected data according to the protocol in this prospective study was completed in children, aged from weeks to . years. the protocol included general patient information, indication for eevus, duration of eevus in minutes, and the presence of vesicoureteric reflux. extensive data about sonovue were recorded: charge number, expiration date, time since opening, amount of initially administered contrast (ml sonovue/ml saline solution), grading of the initial contrast opacification of the bladder, the need for immediate readministration of contrast (dose), grading of contrast opacification during examination, and the need for readministration of contrast later in the course of the examination (dose). in addition, the data regarding bladder (ratio real/predicted bladder volume, wall thickness, ureter dilatation), saline solution, the size of urine catheter (french), and the type of antibiotic prophylaxis were collected. child observation included grading of crying and muscle stiffness. normal contrast opacification of urinary tract during examination was found in / children, while in / ( . %) the contrast opacification was insufficient. in / ( . %) microbubble destruction occurred during the first minute, in ( . %) in minutes, and in in minutes after the beginning of contrast administration. the reason for unsatisfactory contrast opacification at the beginning of the eevus is probably due to small urine catheter size ( % of children with fr catheter had insufficient opacification compared to . % with fr in whole cohort), time since the contrast is opened (more than hours in children), and insufficient bladder emptying at the beginning of the procedure. the reason for microbubble destruction later in the course of the examination is bladder overfilling in combination with increased muscle stiffness and strong crying, which led to increased bladder pressure. there was no correlation between the type of antibiotics and microbubble destruction. we should be aware of possible false negative vur results during eevus caused by premature microbubble destruction. patients with fontan-type palliation of univentricular congenital heart disease have elevated central venous pressure due to their passive pulmonary flow. the altered circulation has a negative impact on several visceral organs, and these patients have chronic liver congestion. they are at risk of developing hepatic fibrosis and cirrhosis with potential malignant transformation. these changes can occur from only a few years after fontan palliation, making early detection and grading of major importance. the patchy pattern of hepatic changes makes liver biopsy an unreliable diagnostic tool. magnetic resonance imaging (mri) t mapping has been suggested as a technique for non-invasive assessment and quantification of hepatic fibrosis/cirrhosis. the aim of this study was to compare two different t mapping sequences of the liver in adolescents with fontan palliation, and in healthy controls. materials: adolescents ( - y) with fontan circulation and young healthy adults ( - y) were included as a part of an ongoing national population-based study. all underwent mri ( . tesla) pre-and post-gadolinium contrast, including two types of t mapping of the liver. a d t volumetric interpolated breath-hold examination ( d vibe) sequence with dual flips with b correction and a modified look-locker inversion recovery (molli) sequence. t relaxation times (ms) were measured by placing five standardized circular regions of interest (roi) in the mid-section of the liver and one in the spleen (fig ) . statistical analysis was performed comparing measurements pre-and post-contrast, between sequences, and patients and controls. there was a significant difference in the measurements between molli and d vibe with increased values for the latter. within each sequence there were small, but significant regional differences in relaxation times (table ). the same pattern was seen in pre-and post-contrast images in both groups. there were significantly increased native t times on both sequences in all regions in the fontan group as compared to the controls, but not post contrast. t relaxation times differ between the t mapping sequences, molli and d vibe, pre-and post-contrast. t mapping of the liver revealed significantly increased native t times in adolescents with fontan palliation compared to healthy slightly older controls. these findings suggest hepatic fibrosis/cirrhosis, but may also represent a component of congestion. diagnostic accuracy of ultrasound, computed tomography and wedge portography in the work-up for mesenterico-rex bypass in children with extrahepatic portal hypertension s. toso, r. breguet, m. annoshiravani, s. terraz; geneva/ch to identify the diagnostic accuracy of ultrasound (us), computed tomography (ct) scan and portography (wedge hepatic vein portography or direct portography) in the pre-operative work-up of mesenterico-rex bypass performed for extrahepatic portal hypertension in children. we conducted a retrospective analysis of pre-operative imaging for mesenterico-rex bypass in our tertiary hospital over the last years. we analyzed all patients between the ages of - years, with extrahepatic portal hypertension necessitating surgical treatment that underwent us, ct and portography. three reviewers independently analysed the patency of the left portal vein, mesenteric vein, splenic vein and the presence of communication between the left and right portal vein on preoperative imaging with correlation to surgical findings. statistical analysis of diagnostic accuracy was performed. eleven patients underwent mesenterico-rex bypass for portal hypertension secondary to portal vein thrombosis. two patients had partial liver transplant. ct with ultrasound correlation was sufficient in responding to the preoperative criteria in % ( / ) cases. portography was useful in the % ( / ) cases where ct could not respond to preoperative criteria, in particular the presence of left-right communication. there was good inter-rater correlation for each modality and good correlation of findings between modalities. in the majority of cases the use of ultrasound and ct is sufficient for preoperative planning for mesentrico-rex bypass. portography is mandatory in cases with large intra-hepatic cavernoma, where the left-right communication could not be confirmed on ct. contemporaneous clinical data was reviewed where available, and a clinical decision on disease severity and activity on a likert scale made with and without imaging. fifty-three patients underwent mre and bowel us in the specified timeframe ( male; median age . years, range - years). twenty patients had sufficient contemporaneous clinical information to be analysed. inter-observer variability for the imaging scores was assessed using bland-altman plots. where variability was beyond pre-determined limits, the studies were consensus reviewed. mean scores were used for the studies within accepted limits of variability. there was no significant difference between total mre and us scores (wilcoxon signed-rank test z= . , p= . ). at the bowel segment level, there was no significant difference between the mre and us segment scores for the ileum and terminal ileum (wilcoxon-signed rank test, z= . , p= . ), but significant differences were present between the imaging scores for other bowel segments, with mre identifying more abnormalities. there is a significant positive correlation between mre and clinical consensus scores (spearman's rho= . , p= . ) and between us and clinical consensus scores (spearman's rho = . , p= . ). imaging caused a refinement to the original clinical assessment in of the cases, with jejunal and ileal disease the most common reason for 'upgrading' a score and absence of any detectable abnormality on us and mre the most common reason for 'downgrading' a score. we found good agreement between mre and us for total patient imaging scores, ileal and terminal ileal scores. both mre and us scores correlated well with the gold standard clinical consensus, with imaging altering the original clinical decision in % of cases. although us detected fewer abnormalities than mr, it correlates marginally better with the clinical consensus, suggesting it is at least equally clinically valuable. background: differentiating between acute osteomyelitis (om) and acute bone infarct (bi) in children with sickle cell disease (scd) is a challenge for clinicians and radiologists, particularly when blood cultures are negative. although bone aspiration is the gold standard test for om diagnosis, it is an invasive procedure and infrequently performed. magnetic resonance imaging (mri) has shown a potential role in differentiating between acute bi and acute om. the goal of this case series is to evaluate the utility of fluid signal on unenhanced fat-suppressed (fs) t -weighted mr sequence in distinguishing acute bi and om in children with scd. methods: we reviewed a total of children with scd admitted with long bone pain during the one -year study period - attributed to either an acute bi or an acute om. twelve of patients with available bone aspiration, blood culture, and mri data were evaluated for fluid signal, marrow signal and other criteria. of patients, nine patients were diagnosed as acute bi and two patients had acute om and one with coexisting bi and om. the diagnosis was based on the fluid signal on t unenhanced t fs mr images as compared to aspiration cytology in which eight of nine patients with bi had hyperintense fluid signal on non-contrast t fs mr images while one of two patients with om demonstrated hypointense fluid signal. the last patient was diagnosed as a probable coexisting lesion (om&bi) based on a giant well demarcated hypointense marrow signal with an extraosseous hyperintense fluid signal. in acute bi, an abnormal hyperintense subperiosteal or paraosteal fluid signal is frequently observed on unenhanced t -fs weighted images. this finding was present in the majority of cases in our study population regardless of age, sex or site in the appendicular skeleton. mri fluid signal characteristic on unenhanced t fs shows promise as a criterion to differentiate between acute bi and om. role of mri to assess skeletal age in pediatric celiac disease s. bernardo, m. martino, a. laghi, e. tomei; rome/it objective: coeliac children are often subject to weight loss and lower somatic growth rate, compared to healthy children of the same age. the purpose of this study was to asses the feasibility of magnetic resonance imaging (mri) of the hand and the wrist to assess skeletal age and growth delay. we enrolled in our study coeliac children ( males and females) affected by histological proven coeliac disease, with a chronological age ranged between years and month and years and months (mean age of years, +/ years and months standard deviation). a single mri sequence (t d se, acquisition time: minute seconds) of the hand and wrist in coronal plane was performed of each patient to estimate the skeletal age. patients' data were compared with a population of normal subjects. the preliminary results showed a delay in skeletal age in children affected by coeliac disease in , % of the simple study, with a delay of maturity of . years (+/- , years of sd). only children showed advance mri skeletal age when compared to normal subjects. mri of hand/wrist to assess skeletal age may be considered as a reliable indicator of somatic growth. mri, without radiation exposure, can be an used as a diagnostic tool in skeletal delay. it could play an important role in the follow up of coeliac children, after glutenfree diet. the prevalence of metaphyseal injury and its mimickers in otherwise healthy children under two years of age p. eide, Å. djuve, r.e. gjøsaeter, k.f. forseth, a. nøttveit, c. brudvik, k. rosendahl; bergen/no objective: metaphyseal lesions in infants and toddlers are believed to have a high specificity for inflicted injury, however, normal metaphyseal irregularities may mimic pathology and lead to overdiagnosis. during the period - all children between and years, seen at the a&e department in bergen (bergen legevakt) due to an injury, and who had radiographs taken, were included. data on previous injury, age, sex and injury mechanism were drawn from the medical notes and pacs archive. all radiographs were reviewed by two researchers and an experienced paediatric radiologist, registering the following: number, site and type of fractures, signs of healing (yes, no), bone structure (normal, pathological) and metaphyseal appearances (shape (normal, metaphyseal collar, metaphyseal irregularity), injury). the study was approved by the institutional review board. six hundred one children ( girls) between and months of age (mean . months) were included, of whom ( girls) had a total of fractures. one hundred eight of the fractures ( . %) involved the forearm, followed by leg-fractures ( / , . %) and fractures to the clavicle ( / , . %). one epiphyseal separation and one metaphyseal lesion (without a history of trauma) were seen. one thousand three hundred twenty metaphysis were analysed, of which ( . %) were defined as either irregular ( / , . %) or demonstrating a metaphyseal collar ( / , . %). metaphyseal lesions with a history of trauma did not occur in otherwise healthy neonates and infants under years of age, indicating that this type of fracture has a particular mechanism. metaphyseal irregularities are frequent, particularly around the knee, and should not be mistaken for clms to evaluate whether mri might be used for age estimation, based on greulich-pyle (gp) atlas criteria. . tesla mri of the left hand was conducted in adolescents, and subjectively evaluated by two blinded radiologists. for sequence optimization, coronal mri sequences (t tirm, t vibe- d-we, and t se) and a left hand x-ray were compared in ten patients (eight male, two female; mean age, . years). the ages of healthy volunteers ( s ( ) (suppl ):s -s pediatr radiol male, female; mean age, years) were assessed from coronal t vibe- d-we. bland-altman plots, intraclass correlation coefficients (icc), and logistic regression models were calculated. coronal t vibe- d-we achieved the best image quality. the correlation between estimated patients' ages on x-ray and mri was high. icc showed high inter-observer agreement ( . for x-ray, . for mri). the estimated age of the healthy volunteers tended to be older than their chronological age. the probability of overestimation was higher in girls than in boys. coronal t vibe- d-we of the left hand is feasible for skeletal age estimation by gp criteria with a high readers' agreement. the likelihood of overestimation of healthy children makes it necessary to develop a new hand atlas representing changes since the s. to assess the relationship between the radiographic findings of metabolic bone disease (mbd) and serum biochemical markers in preterm infants. preterm infants in our neonatal intensive care unit between january and september were included. two readers retrospectively reviewed the wrist radiography for grading according to mbd severity. we recorded the levels of alkaline phosphatase (alp) and phosphorous (p) immediately after birth, on the same day of the first wrist radiography (alp-s, p-s), the highest alp levels before the first wrist radiography (alp-hb) and during follow-up (alp-h), and the lowest p levels before the first wrist radiography (p-lb) and during follow-up (p-l). patients were subdivided into four groups according to mbd severity determined by wrist radiography for the first analysis, and were divided into two groups according to mbd presence for the second analysis. one-way analysis of variance with a tukey multiple comparison and the student's t-test were used for statistical comparisons in the two analyses, respectively. a receiver operator characteristic (roc) curve was constructed to determine the optimal cut-off values of the biochemical markers for the radiological prediction of mbd. of the patients, , , , and infants were classified into grades , , and , respectively. in the first analysis, alp-s, alp-hb, and alp-h were significantly different between grades - and - (all p< . ). plb was significantly different between grades and (p= . ) and p-l was significantly different between grades and or (p< . or p= . ). in the second analysis, alp-s, alp-hb, alp-h, p-s, p-lb, and p-l were all significantly different between the two groups (p< . ). the roc curve of alp-h showed the largest area under the curve values ( . , % confidence interval= . - . ; p= . ) for detection of a radiographic change. the optimal cut-off value of alp-h was . u/l, and the sensitivity and specificity were . % and . %, respectively. the first wrist radiography was obtained at . ± . weeks after birth, and alp-h was measured at . ± . weeks after birth. the cut-off value of alp for the prediction of abnormal radiological changes in wrist radiography was determined to be was . u/l. our findings indicate that the highest alp level at around . weeks after birth could be a valuable predictor of radiological mbd in preterm infants, including those with very low and extremely low birth weights. quantitative grading of tmj synovitis in children with jia-influence of mr-coil, timing after contrast-injection and location of measurements on joint-to-muscle enhancement ratio a. hamardzumyan schmid, c. kellenberger; zurich/ch objective: assessment of signal intensity ratio between joint space and longus capitis muscle on contrast-enhanced mri has been proposed as reliable method across different mr-scanners and protocols for grading temporomandibular joint (tmj) arthritis in juvenile idiopathic arthritis (jia) with a cut-off of . for diagnosing synovitis. the aim of this study was to investigate potential influences on such enhancement ratios (er). retrospective evaluation of contrast-enhanced mr-studies of tmjs in girls with jia (age . ± . y) obtained at two occasions with two different coils on a . t scanner. joint-to-muscle er were calculated from signal intensity measurements in different joint compartments, muscles and sequences obtained with varying delay after contrast-injection, and compared with paired sample t-test. er of tmjs without synovitis (n= ) and tmjs with synovitis (n= ), determined by qualitative criteria, were compared to er reported in the literature. superior and inferior joint space to longus capitis muscle er for normal tmjs ( . ± . ; . ± . respectively) exceeded . in all but one case (figure) and for tmjs with synovitis ( . ± . , . ± . ) were significantly higher than in cases with synovitis from the literature ( . ± . , p≤ ). the same er were higher when obtained with dual-ring coil ( . ± . ; . ± . ) than with multichannel surface coil ( . ± . ; . ± . ; p≤ . ). while er to longus capitis muscle were higher than those to pterygoideus muscle for both coils (p≤ . ), er to pterygoideus muscle did not differ between coils (p> . ). not considering the timing of the scan, er to pterygoideus muscle were highest in the inferior joint space ( . ± . ), followed by the anterior joint recess ( . ± . ) and superior joint space ( . ± . ). comparing images acquired immediately after contrast injection to later images (median delay min, range - min), pterygoideus muscle er in the superior ( . ± . to . ± . ) and inferior ( . ± . to . ± . ) joint space increased substantially (p< . ), while er in anterior recess showed no significant increase ( . ± . to . ± . , p= . ). conclusion: joint-to-muscle er are clearly dependent on ) the signal profile of the mr coil with muscles located further away from the coil providing higher er, ) the time of image acquisition after contrast-injection with later obtained images providing higher er, and ) where the joint signal intensity is measured. as these factors need to be accounted for, the described normal and pathologic ranges of joint to longus capitis muscle er cannot be generalised for every mr-system and imaging protocol. integration of d c-arm ct images with navigational software provides real-time fluoroscopic guidance during percutaneous interventions in the interventional radiology (ir) suite. a trajectory, drawn from skin entry point to the target lesion on the d c-arm ct data, is overlaid on intraprocedural fluoroscopy for real-time needle guidance. this study describes our experience with syngo iguide (siemens) needle guidance software in a range of clinical applications in the pediatric ir suite, including technical success, radiation dose and procedure time. in this irb approved study, all percutaneous interventions performed in the ir suite using syngo iguide over a -year period were included. cases were classified by procedure type; for each type, mean effective radiation dose (msv) was estimated using pcxmc program (v . . . , stuk) and procedure times were evaluated. forty-five patients ( male, female; mean age: ± years) underwent iguide-assisted interventions including: bone biopsies - / ( pelvic, lumbar, and lower extremity), intra-articular steroid injections - / ( sacroiliac, and temporomandibular joint), lumbar punctures - / , percutaneous catheter placements - / (cecostomy, and chest tube placement) and bone biopsy with radiofrequency (rf) ablation - / . iguide was used in particular for the cecostomy procedure due to high sub-hepatic cecal pole position, and in the chest tube procedure due to the presence of loculated pneumothoraces. all procedures were technically successful. the diagnostic bone biopsy rate was . %. the mean estimated dose and procedure times for each procedure type are listed in table . sonography of neonatal spine (sus) is a simple, non-invasive, quick, relatively inexpensive method to evaluate lumbar spine anomalies in infants less than months of age. unossified posterior neural arches allow beam penetration to obtain high-resolution images of the intra-spinal contents. sus is carried out at the bedside, does not utilize radiation & requires no sedation. linear array transducers with extended field-of-view permit diagnostic sensitivity equal to mri. factors affecting mri resolution like patient movement, pulsation & vascular flow do not affect sus. we use sus as first-line screening test in neonates with lumbosacral cutaneous stigmata & spinal dysraphism (sd) associated syndromes. this was a prospective study approved by the institutional ethics committee. thirty five children (age range of to years) with clinically suspected and complicated pulmonary tb were enrolled in the study. lung mri and ct scan was performed in all the patients. the sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of lung mri in detection of radiological findings that were considered highly suggestive or diagnostic for tb, were calculated, with ct as the standard of reference. lung mri performed equivalent to ct in detection of pleural effusion, mediastinal/hilar lymphadenopathy and lung cavitation with sensitivity and specificity of %. agreement between ct and mri in detection of each finding was almost perfect (k: . - ). lung mri was found to be comparable to ct scan for detecting various radiological abnormalities which were highly suggestive for tuberculosis. being a radiation free imaging modality, it has the potential, particularly in children, to replace chest radiographs and ct scan in the coming years. to evaluate differences of myocardial strain assessed by feature tracking using ssfp cardiac mri sequences between pectus excavatum (pe) patients and healthy volunteers. in this prospective study, cardiac mri was performed in pe patients (with a pathologic haller-index above . ) and healthy volunteers ( males and females, respectively; age range - years) including short-and long-axis cine-ssfp sequences on a t scanner. post-examination analysis included standard cardiac volumetry with measurements of the biventricular ejection fractions (ef). additionally, manual biventricular contouring by an experienced radiologist, and subsequent automated strain assessment using dedicated software (circle cvi ®) was performed. longitudinal, radial, and circumferential peak systolic strain and strain rates were analyzed for both ventricles. left-ventricular ef was normal in all patients. five pe patients had a normal right-ventricular ef, in pe patients rvef was slightly impaired ( - %), all healthy volunteers had a normal rvef. compared with healthy volunteers, pe patients showed a significantly higher apical left-ventricular strain (radial: ± . vs. ± %, p< . ; circumferential: - . ± . vs. - . ± %, p= . ) and strain rate (radial: . ± . vs . ± . s - , p< . ; circumferential: - . ± . vs. - . ± . s - , p= . ). mid right-ventricular strain (radial: . ± . vs. . ± . %, p= . ; circumferential: - . ± . vs. - . ± . %, p= . ) and strain rate (radial: . ± . vs. . ± . s - , p= . ; circumferential: - . ± . vs. - . ± . s - , p= . ), as well as apical right-ventricular strain (radial: . ± . vs. . ± . %, p= . ; circumferential: - . ± . vs. - . ± %, p= . ) and circumferential strain rate (- ± . vs. - . ± . s - , p= . ) were also significantly higher in pe patients than in healthy volunteers. left-and especially right-ventricular radial and circumferential strain and strain rate increased from the bases to apices in pe patients. longitudinal strain and strain rate did not differ significantly between pe patients and healthy volunteers. myocardial strain assessed by cardiac mri differs significantly between pe patients and healthy volunteers. as the chest wall deformity usually leads to a compression of the basal parts of the ventricles, higher values of myocardial strain in the mid and apical ventricles in pe patients might indicate a compensation mechanism to enhance especially right ventricular output against sternal compression. to determine the normal range of the haller index (hi) value, and its dependence on the age, sex, and respiratory phase. evaluate the possibility of reduction of the effective dose (ed) of ionizing radiation using a single-slice ct scan technique. the retrospective-prospective study included patients (av. y, sd y). it consisted of parts. the prospective study included evaluation of ct scans performed by single-slice technique in patients with pectus excavatum both in inspiratory and expiratory phase, without topogram. hi was measured in each patient in both respiratory phases. in retrospective study, ct scans of the chest in children without pectus excavatum were analyzed to determine normal range of hi values depending on the age ( - y, - y, - y, - y) and gender. the retrospective study also included the analysis of another ct scans in patients who were operated or diagnosed with pectus excavatum. in the latter group of patients the average value of ed of ionizing radiation was calculated, and the values were compared with the average ed obtained using low-dose ct examinations applied in the new protocol (single-slice technique). the normal value of hi was . ± . . a significant positive correlation between age and value of hi was found. older patients had higher hi ( - y: . ± . , - y: . ± . ). results of mann-whitney test did not demonstrate any difference between gender in the observed group, however girls had generally higher hi in all age groups. in the group of patients who were operated/diagnosed with pectus excavatum, hi was . ± . . the average value of hi in inspirium in children with diagnosed deformity was . ± . , while in expirium it was . ± . . only / ( %) patients had hi value over . (a boundary value for surgical treatment) during inspirium, while / ( %) patients had it in expirium, which showed statistically significant difference (p= . ). single-slice ct technique during the inspiratory and expiratory phase showed average ed of . msv, which is an equivalent of chest xray. it reduced ed more than times in comparison with low-dose whole chest ct. the value of haller index increases with the age and in expiratory phase. we propose the single-slice ct technique without topogram in expiratory phase, as a sufficient and reliable technique in evaluation of haller index and preoperative preparation. mps iva is a lysosomal storage disorder caused by a deficiency of nacetylgalactosamine-sulfatase. main symptom is a systemic skeletal dysplasia. affection of the vascular system has not been described yet. our goal is the analysis of the vascular system in patients with mps iva, based on the example of the aorta. in a retrospective study, patients with mps iva were included. the aorta in its course from th thoracic vertebrae to th was analyzed on the basis of craniospinal mr and ct examinations. to describe the course of the aorta, the area around the vertebral body was devided into equal parts (fig. ) . high buckled arteries in relation to the length of the affected aortal part were indicated as aortal kinking, and a moderate twist in relation to the length of the affected aortal part as aortal tortuosity. results: twelve of patients had an aortal kinking, of patients an aortal tortuosity, of these had moderate and strongly tortuous aortae. seven patients had a normal aortal course, couldn't be analyzed. one patient revealed both, aortal kinking and tortuosity. this study reveals the occurrence of aortic tortuosity in patients with mps iva. we suggest that this complication could be due to glycosaminoglycane deposition in the aortic intima, which may be s ( ) (suppl ):s -s pediatr radiol associated with an increased vulnerability of the vascular wall. we conclude that the examination of the vascular system should be included in regular follow-up protocols. lung ultrasound in the diagnosis and follow-up of pneumonia in children -is it really as reliable as chest x-ray? s. balj-barbir, j. lovrenski, s. petrović; novi sad/rs to investigate the role of lung ultrasound (lus) both in the diagnosis and follow-up of pneumonia in children. a prospective study was carried out in the regional children's hospital, and included children (av. . y, sd . y) with clinically suspected pneumonia, in whom initial lus and subsequent chest x-ray (cxr) were performed within h. the final diagnosis of pneumonia at discharge was used as a reference test to determine the reliability of lus, cxr, clinical and laboratory findings in the diagnosis of pneumonia. children with pneumonia formed a study group, while the control group consisted of children without diagnosed pneumonia. lus finding of subpleural lung consolidation was considered a diagnostic sign for pneumonia. the children with lus signs of pneumonia were followed-up until complete resolution of the lus findings. there were from one to five follow-up lus examinations performed. a final diagnosis of pneumonia was confirmed in / ( . %) patients, and / ( . %) were hospitalized (av. . , sd . hospital days). in diagnosis of pneumonia lus, cxr, auscultation, elevated crp, and tachypnea showed sensitivity of . %, . %, %, % and . %, and specificity of %, %, %, % and % respectively. lus detected lung consolidations in of children with final diagnosis of pneumonia, and in / patients lus showed air-bronchogram (figures , ) . lus was superior to cxr in the detection of lung consolidations smaller than mm. interstitial lung changes were detected by lus in / ( . %) patients, and by cxr in / ( %). lus and cxr detected pleural effusion in / ( . %) and / ( . %) patients respectively. mcnemar's test showed no statistically significant difference, and cohen's kappa coefficient showed almost perfect agreement ( . ) between us diagnosis of pneumonia and final diagnosis of pneumonia. during the follow-ups, moderate to substantial agreement between lus and clinical evaluation of the course of the disease was obtained (k= . - . ). in children with complete clinical and incomplete us regression of pneumonia, consolidations of less than mm were the most prevalent finding. the average time period until complete resolution of the lus findings was . ± . days. children with us detected pulmonary consolidations larger than mm were statistically significantly longer hospitalized than others. lung ultrasound in the diagnosis of pneumonia in children is just as reliable as radiography, and should be included in the standard diagnostic protocol. the latest uk nice guidelines for childhood tb contact screening require that a chest x-ray (cxr) be requested only when mantoux or igra testing is positive or if there is a documented reason e.g. clinical concern. nice clarifies the role of cxr in determining treatment choice. we aimed to review cxr referral and treatment in the current climate of european migrant screening. retrospective review of paediatric referrals to the infectious diseases clinic for tb contact screening of whom had cxrs, from october to august and correlation with the medical notes. a panel of paediatric radiologists independently interpreted radiographs in the clinical context of tb contact screening and a majority decision was reached. of patients referred to the infectious diseases unit, underwent cxr in addition to a mantoux and igra test. of those cxr's, were reported as having features of pulmonary tb but only / ( %) were treated as active tb. eighteen of the ( %) cxr's which were reported as having no features of pulmonary tb, were treated as active tb. of those , only / ( . %) had a clearly documented reason. review of the radiographs (mean age years) by the panel of radiologists noted that all were of readable quality, radiographs were in keeping with a diagnosis of tb, were inconclusive and were normal. the diagnosis of tb was based on lymphadenopathy in and ( ) (suppl ):s -s pediatr radiol milliary nodules in . parenchymal abnormality was seen in patients [one was the milliary] and effusion was seen in . this correlated well with the initial radiology reports of duty radiologists. of the who underwent cxr, referral information was available in . ( . %) of these had been appropriately referred because of a +ve mantoux/igra. only out of ( . %) of those who had cxr despite a -ve mantoux or igra, had a documented reason. according to nice guidelines, % of cxr reported positive for tb were not treated for active tb. this may represent a lack of clarity regarding the definition of 'latent tb'. furthermore, only a third of the % of patients who received treatment despite negative radiographs had a documented reason. the current migrant crisis requires clarity of x-ray definitions of latent tb to avoid the % under-treatment and % overtreatment identified in our population. is there really no cardiac problem for performing sports Ö.İ. koska, p. bayindir, h. alper; izmir/tr objective: sudden death in young is a rare condition excluding known anomalies and sudden infant death sydrome; but its consequences are devastating because they are so unexpected. % of them occur in a context of sports event. everyday parents of millions of children admit hospitals in order to get permission for participating in sports events. and after physical examination and ecg, physicians are expected to decide such an important issue. however there are a number of silent reasons that may lead to child to sudden death. altough we don't perform ct scans for such indications, we have encountered several cases with abnormalities that can lead to sudden child death and while reporting an examination, awareness of these devastating conditions may be usefull. we searched our database from . . to . . in order to see how often we diagnosed such a silent reason from the ct images that are performed for some reasons. as our center is a tertiary center we have performed cardiac ct examinations in that period that are mainly for excluding or defining complex cardiac anomalies. in order to prepare a pictorial review of unexpected but ct detectable sudden cardiac death reasons, we excluded congenital heart diseases (namely obstructive, shunting or complex anomalies) and ecg detectable arrhytmic diseases. the non arrhytmic, non traumatic reasons for sudden cardiac death excluding congenital heart diseases in the papers are: hypertrophic cardiomyopathy (cmp) (% ), some coronary artery path and origin anomalies (mainly abnormal left coronary artery from pulmonary artery (alcapa), and interarterial path)(% ), increased cardiac mass (% ), dilated cmp (% ), marfan disease (% ), myocarditis (% ), ischemic heart disease (% ). we detected examinations according to our inclusion criteria and selected one cases of each; rca path anomaly, alcapa, dilated cmp, hypertrophic cmp, subaortic discrete membrane and increased cardiac mass for presentation although sudden cardiac death is rare in young children it is a so devastating condition that it must be taken into account for every situation. awareness of silent conditions and active search of them may protect professionals from medicolegal issues and unpleasent results. to describe the spectrum of chest ct scan findings of pulmonary involvement in childhood langerhans cell histiocytosis (lch) and propose a simple scoring system to evaluate the profusion and distribution of the main lung lesions. one hundred forty-six chest ct scans of the pediatric patients with pulmonary lch enrolled in the french national database for lch until april , could be retrospectively and independently reviewed by pediatric radiologists. for each ct scan a semi-quantitative analysis was performed for nodular opacities and cystic abnormalities. the chest was divided in fields (upper, medium and lower field of the left and the right lung) and for each field, both for the nodules and the cysts the score was =no lesion, = lesions involving up to % of the parenchyma, = - %, = - % and =more than %. of patients evaluated at diagnosis, patients ( %) presented with nodules, patients ( %) presented with cysts and patients ( %) presented a combination of both nodular and cystic lesions. on the initial ct scan, median nodules total score was and median cysts total score was . during subsequent ct scans almost the same percentage of patients with nodules ( patients, %) was found but we observed an increase number of patients with cysts ( patients, %), median nodules total score was and median cysts total score was . the distribution of nodules and cysts was symmetric in the upper, medium and lower fields with an involvement of costo-phrenic angles in % of the cases. patients with pneumothorax ( patients, %) had a higher cysts median score ( ) than patients without pneumothorax ( ). we found alveolar condensation in patients ( %). none of them showed signs of infection at bal examination or any improvement after a treatment with a standard antibiotic therapy while they did show regression under the lch standard regimen of chemotherapy. we proposed a score for semiquantitative analysis of distribution and profusion of nodular and cystic lesions on chest ct scans that can be a useful tool in pediatric population to monitor lung involvement. we found a significant correlation between pneumothorax and a high cysts median score. alveolar condensation could be considered as a possible manifestation of plch in children. lung bases involvement was found in % of the cases, representing an important different imaging feature from adult plch. high resolution computed tomography for chronic small airway disease in hiv infected adolescents a.-m. du plessis , s. andronikou , h. zar ; cape town/za, bristol/uk early treatment with antiretroviral therapy (art) and decline in infected infants due to prevention of mother-to-child transmission has resulted in an increase in the population of hiv-infected adolescents. pulmonary disease is common among them. cxr is considered insensitive and terminology inconsistent. therefore, despite concerns related to radiation dose in paediatric patients, high resolution computed tomography (hrct) is the modality of choice for the evaluation of small and large airway pathology, prominent in chronic pulmonary disease. hrct findings are used for prognosis, treatment decisions and defining anatomic extent of bronchiectasis for surgical intervention. the aim of this paper is to demonstrate the spectrum, frequency and extent of airway pathology in hiv-infected adolescents using hrct. a nested sub study was undertaken within the cape town adolescent antiretroviral cohort (ctaac); a prospective, descriptive cohort study of hiv-infected adolescents on art and age matched hiv-s ( ) (suppl ):s -s pediatr radiol negative controls. hrct was performed on patients who demonstrated abnormal lung function (defined by forced expiratory volume in second (fev ) of < % and/or low lung diffusion capacity (dlco)). single phase, contrasted multi-detector volume acquisitions were performed from the thoracic inlet to the diaphragms at full inspiration and image data postprocessed to yield thin ( , mm) and thicker slice images ( mm). three mm slices at cm intervals were performed in full expiration. three radiologists interpreted the c t scans independently, with strict imaging definitions, and a majority decision was generated for each finding. ages of patients ranged from between to years with a mean of , . there were females and males with a ratio of : , . bronchiolitis obliterans was seen in % of patients and bronchiectasis was demonstrated in %, % of which was classified as severe (involving either an entire lobe or more than % of at least lobes). there was an absence of lymphadenopathy (a sign of primary tuberculosis (tb)), lymphocytic interstitial pneumonitis (lip) and post tuberculous apical architechtural distortion. miliary tb was identified in a single patient. ground glass was seen in % and consolidation in %. the majority of hiv infected adolescents with poor lung function demonstrated bronchiolitis obliterans strongly emphasizing the use of hrct for confirming small airways disease. hrct was also useful for demonstrating extent of associated bronchiectasis in %. hrct allows classification of patients into those with diffuse small airways disease requiring medical management and those with local disease requiring surgery. background: chronic recurrent multifocal osteomyelitis (crmo) is an autoinflammatory paediatric non-infectious bone disease. presenting symptoms are non-specific, prolonging diagnosis, and leading to deformity, morbidity and unnecessary procedures. imaging is critical to diagnosis, with whole-body mri (wbmri) commonly used in all stages of care. in our institution, a baseline whole-body coronal stir sequence is routinely obtained. aim: to determine lesion distribution and extent on baseline wbmri via retrospective panel review of all patients clinically diagnosed with crmo, and to determine any patterns of involvement that could facilitate earlier radiological diagnosis. method: all patients diagnosed with crmo since december using published bristol criteria were identified and baseline whole body mris reviewed. the reviewing radiologists were blinded to the original report and previous investigations. each mri was reviewed for focal lesions consistent with crmo. the extent of metaphyseal and epiphyseal lesions was categorized into involvement of thirds of the width of the structure. the wbmri of forty children ( girls, boys), averaging years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) were reviewed by the panel using a majority decision rule. three hundred three lesions were recorded, averaging . lesions ( - ). the tibia was most affected ( lesions), most commonly the distal tibial metaphysis ( lesions in patients, bilateral). rib, metatarsals and distal femoral epiphyseals lesions were common. humeral, hand and skull lesions were few. complete metaphyseal involvement, the 'smouldering physis', was most prevalent within the proximal and distal tibial metaphyses. although ranked seventh, the reportedly more common clavicular lesions were the site of the most florid lesions, demonstrating bone expansion and periosteal reactions. two clear patterns of involvement emerged. in patients with clavicular lesions, fewer overall lesions were observed (average . ), mainly affecting the axial skeleton and feet. patients with tibial involvement had a higher number of overall lesions (average . ), but few lesions outside the lower limbs. only four patients had a both clavicular and tibial lesions. twelve vertebral lesions and four cases of spodylo-discitis were identified; two at t / level, one at t / level, and one involving both t / and t / . our series of cases of crmo with baseline wbmri, one of the largest in the published literature, identifies the common sites that should be interrogated for involvement. this study also demonstrates potential as-yet undescribed patterns of skeletal involvement that can be used to aid radiological diagnosis and highlights a non-infective cause for spondylo-discitis. hepatic hemangiomas (hh) are the most common benign vascular tumors encountered in the pediatric population. two main types have been described congenital and infantile, which both display distinct clinical courses and biological features. hemangioendothelioma in differential diagnosis of hh is controversy. recent literature suggests that congenital hepatic hemangiomas present in a focal form, whereas infantile hepatic hemangiomas present in either a multifocal or diffuse form. the goal of this study is to evaluate the features associated with focal and multifocal hh. the records of patients diagnosed with a hepatic hemangioma at a tertiary pediatric hospital from to were reviewed. we divided our series into groups: focal or multifocal including diffuse form. clinical endpoints are: age of diagnosis, presence of cutaneous hemangioma, alpha-fetoprotein, thrombocytopenia, cardiac insufficiency. imaging endpoints were echogenicity on us (hypoechoic, hyperechoic or mixed), vessels density on color doppler (< ; - , > cm ). presence of calcifications, venous lakes, visible vessels and aortic tapering were assessed on us, ct-scan, mr and angio. treatment and outcome were analyzed. univariate and bivariate analysis were done. this study included focal ( m, f) and multifocal ( m, f) hh. antenatal diagnosis was done in focal and multifocal hh. focal lesions were associated with the presence of cutaneous hemangiomas (p< . ) and calcifications (p< . ). no other variable was significant. conservative management was decided in focal and multifocal hh. steroids (focal: , multifocal: ), steroid-interferon (focal: , multifocal: ), propranolol-steroid (focal: , multifocal: ) and surgery in one focal form. complete regression was observed in most lesions (focal: n= , multifocal n= ), whereas incomplete regression < % was observed in patients (focal: n= , multifocal: n= ) and patients in the focal group with the pathology diagnosis of hemangioendothelioma. hepatic hemangiomas demonstrate a wide range of radiological features, with important overlaps in focal, multifocal or diffuse forms. focal and multifocal hh can be seen in congenital hemangioma, infantile hemangioma or hemangioendothelioma. the association of cutaneous infantile hemangioma in the focal group confirmed that the focal lesion can be seen in infantile hemangioma. however, calcifications are more frequent in focal hh which is described in congenital hemangioma or hemangioendothelioma. ( ) (suppl ):s -s pediatr radiol symptomatic and asymptomatic meckel's diverticulum in the pediatric population -a retrospective analysis of imaging findings with histopathologic correlation n. abu ata, r. cytter-kuint, j. bar-ziv, i. hadas-halpern; jerusalem/il objective: though meckel's diverticulum (md) is a relatively common gastrointestinal anomaly, many of the symptomatic and most of the asymptomatic md's are often missed on abdominal imaging. our purpose is to describe the radiologic appearance of symptomatic and asymptomatic md in the pediatric population and to correlate the radiologic findings with histopathology. a retrospective analysis of all children diagnosed with md between / - / and had relevant imaging (n= ) was done. imaging studies-ultrasound (us), computed tomography (ct) and magnetic resonance imaging (mri) were retrospectively reviewed and evaluated for visualization of md in symptomatic and asymptomatic patients. findings were compared with the preoperative radiology report and the pathology specimen. symptomatic group (n= ): mean age . ± years, nineteen males. md presented with abdominal pain in patients, small bowel obstruction (sbo) in patients, gastrointestinal bleeding or anemia in patients and intussusception in cases. md was identified in preoperative reports ( . %) and retrospectively identified in more cases (overall patients, . %). in cases, an inflamed or perforated md were found. in cases, mucosal lining resembling gastric folds was seen. inverted meckel and prominent tissue surrounding the diverticula were seen in patients. in a single case md was misdiagnosed as a duplication cyst. asymptomatic group (n= ): mean age . ± , eight males. md was not mentioned in any of the original reports and only md's were identified retrospectively ( . %). no mucosal abnormality or irregularity were noted. histopathology: ectopic gastric mucosa was found in / ( . %) of the symptomatic patients vs. / ( . %) in the asymptomatic group. all patients with sonographic appearance of gastric mucosa had gastric mucosa in pathology (specificity- %, positive predictive value- %). md has a variety of radiologic appearances. it can be detected in most of the symptomatic patients but is almost undetectable in asymptomatic patients. heterotopic gastric mucosa is more common in the symptomatic group. inflamed gastric mucosa may have a typical appearance resembling a small stomach, a sign that was not described before and has both high specificity and high positive predictive value for gastric mucosa within a meckel diverticulum. preliminary results on dna damage from ct irradiation in pediatric patients i. dilevska, e. nagy, w. schwinger, e. sorantin; graz/at the increased radiation sensitivity in children, compared to adults, is a well-recognised fact in the pediatric radiology community. the high-dose irradiation induced dna damage has been well established, however the dosages that are clinically used in everyday ct procedures are so low, that it remains unclear how this severely affects the dna and can induce cancer in the long run. the aim of this study is to assess the effects of lowdose ionizing radiation from ct in children by establishing a standardization curve ranging from the high to the low, medically significant ctdi values. this is done by measuring the phosphorylation of the h ax histone (γh ax), which is considered a biomarker for quantification of radiation induced dna double-strand breaks (ddsbs). the detection of the γh ax histone was done by two methods: immunofluorescence microscopy (im), which is an established method for detection and quantification of this histone and the new, promising flow cytometry technique (facs). for this study, leucocyte samples ("buffy coats") were provided by the local transfusion department and these samples were irradiated with a clinical ct scanner (aqilionone, tmse) with values ranging from , to , mgy ctdi. afterwards the samples were processed with both methods. for the immunofluorescence, twostep immunostaining was used with two different antibodies and the cell and foci counting were done on an olympus xc microscope, while the facs staining was done with a one-step antibody and the samples were measured on a navios flow cytometer (beckman coulter). comparable results were detected with both methods, with a good correlation between the facs and im, with a linear incline (r > . ) in the high and in the low dosages from , to . mgy ctdi. however, in the samples irradiated with doses below . mgy ctdi, there seems to be less phosphorylated h ax than in the native samples. two possible explanations arise: a) low dose irradiation initiates repair that extends to ddsbs occurring naturally or b) low dose irradiation doesn´t cause phosphorylation of this histone, but affects other dna damage and repair pathways. the preliminary findings show that the facs analysis can be used as a valid replacement method for the labor-intensive if method in the higher dosages. however more analysis should be done to establish its accuracy in the lower regions since underlying mechanisms are not clear yet. a. turkaj , g. cicero , e. sorantin , r. coroiu ; graz/at, mesina/it, cluj-napoca/ro there is only little information available regarding imaging procedures in the trauma setting of pediatric patients. such data can serve as a rational basis for running pediatric trauma units. the purpose of the paper is to investigate the number, types and distribution of imaging procedures in a tertiary pediatric trauma center serving children of about . million inhabitants with approximately . children. all trauma-caused admission to the emergency room and their imaging procedures were analyzed retrospectively occurring within a period of months. a cohort of patients (m:f= . : ) were analyzed. patients were grouped according to age into the following categories: neonates, infants, middle childhood, early adolescence, late adolescence. imaging procedures were classified into plain films, us, ct and mri. furthermore, the time of admission was noted and categorized in time slots : - : , : - : , : - : and : - : . referral cause was divided in domestic accidents, motor-scooter-bicycle accidents, car accidents, sport injuries, falls from height and others. the average age was . ± . years, aligned in the following age-groups neonates ( %), infants ( %), middle childhood ( %), early adolescence ( %), late adolescence ( %). a total of imaging procedures were performed, of which plain films ( %), us ( %), ct ( %) and mri ( %). there was a statistically significant difference of imaging procedures due to age in particular in us and ct. regarding the timeslots: : - : ; patients ( %), : - : ; patients ( %), : - : ; patients ( %), : - : ; patients ( %). domestic accidents were the leading referral cause with cases ( %) prevailed age groups were infants and middle childhoods corresponding for more than %. motorscooter/bicycle accidents accounted for cases ( %) of which most were early and late adolescence (more than %), s ( ) (suppl ):s -s pediatr radiol sport's accidents ( . %) equally shared among middle childhood, early and late adolescences. car accidents ( . %) cases and fall from height ( , %) did not show any prevalence according to the age groups. for the first time detailed data about imaging procedures at the emergency room for pediatric patients are now available. over half of the admissions ( %) occur outside regular work hours thus representing a challenge for the staff in duty and this fact should be considered in working schedules. due to strict interdisciplinary developed diagnostic pathways the number of ct examinations was reasonable low. head ct in a regional children's hospital without mri -effective doses and justification of clinical indications j. lovrenski, n. milenković; novi sad/rs to calculate effective doses (ed) for pediatric head computed tomography (ct), to determine the most common referral diagnoses, and the share of normal and pathological ct findings. a retrospectiveprospective study comprised all the children with performed ct examination ( -slice scanner) within a one-year period. pediatric ct protocols were used. the values of ed for head cts were calculated based on the two different models (shrimpton's and icrp publication ). average ed for different age groups was expressed as the number of chest x-rays (cxrs) ( cxr . msv). the most common non-traumatic referral diagnoses for head cts were determined, as well as percentage and type of pathological ct findings. a share of pathological ct findings was also determined for trauma as a referral diagnosis. head cts were represented with ( %) in total number of ct examinations within a one-year period. the different calculation models have shown the difference in ed values of up to . %. eds for head cts were equivalent of ( years of age and older) to (younger than months of age) cxrs for one sequence of scanning. the most common non-traumatic referral diagnoses for head cts were: loss of consciousness, epilepsy, headache, convulsions, and vertigo. in this group of patients, % of completely normal ct findings were found. pathological findings in this group consisted of the patients with the most common non-traumatic referral diagnoses were as follows: cortical atrophy ( patients), arachnoid cyst ( ), ischemia ( ), porencephalic cyst ( ), agenesis of the corpus callosum ( ), chiari malformation -type i ( ), open-lip schizencephaly ( ), and tumor of the posterior cranial fossa ( ). most common incidental, extracerebral pathology discovered included sinusitis and otomastoiditis. in patients with trauma as referral diagnosis, the share of pathological ct findings was . %. it is necessary to get clinicians familiar with the extent of ionizing radiation that children are exposed to during the head ct examinations. a more careful selection of children for head cts is necessary in an every-day clinical practice, especially for patients with non-traumatic referral diagnoses. diffuse and symmetric diffusion restriction involving the white matter of the brain in patients with neonatal seizures j.-y. hwang , y.j. lee , y.-w. kim ; yangsan-si, gyeongsangnam-do/ kr, yangsan-si/kr this study aimed to evaluate magnetic resonance (mr) imaging findings in patients with neonatal seizures focused on the diffuse white matter lesions on diffusion weighted image (dwi) in addition to clinical manifestations. a total of neonates aged less than -week old underwent brain mr imaging for evaluation of neonatal seizures between november and december . among them, patients showed diffuse and symmetric pattern of high signal intensity on dwi. clinical, laboratory, and mr images were analyzed retrospectively. nine patients were males and three patients were females. patient age was . ± . days (range, - days). all the patients were born at full term. the most frequent month of the hospital visit was march (n= ) and january (n= ). eight patients showed generalized clonic seizure and four patients showed partial clonic seizure. stool viral test was performed in nine patients. among them, five patients were positive for the rotavirus and one patient was positive for the astrovirus. nine patients underwent cerebrospinal fluid analysis, however, all showed negative results. mr imaging was performed at . ± . days after onset of seizures. diffuse and symmetric diffusion restriction were distributed along the cerebral white matter tracts and both thalami (fig ) accompanied with high signal intensity on either t -weighted images or on the fluid-attenuated inversion recovery (flair) sequence. multiple foci of high signal intensity on t -weighted images at the centrum semiovale that was affected on dwi were also observed. follow-up period was . ± . months (range, . - . months) and developmental delay was encountered in three patients. six patients underwent follow-up mr imaging at the age of . ± . months (median, . months; range, . - months). five patients showed volume loss in cerebral white matter on both sides of the brain and four patients showed high signal intensity of the periventricular white matter on either t weighted images or flair sequences (fig ) . myelination delay was not observed in follow-up mr images. diffuse and symmetric diffusion restriction involving the cerebral white matters can be seen in patients with neonatal seizures on mr imaging. our study shows that rotavirus is commonly encountered, but not exclusively detected in these patients. nevertheless, viral infection-associated encephalopathy should be considered when a patient is presented with characteristic clinical and mr findings. whole body mri on diagnosis and follow-up of neurofibromatosis type d. grassi, v. tostes, e. caran, h.m. lederman; sao paulo/br demonstrate that whole body mri is effective on showing neurofibromatosis type involvement of different regions of the body not known by the clinicians. review of patients with neurofibromatosis type (nf ) who underwent whole body mri throughout their follow-up with the majority of them had only brain and spine imaging studies. it was possible to demonstrate that whole body mri provides an overview of nf systemic manifestations and neurofibroma's extension beyond the clinic expectation. despite being rare, sarcomatous degeneration was suspected when there was any difference on the characteristics of the neurofibromas. whole body overview where its possible to see the neurofibroma's extension in right cervical region, scoliosis and multiple plexiform neurofibromas. only the biggest neurofibroma was detected by clinical exam. however it is possible to identify two others neurofibromas. whole body view of multiple plexiform neurofibromas. whole body mri is a radiation-free exam and it is useful on the diagnosis of nf and on patient's follow-up. it provides an overview of the systemic s ( ) (suppl ):s -s pediatr radiol involvement and neurofibroma's extension beyond the clinical expectation. during patients follow up, it could also show tumor's characteristics modification, which was considered as a possible sarcomatous degeneration. accuracy of non-radiologists and lay-persons for identifying children with cerebral cortical atrophy from 'mercator map' curved reconstructions of the brain s. vedajallam , a. chacko , s. andronikou , e. simpson , j. thai ; east london/za, bristol/uk objective: background: communication of cortical brain atrophy in children with term hypoxic ischaemic injury (hii) to parents and the legal fraternity contesting compensation rights can be very difficult using text and standard cross-sectional images. when demonstrating the cortex in hii, a single image of the brain surface, much like the way a map of the earth is derived from a globe, can be generated from curved reconstruction of coronal magnetic resonance imaging (mri) scans i.e. a mercator map. lay people's ability to identify abnormal scans from such maps without prior training requires evaluation before routine use. aim: to determine the sensitivity and specificity of lay people in detecting abnormal brain scans through review of mercator flat-earth maps of the brain, without prior training. ten mercator map images were provided to participants with a distribution of hii, cortical dysplasia and reported normal. participants were required to identify abnormal scans. sensitivity and specificity overall and for sub-groups were derived by averaging true positives and negatives; false positives and negatives. the results show a strong ability for lay-people to identify normal versus abnormal mri brain studies using mercator maps. the sensitivity and specificity in this group is % and % respectively. non-radiologist physicians and radiographers performed slightly better than lay people as expected. radiologists of course had very high sensitivity and specificity of % and %. the mercator map is therefore a viable tool in the communication of complex mr imaging to the lay-person. safety and efficacy of sphenopalatine ganglion blockade in childreninitial experience l. dance, c. schaefer, d. aria, r. kaye, r.b. towbin; phoenix/us objective: sphenopalatine ganglion (spg) blockade is known to be a safe and effective migraine headache treatment among adults. this paper will report the initial experience in the pediatric population with spg blockade. one hundred thirty-three procedures were performed in patients ages to from february through november . pre-intervention headache scores were recorded on a scale of to . the procedure was performed supine with neck in hyperextension. anesthesia of the bilateral nares was accomplished with lidocaine spray and gel. contrast was injected using a sphenocath confirming catheter position. % lidocaine was injected. patients remained supine with neck in hyperextension for minutes. post-intervention headache scores were recorded. mean pre-treatment score of . decreased to . post-treatment (Δ . , % ci . - . , p< . ). there were no complications. spg blockade is a safe and effective treatment for migraine headaches in children which results in decreased reliance on intravenous drug therapy. orbital masses represent a spectrum of benign and malignant lesions in children that can be challenging to diagnose and treat. imaging plays an important role in diagnosis, due to a potentially limited clinical examination and risks associated with biopsy. mr imaging is a powerful tool for imaging the orbit, due to the excellent tissue contrast it provides. yet conventional mri has a limitation in discriminate the benign from malignant lesions. diffusion-weighted imaging (dwi) is non-invasive rapid technique uses the water diffusibility to produce contrast among different kinds of tissues. our propose was to assess the role of dwi and calculated apparent diffusion coefficient (adc) values in characterization of the pediatric orbital masses regarding benignancy or malignancy. one hundred and thirty patients with recently diagnosed orbital masses and who underwent preoperative conventional mri and dwi were included in this study. the orbit was divided into six compartments: the eye globe, retroocular fat, optic nerve, lacrimal system, bony boundaries and extra-ocular muscles. the average adc obtained from each tumor was compared with the histopathological diagnosis determined from subsequent surgical sample. seventy girls and sixty boys with orbital masses were included in this study. their age was ranged from month to years. the globe is the seat of lesions in / cases, optic nerve in / case. seven cases have lesions in the lacrimal gland. forty-five of cases was diagnosed as having benign masses & of cases have malignant lesions. there is a statistically significant difference between the mean adc value of the benign lesions ( . ± . x - mm /s) and the mean adc value of the malignant lesions ( . ± . x - mm /s) (p< . ). the optimal adc cutoff value that was determined for discrimination between these lesions is: . x - mm /s), with sensitivity of . % and specificity of %. using conventional mri alone in predicting benign and malignant lesions has the sensitivity of % and specificity of % with % positive predictive value and % negative predicative value. combining dwi and conventional mri has increased accuracy, as the sensitivity and specificity were %, % respectively with % positive predictive value and % negative predicative value. ( ) (suppl ):s -s pediatr radiol adc values provide an accurate, sensitive, fast, and non-invasive mean of characterization of pediatric orbital tumors. a priori tumor characterization is useful in timing and treatment planning for orbital tumors. utiliy of resting state fmri in children for preoperative language mapping l.-m. leiber, m. delion, a. ter minassian; angers/fr to assess if resting state fmri is able to detect language eloquent areas in childrens. six children, from to years old suffering from brain lesions were enrolled in this study. they underwent mri with one dt morphology session and three minutes fmri sessions, including one resting state fmri and two language task induced activity fmri sessions. analysis was performed using a generalized linear model for the first one and a spatial independent component analysis approach for the two others. language maps were compared with cortical mapping obtained by intraoperative direct stimulation. language network was identified systematically by resting state session but not by task induced activity sessions. moreover, in two of the six patients, resting state fmri was able to detect eloquent areas found during intraoperative cortical mapping that were not present in task induced activity sessions. resting state fmri appears superior to task induced activity fmri in detecting language eloquent areas. is sclerotherapy an effective treatment option for ranulas or thyroglossal duct cysts in children? d. aria, l. dance, c. schaefer, r. kaye, r.b. towbin; phoenix/us to assess the utility of sclerotherapy in the treatment of ranulas and thyroglossal duct cysts materials: from - , patients varying in age from months to years were referred to the ir department for sclerotherapy. of the patients, had a diagnosis of ranula while had the diagnosis of thyroglossal duct cyst by either mr, ct, or us. sclerotherapy treatments were performed with standard sclerosing agents, i.e. sotradecol % foam, absolute ethanol, and bleomycin. in the subset of patients with ranulas, sclerotherapy was commonly performed in accordance with salivary (submandibular and/or sublingual) gland botox injection or ethanol ablation. -gauge or f sheathed needles were used for us-guided access to the lesions, with ranula sclerotherapy being performed after placement of side-hole drainage catheters ( - f) due to their increased viscosity. the preferred sclerosing agent was injected with dwell times ranging from mins to hours. salivary gland injection/ablation was performed under usguidance using a -gauge needle with volume injection targeted centrally within the gland or in the portion of the gland abutting the ranula. after treatment, all patients were scheduled for follow-up ultrasounds at a minimum of weeks to assess lesion response or residual disease. a total of sclerotherapy treatments were performed. of the patients, were lost to follow-up after single sessions for ranula and thyroglossal duct cyst. the other patients all had follow-up ultrasounds after each of the remaining sclerotherapy sessions. four of these patients showed initial improvement with either decreased size of lesion or lesion resolution while the other showed no improvement with either stable or increased size on initial follow-up. the patients who initially showed promising response unfortunately had recurrence on follow-up imaging and ultimately, demonstrated no favorable response to sclerotherapy after subsequent treatments regardless of whether treatment was combined with ethanol/botox salivary gland injection. in summary, all patients who were successfully followed show no appreciable response to treatment for ranula or thyroglossal duct cyst. despite the emergence of clinical requests for sclerotherapy of ranulas and thyroglossal duct cysts, in our case series, sclerotherapy has not proven to be an effective treatment option using our current drug regimen. role of the susceptibility-weighted imaging (swi) in the neuroimaging of term newborns g. rudas, e. varga, p. barsi, l. kozák, Ü. méder; budapest/hu objective: susceptibility-weighted imaging (swi) was introduced in the neonatal neuroimaging only a few years ago. we can find only a few publications about its advantages and disadvantages. according to our experience, swi is extremely useful not only for detecting bleedings but for the diagnosis of other diseases as well. during the last year we had mri examinations on term newborns ( - days of life) and the swi gave additional information in cases. we used a t philips insignia scanner. in the case of the questionable hypoxic-ischemic-encephalopathy ( cases) and the metabolic diseases ( cases) we could find increased signal intensity in the cortex; in the case of stroke we could find the thrombus itself in cases; the avm were much clearer using the swi in cases; at the pvl in cases we could visualize the cysts better using swi; in the case of congenital heart disease ( cases) and in the case of sinus thrombosis ( ) we could find microbleedings and/or dilated veins; in cases the position of the lateral ventricle drain or shunt was much clearer using the swi. the swi gave important additional information in / ( %). the swi is a strongly recommended new sequence at the mri examination of the term newborns' brain. a disadvantage of swi is that it requires ca. three minutes examination time (in contrast to t * which is only minute long). mechanical birth-related trauma: imaging of the "accidents of birth" a. chaturvedi, j.g. blickman; rochester/us objective: . to discuss definition, incidence and risk factors leading to "mechanical birth-related trauma" and compare these with existing literature. offer an organ-system based classification scheme encompassing the varied manifestations of birth-related trauma and describing the implications on care decisions. materials: the hospital imaging department database was searched for neonates who presented with history of difficult/traumatic birth at our obstetric center between january , -june , . search software used was primordial customised radiology solutions, san mateo, ca. the search terms used were "macrosomia", "shoulder dystocia", "instrumental delivery", "malpresentation", "cephalopelvic disproportion", "forceps" and "vacuum". initial and follow-up imaging and clinical data on these neonates was reviewed and compiled by two board-certified pediatric radiologists. the relevant literature was reviewed and findings compared. organ-system based classification scheme for birth-realted trauma. in our study, mechanical trauma of birth was seen to manifest within different organ systems, which have been listed below in the order of occurrence within our sample. injuries to the skull (sutural overlap, dents and fractures), scalp hemorrhages (subgaleal hematoma, cephalhematoma, caput). intracranial intraand extra-axial hemorrhages (subdural, subarachnoid, epidural, intraparenchymal). clavicle fractures neonatal brachial plexus injury. sternocleidomastoid hematomas. adrenal hemorrhages. cervical spinal cord contusions. schematic diagram depicting intra-and extracranial hemorrhages by location. -year-old with history of calvarial fracture at birth-fracture did not heal but enlarged secondary to leptomeningeal entrapment at the fracture sitean entity called "growing fracture" or "leptomeningeal cyst". multiple newborn organ systems can be injured from mechanical trauma of birth. our numbers compare favourably with the existing literature. mechanical birth-related trauma can occur simultaneously with hypoxic-ischemic birth injury. although most of these injuries spontaneously and completely resolve, long-term complications can be seen in some cases. few of these injuries are life-threatening. imaging plays a crucial role in diagnosis and follow-up, and can assist in decision making as well as in counselling the parents. ewing sarcoma of tibia in an infant girl a. seehofnerova, j. skotáková, i. Červinková; brno/cz objective: ewing sarcoma (es) is the second most common primary bone malignancy in children. it histologically originates from neuroectodermal tissue and consists of small round blue cells. ewing tumour family is very close to primitive neuroectoderm tumour (pnet) family with diverse stage of differentiation, ewing sarcoma being less differentiated. approximately % of the cases occur between ten and twenty years of age with slightly higher prevalence in male gender. nine-month-old caucasian girl presented to local surgery department after she had wedged her lower leg in a bed. the right lower leg was swollen and painful. she was initially diagnosed with a ligament injury and underwent standard treatment. oedema gradually disappeared, but swelling and pain increased after three weeks. she also suffered from a fever of . °c ( . o f). at that point x-ray of her right lower leg was performed with report describing pathologically changed structure of tibia and she was referred to our university centre. ( ) (suppl ):s -s pediatr radiol we made a second reading of the plain film, reporting sclerotic heterogeneous bone structure of the right tibial diaphysis and distal metaphysis, onion-like periosteal reaction with sunburst spiculation and cortical bone destruction. her laboratory results were: crp . mg/l, ld . μkat/l, nse . μg/l, ferritin μg/l. crp has been raising for a week to mg/ l, then decreased to normal level. differential diagnosis was established as a primary bone malignancy (especially es) or, less likely, an osteomyelitis. mri revealed pathological signal of bone marrow of diaphysis of the whole tibia with cortical scalloping and periosteal spiculated apposition. epiphyses were spared. dorsal cortical bone was interrupted with extraosseal spread of the process. intraosseal part enhanced heterogeneously, whereas extraosseal component enhanced almost homogeneously after contrast medium administration. total size of the tumour was assessed as x . x mm ( . ml) . adjacent muscles were oedematous with post-contrast enhancement. there were also few enlarged lymph nodes in popliteal region. results from the biopsy confirmed ews with positive ews/fli gene. tumour was assessed as a localized disease, enneking iib. patient underwent chemotherapy according to aews doc protocol and a knee-exarticulation with no traces of tumour in resection lines. nowadays she is in the first complete remission. x-ray: ap view mri: etw _tse postcontrast, sagittal view, pre-treatment mri: etw _tse postcontrast, sagittal view, after initial treatment unique teaching points: ewing sarcoma belongs to common primary bone tumours in children but is a very rare unit in infants. despite the age predilection it is necessary to consider this diagnosis even in children younger than one year of age. scimitar syndrome together with pulmonary sequestration and horseshoe lung: congenital pulmonary venolobar syndrome b.e. derinkuyu, h.n. Özcan, y. tasci-yildiz, h g. cınar, u.a. orun; ankara/tr objective: congenital pulmonary venolobar syndrome (cpvls) comprises of a spectrum of pulmonary developmental anomalies. the main components of cpvls are hypogenetic lung partial anomalous pulmonary venous return, absence of pulmonary artery, pulmonary sequestration, systemic arterialization of lung, absence of inferior vena cava. minor components of cpvls include tracheal trifurcation, eventration and partial absence of the diaphragm, phrenic cyst, horseshoe lung, esophageal and gastric lung, anomalous superior vena cava, and absence of the pericardium. in this case presentation, we present a baby with scimitar syndrome, pulmonary sequestration, horseshoe lung and right aberran subclavian artery. a month-old girl was admitted to our hospital with the suspicion of scimitar syndrome from a different hospital. she did not have any symptoms. the physical examination was unremarkable. on plain radiograph, the baby had dextrocardia. there was a doubtful tubular structure with the shape of scimitar and a nodular radioopacity behind the heart (figure ). transthoracic echocardiography demonstrated the dextrocardia, atrial septal defect and the right pulmonary artery hypoplasia. afterwards, the ct angiography was done for confirmation of scimitar syndrome and other accompanying abnormalities. on the ct angiography, there was a partial anomalous pulmonary venous return to the suprahepatic inferior vena cava known as scimitar syndrome. besides this, there was a right pulmonary extralobar sequestration in the lung base. the arterial supply was arising from the celiac trunk, while the venous drainage was going directly to the inferior vena cava. therefore, the right lung was hypoplastic of which the tongue of the right pulmonary parenchyma passing between the aorta and heart, appearing confluent with the left lung in a horseshoe configuration. there was dextrocardia and right aberran s ( ) (suppl ):s -s pediatr radiol subclavian artery. the patient was subjected to catheterization and angiography for treatment. on plain radiograph, the baby had dextrocardia. there was a doubtful tubular structure with the shape of scimitar and a nodular radioopacity behind the heart unique teaching points: the term cpvls is an umbrella to a group of pulmonary parenchymal and vascular anomalies that may present in combination. mdct is a helpful diagnostic tool in the preoperative evaluation for delineation of the components of this syndrome. congenital pulmonary venolobar syndrome refers to a wide spectrum of pulmonary developmental anomalies that may appear single or in combination. the main components of congenital pulmonary venolobar syndrome are hypogenetic lung (including lobar agenesis, aplasia, or hypoplasia), partial anomalous pulmonary venous return, absence of pulmonary artery, pulmonary sequestration, systemic arterialization of lung, absence of inferior vena cava, and accessory diaphragm. in this case presentation, we describe a child with scimitar syndrome, bilateral sequestration, hypogenetic lung (single lobed left lung) and right aberran subclavian artery. an year-old syrian girl was admitted to our hospital with the history of heart defect. she did not have syncope or ciyanosis whereas she has easy fatigue and palpitation. on plain radiograph the anomalous draining vein was seen as a tubular structure paralleling the right heart border in the shape of a turkish sword ("scimitar") ( figure ) . transthoracic echocardiography demonstrated the scimitar vein as well as large patent ductus arteriosus (pda), atrial septal defect and left pulmonary hypoplasia. afterwards, the ct angiography was done for confirmation of scimitar syndrome and other accompanying abnormalities. on the ct angiography, there was a partial anomalous pulmonary venous return to the suprahepatic inferior vena cava known as scimitar syndrome. besides this, there was a bilateral intralobar pulmonary sequestration in the lung bases. the arterial supply of the right side was arising from the celiac trunk, while the left side feeding artery was originating directly from the descending aorta. therefore, the left lung had a single lobe with single pulmonary vein draining to left atrium. there was a large pda and right aberran subclavian artery. the patient was subjected to catheterization and angiography for treatment. the right sided anomalous draining pulmonary vein and the feeding artery of the right sequestration were closed in the first session. the procedure was completed without any complication. afterwards, the closure of the feeding artery of the left pulmonary sequestration and the pda were planned in the next sessions. on plain radiograph the anomalous draining vein was seen as a tubular structure paralleling the right heart border in the shape of a turkish sword ("scimitar") unique teaching points: congenital pulmonary venolobar syndrome comprises a heterogeneous group of uncommon abnormalities that may occur in combination. diagnosis of congenital pulmonary venolobar syndrome can be confirmed by ct angiography that allows detailed evaluation of vascular, tracheobronchial, and pulmonary parenchymal abnormalities with a single short, noninvasive procedure. neck infection disclosing diagnosis of congenital fourth branchial arc anomaly in a girl h.n. Özcan, z. aycan, b. ardıclı, m. haliloglu; ankara/tr objective: congenital branchial arc anomalies are rare entities. herein, we describe the imaging findings of acute suppurative infection of the neck caused by fourth branchial fistula in a child. case presentation: an -year-old girl presented to our pediatric emergency department with fever, left sided neck swelling and redness. her complaints were started five days ago. on her physical examination, there was a x cm, stiff, painful mass lesion with redness on the left side of the neck. blood count and thyroid function tests were in normal range; however, c-reactive protein level and erythrocyte sedimentation rate were elevated. neck ultrasonography revealed diffuse soft tissue swelling, a hypoechoic mass consistent with abscess in the left thyroid lobe and perithyroid tissue. the left lobe of the thyroid gland had poorly defined margin and a focus of air. contrast-enhanced neck mr imaging demonstrated an abscess in the left thyroid and perithyroid tissue ( figure ) and enhancement of the soft tissue plane around the left pyriform fossa (figure ). barium swallow revealed the sinus tract originating from the left pyriform sinus apex. the patient was operated after antibiotic treatment and sinus tract was surgically excised. the aim of this report is to describe three cases of right kidney wilms' tumor with cavoatrial tumor extension, referred to our institution between january and september . case presentation: three children, two girls ( and years old) and one boy ( years old) were admitted at the emergency service with cardiac failure symptoms; the latter had also liver failure. echocardiography showed right atrial thrombus in all three patients, as an extension of massive obstructive thrombosis of the inferior vena cava (ivc). abdominal ultrasonography revealed in all patients a right renal mass, associated to right renal vein thrombosis that extended to the ivc and to the right hepatic vein. contrast enhenced computed tomography confirmed findings. patients were treated primarily with chemotherapy before surgery, with partial regression of the thrombus in two patients and no response in one. ( ) (suppl ):s -s pediatr radiol unique teaching points: wilms' tumor is the most common renal malignancy in children and its intravascular extension is a well-recognized event. incidence of tumor extension to inferior vena cava (ivc) is reported to be of - % and intra-atrial extension of , - , %. it occurs most commonly in tumors located in the right kidney (probably due to the shorter path of the right renal vein compared to the left one). this complication does not directly influence the prognosis of malignancy, but the degree of intravascular extension determines technical surgical strategy and increases difficulty of the surgical procedure, especially when there is intracardiac involvement, which increases morbidity. several classifications have been proposed in the adult age group, but due to the similarity of the degree of intraoperative difficulty, the same classifications are used in children. pritchett et al. ( ) described the relation between thrombus and hepatic vessels: level i -intrahepatic intravascular extension; level iiintrahepatic extension; and level iii -suprahepatic or atrial extension. staehler et al. ( ) proposed a different classification that was posteriorly modified and detailed by daum ( ) : stage i -small extension (thrombus size within ivc < cm); stage iilarge thrombus (> cm within the ivc), but still below the hepatic vessels; stage iii -thrombus extending to the level and above the hepatic vessels; stage iv -intra-atrial thrombus. a year old boy presented with a soft tissue mass in his forearm which appeared to have grown quickly in size over a period of three to four months. physical examination demonstrated a welldefined mass in the dorsal aspect of the forearm with no pulsatile bruit. intial differentials included a vascular anomaly or a sarcomatous lesion. the patient proceeded to have an ultrasound examination which revealed a very well-defined heterogenous subcutaneous mass, mostly solid in substance. the lesion measured . cm x . cm x . cm (transverse x length x depth). there was no evidence of muscle invasion. prominent internal arterial vascularisation was demonstrated and the mass was classed as inderminate in nature. subsequent mr findings demonstrated a mass with t signal isointense to muscle, hyperintense t signal and marked homogenous enhancement. small foci of intralesional t hyperintensity and larger areas of t * gradient hypointensity were noted, in keeping with small areas of intralesional blood. vessels were seen to extend from the subcutaneous fat into the lesion. the mass slightly distorted the underlying extensor muscles and tendons of the forearm but there was no deep extension across the fascia. findings deemed the lesion to be more malignant in nature. the patient underwent incisional biopsy and histological findings confirmed a diagnosis of angiomatous fibrous histiocytoma. these tumours are rare soft tissue tumours which most commonly occur in children, adolescents and young adults. while it is rare, there is a potential for local recurrence and metastasis. therefore, it is essential to identify these tumours where possible or at least consider them as a differential for a soft tissue mass in a child. the surgeon commented that the imaging findings and report were essential in making the initial decision about whether to perform an incisional or excisional biopsy as the best treatment for the tumour is wide surgical excision with clearance of margins. unique teaching points: angiomatous fibrous histiocytomas are rare lesions with potential for recurrence and metastasis and therefore should be identified and managed appropriately as a malignant tumour. they are often confused as soft tissue haemangiomas or complex haematomas. it is very important to be aware of the presentation and imaging findings, remembering this form of tumour as a key differential for a soft tissue mass. nasopharyngeal anlage tumor in a neonate with the initial presentation of respiratory difficulty: correlation between imaging and clinicopathologic findings p.-s. tsai, d.-c. lin, s.-l. shih; taipei/tw the etiologies of nasal or nasopharygeal obstruction are variable in neonates. the respiratory symptoms are varied in these cases. mass lesions in nasal cavity or nasopharynx are extremely rare during the neonatal period. however, we must keep it in mind when respiratory problems occur in the neonatal period. here, we report a case presenting with sleep apnea resulting from nasal obstruction by a rare benign salivary anlage tumor in nasopharynx and discuss the imaging findings as well as clinicopathologic characteristics. the -day-old female infant had loud breathing sound, slow feeding and sleep apnea since birth. nasal endoscope and laryngoscope disclosed a polypoid tumor in nasopharyngeal cavity with a stalk connecting with posterior nasal septum. further magnetic resonance imaging (mri) revealed a lobulated mass about . cm in greatest diameter occupying posterior nasal cavity to the nasopharynx that was intermediate signal intensity on t -weighted/t -weighted images and heterogeneous gadolinium enhancement. the patient then received endoscopic resection. the tumor was shown locating in nasopharyngeal cavity and having a stalk from posterior nasal septum, partially occluding the choanae as well. resected tissue fragments displayed tan and whitish in color grossly. microscopic examination demonstrated duct-like structures and mesenchymal elements in a nodular pattern which are typical features of salivary gland anlage tumor. until now, there is no tumor recurrence for four years. unique teaching points: "salivary gland anlage tumor (sgat)" was firstly introduced in a report by dehner et al in . the tumor that has histologic resemblance to the developing salivary gland, is believed to be a hamartoma originating from minor salivary gland rather than a true neoplasm. congenital sgat displays male predilection and is a rare cause of neonatal airway obstruction. the mass is usual in the midline and attached to the posterior nasal septum or posterior nasopharygeal wall by a delicate pedicle. favorable results with simple excision are obtained. once massrelated airway obstruction is established, further examination with computed tomography (ct) or mri is helpful in anatomic evaluation, size measurement, characteristics definition and intracranial involvement. if mass induced airway obstruction is suspected in a neonate and sgat is considered based on imaging studies, invasive procedure should be careful due to the potential of tumor dislodgement from its fine pedicle resulting in complete airway obstruction. the association of intussusception with malrotation is referred to as waugh syndrome. [ ] malrotation occurs in approximately in live births. [ ] the incidence of malrotation amongchildren with intussusception is %. we hereby present a case report of waugh's syndrome associated with midgut volvulus. case presentation: a month old male child reported to the emergency department with the clinical history of vomiting, abdominal distension, bloody mucoid stools and incessant cry. routine blood examination revealed hb: . gm%, tlc: /cu mm, plt- . lac/cu. mm. ultrasound (us) examination was performed and it revealed dilated fluid-filled small bowel loops with moderate amount of free fluid, right iliac fossa showed bowel within bowel appearance suggestive of target/pseudo kidney sign of bowel intussusception. no pathologic lead point was identified. transverse ultrasound image through the upper abdomen showed superior mesenteric vein noted to the left of the superior mesenteric artery hence malrotation should be considered. in view of surgical emergency non contrast enhanced ct was done and axial image showed target/sausage shaped soft tissue density mass it had alternating areas of low and high attenuation due to bowel wall and mesentry. on emergency laparotomy patient was found to have intestinal malrotation with duodenojejunal junction on the right of the midline and mid gut volvulus in clockwise direction. intussusception with terminal ileum (gangrenous), caecum, appendix, whole of ascending colon, transverse colon were telescoping into descending and sigmoid colon. the volvulus was derotated and the in tussusceptum was reduced. the gangrenous terminal ileum and appendix was resected and ladd's procedure was done, a diverting ileostomy was created. the patient recovered uneventfully after which an ileo-colonic anastomosis was created transverse ultrasound shows a mass with a swirled appearance of alternating hypoechoic and hyperechoic "bowel-within-bowel" appearance (target sign) unique teaching points: on ultrasonography multiple, concentric, target like appearance of wall layers of invaginated segments (target sign) on axial scan, as well as pseudokidney sign (sandwich sign) on longitudinal scans were accepted as diagnostic criteria for intussusception. [ ] it can assess the relative positions of the smv and sma which are mostly abnormal in malrotation. upper gastrointestinal contrast study is the imaging reference standard for diagnosis of malrotation with or without volvulus. abnormal position of the duodeno-jejunal junction. spiral, "corkscrew" or z-shaped course of the distal duodenum and proximal jejunum, and location of the proximal jejunum in the right abdomen. [ ] a high degree of clinical suspicion and radiologist's awareness of this entity is helpful in guiding the surgeons towards diagnosis and prevention of morbidity and mortality. a rare case of epidermal naevus syndrome p. joshi; pune/in to acquaint the radiologists with the entity of epidermal nevus syndromes (enss) which are a group of rare complex disorders characterized by the presence of skin lesions known as epidermal nevi associated with additional extra-cutaneous abnormalities, most often affecting the brain, eye and skeletal systems case presentation: this one and a half year old child was referred to us for neuroimaging. he had multiple hairy naevi over his face, limbs including the palms, since birth, associated with blackish discolouration of his entire trunk. unique teaching points: epidermal nevi are overgrowths of structures and tissue of the epidermis, the outermost layer of the skin. the different types of epidermal nevi can vary in size, number, location, distribution and appearance. neurological abnormalities that can be associated with enss can include seizures, cognitive impairment, developmental delays and paralysis of one side of the body (hemiparesis). skeletal abnormalities can include abnormal curvature of the spine, the term "epidermal nevus syndrome" has generated significant controversy and confusion in the medical literature. originally, the term was used to denote a disorder that was actually several different disorders erroneously grouped together. in the recent past, the term was used to denote a specific disorder now known as schimmelpenning syndrome. however, the term epidermal nevus syndrome could be correctly applied to several different disorders. therefore, the umbrella term "epidermal nevus syndromes" now represents a group of distinct disorders that have in common the presence of one of the various types of epidermal nevi. however, there is so far no general agreement how to classify the types of this diverse group of disorders, adding to the confusion within the medical literature. these disorders are quite different from one another and are not "variants" of each other as is sometimes mistakenly stated in the medical literature. in the future, as the genetic molecular basis of these disorders is better understood, the classification may change or expand. bilateral axillary lump in a newborn diagnosed as hematoma h.n. Özcan, u. aydingoz, m. haliloglu; ankara/tr objective: most birth traumas are self-limiting and have a favorable outcome. injuries to the infant that result from mechanical forces during the birth process are not uncommon. they occur most commonly on head and neck after vaginal breech delivery. however, soft tissue hematomas can be rarely seen after caesarian section (c/s). herein, we describe imaging findings of a newborn with bilateral axillary lump diagnosed as hematoma. case presentation: a -year-old woman was admitted to an outside hospital at weeks' gestation for c/s due to prior caesarean operation. it was her fourth pregnancy (g p ). the pregnancy was unremarkable and she had normal ultrasounds at gestation. there was no history of trauma or fall during antenatal period. according to the c/s reports, the process of operation was uneventful any undue prolongation and without having used any other instrumentation. the weight of the female baby was . kg at birth. on the rd postnatal day, her mother noticed a left axillary swelling, then admitted to a tertiary children's hospital. her physical examination revealed, bilateral axillary asymmetry with a fluctuant, nontender swellings. there was no redness or discoloration of the skin. there was no clinical feature suggestive of trauma or bleeding diathesis. a superficial ultrasonography showed solid heterogeneous, hyperechogenic masses x mm in the left axillary region and x mm in the right side. doppler study did not reveal any flow in the masses. contrast enhanced mr imaging demonstrated, bilateral axillary mass lesions with fluid levels and smooth contours (figure and ) . t w images demonstrated hyperintense component suggesting hemorrhage. after the administration of the gadolinium-based contrast material, lesions showed peripheral enhancement (figure ) . a diagnosis of hematoma was entertained. the child was managed non-operatively. she was monitored clinically and radiologically. follow-up ultrasounds scan revealed significant regression of the swellings. unique teaching points: soft tissue hematomas can be rarely seen in newborns. the formation of axillary hematoma on the background of c/s is a rare complication, which, to the best of our knowledge, has not been previously reported. ultrasonography and mr imaging readily depicts hematoma and aids in the differential diagnosis. colorectal carcinoma (crc) is extremely rare in pediatric age, with an estimated annual incidence of approximately case per million individuals. the majority of reported cases occur in adolescence, while the incidence is further lower for children under years. the distribution between males and females is not equal, with higher prevalence in males (ratio of : ). the etiology in children is unclear as these tumors are often sporadic and not linked to a preexisting adenomatous polyp, unlike adults. predisposing factors such as familial polyposis of the colon, other polyposis syndromes, ulcerative colitis and familial multiple cancer syndromes were reported in % of cases. advanced stage at diagnosis, aggressive histologic subtypes (poorly differentiated, signet ring and mucinous adenocarcinoma) and poor survival are the hallmarks of pediatric crc. case presentation: a -year-old male presented with a history of dyspeptic symptoms (recurrent epigastric-right flank colic pain and heartburn) for the last eight months, without evidence of irregular bowel function. after a prior diagnosis of esophagitis secondary to a gastroesophageal reflux disease, physical and laboratory examinations revealed anorexia, progressive body weight loss, microcytic iron deficiency anemia and positive fecal occult blood test. during an emergency access, abdominal ultrasound identified rounded target liver lesions and circumferential heterogeneous mural thickening of the ascending colon. contrast-enhanced computed tomography scan (cect) demonstrated a marked circumferential wall thickening of the ascending colon and cecum with a longitudinal extension of mm and thickness of mm; the mass contained lowdensity areas and calcifications. furthermore hypovascular hepatic lesions along with lymph node metastases containing calcifications were identified. no lung metastases were found. histopathological analysis confirmed the diagnosis of metastatic colon adenocarcinoma. after chemo-and radio-therapy, only the hepatic lesions showed reduction in size and number. the patient subsequently underwent right hemicolectomy. one month after surgery he is in a rigorous follow-up through ultrasonographic evaluation of pleural effusion and ascites and cect. unique teaching points: crc, although rare, should be suspected in children presenting with unexplained persistent abdominal pain, progressive body weight loss and positive fecal occult blood test. ultrasound imaging can be appropriate in the preliminary detection of abnormal bowel wall thickening, lymph node and liver metastases; cect is mandatory to confirm the radiological diagnosis and complete the staging. to increase awareness of this rare syndrome and its varied presentation in order to facilitate its early diagnosis and treatment to prevent poor prognostic outcomes. case presentation: lemierre syndrome is a rare disease characterized by an initial infection of the head and neck leading to the development of a septic thrombophlebitis which has a propensity to spread and involve the jugular and facial veins. this progressive infection then leads to the development of metastatic septic emboli to the respiratory tract. we present the case of a year old boy who attended with a week history of fever and a cough. initial imaging on admission demonstrated a large left sided hydropneumothorax with multiple cavitating lesions throughout the lung parenchyma in addition to thrombosis of some of the segmental pulmonary veins. the hydropneumothorax was surgically drained and the patient was transferred to the paediatric intensive care unit after further deterioration with the development of a broncho-pleural fistula. following a short course of antibiotics there was no clinical or radiological improvement and sputum cultures grew coliform organisms which raised suspicion for a more distant source. when pus was noted to be discharging from the left ear, a contrast enhanced ct of the head and neck revealed a left mastoiditis with multiple cerebral abscesses and occlusive thrombi in the left jugular vein, transverse venous sinus, sagittal and straight sinuses. following this diagnosis antibiotic therapy was modified and targeted at anaerobes, which was vital in assisting the patients recovery and successful discharge home. unique teaching points: clasically the majority of lemierres syndrome begins in the oropharynxinvolving the palantine tonsils and peritonsillar tissue often presenting with fever, sore throat and neck pain. our case demonstrates an atypical presentation with sepsis and respiratory symptoms as a result of the septic emboli which delayed diagnosis. we have learnt from this case the importance of considering lemierres syndrome in patients presenting with signs of a respiratory infectionin particular cavitating pulmonary lesions-that have not improved with conventional therapy and to have a low threshold to investigate the head and neck as a potential source of infection. when the working hypothesis of meningitis could not help e. kovacs , n. pinter , g. balázs , a. machovitsch , a. arany , z. liptai , l. fonyad , p. benke ; budapest/hu, amherst/us objective: neuroinfection still represents a diagnostic challenge in the everyday practice, where clinical evaluation, imaging, laboratory and pathological workup and treatment goes hand in hand under the pressure of time. we summarized a case in which, despite the extensive multilateral collaboration the battle was lost, to bring attention to the possible causes. a two year old, previously healthy female was taken to the emergency department for altered state of consciousness and fever. she also suffered from gingivitis. the unconscious child underwent an emergency ct scan: hydrocephalus with signs of raised intraventricular pressure was detected. subsequently mri of the head and spine was performed, and showed signs of diffuse leptomeningeal enhancement with basal predominance. multiple dwi restricted parenchymal lesions with basal predominance were also found. repeated csf and blood tests did not reveal any causative organism, although the gradually increasing crp suggested infection. two weeks after the onset of symptoms a follow up mri study showed extensive cerebral and spinal swelling with no focal lesion. the child passed away three days later due to cardiac failure. autopsy and neuropathological evaluation could not reveal the cause of the disease, which was identified only weeks after the child died, by culturing sputum and csf. unique teaching points: an overview of the clinical and radiological presentation of meningitis basilaris is carried out. attention is given to the circumstances, when tuberculotic infection should be suspected, and antituberculotic treatment should be started, even before the confirmation of the presence of mycobacteria can be obtained. to describe the clinical, laboratory and mri findings of chronic nonbacterial osteomyelitis(cno) in a patient with a negative radiograph and emphasize useful imaging findings, including an unusual radial pattern of edema in both femoral heads. case presentation: a -year-old adolescent, was referred with progressive debilitating hip pain and inability to walk since days, that was unsuccessfully treated with non-steroidal anti-inflammatory drugs. during hospitalization he developed fever up to ο with normal full blood count and smear, elevated esr ( mm/h) and crp ( . mg/dl), positive serologic markers for streptococcus (asto) and ebv and received antibiotics with relative good response. blood cultures did not grow any pathogens, the rest of serology was negative for acute infection, tuberculin skin test was negative and immunological investigation was unremarkable. pelvic radiographs were negative. mri showed a symmetric pattern of bone marrow involvement around both triradiate cartillages, at both femoral heads and ( ) (suppl ):s -s pediatr radiol major trochanters. complementary evaluation of tibial areas with a limited protocol disclosed asymptomatic involvement of tibial epiphyses and apophyses. a radial pattern of edema was seen at the femoral heads with alternating stripes of involved and uninvolved areas. clinical course and imaging appearances were highly suggestive of cno. rapid clinical improvement occurred during hospitalization while a repeat mri months later showed complete resolution of hip findings and the patient was free of any symptoms or signs. coronal stir sequence at presentation showing the radial pattern of bone marrow edema (arrowheads) alternating with stripes of normal marrow (*) at both femoral epiphyses. note hyperinense edema (arrows) around triradial cartillages. coronal stir sequence showing the predilection of bone marrow edema symmetrically around triradial cartillages (arrows) and at major and minor trochanters (arrowheads). coronal stir sequence at -months follow-up shows resolution of edema. unique teaching points: cno is a not well known chronic autoinflammatory bone disorder affecting primarily children and adolescents. positive serology for streptococcus or other infectious agents has been previously reported as in our case and may be a triggering factor. striking mri findings with a negative radiograph may occur at initial stages. symmetrical distribution of non-specific bone marrow edema around epiphyses and apophyses is highly suggestive of the diagnosis in the appropriate clinical setting and following exclusion of suppurative bone infections as well as bone or hematologic malignancies. the radial pattern of edema in our patient is unusual and considered to comply with the direction of main trabecular systems in femoral heads. in / chest cts, nodules (median size . mm) were detected. display mode a with mm mip yielded the best interreader variability (κ= . ) and the highest sensitivity ( . %) compared to mode b and c (κ= . , sensitivity . % and κ= . , sensitivity . %, respectively). perifissural nodules were detected in all subgroups. conclusion: mip improves the detection of pulmonary nodules in chest cts of young children, but overall interreader agreement is only fair. nodules, including perifissural nodules, occur in children with and without malignancy. images were subsequently read and interpreted by board-certified radiologists and nuclear medicine physicians in communal reading. in case of identifying suspicious lesions in cect additional imaging (mri) or biopsy was performed. compared to pet/ct employing only low dose ct (ldct), the use of cect resulted in the identification of additional suspicious lesions in patients. furthermore the use of cect allowed us to qualify lesions as benign/ physiologic which in pet/ldct were identified as suspicious and lesions suspect for metastases or tumor. in those patients who received combined integrated fdg pet/ct including both ldct and cect the ctdi ranged in between , - . mgy (n= . mgy) and the dose length product (dlp) ranged in between . - mgy *cm (n= . mgy *cm) specificity was significantly higher combining pet and ct compared to stand-alone ct and pet. our study showed that the acquisition of cect in combined integrated pet/ct leads to an increased specificity and thus represents an essential component of a good fdg pet/ct in pediatric oncology. in assessment of lymph nodes, inflammatory foci and liver lesions diagnostic contrast enhanced ct is essential. comparison of the detectability of ubos in neurofibromatosis type i patients with proton density-weighted and flair sequences in t mri l. porto, s. lescher, n. hillenbrand; frankfurt/de objective: neurofibromatosis type (nf ) is an autosomal-dominant congenital disease. in nf patients, significant numbers of so-called unidentified bright objects (ubos) can be found in brain imaging, with predilection sites at the basal ganglia and the dentate nucleus. ubos seem to develop at a very early age, contrary to other criteria leading to diagnosis. the detection of ubos might therefore prove helpful in the early diagnosis of nf , complementing the clinical diagnosis based on criteria of the "national institutes of health consensus development conference". the aim of the study was to investigate whether the detectability of ubos increases at t by comparing proton density-weighted images (pdw) with fluid-attenuated inversion recovery (flair) sequences. a total of nf patients ( male, female, between and years old, mean age . years) were examined by a t magnetic resonance scanner. the presence of ubos was evaluated on pd-w and flair images by evaluators ( experienced neuroradiologists, junior radiologist and student in his final year). detectability was rated by a three-point scoring system for dedicated regions: lesions which were "well defined/detectable", "suspicious" or "detected after a second look". the wilcoxon signed-rank test was used for comparisons between the raters. the level of significance was p< . . significantly more lesions were marked as "well defined/detectable" in the pd-w sequence compared to flair (p< , for all four evaluators together, as well as for each evaluator separately). in particular, pd-w proved to be superior for detecting ubos located in the medulla oblongata (p= , ) dentate nucleus (p= , ) and hippocampal region (p= , ), regardless of the level of the raters' experience. this is the first study that compares flair and pd-w at t for the diagnosis of ubos in nf . significantly more ubos are detected in the pd-w compared to flair sequences, especially for the infratentorial regions. as ubos occur at very early stages of the disease in patients with suspected nf , pd-w might aid an early diagnosis in these patients. assessment of radiation doses from diagnostic imaging in the followup of paediatric oncology patients p. logan , r. harbron , k. mchugh ; london/uk, newcastle-upon-tyne/uk objective: previous literature ( , ) has suggested paediatric oncology patients accumulate a large radiation burden as a consequence of routine diagnostic imaging examinations during therapy. we retrospectively looked at the effective doses from routine ct and nuclear medicine in three cohorts of children, namely patients with hepatoblastoma, wilms' tumours and rhabdomyosarcoma (rms). of note, in our centre we rely on repeated mris of the primary site for many tumours. effective doses (e), in millisieverts (msv), were estimated using the ncict dose estimation tool (lee et al ) , based on details specific to each procedure: patient age, scan region, scanner type and ct dose index (ctdi -an indicator of radiation exposure recorded at the time of each scan). doses for general radiography were estimated using pcxmc v . monte carlo simulations, assuming standard exposure factors and field size. there were patients in total ( hepatoblastoma, wilms', rms). there were boys. the mean age was years months (ranging from days - years months). the mean and median cumulative effective doses from ct for the whole cohort were . msv and . msv respectively. four patients in the wilms' cohort had a dmsa nuclear scintigram ( . - . msv), no hepatoblastoma patient had any nuclear medicine imaging, and patients with rms received a bone scan ( - . msv) or a pet scan (approximately msv). cumulative radiation doses from routine radiological investigations in paediatric oncology can be kept in a much lower range than reported in the literature ( , ). in our institution, the followup of solid intra-abdominal tumours with mri, with additional ct or nuclear medicine only when clinically justified, has resulted in a significantly low radiation exposure in these patient cohorts. mri of the primary tumour site should be implemented as a replacement for ct imaging when there is no significant detriment to the diagnostic information obtained. ( ) (suppl ):s -s pediatr radiol mri-based evaluation of multiorgan iron overload is a predictor of adverse outcomes in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation f. zennaro , d. zanon , r. simeone , g. boz , f. degrassi , m. gregori , g. schillani , c. boyer , n. maximova ; nice/fr, trieste/it objective: iron overload is associated with poor clinical outcomes in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). although the effects of hepatic and cardiac siderosis on patient outcomes have been extensively studied, less is known about the effects of siderosis in other organs. the medical records of consecutive pediatric patients who underwent allogeneic hsct in our institute from to were retrospectively reviewed. mri was used to measure iron concentrations in the liver, spleen, pancreas and bone. these patients were divided into two groups, with non-elevated (< μmol/g; group ) and with elevated (> μmol/g; group ) liver iron concentration (lic) at baseline. in group , only two patients had normal iron concentrations in all organs. none of the patients of group presented with pathological iron concentrations in only two organs. comparisons of baseline data with results of the first follow-up mri performed - months after hsct, showed a general worsening of iron accumulation. in group , none of the patients showed complete absence of iron overload in a single organ. in group , none of the patients showed a total absence of siderosis involving fewer than three organs. this study confirms the correlations between iron overload and the risks of transplant-related complications, such as transplant related mortality, sinusoidal obstruction syndrome, infections, pancreatic insufficiency, and metabolic syndrome, in transplant recipients with systemic siderosis. another important finding of this study was the close correlations between pre-transplant bic and times to neutrophil and platelet engraftment (p< . each). ( ), ganglioneuromas (gn, ) and ganglioneuroblastoma (gnb, ), examined by t mri were retrospectively grouped according to tumor entity, risk factors (bone marrow metastasis, mycn amplification or p deletion) and therapeutic regime (observation versus chemotherapy). dw (b values , and ) and conventional mri images (t , t pre and post contrast) were analyzed for tumor size, relative si-and absolute adc-values at baseline (base; no therapy), and after (fu ) and (fu ) months. adc values in nb were lower than in gnb and gn ( . * - mm /s versus . * - mm /s; p< . ). there was a tendency towards lower adc values in tumors with risk factors (n= ) versus no risk factors (n= ) at baseline, which did not reach statistical significance (p= . ). during follow-up shrinkage of tumor volume was noted (baseline ml, fu ml, fu ml; p< . baseline vs. fu ; p= . baseline vs. fu ). in the observation group, tumor adc values rose without relapse ( . * - to . * - mm /s). only in eventually relapsing tumors adc values tended to decrease further ( . * - to . * - mm /s, p= . ), despite initial reduction in tumor size. to establish inter and intra-observer variability in the radiological detection and assessment of pulmonary nodules at diagnosis in children with wilms tumours. a test set of ct thoraxes at diagnosis from patients enrolled in the multicentre 'improving population outcomes of renal tumours of childhood' (import) study in the uk were assessed. five radiologists ( chest, paediatric) from different centres ( uk, netherlands) completed a scoring sheet for nodule assessment on the same studies on two occasions, months apart. the readers were blinded to patient respiratory symptoms, the original radiology reports and also that they were scoring identical cases. descriptive statistics, modified bland altman graph and fleiss kappa scores were used for statistical assessment. in total, different pulmonary nodules across the ct thoraces at both rounds were scored by at least one reader. ( %) were seen by at least one reader in round and ( %) in round , ( . %) nodules were seen by at least one reader in both rounds. only ( %) nodules were scored by all readers in round , ( %) by all readers in round , and ( %) nodules by all readers in both rounds. of the nodules seen in the first round, were measured to be > mm in at least one dimension and of these, were classified as malignant by all readers. the limits of agreement for mean difference in nodule size in anterior-posterior, transverse and longitudinal measurements were ± . mm, ± . mm and ± . mm respectively. the fleiss kappa scores ranked from poor to fair agreement for nodule border smoothness ( . ), nodule shape ( . ), solidity ( . ) and impression of malignancy ( . ). within the same readers for both rounds, nodule detection rates of agreement were between . - . %. the average intra-reader percentage of observed agreements for nodule border smoothness, shape, solidity and impression of malignancy were . %, . %, . % and . % respectively. conclusion: detection and characterisation of pulmonary nodules on ct thorax shows both intra-and inter-observer variability. this has important implications for the interpretation of metastatic disease at presentation. fever without a focus is defined as febrile illness without an initial obvious cause or localizing signs. our aim is to assess the diagnostic value of whole-body mri (wb-mri) in the diagnostic work-up of children with fever without a focus. we retrospectively searched for subjects who underwent wb-mri for fever without a focus. a total of children (m= , f= ), mean age . years (range: . - . ) were included. / ( . %) subjects were immunosuppressed and / ( . %) subjects were hospitalized at onset of fever. the reference standard was based on positive cultures, biopsy or surgery. when this was not possible, a probable diagnosis was made based on clinical follow-up or serology. initially, the wb-mri images were reviewed independently by pediatric radiologists blinded to all clinical information. at the end of each case the final diagnosis and the diagnostic category ( categories: a. normal, b. infection, c. oncologic, d. rheumatologic, e. miscellaneous) was recorded. this was followed by a consensus read for comparison with the reference standard. for statistical analyses all subjects were treated as fever without a focus. results: reference standard: the diagnostic category of the reference standard was as follows: infectious / ( . %), oncologic / ( . %), rheumatologic / ( . %), miscellaneous. / ( . %). even after extensive work-up in / ( . %) no clear cause for the fever was found table . wb-mri: wb-mri diagnosed the cause of fever without a focus in / subjects ( . %) ( table ). in subjects ( . %) wb-mri results were falsely positive ( jia and myositis), and in the remaining subjects no imaging findings compatible a cause of febrile disease were found. interobserver agreement was fair (kappa . ). in children with fever without a focus wb-mri provided the diagnosis in in almost a quarter of the cases. given the multiplicity of causes of fever without a focus, some of them not possible to visualize on mr imaging, wb-mri may be considered in routine imaging practice when evaluating pediatric patients with fever without a focus. to compare linear measurement/volume to direct volumetric measurements using dimensional( d) post-processing software. for this irb approved study initial diagnostic ct or mr exams in patients( mo- yr) with solid tumors were reviewed by radiologists and technologists. radiologists recorded measurements in axes in their routine method, described tumor shape (sphere, ellipse, cone) and surface texture (smooth, almost smooth, or mildly, moderately, markedly irregular). three technologists individually, and radiologists by consensus, used d processing software (intellispace portal, philips, cleveland, oh) to directly measure tumor volume. tumor volume (v) was calculated from linear measuments using the following equations: sphere v= / πr , ellipsoid v= πr or πr , conicalv= πr or πr , and cuboid v=(xyz). inter-reader variability in tumor measurement in all tumors and for tumors divided by surface characteristics was assessed amongst radiologists and technologists, and radiologist consensus using coefficient of variation (cov). tumor shape analysis was reported as sphere, ellipse, cone, and surface texture smooth, almost smooth, mildly irregular, moderately irregular, markedly irregular. inter-reader variability of as much as , cc above to cc below the mean tumor volume was found when using radiologist determined linear measurements, with standard deviation (sd), range . - . inter-reader variability amongst technologist derived volumes was considerably less, range cc above to cc below the mean, with sd, range . - . cov analysis shows a greater degree of variation in tumor volume calculated from linear measurements [smooth( %), almost smooth( %), mildly( %), moderately( %), markedly( %) irregular] than direct volume determination [smooth( %), almost smooth( %), mildly( %), moderately( %), markedly( %) irregular]. variation was significant only for tumor with irregular surface texture [smooth (p= . ), almost smooth (p= . ), mildly (p= . ), moderately (p= . ), or markedly (p= . ) irregular]. variation in linear/volume measurements in very irregular tumors. light blue=middle % tumor volume measurements by pediatric radiologists. whiskers mark limits of range. ▲♦ • markers =measurements by technologists. note broad degree of variation. ( ) (suppl ):s -s pediatr radiol variation in linear/volume measurements in almost smooth tumors. light blue=middle % tumor volume measurements by pediatric radiologists. whiskers mark limits of range. ▲♦ • markers =measurements by technologists. note narrow degree of variation. both graphs show the same informationthe % relative variation in tumor volume measurements determined by dimensional linear measurements ( pediatric radiologists) v. volumetric processing (technologists & consensus group). radiologist generated measurements are subjective and unreliable. variation in measurement technique leads to differences in calculated tumor volume which significantly over or under estimate volume in tumors with irregular texture and is not significant in smooth tumors. quail-quantitative mri-based evaluation of pancreatic iron overload in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation f. zennaro , m. gregori , f. degrassi , e. cattaruzzi , y. diascorn , c. boyer , n. maximova ; trieste/it, muggia/it, nice/fr objective: iron overload (io) is a relatively common but often neglected transplantrelated complication and has been associated with poor prognosis in patients undergoing allogeneic hsct for hemato-oncological disease. pancreatic io is frequent among patients with transfusion-dependent anemias, but is uncommon among patients with hematologic malignancies. the causes of pancreatic io and the potential effects of pancreas iron deposits on transplant outcomes or on the risk of developing significant late effects in long-term hsct survivors have not been yet determined. our institute routinely uses magnetic resonance imaging (mri) with various gradient-recalled-echo (gre) sequences to quantitatively measure the iron concentration in abdominal parenchymal organs in all pediatric patients before and after allogeneic hsct. this study retrospectively analyzes the correlations of pancreas io with the type of conditioning regimen and pretransplant liver iron concentration (lic) in pediatric patients who underwent allogeneic hsct in our transplant unit over the last years. we enrolled patients, age - years. pre-transplant mean lic was , μmol/g (normal values μmol/g). ( %) patients had mild liver io and ( %) had moderate or severe io. pretransplant mean pancreatic iron concentration (pic) was , μmol/g, whose only ( %) had mild pancreatic io and none had severe io. post-transplant mean lic was , μmol/g, only one patient had mild liver io but patients ( %) had moderate or severe io. post-transplant mean pic was , μmol/g, ( %) patients had moderate or severe io. mean pre-transplant pancreatic volume was , cm , while mean post-transplant pancreatic volume (evaluated days after transplantation) was , cm . ( , %, p< , ) patients with post-transplant moderate or severe pancreatic io underwent tbi-based conditioning. mean reduction of pancreas volume in tbi group was , cm (p< , ). no pancreatic volume reduction was observed in chemotherapy-based group. all patients with pancreatic io have had exocrine pancreatic insufficiency and ( , %) patients have had metabolic syndrome. volume reduction well correlate (mean , %, p< , ) with pancreatic io. this study confirms that pancreatic iron overload is not so rare in patients with hematologic malignancy underwent allogeneic hsct, with increased risk of metabolic syndrome and total deficit of exocrine pancreatic activity, but not of endocrine activity. iron overload monitoring allows for chelation therapy optimization. mr is fast, reproducible and more reliable compared to serum ferritin and transfusional history and allows a multi organ evaluation. pulmonary tb is common in south africa, with many children affected. diagnosis can be challenging and chest x-ray remains fundamental for diagnosis. interpretation is difficult and shown to have wide inter-reader variability. no study however has compared cxr findings and interreader agreement between ambulatory and hospitalised patients. this study compares the frequency of cxr changes, as well as interreader agreement in ambulatory compared to hospitalised children with suspected tb. from nolungile clinic and red cross children's hospital respectively was done. each sample contained % proven tb and % negative controls. two paediatric radiologists and one paediatrician served as blinded, independent readers for the database using standardised ticksheets. our study demonstrated no significant difference in lymphadenopathy, but an increase in parenchymal change in the hospitalised group. we otherwise showed similar results to literature regarding finding frequency, but poor inter-observer agreement. if the least expert reader were removed, results were comparable with available literature. this highlights the need for development and study of explicit cxr criteria for lymphadenopathy to improve the value of cxr for paediatric tb in all settings. lung ultrasound in pediatric pneumonia -why is it necessary to use the additional trans-abdominal approach? j. lovrenski; novi sad/rs objective: to emphasize the need of lung ultrasound (lus) technique modification, which enables detection of pneumonia in children not visualized by using solely the standard trans-thoracic approach. a prospective study was carried out in the regional children's hospital, and comprised a -year period. the inclusion criterion was us finding of pneumonia detected by trans-abdominal, and not with trans-thoracic approach. lus examinations were performed using a combined, trans-abdominal and trans-thoracic approach. longitudinal, transversal (intercostal), and oblique sections were used. trans-abdominal examination included transhepatic and trans-splenic approach. the ultrasound probe was angulated from the most anterior to the most posterior sections while examining the lung bases by trans-abdominal approach. a pneumonia-positive lus finding included subpleural consolidation with air-bronchogram, or with an adjacent area of interstitial/ alveolar-interstitial edema. lus was always performed before the other diagnostic modalities (chest x-ray (cxr) and computed tomography (ct)), if they were indicated by pediatrician or radiologist. within a -year period in children (mean age . y, sd . y) the pneumonic focus was discovered using the trans-abdominal approach, while the trans-thoracic approach showed a normal lus pattern. all the children had the clinical symptoms of pneumonia (fever and cough, with or without dyspnea/tachypnea). the auscultatory finding was positive in children. cxr was performed in three children, showed a right-sided pneumonia in two, and was negative in one patient. one child had a contrastenhanced chest ct, which confirmed a left-sided pulmonary base abscess detected during lus examination by trans-splenic approach only (figures , ) . apart from pulmonary symptoms, there has not been any other associated diseases found, apart from otitis media in two children. each child responded to the antibiotics treatment with resolution of infection and us signs of pneumonia. in this oral presentation we will explain and give anatomical and technical reasons for pneumonia-positive us findings within lung bases, that remained undetected by the trans-thoracic approach. left-sided abscess abutted on the spleen (s), and was detected by trans-splenic us approach. it did not contact the pleural surface approachable by trans-thoracic ultrasound (black semi-lunar mark). l-liver. conclusion: trans-abdominal (trans-hepatic and trans-splenic) approach should become an inseparable part of each lus examination, along with a standard trans-thoracic approach. this modification of technique is expected to result in a further increase of lus sensitivity in diagnosing pneumonia. is thoracic ultrasound really competitive to computed tomography in children -a two-year retrospective study j. lovrenski, k. antolović; novi sad/rs to compare diagnostic accuracy of thoracic ultrasound (us) and computed tomography (ct) in children. a retrospective study was conducted in the regional children's hospital, and comprised a -year period. the inclusion criteria were: chest ct performed within h after the us examination of thorax, and us and ct examinations in the same patient performed by different pediatric radiologists. all us examinations were performed using a combined transabdominal-transthoracic approach. ct examinations were done ( ) (suppl ):s -s pediatr radiol according to the body mass based pediatric ct protocols. each hemithorax was analyzed separately in terms of comparison between us and ct findings. statistical analysis included the calculation of sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of ultrasound in diagnosis of pulmonary pathological entities. out of children with chest ct, of them (mean age , y, sd , y) fulfilled the criteria to enter the study group. lung us showed sensitivity, specificity, ppv and npv in diagnosis of pleural effusion: %, , %, %, %; lung consolidation: %, %, %, %; lung abscess: %, %, %, %; and interstitial lung disease: %, %, %, %, respectively. within hemithoraces multiseptation of pleural effusion was observed by us only. air bronchogram within lung consolidation was observed in hemithoraces both by us and ct examinations. necrotic areas within pulmonary consolidations were detected by us in hemithoraces, which was later confirmed by ct examination. lung abscesses were diagnosed in hemithoraces by both us and ct. two small lung abscesses filled with air ( hemithorax) and bronchiectasis ( hemithoraces) were detected only by ct examinations. other pathological findings detected both by us and ct examinations were: congenital pulmonary airway malformation (cpam) ( hemithorax), pulmonary sequestration ( hemithorax), partial pneumothorax ( hemithoraces), hidropneumotorax ( hemithorax), inflamed pneumatocele ( hemithorax), hydatid cyst ( hemithorax), pericardial effusion ( patients), soft tissue masses of thoracic wall with initial bone destruction ( patients), and lymphomas ( patients) (figures - ) . in one patient us and ct revealed cysts and an extremely dilated bronchus within lung consolidation (pathohistological finding: cpam type combined with subsegmental bronchial atresia, and extensive bronchopneumonia). us examination, unlike ct, could not differentiate between eventration of the left hemidiaphragm and diaphragmatic hernia in one patient. to determine and compare the accuracy of frontal cxrs alone and 'combination frontal-lateral' set of cxrs for diagnosing lymphadenopathy in children with tb using patients with confirmed tb and controls without tb, and to compare findings in hiv-infected and hiv-uninfected children. a total of children (ie: children with gene xpert confirmed tb and control patients admitted with lower respiratory tract infections), which were part of a larger south african study, who had both frontal and lateral cxrs, were included. three qualified radiologists read the cxrs in separate sittings one month apart (one for the frontal x-ray alone and one for the 'combination frontal-lateral' cxrs) for the presence of lymphadenopathy. odds ratios and % confidence intervals were calculated to determine the presence of lymphadenopathy using a consensus reading on the frontal cxr and frontal-lateral cxr combination according to the final diagnosis of tb. inter reader agreement was determined using the kappa statistic. lymphadenopathy was reported in ( %) patients on the frontal cxr alone and in ( %) patients on the frontal-lateral cxr combination. ( %) of the patients with lymphadenopathy on the frontal cxr alone were gene xpert positive versus ( %) of the patients with lymphadenopathy on the frontal-lateral cxr combination. in all patients, the consensus reading using a frontal-lateral cxr combination resulted in a -fold increase (or , ; % ci , ) in calling lymphadenopathy compared to using a frontal cxr only in the gene xpert positive group, the consensus reading using a frontal and lateral cxr combination resulted in a fold increase (or , ; % ci , - , ) in calling lymphadenopathy compared to a frontal cxr only. overall inter reader agreement for all readers when evaluating for lymphadenopathy was 'fair' on both the frontal cxr (k= , ) and the frontal-lateral cxr combination (k= , ). the addition of a lateral view to the standard frontal cxr increased the rate of calling lymphadenopathy. however, the accuracy of diagnosing lymphadenopathy on chest x-ray as a marker for tb was poor. this poor accuracy was further hampered by only 'fair' inter reader agreement for the presence of lymphadenopathy on chest x-ray. dynamic d ct imaging in children has significant advantages over routine ct scanning, bronchography and bronchoscopy for diagnosing trachebronchomalacia because it can be performed during free breathing without anaesthesia or invasive airwayaccess.itcanalsodemonstratevascularcausesoftracheo-bronchomalaciain the same sitting. the technique is currently performed in paediatric center in the uk. we aimed to report pitfalls encountered while setting up a dynamic d ct imaging service for children and report the findings of studies performed. materials: dynamic d ctscanning was introduced after installation of a large array ( slice) ct scanner, applications specialist training and review of the literature. imaging parameters in use by greenberg and colleagues (arkansas children's hospital, usa) were applied. referral indications, pitfalls encountered, quality of scanning and imaging findings/diagnoses were reviewed and enumerated. results: nineteen paediatric dynamic d ct scans ( females, males; days - years months; mean months) were performed over months. the first studies were performed without ivi contrast due to lack of experience and subsequent studies were performed with contrast ( figure major pitfalls included initial failure to perform contrasted studies for simultaneous evaluation of vessels, initial failure to withdraw the endotracheal tube, patient motion under care of nurses and clinicians, failure to appreciate the value of imaging the full lung volume while trying to keep dose length product to a minimum and failure to appreciate that collapse of the airway is often in the ap plane and not appreciated on coronal slab projections -rotating d volume rendered images is a requirement ( figure ). additional obstacles were initial clinician and radiologist lack of support after early failures and colleague concerns regarding the radiation dose. objective: diagnosis of pulmonary tuberculosis (ptb) in children relies on chest radiography, however there is wide inter-observer agreement in detecting lymphadenopathy, the hallmark of ptb. paediatric airways are pliable, thus detection of airway compression may be a more objective criterion for the presence of lymphadenopathy. thus the objctive was to assess the usefulness of airway compression on chest radiographs for diagnosis of ptb in children. chest radiographs of children admitted to red cross children's hospital with suspected ptb were read by two readers according to a standardised format and a rd when there was disagreement. radiographs of children with definite ptb were compared to those with lower respiratory tract infection (lrti) from another cause. the prevalence and location of airway compression was evaluated. findings were correlated with hiv status and age. inter-observer agreement was assessed using kappa statistic. . % in older children (or . ; %ci . - . ). no association with airway compression and hiv infection was found. inter-observer agreement ranged from . - . . eighteen-month-old male patient diagnosed with ptb; hiv negative. majority agreement of airway compression at lmb indicative of lymphadenopathy. left upper lobe opacity is in keeping with a ghon focus. there is a strong association between airway compression on chest radiographs and confirmed ptb, particularly in infants, irrespective of hiv status. however, clinical use is limited by poor inter-observer agreement. paediatric ultrasound-guided biopsies in a tertiary oncology centre: five years experience n. parvizi, m. smedley, s. chakraborty; oxford/uk objective: histological diagnosis is almost always essential to guide appropriate therapy for children diagnosed with cancer. tissue can either be obtained by surgical/open or image-guided percutaneous biopsy. the aim of this study is to assess the safety and diagnostic accuracy of ultrasound-guided biopsies in a tertiary oncology referral centre. a retrospective analysis of clinical data, imaging findings and histological diagnosis of patients aged to years between january and december was carried out. a total of ultrasound-guided biopsies were performed in our institution on patients. most of the biopsies were performed in theatre with the patient under general anesthetic and with an -gauge spring-loaded core biopsy needle with a minimum of two cores per patient. in % of lesions the needle biopsy was diagnostic. the single nondiagnostic case did not have sufficient material to make a full diagnosis and a surgical biopsy was required. eighty-two of the biopsied lesions were malignant and were benign. in no cases was a repeat biopsy required. the vast majority of the biopsies were performed within one week of request with over half performed within days. all biopsies were performed without complication and in the majority of cases the patients were discharged the same day or following an overnight stay. ultrasound-guided percutaneous biopsy is an accurate and safe technique in order to acquire tissue from suspected malignant lesions in children. these can be performed instead of or in addition to open biopsy and will often result in a shorter hospital admission and recovery time. the role of imaging in the diagnosis of thymoma in paediatric patients with myasthenia gravis j. adu, t. a. watson; london/uk thymomas are exceedingly rare tumours in the paediatric age group, with only very few cases having been reported in the literature. thymomas are commonly associated with myasthenia gravis (mg), with thymectomy being potentially curative. ct is the mainstay imaging modality for thymoma diagnosis in the adult population. while, chemical shift mr imaging can be helpful to distinguish thymoma from other anterior mediastinal abnormalities. currently, there is no consensus on the imaging pathway for children with mg with suspected thymoma. our aim is to review the imaging of patients who were referred to our institution for management of mg, and suggest an imaging pathway in cases where thymoma is suspected. we performed a retrospective search of the local pacs system of cases between and using the search terms "thymoma" and "myasthenia gravis" in the clinical indication for the study and the body of the final report. forty-three cases were identified using the search criteria. eight cases were excluded owing to an absence of cardiothoracic imaging. / of all cases ( %) had chest x-rays (cxr's), of these / ( %) were normal. the three remaining patients who had abnormal cxr's went on to have ct scans, which confirmed an anterior mediastinal mass (amm) in all three cases. / of all cases ( %) had cross-sectional imaging (mri / cases, ct / cases). of those, / of cases ( %) had normal studies. specifically, all mri studies ( % of cases) were normal, while only / ct scans ( %) demonstrated an anterior mediastinal abnormality. / of all cases ( %) had both cxr and cross sectional studies. / of these cases ( %) had a normal ct or mri. in the remaining three cases, the amm was clearly demonstrated on both cxr and the crosssectional imaging. in our series, radiography, ct and mri studies were normal in the vast majority of cases. however, given that thymectomy is potentially s ( ) (suppl ):s -s pediatr radiol curative, it is appreciated that clinicians may still be keen to radiologically investigate paediatric patients with myasthenia gravis. cxr is not an efficacious imaging modality in this context, as patients with a normal cxr may be falsely negative, and patients with an abnormal cxr may undergo cross-sectional imaging regardless. we propose that mri should be used as first line investigation for patients in this population. this approach will negate the need for ionizing radiation, maximize diagnostic yield, and facilitate surgical planning if deemed clinically appropriate. increased risk of venous thrombosis of the arm with multiple peripherally inserted central catheters insertion in paediatric patients r. gnannt , n. waespe , j. donnellan , k. liu , l. brandao , b. connolly ; zurich/ch, toronto/ca objective: peripherally inserted central catheters (piccs) are associated with superficial and deep venous thrombosis of the arm. the impact on the incidence of developing a thrombosis of the arm when inserting a subsequent picc remains unclear. the purpose of this study was to analyze the incidence of deep, upper limb thrombosis of repeated upper limb piccs in children. the study population included all patients who underwent their first successful arm picc insertion between january and december . subsequent ipsilateral arm piccs were included in the analysis. patients were followed until march or until any alternative central venous line insertion (jugular, femoral, saphenous or umbilical vein lines -because of their thrombogenic effect). for each picc insertion the following data were collected: date of insertion and removal, weight of the patient, type of picc ( . fr, . fr, fr, fr, fr), left or right arm, and vein cannulated (basilic, brachial, cephalic). all symptomatic deep and superficial thrombosis of the arm were correlated with the picc database. four thousand one hundred thirty-eight piccs were inserted. applying inclusion and exclusion criteria, piccs remained for analysis. first, nd , rd , and th picc insertions in the same arm were identified in , , and patients, respectively. in total there were upper body deep symptomatic thrombotic events diagnosed with ultrasound. a st , nd , rd , and th picc insertion was associated with / (incidence . %), / ( . %), / ( . %), and / ( . %) thrombotic events, respectively. an increasing hazard ratio was seen with higher numbers of picc insertions, which was significant when comparing the st with the rd picc insertion in the same arm (hr . , ci % . - . , p= . ). after excluding any confounder, double lumen piccs were associated with a significantly higher risk of thrombosis than single lumen (or . , ci . - . , p= . ). repetitive picc insertions in the same arm are associated with an increased risk of thrombosis. double lumen piccs are associated with a higher risk of thrombosis compared to single lumen lines. diagnostic performance of lung ultrasound for the detection of community acquired pneumonia in children j.a.m. stadler , s. androunikou , h. zar ; paarl/za, bristol/uk, cape town/za objective: chest radiographs (cxr) are considered the first line imaging modality when investigating cases of suspected community acquired pneumonia (cap) in children. however, cxr interpretation is limited by moderate sensitivity and specificity and poor inter-and intra-rater reliability and expose children to potentially harmful ionizing radiation. point-of-care lung ultrasound (lus) has been proposed as alternative to cxr for diagnosis of pneumonia in children and some published data suggest accuracy and reliability as good as or better than cxr. most of these studies however, were performed in in-hospital settings creating a bias for selceting more severe disease and consequently more overt radiological findings. the mean age of children in most of these studies were also well above one year, while the highest incidence and risk of complicated pneumonia occurs during the first year of life. the purpose of our study was to assess the diagnostic performance of lus for the diagnosis of pneumonia in both hospitalised and non-hospitalised children in an age group representative of the most at risk segment of the population. we performed a lus on children who presented with clinical signs consistent with the who case definition for childhood pneumonia. one hundred of these patients also had chest radiographs performed as part of routine clinical care. inter-rater reliability (irr) between a general practitioner and an expert paediatric radiologist were assessed for the interpretation of lus findings consistent with pneumonia. where radiographs were available concordance between lus and cxr findings of pneumonia were also assessed. results: seventy-four hospitalised and non-hospitalised clinically defined pneumonia cases were included with a median age of . years. our general practitioner reported lus findings consistent with pneumonia in / ( %) compared with / ( %) by the paediatric radiologist. substantial overall agreement between the reporters was found with an overall agreement proportion of . and kappa= . . agreement for the presence of lung consolidation or for a normal scan was also substantial with kappa of . and . respectively. agreement on the finding of interstitial syndrome was moderate with kappa= . . agreement was higher in hospitalised than in non-hospitalised cases with kappa of . and . for the respective categories. results showing concordance between lus and cxr findings are pending. conclusion: lus shows substantial irr for the diagnosis of pneumonia in children. irr are higher for the detection of consolidation or for no pathology than for interstitial syndrome. irr also appears to be lower in clinically less severe disease. 'white-out' on plain chest radiograph-a late presentation of congenital diaphragmatic hernia a. fagan , c. stewart , k. halliday , s. rao , d.t. chang kwok ; peterborough/uk, lincoln/uk, objective: awareness of the limitations of plain radiograph and computed tomography in diagnosis of late presentation of congenital diaphragmatic hernia. case presentation: a year old boy presented with a day history of pyrexia, vomiting and respiratory distress. he was haemodynamically stable, and had no audible air entry over his upper left thorax with occasional wheeze over the left base. he had bronchiolitis previously but did not require ventilatory support. he was otherwise well with unremarkable antenatal scans. initial chest x-ray showed a large air collection with fluid or soft tissue density within the left hemi-thorax and mediastinal shift to the right. repeat x-ray (figure ) demonstrated the nasogastric tube below the diaphragm. complicated pneumonia was suspected but as the findings were atypical a non-contrast ct was performed. this was interpreted as showing a large hydropneumothorax. (figure ) . a chest drain was inserted which drained only a small volume of fluid, and a repeat chest film showed no change. ct chest and abdomen with oral and intravenous contrast revealed a bochdalek diaphragmatic hernia (figure ) . fortunately the chest drain had not entered the herniated stomach. the hernia was surgically corrected and the child recovered well. ( ) (suppl ):s -s pediatr radiol bochdalek is the most common congenital diaphragmatic hernia (cdh). it is often diagnosed on prenatal ultrasound, with mri used for confirmation. cdh which is not diagnosed in the perinatal period may be asymptomatic and imaging findings can be confusing. postnatal x-ray typically shows an opacified hemi-thorax with or without gas bubbles. there can be mass effect with mediastinal shift. the position of an ng tube can be helpful in localising the stomach, but in this case the infradiaphragmatic position of the tube gave false reassurance. in neonates, the position of an umbilical venous catheter may demonstrate abnormal location of the liver. computed tomography generally demonstrates a posterolateral defect (foramen of bochdalek), which is located on the left in % of cases. ct is useful for excluding lung masses or bronchopulmonary foregut malformations, which may appear similar to cdh on x-ray. ct can also identify anatomical abnormalities associated with cdh. late presenting cdh is often misdiagnosed as pleural effusion or pneumothorax. there are other case reports published where chest drains were inserted before cdh was diagnosed. it is important to keep cdh in mind as a potential cause of unilateral hemithorax opacification, even in previously asymptomatic older children. ct with oral contrast can be useful in diagnosis. ovarian tuberculosis with peritoneal dissemination mimicking ovarian tumor with peritoneal seeding d. grassi, v. tostes, a. duarte, s. abib, h.m. lederman; sao paulo/br consider tuberculosis (tb) as a differential diagnosis whenever the case enrolls in an endemic region. case presentation: female, years old adolescent, who presents with abdominal pain and weight loss. abdominal sonography was performed in a public family practice location and bilateral ovarian masses were detected. she was referred to an oncology pediatric facility for further investigation. abdominal mri and chest ct were performed where dissemination through the peritoneal and mesenteric lymph nodes could be detected; chest ct was normal. the patient underwent surgical intervention for diagnosis and on pathology the findings in the bilateral ovarian masses were secondary to tb involvement. sonography showing bilateral pelvic masses. t -weighted coronal overview bilateral ovarian masses. unique teaching points: whenever a case enrolls in an endemic region of tuberculosis, it is important to consider it as a possible differential diagnosis. in this case, the initial presentation mimicked ovarian tumor with mesenteric seeding. however, only after surgical approach was possible to diagnose ovarian tuberculosis with mesenteric lymph nodes and peritoneal involvement. retrospectively, patient's uncle was discovered as having pulmonary tb. langerhans'-cell histiocytosis with thoracic involvement in infant and young child: ct findings s.-l. shih , k. tsai , w. huang , f.-s. yang ; taipei/tw, taitung/tw the purpose of the study was to evaluate the ct changes of thorax in the patients with langerhans'-cell histiocytosis. the -month-old female infant presented with generalized hemorrhagic macular rash over the skin for months. the laboratory findings showed hemoglobin . gm/dl (normal . ~ . gm/dl). the chest radiograph showed bilateral reticulonodular infiltration. high-resolution computed tomography (hrct) of chest showed multiple cystic-like lesions ( - mm) in the right middle and bilateral lower lobes. the pathological report was langerhans'-cell histiocytosis after skin biopsy from upper chest. then she was on scheduled chemotherapy. she was in remission after one-year treatment. the y m-old girl presented with fever for months. the physical examination revealed hemorrhagic-macular rash over the skin in the anterior chest wall and hepatosplenomegaly. the laboratory findings revealed albumin . g/dl (normal . - . g/dl) and hemoglobin . g/dl (normal . - . g/dl). hrct of chest showed multiple cystic-like lesions ( - mm) in the bilateral lower lobes with left pleural effusion as well as multiple osteolytic lesions in the vertebral bodies of t , t , t and t . the pathological report was langerhans'-cell histiocytosis after skin biopy from anterior chest wall. then she was on scheduled chemotherapy. she was doing well years after treatment. the y m-old girl presented with yellowish discoloration of skin for one month. the laboratory findings revealed direct/total bilirubin . / . mg/dl (normal . - . / . - . mg/dl), got iu/l ( - iu/l) and gpt iu/l ( - iu/l). the chest radiograph revealed enlargement of upper mediastinum. the ct scan of chest and upper abdomen showed punctuate calcification with heterogeneous enhancement in the upper mediastinum and several minute cysts in the lower lobes of lung (hrct) as well as dilatation of bilateral intrahepatic bile ducts in the liver. the pathological report was langerhans'-cell histiocytosis after biopsy from thymus and liver. then she was on scheduled chemotherapy and got initial response. unique teaching points: langerhans'-cell histiocytosis affecting the lungs and thymus may be in isolation or as part of a multiorgan disease. the pulmonary changes on ct scan may not have corresponding respiratory symptoms. ct scan of thorax may have multiple minute cysts ( - mm) in the lungs, pleural effusion, calcification in the thymus and osteolytic lesions in the thoracic spine. case of fungal infection of the soft tissue in a child with acute myeloid leukemia (ultrasound aspects of diagnosis) i. begun, s. kondaurova; minsk region/by objective: early diagnosis of fungal infections of the tissues is essential for a successful and complete recovery. we describe a clinical case of fungal infection of the soft tissue in a child with acute myeloid leukemia (aml). ultrasound were made for the characteristics of the structural changes in the area of interest to perform biopsies followed by bacteriological culture studies. case presentation: patient k., years old, diagnosed with aml, from which after a course of induction chemotherapy with neutropenia about weeks on the skin of the foreskin appeared removable hard white coating. cultures of plaque it possible to establish the presence of fungi of the genus trichosporon spp. after days, there were hyperemia, compaction and ulceration of the glans penis, which led to extensive tissue defects. with help ultrasound were determined the structural deformation of the glans penis with the pronounced around changed tissues vascularization. after days in the rear surface projection of the left thigh and the lateral surface of the left calf were defined erythematous papules which progressed to ulceration with central black scab. by standard ultrasound were visualized: subcutaneous nodal education oval , х , sm on hip and echogenic skin thickened portion having an average degree of severity of dorsal acoustic shadow on the lower leg (weakening of the signal behind scab). in cultures of biopsies of subcutaneous foci were revealed fungi of the genus trichosporon spp too. the patient received the combination treatment (intravenous liposomal amphotericin b and surgical rehabilitation of lesions of glans and corpus cavernosum of penis). after the stabilization of patient state the treatment of the underlying disease was continued. unique teaching points: for some patients, lesions of superficial tissues may be the only sign of systemic fungal infections, and rapid recognition of these lesions may contribute to early diagnosis and treatment. ultrasound examination in such a situation naturally becomes an main imaging tool and by choice method. the scanning high-resolution of foci of the thigh of the patient k. in grayscale made possibility to determine the configuration consisting of the central echogenic focus surrounded by a hypoechoic rim (fig. ) with peripheral changes in the type of "infiltrative" according by the active fungal infection at the exit of cytopenia. duplex and triplex ultrasound scanning were indicating to the perifocal vascularization with low level vascular resistance around of the affected area (see fig. - ) . to increase knowledge and awareness of rare cases and diseases in order to be able to better manage and treat patients in the future. case presentation: an -month-old female was presented to our hospital with abdominal distention that increased in the past months associated with low-grade fever, loss of weight and mild respiratory distress. abdominal ultrasonography revealed an enlarged liver with multifocal hypoechoic lesions scattered all over the liver (fig ) . a ct scan with iv contrast (mri was not available at that time in our district) revealed severe hepatomegaly with the presence of multiple, variable in size, hepatic hypodense lesions which had peripheral (ring) enhancement after contrast injection in the arterial phase (fig ) . progressive centripetal filling in portal phase is seen and in the delayed images many of the lesions were completely filled (fig ) . reduction in the aortic caliber (mid-aortic syndrome) below the level of celiac branch was noted. a diagnosis of hemangioendothelioma was made although liver biopsy was not done due to fear of hemorrhage. alternative diagnosis to infantile hemangioendothelioma in this age group is hepatoblastoma, mesenchymal hamartoma and metastatic neuroblastoma. the patient was transferred to another city to a hospital with pediatric oncology department for follow up and treatment. unfortunately the lack of experience and knowledge of such rare cases led to mismanagement and delayed treatment and after less than months the patient was brought back to our hospital to the pediatric icu due to deterioration of her status due to congestive heart failure. unfortunately the patient died shortly afterwards. hemangioendothelioma is twice as common in girls and can have complications due to high output chf secondary to arteriovenous shunting hemangioendotheliomas tend to involute spontaneously without therapy over a course of months to years. they are followed with sequential ultrasounds. medical therapy is reserved for severely symptomatic lesions (e.g. anemia, consumptive coagulopathy, high-output chf) and includes high-dose steroids or alpha-interferon. in cases of failed medical management, surgical resection should be performed. if partial hepatectomy is not technically achievable, transarterial embolization should be used either as definitive therapy or as a temporizing measure until liver transplantation is possible. the sad outcome of this case was mainly due to mismanagment due to lack of medical experience and knowledge of such rare cases so we suggest that such rare cases should be catalogued in a national data bank for future consultation and teaching purposes. fatal outcome of acute gastric dilatation causing acute abdomen compartment syndrome in a child: a case review c.s. yoon; seoul/kr to describe and review presumed acute abdominal compartment syndrome in a child. case presentation: a years and months old boy was admitted to emergency room due to abdominal distention. he suffered abdominal pain and vomited since yesterday after lunch. on physical examination, his abdomen was rigid and distended. body temperature is . °c. the white cell count was increased ( , /μl). esr is mm/hr and c-reactive protein was . mg/l. creatinine was increased ( . mg/dl). amylase and lipase were increased ( u/l and u/l respectively). prothrombin time was prolonged ( . sec). plain abdomen radiograph shows markedly distended stomach with air-fluid level (fig. ) . first trial of nasogastric tube insertion was failed due to kinking of tube at gastroesophageal junction. contrast-enhanced abdomen ctscan shows marked distensionofstomachwithlargeamountoffoodmaterialsandintraluminalairwith prominent external compression in the duodenal rd- th junction. esophageal air distention is also markedly noted with l-tube insertion. no opacification of large vessel with contrast media, without contrast enhancement of spleen, pancreas and left kidney is noted (fig. ) . prob. markedly compressed and poorly defined lower abdominal aorta with faintly visible both common iliac arteries and femoral arteries. after ctscan, nasogastric tube exchange was performed due to poor drainage of gastric fluid. about cc of gastric fluid was drained. however, sudden cardiac arrest of the patient was developed. although vigorous cardiopulmonary resuscitation was performed, the patient was died. ( ) (suppl ):s -s pediatr radiol unique teaching points: acute abdomen compartment syndrome is a very serious and lifethreatening disease. as soon as possible, rapid diagnosis and adequate treatment are necessary for good prognosis. delayed diagnosis and treatment may result in fatal outcome. pleuroperitoneal fistula in a pediatric patient with primary hyperoxaluria type w.p. chu; hang hau/hk to illustrate the imaging features of pleuroperitoneal fistula in a pediatric patient suffering from primary hyperoxaluria type case presentation: an -year-old girl with the history of primary hyperoxaluria type was repeatedly admitted to the hospital for recurrent right pleural effusion despite chest drain insertion. the right pleural fluid was transudative in nature and the microbiological cultures for bacteria and mycobacterial species were negative. the radiographic examination [ figure ] showed moderate right pleural effusion a n d f e a t u r e s o f o x a l o s i s i n c l u d i n g b i l a t e r a l c o r t i c a l nephrocalcinosis and generalized increased in bone sclerosis. delayed planar images of the peritoneal scinitigraphy [ figure ] obtained and hours after injection of technetium- m suphlur colloid found diffuse tracer activity at the right hemithorax, suggestive of pleuro-peritoneal fistula. the patient subsequently required thoracoscopy and surgical decortication at the right hemithorax and renal transplantation. primary hyperoxaluria is due to defective glyoxylate metabolism and results in increased synthesis of oxalic acid. cortical nephrocalcinosis and diffusely increased bone sclerosis are characteristic radiographic features. pleuroperitoneal fistula is unusual in patients without peritoneal dialysis. possible cause in this patient is increased intra-abdominal pressure related to portal hypertension and cirrhosis. osteosarcoma with pulmonary intra-arterial tumor embolism metastasis a. alzaher, f. alzaher; dammam/sa objective: osteosarcoma rarely invade the veins and small number of cases has been reported with venous invasion at the presentation. however, to our knowledge, no case has been reported with venous invasion and isolated distal metastasis as intra-arterial pulmonary embolisms. we are presenting a case of pediatric pelvic osteosarcoma with venous invasion and pulmonary arterial tumor embolisms as isolated distant metastasis at the presentation. the purpose of this case report is to describe the rare presentation of distant metastasis as isolated pulmonary arterial embolism that might be overlooked radiological. additionally, such tumor embolism might cause respiratory symptoms and differentiating tumor emblism from pulmonary thromboembolism is crucial to avoid the unnecessary anticoagulation. case presentation: fourteen year old boy who presented with months history of right hip and lower limb pain after trauma. this was associated with lower limb swelling. the plain radiography showed right pelvic iliac bone aggressive mass, along with lobulated, soft-tissue components, extensive areas of osseous matrix, and malignant periosteal reaction. the patient could not tolerate the mri and ct scan was performed and it showed that the mass was invading the right external and internal iliac vein with imaging appearance was most consistent with osteosarcoma. patient staging was then carried on with mri under anesthesia and chest, abdomen and pelvic ct scan. the unenhanced and iv contrast enhanced chest ct scan showed multiple beaded expansion of sub segmental pulmonary arteries with soft tissue destinies and calcification suggestive of intra-arterial pulmonary tumor embolisms. there was no isolated pulmonary nodule or any other site of distant metastasis. unique teaching points: we present this case to increase the awareness of isolated intra-arterial pulmonary tumor embolisms as osteosarcoma metastasis especially with the present of venous invasion. additionally, such condition might be with respiratory symptoms and differentiating the tumor embolism from pulmonary thromboembolism is crucial to avoid the unnecessary anticoagulation. case presentation: a -year old boy with acute myelodysplastic syndrome presented with recurrent, acute severe anemia (hemoglobin g/dl) and melena. his past history was significant for bone marrow transplant twice followed by graft-versus-host-disease of intestines, bilateral lung transplants for bronchiolitis obliterans, renal failure, scleroderma and acute pancreatitis. ct angiography performed previously did not identify active extravasation. several days before, upper gi endoscopy had demonstrated ulceration of the greater curvature of the gastric wall that was initially treated with epinephrine injection and surgical clip placement. at the time of referral, endoscopic interventions were unsuccessful leading to progressive clinical deterioration. a decision was taken to proceed to angiography to isolate the arterial source of hemorrhage, with an intention to embolize, if feasible. catheter angiography via transfemoral fr access revealed a left gastric artery pseudoaneurysm with active extravasation into the gastric lumen through the ulcer. after selecting the feeding pedicle of the left gastric artery with a microcatheter, the pseudoaneurysm was embolized using % nbca in lipiodol, resulting in complete angiographic obliteration of the bleeding source. on repeat cbc hours post-procedure, the hemoglobin had increased from to g/dl. the patient remained hemodynamically stable in the intensive care unit. there is no evidence of bleeding recurrence days later. unique teaching points: catheter angiography can define the bleeding source with greater accuracy than cta in children. there should be a low threshold to perform catheter angiography, with an intention to proceed to treatment. nbca embolization is a feasible and effective option for treatment of acute gi bleeding in children. case presentation: an infant born by cesarean section at weeks of gestation, after nonreassuring cardiotocoghraphy, with meconium aspiration at birth, severe hepatocellular failure with hyperbilirubinemia, signs of hemorrhage, edema, ascites, hypoglycemia, increased ferritin values, and lactic acidosis was referred for ultrasound and magnetic resonance. both examinations showed signs of liver cirrhosis with portal hypertension; in addition, on t -weighted images and gradient-echo images, the signal intensity of the liver and the pancreas was lower than that of the spleen and skeletal muscle, a finding consistent with abnormal iron deposition in those organs. a biopsy of the lower lip confirmed the diagnosis of neonatal hemochromatosis. unique teaching points: although the diagnosis may be suspected clinically, it must be confirmed by demonstrating the generalized iron overload affecting, among other organs, the salivary glands, liver and pancreas, with sparing of the reticuloendothelial system. the underlying cause may be associated with an an alloimmune mechanism; thus, intravenous immunoglobulin during gestation is administered in selected cases to prevent the severity of neonatal hemochromatosis. diagnosis is then crucial not only for management of the affected infant, but also for prevention in the future offspring. fishing for the answer -a rare case of paediatric exogenous lipoid pneumonia secondary to fish oil aspiration h. moodley, d. white, g.d. baker; johannesburg/za objective: lipoid pneumonia is a rare condition caused by the intrapulmonary accumulation of endogenous or exogenous fat containing substances. in the acute exogenous form secondary to aspiration of oil, it is important to make the diagnosis and remove the causative agent to prevent or arrest the progression of pulmonary fibrosis. radiopathological findings usually prompt the diagnosis, as aspiration of mineral oils is usually unnoticed due to the lack of reactive airway symptoms and patients present with vague chronic respiratory symptoms. case presentation: we present the clinical, radiological and pathological correlation of exogenous lipoid pneumonia in a -month-old male patient with recurrent respiratory tract infections. a ct chest demonstrated an extensive crazy paving pattern of the dependent lung segments bilaterally. the lung biopsy findings of occasional intra -alveolar macrophages with larger ( ) (suppl ):s -s pediatr radiol foamy cytoplasmic vacuoles, raised the possibility of an exogenous lipoid pneumonia secondary to aspiration. on further history, the patient was found to have been fed fish oil by his mother, confirming the diagnosis. unique teaching points: the rare diagnosis of exogenous lipoid pneumonia can be confirmed on ct chest by measuring the hounsfield units in the most hyperdense components of consolidation (typically - to - hu). histopathological confirmation can be obtained provided that the specimens are not embedded in paraffin. the possible role of visual evaluation of dwibs in childhood renal masses based on our five cases e. varga, g. rudas; budapest/hu objective: nowadays, the diffusion-weighted mri has a great importance not only in the differential diagnosis and follow-ups of childhood renal tumors, but in the early detection of recurrence of the disease as well. the dwibs with appropriate b-values and the adc calculation can be helpful in distinguishing between benign and malignant processes. however, the adc calculation is a time consuming method and in addition, there are cases when we cannot use this technique, but we can still apply the visual evaluation of diffusion. case presentation: between - , we had cases in which the visual assessment of dwibs was the best method which helped to make the appropriate therapeutic decisions. left kidney of an infant with nephroblastomatosis was removed because of an arising wilms tumor. , years later, in the contralateral kidney, a small area of diffusion restriction appeared on the dwibs in one of the cystic residual lesions, but the anatomic sequences haven't showed any changes comparing with the previous examinations. in another patient with beckwidt-wiedemann syndrome, the follow-up ultrasound examination showed a little bulging of the surface of the left kidney. accordingly, the mri showed a barely distinguishable nodule, but the dwibs referred to a wilms tumor. in a -month-old child, more nodules were visible in both kidney on the dwibs than on other sequences. with the help of visual evaluation of dwibs, we were able to detect the malignant lesion easily and quickly, among a lot of cystic and solid nodules of the kidneys in a seven years old patient with sclerosis tuberosa. an -month-old infant was followed with a benign cystic renal disease and a new small solid nodule was found on the last ultrasound examination. instead, the visual assessment of dwibs indicated a multilocular cystic wilms' tumor. unique teaching points: the diffusion-weighted mri is suitable for differentiate benign and malignant renal lesions in children. the dwibs (with appropriate b-values) and the adc calculation are very sensitive methods in pediatric oncoradiology. the adc calculation is a long process andas our cases demonstrated -we cannot apply in every cases. the visual evaluation of dwibs is a time saving method which is spared from limitations of adc histogram-based assessment, so it may become very useful in the everyday practice. we can use it in the differential diagnosis and follow-ups of childhood renal tumors and we can detect the recurrence of the malgnancy very early and easily. mr urography in a -years-old female with unusual urinary dribbling m.c. terranova, c. tudisca, d. narese, g. li voti, s. salerno; palermo/it objective: congenital anomalies of kidney and urinary tract (cakut) occurs in up to . % of infants, and clinically they can range from asymptomatic patients, in which anomaly is detected incidentally even in adulthood, to ante-natal or post-natal mortality due to bilateral kidney agenesis or acute renal failure. dmsa renal scintigraphy is considered gold standard, for evaluation of those cases electable for surgery, in order to assess renal function, depict and locate ectopic kidney and guide surgical management, but has the important limit of radiation exposure and may undetect poorly functional renal moieties. the advent of modern magnetic resonance technics proven to be able to assess anatomical malformations and renal function, overcoming the limits of dmsa scintigraphy, may be used as a valid alternative, especially in vulnerable pediatric population. we herein describe a case of a young girl with small left renal bud and ectopic ureter, draining in vagina, discovered by mr and undetected by previous dsma scintigraphy. case presentation: a years old girl was referred for continuous urinary dribbling, after starting toilet training, with normal bladder voiding pattern, unrelated to any physical and psychological events, and no history of urinary tract infections. physical examination revealed vaginal septa and micturition training was practiced, with no symptoms improvement. abdominal us study was performed, reporting empty left renal fossa and hypertrophic right kidney; no ectopic kidney nor sign of urine stasis or other urogenital anomalies were detected, and dmsa renal scintigraphy was planned. it depicted only normal right kidney radionuclide uptake but no evidence of left renal or ectopic renal tissues activity. patient then underwent mr evaluation for suspected genito urinary malformation, that revealed a small cystic formation, with a slight cortex, at the level of the iv lumbar vertebra -that represented the left immature renal bud -supplied by a short fluid-filled tubular structure, located postero-medially to the bladder -that configured the left ectopic ureter, draining in left vaginal wall. bladder was normal, and regularly connected with the right orthotopic ureter (fig ) . pre-surgical cystoscopy and vaginoscopy, followed by left ascending urethrogram were performed, confirmed previous mr findings, and patient underwent successfull laparoscopic left nephron-ureterectomy. unique teaching points: mr urography has proven to be a rapid, safe, radiation free, systematic diagnostic tool especially in the evaluation of poorly functioning renal systems, and of collecting system, bladder and ureteral abnormalities, overcoming the limits of conventional imaging technics agenesis of the dorsal pancreas: case report c. lanza, g. pieroni, l. amici, a. giovagnoni; ancona/it objective: agenesis of the dorsal pancreas (adp) is a rare malformation. since and until , cases have been reported. majority of the patients with this anomaly are asymptomatic or associated with abdominal pain, hyperglycemia, diabetes mellitus, and acute or chronic pancreatitis. case presentation: we present a case report of a -year-old girl with adp, diagnosed incidentally during radiological evaluation for abdominal pain. she was hospitalized in the pediatric department for recurrent abdominal pain for the past months. there was no history of nausea, vomiting or trauma. biochemical investigations showed a normal random serum glucose, serum amylase levels slightly increased ( u/l; reference value - u/l) and slightly elevated serum pancreatic lipase levels ( u/l; reference value - u/l). the day after serum amylase levels decresed up to u/l and lipase levels to u/l. us revealed increased -size pancreatic head with normal contour and echotexture with no parenchymal calcification or duct dilatation. the body and the tail of the pancreas were poorly visualized. mr imaging examinations revealed only a partial visualization of the pancreas: the pancreatic head and the uncinate process were visualized with defined margins with peripancreatic fat stranding, but the distal neck, body, and tail of the pancreas were absent. on mrcp, the dorsal pancreatic duct of santorini and the minor duodenal papilla could not be visualized. the ventral pancreatic duct of wirsung and the common bile duct were normal and clearly visualized. these findings were compatible with complete adp, eliminating the need for ercp. unique teaching points: the clinical presentation of dpa varies greatly ranging from incidental detection on x-ray, surgery or autopsy through to the development of a ductal adenocarcinoma of the pancreas. abdominal pain and diabetes are the most frequent clinical manifestations reflecting exocrine and endocrine insufficiency as most of the islands of langerhans are located in the tail of the pancreas. there have also been reports of an increase in the size of the remnant pancreas and recurrent acute pancreatitis as a form of presentation. diagnosis requires confirmation of the absence of the neck, body and tail of the pancreas and duct of wirsung using endoscopic retrograde cholangiopancreatography (ercp) or mrcp. one hundred four mr images of foetal cns with a us suspicion of acc were retrospectively reviewed. foetal mri was performed at . t magnetom avanto (siemens, erlangen, germany) without motherfoetal sedation. polymicrogyria, lissencephaly, schizencephaly, subependymal heterotopias and migration disorders were evaluated. cortical findings were compared to three types of acc (complete agenesis, partial agenesis and hypoplasia). genetic tests were collected. postnatal mri or foetopsy for diagnostic confirmation were collected. on foetuses, fetal mri was able to detect cortical malformations in cases even in early gestational ages (< gw). the mean gestational weeks (gw) at mr diagnosis was (range: - gw). mr imaging found / polymicrogyria, / lissencephaly, / schizencephaly, / subependymal heterotopias and / neuronal migration disorders. / had complete acc, / had partial acc and / had cc hypoplasia. statistically significant correlations (p< . ) between complete acc, focal polymicrogyria and cortical dysmorphism affecting frontal lobes were found. fetal cns mri can detect cortical development malformations in complex acc, providing further information for the clinician to assess the severity of perinatal outcome. mri is a useful tool in improving obstetrical genetic prenatal counselling to predict pregnancy and foetal prognosis. clinical signs of the neonatal lymphatic flow disorder (nlfd) are a combination of the congenital chylothorax, chylous ascites and body edema. it can present as neonatal chylothorax (nc), neonatal chylous ascites, or congenital lymphatic dysplasia (cld). the prenatal appearance of lymphangiectasia has been described as nutmeg lung. the purpose of this study is to describe prenatal and postnatal imaging features and outcomes of neonates with nlfd. materials: this is a retrospective case series of neonates in our institution that had pre-and postnatal lymphatic imaging and nlfd. all patients had prenatal imaging (fetal mri and us) and underwent postnatal dynamic contrast mr lymphangiography (dcmrl) with a three-dimensional ( d) t space. conventional lymphangiography (cl) when performed was also reviewed. six patients with nlfd were identified ( with nc and with cld). one patient had congenital heart disease. nutmeg lung was seen in all patients on fetal mri and patients on fetal us. / patients had pleural effusions, / had ascites and / had body wall edema prenatally. postnatal mri with d t space revealed soft tissue edema in the upper chest and neck ( / patients), mediastinal edema ( / patients), interstitial lung edema ( / patients), retroperitoneal edema ( / patients), and ascites ( / patients). dcmrl demonstrated lymphatic flow to the pleural space ( / patients) and to the abdominal cavity ( / patients) and dermal backflow ( / patients). cl was performed in patients, all of which had collateral lymphatic flow to the lung. lymphatic intervention was performed in patients, lipiodol injection for patients with nc and thoracic duct embolization (tde) for patient with cld. mean hospital duration in the first months of life was days (range - ) for nc and days (range - ) for cld. all patients with cld died after months of age due to respiratory distress including the patient that had tde and both with findings of dermal backflow. the pleural effusions in the patients with nc resolved post lipiodol injection and in the other patient with nc it resolved with conservative therapy. conclusion: nlfd is a disorder that can be recognized on prenatal and postnatal imaging. in this small series, nutmeg lung was present in all patients with nlfd and may be easier to recognize with fetal mr than us. dermal backflow on dcmrl suggests a poor prognosis. both prenatal and postnatal imaging may guide treatment and interventions in nlfd. fetal mri and postnatal ct scans of prenatally diagnosed bpms from patients with available histology were analyzed retrospectively. the fetal mri and ct images were reviewed by two radiologists blinded to histological findings. specific diagnosis was assigned based on predetermined criteria. the accuracy of fetal mri was evaluated. the agreement rate in fetal mri diagnosis between two radiologists was %. an overlap of % in fetal mri and histopathological diagnosis was reached. when comparing fetal mri and postnatal ct examinations, the agreement of the results was also %. the least matching histological diagnosis was bronchopulmonary sequestration (bps). fetal mri is very accurate in characterizing the bpm spectrum and provides important information on lesion type and structure when compared with histology. with relatively small number of patients high correlation between prenatal mri and postnatal ct was reached. therefore, further investigation with more patients is needed. we hypotethise that fetal mri in late pregnancy could in the future replace early (neonatal) ct examinations if fetal mri provides sufficient inforfmation for clinical management. real time virtual sonography: a new integrated approach for the evaluation of fetal cerebral pathologies? s. bernardo, a. antonelli, v. vinci, m. saldari, c. catalano, l. manganaro; rome/it objective: real-time virtual sonography (rvs) is a new technique that uses magnetic navigation and computer software for the synchronized display of real-time us and multiplanar reconstruction mri images. the purpose of this study was to evaluate the feasibility and ability of rvs to assess the main cerebral pathologies in fetuses with suspected us anomalies. materials: this is a prospective study. fusion imaging (hitachi hi vision ascendus) was offered to patients undergone fetal mri for a us suspicion of cerebral pathology. the mri image dataset acquired was loaded into the fusion system using a cd support and displayed together with the us image. both sets of images were then manually synchronized and images were registered. the possibility to record the images in a video format allowed, however, the possibility to re-evaluated the examination. results: rvs was technically possible in all cases. data registration, matching and fusion imaging were performed in minutes at the beginning and in less than - minutes after practice. the ability of rvs imaging to assess the main anatomical sites and fetal anomalies was evaluated and compared with standard us and mri images. the principal application of rvs was the study of midline, cerebral gyration and vascular malformations because it also allowed adding a real time doppler signal on mri images. fusion imaging helped the diagnosis in %. in the / cases of encephalic pathology, fusion imaging improved the diagnosis; in the other cases mri was superior to us even using the rvs. this is a preliminary study on the feasibility and practical use of a fetal mri-us real-time fusion imaging. both techniques are complementary but still independent and the retrospective synthesis of these exams allows optimal analysis of fetal cerebral anomalies. this technique has many advantages especially on the pedagogic plan. however, rvs is currently limited to the research area. role of foetal mri in the evaluation of ischaemic-haemorrhagic lesions of the foetal brain s. bernardo, a. antonelli, v. vinci, m. saldari, c. catalano, l. manganaro; rome/it the aim of this study was to define the role of fetal magnetic resonance imaging in the evaluation of cerebral ischaemic-haemorrhagic lesions and the extension of parenchymal injuries. from september to december we performed fetal mri of cerebral region in foetuses with suspected abnormalities on ultrasound or cmv infection and toxoplasma serum conversion. fetal mri was performed with a . -t magnet system without materno-fetal sedation. fetal mri detected ischaemic-haemorrhagic lesions in / fetuses, revealing a % pathology incidence. mri confirmed the diagnosis in / cases with us suspect of ischaemic-haemorrhagic lesions associated with ventriculomegaly. in / cases with us findings of cerebellar haemorrhage, mri confirmed and provided additional information regarding the parenchymal ischaemic injury. in / cases with us suspect of ventriculomegaly (n= ), corpus callosum agenesis ( ), cerebellar vermis hypoplasia ( ), holoprosencephaly ( ), spina bifida ( ) mri detected ischaemic and haemorrhagic lesions unidentified at us examination. in / fetuses with us suspicion of intracerebral tissue space-occupying lesion, mri modified the diagnosis to extra-axial haematoma associated with dural sinus malformation. results were compared to fetopsy or after-birth follow up. fetal mri is an additional imaging modality in the diagnosis of cerebral ischaemic-haemorrhagic lesions and it is useful in providing further information on the extension of parenchyma injury and associated abnormalities and in improving delivery management. the contribution of mid-trimester virtual autopsy with mr imaging a. d'hondt, n. d'haene, j. rommens, m. cassart, f.e. avni; brussels/be the aim of the study was to assess the potential contribution of fetal virtopsy (post-mortem mr imaging (pm-mri)) in the second trimester of pregnancy. during a one-year period, post-mortem mr imaging (pm-mri) was performed in all fetuses who died in utero or whose pregnancy was interrupted due to major malformations. the study was performed in agreement with the local ethical committee. fetuses of < weeks that underwent obstetrical ultrasound and pm-mr were included. mr imaging examination was performed on a . tesla magnet with a standardized protocol. the findings on pm-mri were compared to obstetrical sonographic findings (and to pathology when available). we have analyzed separately the findings in the central nervous system and those in the rest of the fetus (chest, abdomen and skeleton). the results were classified in three categories according to the diagnostic accuracy: ultrasound>pm-mri, ultrasound=pm-mri and pm-mri>ultrasound. the us and pm-mri data of ten fetuses were analyzed. their gestational age ranged from . - weeks and their bodyweight ranged from - g. for the cns malformation: pm-mri offered a better diagnostic accuracy than us in cases ( %) (e.g. agenesis of the corpus callosum and holoprosencephaly). in cases ( %) us offered the same information than pm-mri. there was no case where us was more accurate than pm-mri. for the rest of the body malformations: pm-mri offered a better diagnostic accuracy in cases ( %) (e.g. heterotaxy anomalies or vertebral segmentation anomalies). in cases ( %), us offered the same information as pm-mri. there were cases ( %) where us showed major malformations that were not diagnosed on the pm-mri (two cases of cardiac malformation). post mortem mr imaging is more accurate than obstetrical ultrasound in detecting major malformations in the cns as well as in the rest of the body. the present exceptions are cardiac malformations. the examination offers an easy evaluation of the deceased fetus. it provides, in most cases, important additional information. diffusion coefficient and perfusion fraction parameters correlate with gestational age in normal human in vivo placenta: a preliminary study a. antonelli, m. guerreri, s. bernardo, s. capuani, c. catalano, l. manganaro; rome/it to investigate the potential of diffusion parameters derived from a biexponential analysis as marker to evaluate the perfusion quality of normal in vivo placenta. eighteen normal pregnancies, fulfilling the study inclusion criteria, have been analysed at . t magnetom avanto (siemens, erlangen, germany) without mother-foetal sedation. dw imaging was collected using seven b values: , , , , , , (s/mm ). three regions of interest (rois) have been considered -central (c), peripheral (p) and umbilical (u) regions. a bi-exponential model was used to obtain perfusion fraction (f), pseudo-perfusion (d*) and apparent diffusion (d) coefficients. pearson test was performed to investigate correlation between diffusion parameters and gestation weeks (gw), body mass index (bmi) and basal glycaemia (bg). the average values on all rois were d= . ± . • - (mm /s), d*= . ± . • - (mm /s), f= . ± . • - , in good agreement with the literature. in the c roi, a positive correlation (p< . ) was observed between f and gw. after gw in the p roi a positive correlation between f and gw (p< . ) and a negative correlation between d and gw (p< . ) were found. no correlation was found between d, d*, f, bmi and bg. conclusion: the f increase reflects normal placenta perfusion physiology. on the other hand, the decrease of d highlights placental parenchyma maturation becoming more fibrotic during late gestational age. bi-exponential model provides more and useful information about placental morphological changes compared to mono-exponential diffusion model. to demonstrate the diagnostic value of fetal mri in the detection of fetal central nervous system (cns) impairment in prenatally echocardiographic diagnosed congenital heart diseases. we retrospectively examined fetuses between gestational weeks and gestational weeks who performed a fetal mri in our institution after a second-line ultrasonography, between april and october . fetal heart and cns studies were performed with a . tesla magnet (siemens magnetm avanto) without maternal sedation. prenatal findings were compared to fetopsy results, fetal mri after gw or postnatal mri. in our sample of cases, / had interatrial septal defect (iasd),intervertricular septal defect (ivsd), and atrioventricular canal defect (cavc), / had cardiac rhabdomyomas, / had hypoplastic left heart syndrome and hypoplastic aorta, / had transposition of the great vessels, / had fallot tetralogy, / had aorta coartation and / had intracardiac masses of uncertain significance. magnetic resonance imaging was able to detect cns impairment: we recognize / corpus callosum (cc) dysgenesis ( / cc hypoplasia, / complete cc agenesis, / partial cc agenesis), / ventriculomegalies or hydrocephalus, / subtentorial anomalies (dandy-walker, vermian hypoplasia and vermian malrotation) and / gyration anomalies. due to the high risk of cns involvement in prenatal congenital heart diseases, it is essential to suggest an mri study of the evolving fetal brain especially in complexes forms that suggest a syndromic background. fetal mri of the cns is mandatory in the study of congenital heart disease due to the high rate of encephalic anomalies associated, particularly in iasd, ivsd and cavc. first experiences and diagnostic utility of micro-ct for fetal autopsy j.c. hutchinson , x. kang , s.c. shelmerdine , m. cannie , v. segers , n. sebire , j. jani , o.j. arthurs ; newcastle upon tyne/uk, brussels/ be, london/uk perinatal autopsy remains poorly accepted by parents, despite yielding information that affects the management of future pregnancies in around % of cases. microcomputed tomography (micro-ct) has shown promising results in the examination of ex-vivo fetal organs, and may provide diagnostic imaging in cases where traditional autopsy is challenging, and s ( ) (suppl ):s -s pediatr radiol existing post mortem imaging techniques (ct and mri) provide insufficient diagnostic resolution. our objective was to examine whole fetuses non-invasively using micro-ct, and compare the findings with standard autopsy as the gold standard. in this ethically approved double blinded study, terminated fetuses or miscarriages underwent iodinated micro-ct examination followed by conventional autopsy. images were acquired using a nikon xth st microfocus-ct scanner with individual specimen image optimisation. forty indices normally assessed at perinatal autopsy were evaluated for each imaging dataset by two independent reporters and a consensus report produced. autopsies were performed blinded to the imaging findings by one of two perinatal pathologists. we examined fetuses, with a gestational age range of - gestational weeks. / indices were non-diagnostic ( %), but there was agreement for / diagnostic indices (overall concordance of . % ( % ci . , . %). in seven out of eight fetuses ( . %), the same final diagnosis was made following micro-ct examination and autopsy examination; in one case, micro-ct was non-diagnostic. ten false negatives indices included a vsd, laryngeal anomaly, ambiguous genitalia and incomplete bowel rotation, none of which changed the overall diagnosis. three apparent false positives on micro ct were a cloacal anomaly, incidental cystic neck lesion and thymic atrophy, which were not detected at autopsy. micro-ct of early gestation whole fetuses can provide highly accurate datasets with three-dimensional renderings of complex disease processes. this approach confirms the potential of this technology for non-invasive examination of small fetuses. investigation of perinatal body organ diffusion-weighted post mortem mri s.c. shelmerdine , m. cheryl , j.c. hutchinson , n. sebire , o.j. arthurs ; london/uk, southampton/uk objective: diffusion weighted magnetic resonance imaging (dwi) uses water molecule diffusion to generate mr contrast images, and can reveal microstructural or functional changes in tissues, quantified by measuring the apparent diffusion coefficient (adc). the application of dwi to the post mortem setting is appealing as it does not require the administration of an exogenous contrast agent. a recent pilot study of paediatric cases suggested that lung adc values at pm mri may be a useful marker of post mortem interval (time since death; pmi) which has forensic relevance, but other body organs have not been comprehensively evaluated. the aim of this study was therefore to evaluate the relationship between pmi and body organ adc values in a larger cohort of subjects across a wider gestational range in the setting of perinatal death. whole body perinatal postmortem mri with dwi sequences were performed at . t, with b values of , , mm /s. mean adc values were calculated from regions of interest (rois) placed in the lungs, myocardium, spleen, renal cortex, liver and psoas muscle. the values were measured by two independent readers, correlated against gestational age and post mortem interval (pmi) using the pearson product-moment correlation coefficient. bland-altman plots were created, and the limits of agreement used to assess the inter-observer agreement of mean adc values. results: eighty fetal deaths and stillbirths were imaged with mean gestational age . weeks (range: - weeks). the mean pmi was . days (range - days). there was a weakly positive correlation between pmi and mean lung adc (r = . ) and spleen adc (r = . ). no correlation was found with between adc and pmi for the other body organs. there was reasonable inter-observer agreement between the two readers, with mean adc difference . mm /s (+/- . mm /s). perinatal lung and splenic adc values show a mild increase with increasing pmi. together with other imaging parameters, this may be useful to evaluate organ-specific changes which occur in the post mortem period, particularly in a forensic setting. further research is needed to understand the organ-specific changes which occur in the post-mortem period. usefulness of combined grey-scale and color doppler ultrasonography(us) findings in the evaluation of acute pyelonephritis in children k. lee, j.h. lee; anyang/kr objective: us diagnosis of apn in children can give a valuable information to the clinicians for the early treatment. but the problem of us in the diagnosis of apn is wide range of sensitivity, which is - %. the purpose of this presentation is to evaluate the usefulness of grey-scale us and color doppler us in the diagnosis of acute pyelonephritis in children. from march to february , children( kidneys), boys and girls, aged weeks to years (mean age, . months) underwent kidney us as an initial diagnostic tool for acute pyelonephritis and follow up dmsa scintigraphy within a week. criteria for acute pyelonephritis on grey-scale image were focal/diffusely increased/decreased echogenicity or loss of corticomedullary differentiation. on color doppler sonography, the criterion was decreased color flow. we classified the us diagnosis of apn into categories. definite, suggestive, possible and normal. when above two grey-scale us criteria and color doppler us criterion are seen, we classified it as 'definite'. when one of greyscale us and color doppler us finding are seen, it was classified as 'suggestive' of apn. 'possible' apn was abnormal finding either on grey-scale or color doppler us. 'normal' was no abnormal findings on grey-scale and color doppler us. we compared above findings with dmsa scan, which is considered as gold standard for diagnosing apn. statistical analysis was performed on all kidneys. the overall sensitivity of our study was %( / ) and specificity was %( / ). the positive predictive value for each definite, suggestive, possible groups were %, %, and % respectively. the negative predictive value for normal group was %, which means the false ppv was %. the p-value of the definite and suggestive was statistically significant, but the possible was statistically insignificant. in the diagnosis of apn in children, abnormal us finding either on greyscale or color doppler us is not optimal. abnormal us findings both grey-scale us and color doppler us showed good association with dmsa scan and statistically significant. combined grey-scale and color doppler us findings can give a more reliable information in the diagnosis of apn in children. the greater degree of gastric and/or duodenal wall thickening and increased echogenicity are helpful sonographic features in differentiating congenital duodenal anomalies from malrotation. our findings confirm the superiority of us vs ugi for evaluation of duodenal obstruction in neonates and evaluation of gastric and duodenal wall must be added to the constellation of other features to be assessed on us examinations. a measure of renal morphology as an indicator for potential renal failure a.c. eichenberger, p. grehten, c. kellenberger; zurich/ch this study introduces a measure of renal morphology, herein labelled split renal volume (srv), that should be applied as an indicator for potential renal failure and eventual surgical treatment of obstructive uropathy in children. current practice applies dynamic contrast enhanced functional renal imaging (fri) with complex post-processing methods. fri generates a measure of split renal function (srf). reduced values of srf under % are currently considered to be an indicator for surgical treatment. this retrospective study compares the accuracy of srv with the accuracy of srf as methods for assessing potential renal failure. materials: srv is a quotient of volumetric measurements. total renal volume is described by the sum of parenchymal volume and intra-renal collecting system volume. srv is designated in this study as the quotient of two ratios: first, the ratio of total renal volume to parenchymal volume of the left kidney; and second, the ratio of total renal volume to parenchymal volume of the right kidney. twenty-two children were studied: (age . ± . y) with unilateral asymptomatic intrinsic uretero-pelvic-junction obstruction (upjo), and normal controls (age . ± . y). all subjects underwent mr urography at . t, which provided estimates of srf and srv for each of the examined kidneys. the sensitivity and specificity of both srf and srv for predicting surgical management were determined by comparing the indicators with an expert review panel's decision to operate. the panel was blinded to values of srv. results: when a cut-off value of % for srf was used, the resultant sensitivity and specificity of srf for the detection of kidneys at risk were found to be % and %. the values of srv ranged between . and . . it was found that a value greater than . indicated kidneys at risk. when the cut-off value of . for srv was used, the resultant sensitivity and specificity of srv for the detection of kidneys at risk were both %. in this small population, srv proved to be % accurate and is superior to srf for detecting kidneys at risk of failure due to obstruction. routine application of srv promises to simplify mr urography by obviating dynamic contrast enhanced imaging studies. further prospective studies are necessary in order to select an optimal cut-off value of srv. factors that can distort the dj flexure mimicking malrotation v. bhalla , s. mohan , k.a. bradshaw , m. thyagarajan ; stoke-on-trent/uk, birmingham/uk to highlight the varied radiological appearances and position of the duodenal-jejunal flexure in children and to discuss its importance in assessing for malrotation materials: retrospective analysis of the multiple fluoroscopic examinations performed in the assessment for malrotation over the past years in a busy tertiary centre results: the classic position of the dj flexure is to the left of left pedicle of l and at the level of the duodenal bulb on frontal views and posterior (retroperitoneal) on lateral views. however variations of the normal location can appear, particularly on frontal views, in the upper gi series that can mimic malrotation which has shown to be more common in neonates. we present cases with examples to illustrate the variability in position due to various causes and its implications in the diagnosis of malrotation and volvulus. our case mix includes patients with excessively distended stomachs, large bowel obstruction, renal pelvic dilatation, repeated naso-jejunal and gastro-jejunal tube insertion and in patients post liver transplantation. malrotation and its assessment have serious management and prognostic implications. this presentation demonstrates that the imaging features can be varied, and knowledge about factors distorting the position of the dj flexure is vital in the accurate management of neonates presenting with bilious vomits. retrospective study of prospectively collected data performed at a single tertiary paediatric institution over a . year period. a total of consecutive patients, aged < years, were reviewed who underwent native renal biopsy. all biopsies were performed within the interventional radiology department. all patients had renal disease requiring a renal biopsy for diagnosis. outcome measures include technical success, early and late complications and the adequacy of histological samples. in addition, age, body weight, glomeruli number, histological data, number of cores, size of the biopsy needle, use of co-axial needle and the rate of tract embolisation/plugging were recorded. results: from september to april , patients (mean age: . years +/- . ; range . - . years) underwent native renal biopsy. one hundred ninety-one patients were < years of age. nine hundred forty-six patients ( . %) had a biopsy of the right kidney, patients ( . %) had a biopsy of the left kidney and patient ( . %) had a biopsy of a horseshoe kidney. five hundred fifteen patients were female ( . %). seven hundred sixtynine patients ( . %) had the procedure performed under general anaes-thetic and of patients ( . %) had the procedure performed under local anaesthetic (+/-sedation/entonox). mean number of passes of the core biopsy needle through the renal capsule was . . a gauge core biopsy needle was used in % of the patients. . % of the patients had three or less passes of the biopsy needle though the renal capsule. the overall complication rate was . % (n= ). . % (n= ) of patients had a non-diagnostic biopsy. fifty-five patients underwent a post biopsy ultrasound due to clinical concerns. twenty patients developed perinephric haematoma ( were treated conservatively; one underwent embolisation and subsequent nephrectomy). four patients developed arteriovenous fistulas. two patients developed post procedure infections (one at the skin site and one a perinephric collection). histology results were reviewed in all patients. the mean number of glomeruli obtained was . (range - ). glomerulonephritis was the most common histological diagnosis (n= ; . %) conclusion: renal biopsy is an extremely useful diagnostic tool for renal disease. there is no published data of this size assessing the outcome of native renal biopsies in the paediatric population. jr usa a. lassrich germany j. sauvegrain france c. fauré france a. giedion switzerland e. willich germany r. astley united kingdom ringertz sweden d.g. shaw united kingdom r. lebowitz usa b. lombay hungary pena spain gold medallists london/united kingdom the dutch group of paediatric radiologists, the hague/the netherlands g. stake ringertz (espr) & d. kirks (spr) chicago/united states future espr meeting italy european courses of paediatric radiology (ecpr) genoa/italy (neuroradiology) r.fotter, graz/austria (abdomen) brussels/belgium (thorax) j-n. dacher paediatric musculoskeletal imaging) references: . -stellungnahme-lnt-modell.pdf [internet]. [zitiert an evaluation of paediatric projection radiography in ireland contrast imaging -> application -dectris background ionizing radiation and the risk of childhood cancer: a census-based nationwide cohort study best practices in digital radiography communicating radiation risks in paediatric imaging kinderradiologie-besonderheiten des strahlenschutzes diagnostic imaging and ionizing radiation -canadian nuclear safety commission epidemiology without biology: false paradigms, unfounded assumptions, and specious statistics in radiation science (with commentaries by inge schmitz-feuerhake and christopher busby and a reply by the authors) european guidelines for ap/pa chest x-rays: routinely satisfiable in a paediatric radiology division? eurosafe imaging together -for patient safety image gently campaign back to basics initiative: ten steps to help manage radiation dose in pediatric digital radiography hostens j, u. a. in-vivo dark-field and phase-contrast x-ray imaging safety commission cn. linear-non-threshold model optimisation of paediatric chest radiography optimizing digital radiography of children radiation exposure in diagnostic imaging: wisdom and prudence, but still a lot to understand radiation shielding for diagnostic radiology strahlenhygienische aspekte bei der röntgenuntersuchung des thorax the image gently pediatric digital radiography safety checklist: tools for improving pediatric radiography the standardized exposure index for digital radiography: an opportunity for optimization of radiation dose to the pediatric population gastroenterology and radiology records were searched to identify ibd patients with colonic strictures. all patients underwent an mre within months of colonoscopy. the following colonic parameters were evaluated: bowel wall thickening with luminal narrowing, pre-stenotic bowel dilatation, bowel wall enhancement, and diffusion restriction (if performed). colonoscopy and operative notes were correlated. results: fourteen patients met the inclusion criteria, one with colonic strictures. bowel wall thickening with luminal narrowing at the site of the reported stricture was present in all cases. pre-stenotic bowel dilatation (> . cm) proximal to the reported stricture was present in / cases. using luminal narrowing and prestenotic dilatation as criteria for diagnosis of a colonic stricture, / cases were therefore positive on mre. when comparing to colonoscopy, mre diagnosed colonic strictures in / cases ( %). in the six patients who had surgery, mre accurately diagnosed colonic strictures in / cases ( %). conclusion: mre is not the primary modality for colonic evaluation, yet diagnosing colonic pathology on mre, particularly strictures, may be beneficial for the referring gastroenterologist in the assessment of these patients. potential strictures on colonoscopy did not agree with mre in all cases, but when correlating with surgery % of colonic strictures were accurately diagnosed in a small subset. although mre is not optimized for the evaluation of the colon, colonic strictures can be recongnized in children with crohn's disease.disorders of sexual differentiations in neonates: standardized sonographic evaluation and proposal of a reading grid h. lerisson, e. amzallag -bellenger, f.e. avni, m. cartigny; lille/fr to propose a systematic and structured sonographic approach in neonates with disorders of sexual differentiation (dsd) materials: review of the us pelvic, external genital and adrenal findings in consecutive patients with clinical suspicion of dsd evaluated in the neonatal period. the us survey included: the uterus (absent or visible -with or without hormonal impregnation), the vagina (absent or present (complete or partial)), the gonads (ovaries, testis or unsetermineddysgenetic ) as well as the adrenals (normal, too small or enlarged). the us conclusions were correlated with the endocrinological and genetical work-up of each patient results: twenty cases of dsd have been included us had correctly identified the presence of a uterus in patients. there was one false positive case; among the patients did not show the physiological hormonal impregnation. the vaginal anomalies were correctly evaluated. the gonads were defined correctly as normal testis in patients, normal ovaries in and dysgenetic gonads in . they could not be visualized in patients. adrenals were considered normal in patients (one false negative), hypertrophied in and small in one patient. to compare hepatic d shear wave elastography ( d swe) in children between free-breathing and breath-hold conditions, in terms of measurement agreement and time expenditure. a cohort of children ( . ± . years) who underwent standardized d swe between may and october were retrospectively evaluated. liver elastograms were obtained under free-breathing and breath-hold conditions and time expenditure was measured. median stiffness, interquartile range (iqr), and iqr/median ratio were calculated based on , six, and three elastograms. results were compared using pearson correlation coefficient, intraclass correlation coefficient (icc), bland-altman analysis, and student's t. median liver stiffness under free-breathing and breath-hold conditions correlated strongly ( . ± . kpa vs. . ± . kpa; r= . , p< . ). time to acquire elastograms with free-breathing was lower than that with breath-holding ( . ± . sec vs. . ± . sec, p< . ). results for median liver stiffness based of , six, and three elastograms demonstrated very high agreement for free-breathing (icc . ) and for breath-hold conditions (icc . ). hepatic d swe performed with free-breathing yields results similar to the breath-hold condition. with a substantially lower time requirement, which can be further reduced by lowering the number of elastograms, the free-breathing technique may be suitable for infants and less cooperative children not capable of breath-holding. abstract: pelvi-ureteric junction obstruction (pujo), classified into intrinsic and estrinsic is one of the most frequent urological diseases affecting the pediatric population. extrinsic causes include the presence of crossing vessels, kinks or adhesions. in cases with extrinsic obstruction of puj, colour doppler ultrasound (cd-us) can detect the presence of crossing vessels. in presence of crossing vessels pyeloplasty or vascular hitch can be performed. the aim of the study is to analyze the sensitivity of cd-us and magnetic resonance urography (mru) in visualizing crossing vessels in extrinsic pediatric hydronephrosis in order to decide the correct diagnostic pathway and evaluate in the pre-operative phase which surgical technique and approach (open, laparoscopic or robotic) is the ideal to be performed. a retrospective review of medical records for patients who underwent surgical treatment for hydronephrosis from august to february was performed. a descriptive statistical analysis was performed. the presence of crossing vessels at surgery was considered the gold standard. the sensitivity was calculated for both the imaging techniques as a measure of accuracy, evaluating the ratio between the positive cases divided by the those with aberrant vessels identified at surgery. results clinical charts were reviewed. crossing vessels identified at surgery were ( , % of pujo). the median age was higher in the group with crossing vessels compared to the group without crossing vessels (p< , ). the sensitivity of cd-us was higher compared to mru ( , % vs , %). before the surgical time knowing which technique and approach have to be managed in hydronephrotic patients with crossing vessels could be very important. according to our preliminary datacollection cd-us has got a higher sensitivity and could be the gold standard technique. study limitations include the absence of specificity, positive and negative predictive values. in the future it could be useful to perform a double blind trial in which children with moderate-severe hydronephrosis will be subjected to both imaging techniques to evaluate not only the sensitivity, but also the specificity, the positive predictive value and the negative predictive value conclusion: conclusions in the pre-operative phase, cd-us could be sufficient for the surgeon to discern between pujo with the presence or the absence of crossing vessels, as it has a higher sensitivity and lower costs compared to mru.urosonography -nonradiant alternative for voiding cystourethrography o.m. fufezan, c.a. asavoaie, s. tatar; cluj-napoca/ro voiding cystourethrography (vcug) was considered the gold standard in the diagnosis and monitoring of vesicoureteric reflux (vur). this method is invasive due to the radiation exposure. in the present the diagnosis of vur can also be established by contrast ultrasound examination, also known as voiding urosnography (vus). the authors will present the role of vus in the diagnosis and grading of the vur and the role of patient position in the detection of vur. the infants and children with congenital anomalies of the urinary tract and/or urinary tract infection have been evaluated with vus. iatrogenic vur, neurogenic bladder and urogenital sinus anomalies were excluded. the presence and the degree of the vur were evaluated. vus has been performed using a protocol similar to the one used for vcug. in conditions of sterile urine, . ml sonovue and saline solution have been introduced into the bladder until voiding started. the patients were examined both in a supine and an upright position and the following structures have been scanned: urinary bladder, distal part of the ureters and both pelvicaliceal systems during bladder filling, during and after voiding. the visualisation of the ultrasound contrast agent in the upper urinary tract represented a positive vur diagnosis. the grading of the vur has been established based on the same criteria as in vcug. sixty five patients ( renal units), ages between weeks and years were evaluated (median age ± sd: years ± years and months) through vus. vcug was performed in patients in a maximum of hours after vus. vur has been identified in patients ( . % renal units). a high vur grade (iv-v) was identified in . % of renal units. for the patients investigated with both methods, the results were concordant in patients. in two patients vur has not been identified by vcug, but was detected during vus. the upright position (in addition to decubitus) revealed vur in renal units in which the reflux was not detected in decubitus. conclusion: vus is extremely useful and reliable in diagnosing and grading vur in pediatrics. the changing of the patient position during examination can improve vur detection.new sonographic features useful in differentiating congenital duodenal anomalies from malrotation: gastric and duodenal wall thickening and hyperechogenicity p. caro dominguez , s. hameed , a. zani , r. moineddin , o.m. navarro kunstmann , a. daneman ; cordoba/es, london/uk, toronto/ca the clinical and plain radiographic differentiation of congenital duodenal anomalies (atresia, web, stenosis) and intestinal malrotation is not always clear. although sonography has been documented as an important diagnostic tool to differentiate these two entities, its role is still not widely appreciated. the purpose of this study was to assess the sonographic features of the gastric and duodenal wall in a large series of neonates with congenital duodenal obstruction as these have not been reported previously. neonates who had surgically proven congenital duodenal anomalies or malrotation were identified from the surgical database in a tertiary pediatric hospital in a period of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . those with an ultrasound performed within hours of surgery were included in the study. imaging was retrospectively and independently reviewed by two readers in chronological order blinded to final diagnosis. a wall thickness of ≥ mm of a distended loop was considered abnormal. hyperechogenicity was recorded when the wall of the stomach or duodenum was brighter than liver or splenic parenchyma. imaging findings in the group with congenital duodenal anomalies was compared to the group with malrotation using fisher's exact test. one hundred eight neonates were included in the study, with a congenital duodenal anomaly, with malrotation ( with volvulus) and with both. ugi was performed in neonates who had us. the correct diagnosis was provided only by us in of these newborns ( %), only by ugi in ( %), by both in ( %) and by neither in ( %). ugi was performed in children with malrotation and volvulus, eight were diagnosed only by us, four only by ugi and nine by both. the gastric and/or duodenal wall was significantly thicker and more hyperechoic in neonates with congenital duodenal anomalies than those with malrotation (p< . ) [fig , table ]. conversely an abnormal relationship between the superior mesenteric artery and vein, abnormal position of the third part of the duodenum and the whirlpool sign were found more commonly in neonates with malrotation than those with congenital anomalies (p< . ). key: cord- -p weif authors: magalhaes, tereza; chalegre, karlos diogo m.; braga, cynthia; foy, brian d. title: the endless challenges of arboviral diseases in brazil date: - - journal: trop med infect dis doi: . /tropicalmed sha: doc_id: cord_uid: p weif in this editorial, we list and discuss some of the main challenges faced by the population and public health authorities in brazil concerning arbovirus infections, including the occurrence of concurrent epidemics like the ongoing sars-cov- /covid- pandemic. the cross-reactivity between denv and zikv serological assays are due to similar antigenic regions of viral proteins of these genetically related flaviviruses that can be recognized by the same antibodies. besides being an issue in serological tests, cross-reactive denv and zikv immunity can have important epidemiological implications in places where these viruses co-circulate. for instance, in vitro, in vivo and epidemiological studies have shown that pre-existing denv immunity can either protect or enhance zikv infection, and consequently impact disease development [ ] [ ] [ ] . other studies suggest that the atypically low dengue incidence observed after the zika epidemics in brazil and other latin american countries was due, in part, to short-term denv protection from zikv infections [ , ] . importantly, this lower dengue incidence was followed by a significant increase in dengue cases [ , ] . the impact of pre-existing denv and zikv immunity in further heterologous infections and, importantly, in clinical diseases, needs to be continuously assessed in endemic areas. it is also possible that zikv or other arboviruses may establish sylvatic transmission cycles in brazil, as discussed by other authors [ ] . if one looks at the map of paulista, for example, a municipality within the recife metropolitan region (rmr) in pernambuco state that was heavily affected by zikv and chikv, forested areas surround all the urban areas where the viruses co-circulated and human cases were concentrated in - ( figure and [ ] ). these forested areas may harbor several sylvatic mosquitoes like aedes albopictus, haemagogus janthinomys, and sabethes tarsopus that feed on non-human primates (nhps) and may serve as vectors of arboviruses [ ] . in addition, nhps like the common marmoset callithrix jacchus are abundant in the area [ ] and found near humans. importantly, zikv rna and antibodies against several arboviruses have been found in nhps in different regions of brazil, including marmosets [ ] [ ] [ ] [ ] . the seriousness of an established sylvatic arbovirus transmission cycle in nhps and sylvatic mosquitoes in brazil is well represented by yellow fever virus (yfv), which causes sporadic spillover human outbreaks leading to hundreds of deaths. although a few studies have found little evidence of sylvatic zikv transmission in brazil [ , ] , the possibility of a sylvatic cycle being established in distinct brazilian regions and at different times cannot be excluded. further governmental or research-related arbovirus surveillance activities should intensify monitoring of sylvatic mosquitoes, nhps and other small mammals, as the establishment of sylvatic cycles will require changes in the design of control programs. the zikv outbreaks that occurred in brazil in - probably ceased due to herd immunity-however, instead of disappearing, the virus is still circulating in areas that were intensely affected, like the rmr, even if at low rates. in addition, virus transmission during the outbreaks was focal across metropolitan regions, where some areas were more intensely hit than others within the same municipality [ ] , corroborating the notion of clustered household/community transmission of arboviruses transmitted by ae. aegypti. the low but constant circulation of zikv, the presence of prior virus foci with surrounding patchy areas containing higher numbers of naïve people, and the possibility of a sylvatic cycle being established in some regions increase the chances of unexpected re-emergence of the virus. it will also be important to assess the importance of sexual transmission among the sustained, low zikv circulation in endemic regions, as the epidemiological relevance of zikv sexual transmission may be higher than previously thought ( [ ] and magalhaes et al., unpublished) . escalating the problem of arboviral disease surveillance and management, concurrent outbreaks/epidemics of arboviruses and non-arthropod-borne pathogens can further complicate clinical diagnosis and completely overwhelm/saturate the health care system, as we may be seeing now with the pandemic of coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ). the number of notified dengue, zika and chikungunya cases in brazil in have reached over , by april [ ], reflecting a difficult year for arboviral diseases in the country (figure a ). although the true incidences of sars-cov- infections and covid- cases are unknown in brazil due to the very limited testing (currently, the brazilian government recommends that only severe cases are tested in health clinics and hospitals), the notified numbers of infections and deaths are starting to increase, indicating a worsening epidemic scenario as of april ( figure b) [ ]. at the moment, health care units like the local rapid-access units (unidades de pronto atendimento-upas), which serve communities like the paulista population (~ , habitants), are working with a reduced number of staff as some individuals have fallen ill and many elderly professionals or those with comorbidities are on leave due to fear of becoming infected with the virus. in a recent serosurvey of sars-cov- antibodies among health professionals in pernambuco state, % have tested positive, confirming these professionals are under very high risk of infection [ ] . although the highest numbers of notified arboviral diseases seemed to have occurred in march , it is very likely that case notification has dropped as a result of fewer people infected with arboviruses seeking health facilities due to the sars-cov- pandemic. in fact, it would be important to see if household mosquito transmission of arboviruses increases because of social isolation during the covid- pandemic, considering the endophilic behavior of ae. aegypti (although social isolation is necessary, it is also important to assess its effects on other health factors). the blunt reality is that health care units have been dealing with a peak in arbovirus infections and covid- cases concomitantly. besides the many troubles inherent to an overwhelmed health care system, concurrent epidemics also can complicate clinical-epidemiological diagnoses. some studies show that dengue cases can be misdiagnosed as respiratory infections and vice-versa [ , ] . coinfections during concurrent epidemics must also be considered as they may worsen clinical diseases. coinfections of influenza virus and denv have been identified in several occasions during concurrent epidemics [ ] [ ] [ ] . future control efforts and programs must consider concurrent epidemics as they will most likely continue to happen in the future (e.g., epidemics of denv and new strains of influenza virus). areas where cases were concentrated: an optimal interface for the establishment of sylvatic cycles of arbovirus transmission (this map was published in [ ] ). effective management of arboviral diseases in brazil requires confronting major challenges. the co-endemicity of multiple and related arboviruses complicates clinical-epidemiological diagnoses, clinical management and case notification, in addition to impacting the epidemiology of arboviral diseases in unclear ways. the possible establishment of sylvatic transmission cycles will represent a significant additional challenge to the development of control programs and should be constantly monitored. lastly, concurrent epidemics like the sars-cov- /covid- or other respiratory pathogens/illnesses can overwhelm health care systems and further complicate clinicalepidemiological diagnoses. efforts to better control these diseases must seriously consider all these issues. the authors declare no conflict of interest. brito effective management of arboviral diseases in brazil requires confronting major challenges. the co-endemicity of multiple and related arboviruses complicates clinical-epidemiological diagnoses, clinical management and case notification, in addition to impacting the epidemiology of arboviral diseases in unclear ways. the possible establishment of sylvatic transmission cycles will represent a significant additional challenge to the development of control programs and should be constantly monitored. lastly, concurrent epidemics like the sars-cov- /covid- or other respiratory pathogens/illnesses can overwhelm health care systems and further complicate clinical-epidemiological diagnoses. efforts to better control these diseases must seriously consider all these issues. the authors declare no conflict of interest. one year after the zika virus outbreak in brazil: from hypotheses to evidence serological tests reveal significant cross-reactive human antibody responses to zika and dengue viruses in the mexican population impact of preexisting dengue immunity on zika virus emergence in a dengue endemic region previous dengue or zika virus exposure can drive to infection enhancement or neutralisation of other flaviviruses dengue virus and zika virus serological cross-reactivity and their impact on pathogenesis in mice impacts of zika emergence in latin america on endemic dengue transmission the decline of dengue in the americas in : discussion of multiple hypotheses human urban arboviruses can infect wild animals and jump to sylvatic maintenance cycles in south america a list of mosquito species of the brazilian state of pernambuco, including the first report of haemagogus janthinomys (diptera: culicidae), yellow fever vector and other species (diptera: culicidae) spatial distribution and exploitation of trees gouged by common marmosets (callithrix jacchus) evidence of natural zika virus infection in neotropical non-human primates in brazil seroprevalence of selected flaviviruses in free-living and captive capuchin monkeys in the state of pernambuco limited evidence for infection of urban and peri-urban nonhuman primates with zika and chikungunya viruses in brazil zika virus surveillance at the human-animal interface in west-central brazil prevalence and incidence of zika virus infection among household contacts of patients with zika virus disease influenza illness among case-patients hospitalized for suspected dengue frequency and clinical features of dengue infection in a schoolchildren cohort from medellin, colombia severe coinfections of dengue and pandemic influenza a h n viruses co-infection with dengue virus and pandemic (h n ) virus the diagnostic challenge of pandemic h n virus in a dengue-endemic region: a case report of combined infection in jeddah, kingdom of saudi arabia this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -iex lr authors: niu, xuefeng; zhao, lingzhai; qu, linbing; yao, zhipeng; zhang, fan; yan, qihong; zhang, shengnan; liang, renshan; chen, peihai; luo, jia; xu, wei; lv, huibin; liu, xinglong; lei, hui; yi, changhua; li, pingchao; wang, qian; wang, yang; yu, lei; zhang, xiaoyan; bryan, l. aubrey; davidson, edgar; doranz, j. benjamin; feng, liqiang; pan, weiqi; zhang, fuchun; chen, ling title: convalescent patient-derived monoclonal antibodies targeting different epitopes of e protein confer protection against zika virus in a neonatal mouse model date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: iex lr the zika virus (zikv) outbreak and its link to microcephaly triggered a public health concern. to examine antibody response in a patient infected with zikv, we used single-cell pcr to clone heavy and light chain-paired monoclonal antibodies (mabs) that bind to zikv envelope (e) proteins isolated from memory b cells of a zikv-infected patient. three mabs ( b , c , and a ) that showed the most potent and broad neutralization activities against the african, asian, and american strains were selected for further analysis. mab b showed an ic value of . ng/ml against the circulating american strain gz . epitope mapping revealed that mabs b and c targeted residue k of the lateral ridge (lr) epitope of the ediii domain, but b has a broader lr epitope footprint and recognizes residues t , g , e , and n as well. mab a recognized residues d , k , and k of the edii domain. interestingly, although the patient was seronegative for denv infection, mab c , originating from the vh - and vk - germline pair, neutralized both zikv and denv . administration of the mabs b , c , and a protected neonatal scid mice infected with a lethal dose of zikv. this study provides potential therapeutic antibody candidates and insights into the antibody response after zikv infection. zika virus (zikv) is a member of the flaviviridae family which includes dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), west nile virus (wnv), and tick-borne encephalitis virus (tbev) [ , ] . zikv is mainly transmitted by aedes mosquitoes but can also spread through sexual contact, blood transfusions, or via mother-to-child transmission during pregnancy [ , ] . zikv was first discovered in africa in [ ] and was confined within the equatorial zone of africa and asia until the outbreak in yap island, which was then transmitted to french polynesia and other southern pacific islands in [ , ] . it is believed that the adaptation and infectivity of zikv in mosquito-vectors contributed to the spread of the virus from asia to the americas [ ] . the zikv outbreak and associated increase in microcephaly cases in brazil increased global awareness [ ] ; to date, more than countries have reported zikv infections [ ] . it is known that zikv can cross the placental barrier, leading to fetal microcephaly, and can cause neurological complications in adults, such as guillain-barré syndrome [ ] [ ] [ ] . currently, there are no approved drugs or vaccines to mitigate the risk of zikv infection. the zikv surface is formed by copies of each envelope (e) glycoprotein and associated membrane (m) protein [ , ] . e proteins are arranged as dimers, with three parallel dimers connected to form a raft, and with rafts covering the viral surface [ ] . the e protein mediates viral entry into host cells and membrane fusion and is the major target for neutralizing antibodies and vaccine immunogens [ ] . the flavivirus e ectodomain consists of three distinct domains, edi, a -stranded beta-barrel that acts as a bridge between edii and ediii [ ] ; edii, a finger-like structure that is responsible for the dimerization of soluble e protein monomers and viral fusion [ ] ; and ediii, an immunoglobulin-like segment that is involved in host cell receptor recognition and viral fusion [ , ] . in recent years, a number of neutralizing antibodies (nabs) have been isolated from individuals infected with zikv [ ] [ ] [ ] [ ] [ ] . these nabs mainly recognize edii, ediii, and tertiary or quaternary epitopes that constitute e ectodomains. although ediii-targeted antibodies represent a relatively small population of e protein-binding antibodies, their presence is associated with serum neutralizing activity against zikv [ , ] . among these nabs, ediii-targeted antibodies and edii/e-dimer epitope (ede)-targeted antibodies showed the most potent neutralization activities. in this study, we cloned and characterized e-targeted monoclonal antibodies (mabs) from a chinese patient who returned to china from a visit to venezuela. selected mabs were evaluated for their neutralizing activities in vitro and in vivo via a zikv-infected neonatal severe combined immunodeficiency (scid) mouse model. the patient was a -year-old male who returned from venezuela in february . he was hospitalized in guangzhou th people's hospital (guangzhou, china). zikv rna was detected in serum, saliva, and urine samples by rt-pcr. the patient manifested relatively mild symptoms including fever, rash, sore throat, and fatigue, and recovered and was discharged approximately weeks after the onset of symptoms with no detectable zikv. the patient tested serologically negative for denv - infection using an ns based elisa kit (euroimmun, lubeck, germany), indicating that the patient had no previous exposure to denv - before infection with zikv [ , ] . single b cell sorting, rt-pcr, sequencing, and cloning freshly isolated peripheral blood mononuclear cells (pbmcs) were stained with a cocktail of antibodies including anti-human cd -fitc (invitrogen, carlsbad, ca), igg-apc-h /cd -pacific blue/cd -percp-cy . (bd biosciences, franklin lakes, nj), and anti-his-pe (miltenyi biotec, bergisch gladbach, germany). for antigen-specific memory b cells, we used zikv e protein (cat. no. -v b ; sino biological inc., beijing, china) as a probe. after washing, cd − cd + cd + igg + his + memory b cells were sorted using a multi-laser ariaii sorter. individual b cells were sorted into -well pcr plates containing µl lysis buffer per cell. the lysis buffer contained . µl rnasin inhibitor (promega, madison, wi), µl x first-strand buffer, . µl . m dtt, and . µl igepal (sigma-aldrich, st. louis, mo). pcr plates with sorted cells were frozen on dry ice and then stored at − °c or subjected to reverse transcription. rt-pcr and cloning into expression vectors was performed as previously described [ ] . briefly, µl random hexamers ( ng/µl; promega), µl dntps (each at mm), and . µl superscript iii (invitrogen) were added to each well, followed by incubation at °c for h. the igg heavy and light chain variable regions were amplified independently by nested pcr [ ] . first round pcr was performed using µl cdna directly following reverse transcription, with hotstart taq plus dna polymerase (qiagen, hilden, germany) and the primer mix. the pcr programme was initiated by min incubation at °c, followed by cycles of °c for s, °c (first round) or °c (second round) for s, and °c for min, followed by °c for min before cooling to °c. using % agarose gels, the pcr products were evaluated, excised from the gel (approximately bp for the heavy chain and bp for kappa and lambda chains), and sent for sanger sequencing after purification. human antibody sequences were analysed using imgt/v-quest (http://www.imgt.org/) [ ] . fulllength igg was expressed by co-transfecting hek- t cells with equal amounts of paired heavy and light chain plasmids based on the backbone of the pci-neo vector [ ] . culture media were harvested four days after transfection and purified using protein a agarose (ge healthcare, chicago, il). zikv particles were successfully isolated from the infected patient's blood plasma or urine. the virus was passaged once in suckling mouse brains and cultured in vero cells to prepare stocks, which were stored at − °c before use. denv (hawaii strain), denv (new guinea-c strain), denv (h strain), and denv (h strain) were prepared in vero cells. recombinant zikv e and ediii protein (cat. no. -v b and -v h, respectively) were purchased from sino biological inc. the e protein was derived from zikv strain sph (ku ), isolated from brazil in . denv ediii protein (cat. no. -v b), denv ediii protein (cat. no. -v y ), and denv e protein (cat. no. -v b ) were also purchased from sino biological inc. nunc maxisorp plates (thermo fisher scientific, waltham, ma) were coated with zikv e or e domain iii protein ( µg/ml) and incubated at °c overnight. after blocking for h, the plates were washed six times with phosphate-buffered saline (pbs) and transfected with antibody supernatants at a : dilution with blocking buffer or a serial dilution of purified mabs. secondary antibody (goat anti-human igg; ab ; abcam, cambridge, uk) was applied at a : dilution in blocking solution and then the plates were incubated at room temperature for h, and tmb substrate. absorbance values were determined at nm using a biotek plate reader (biotek instruments, winooski, vt). neutralization activity of purified antibodies was measured using a flow cytometry-based neutralization assay with vero cells as previously reported [ , ] with minor modifications. briefly, × cells were plated in a -well plate h before the experiment. purified human mabs were serially diluted in dmem (gibco; thermo fisher scientific) supplemented with % feta bovine serum (fbs; gibco) and incubated with zikv ( × pfu) for h at °c under % co . the cells were then incubated with µl of the mixture for h at °c under % co , after which ml/well mem medium containing % fbs was added and incubated for another h. zikv-infected and uninfected cells were used as positive and negative control, respectively. cells were then trypsinized, fixed, and permeabilised with fixation and permeabilization solution (bd biosciences) on ice for min, followed by staining with g antibody ( µg/ml) diluted with x perm/wash buffer and staining with anti-mouse igg fitc ( : in x perm/wash buffer) on ice for min. after washing, the percentage of e-positive cells was measured using bd facscanto ii (bd biosciences). the antibody dilution that neutralized % of the viruses (half-maximal neutralizing inhibitory concentration; ic ) was calculated by nonlinear, dose-response regression analysis with graphpad prism version . (graphpad software inc., la jolla, ca). epitope mapping was performed by shotgun mutagenesis as previously described [ ] . zikv prm-e protein expression constructs (based on zikv strain sph ) were subjected to high-throughput alanine scanning mutagenesis to generate a comprehensive mutation library [ , ] . each residue within prm-e was changed to alanine, with alanine codons mutated to serine. in total, zikv prm-e mutants were generated ( % coverage)which were confirmed by sequencingand arrayed into -well plates. each zikv prm-e mutant was transfected into hek- t cells and incubated for h. cells were then fixed in % (v/v) paraformaldehyde (electron microscopy sciences, hatfield, pa) and permeabilised with . % (w/v) saponin (sigma-aldrich) in pbs plus calcium and magnesium (pbs++). for mapping, cells were sequentially incubated with . µg/ml mabs and . µg/ml alexafluor -conjugated secondary antibody (jackson immunoresearch laboratories, west grove, pa) diluted in pbs++, % normal goat serum (sigma-aldrich), and . % saponin. cells were washed three times with pbs++/ . % saponin, twice with pbs, and then mean cellular fluorescence was recorded using a high-throughput flow cytometer (htfc; intellicyt, albuquerque, nm). antibody reactivity against each prm-e mutant relative to the wildtype (wt) protein was calculated by subtracting the signal from mock-transfected controls and normalizing to the signal from wt prm-e-transfected controls. binding competition between mabs b , c , and a and five other ediii-targeted antibodies was determined using a real-time, label-free, bio-layer interferometry assay on an octet red biosensor (fortebio, fremont, ca) as previously described [ ] . the experiment was performed at °c in pbs buffer with shaking at , rpm. ni-nta biosensors (forte-bio) were first loaded with μg/ml his-tagged-e protein for s and then associated with the first mab ( b , f , d , c , a , b , d , or f ) for s. an irrelevant mab, d , was used as negative control and pbs was used as blank solution. the biosensors were then dipped into the second mab and incubated in the presence of the first mab. the capacity of additional binding was monitored by measuring further shifts for s. all mabs were evaluated at concentration of nm, except for b ( nm), d ( nm), and f ( nm), for saturation measurement. the ni-nta biosensors were regenerated with mm glycine-hcl (ph . ; ge healthcare) and re-charged with mm nicl . the response of mab binding to the e protein was compared and the data were processed using biaevaluation software (biacore, uppsala, sweden). the use of pregnant scid and suckling mice in this study was carried out in strict compliance with the association for the assessment and accreditation of laboratory animal care. the experimental protocol was approved by the guangzhou institute of biomedicine and health (gibh) institutional animal care and use committee. scid beige mice were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china). one-day-old suckling mice (n = - ) were intraperitoneally inoculated with zikv at . × pfu. zikv mabs were administered as a single dose h after virus infection. mouse brain, spleen, and serum samples were then collected days after virus infection for rna extraction ( ; qiagen). zikv rna detection was determined by one-step qrt-pcr ( ; qiagen) on a cfx real-time pcr system (bio-rad laboratories, hercules, ca) using published primers and conditions. briefly, µg rna together with a mixture of . µl sybr green, . µl rt-mix, and . µl each of forward and reverse primers were placed in -well pcr plates in µl reaction volumes. amplification was performed at °c for min, °c for min, followed by cycles of °c for s, °c for s, and °c for s. viral load was expressed on a log scale as viral rna copies per µg after comparison with a standard curve. three replicates were conducted for each sample. flow cytometric data were analysed using flowjo version . (tree star inc., ashland, or). the ec (half-maximal effective binding concentration) and ic values obtained from the elisa assay of zikv e/ediii binding activity to mabs and the neutralization assay, respectively, were calculated using a dose-response inhibition model and sigmoidal curves were generated using graphpad prism version . (graphpad software inc.). statistical significance of the difference in viral loads between groups was determined using one-way anova. p values < . were considered statistically significant. to clone e-targeted mabs from a zikv-infected patient, we used recombinant zikv e protein as a probe to isolate e-specific memory b cells from a chinese patient that had returned from venezuela and was identified as infected with zikv during border entry. thirty-one mabs that bind to e protein were successfully cloned days after the onset of symptoms. we first used a facs-based neutralization assay to screen neutralizing activities in the cultured media of hek- cells transfected with expression plasmids for each heavy and light chain of the mabs. eight mabs with the best neutralizing activities and binding to e protein were selected for further analysis. the binding activities of these eight mabs to zikv e, ediii, and denvi ediii proteins were assessed ( figure and table ). mabs b , c , d , and b bound both zikv e and ediii, while mabs a , a , e , and a bound e protein but not ediii ( figure (a) ). mabs b and c showed strong binding activities to zikv e and ediii protein in the nanomolar range. we then measured the neutralization activities of purified mabs against the zikv gz strain. zikv gz has the same genomic sequence as the current circulating zikv strain in south america and was the same as the zikv strain isolated from the patient [ ] . three mabs, b , c , and a , showed the best neutralization activities with ic values of . , . , and ng/ml, respectively; thus, we further analysed these mabs for epitope identification and in an animal model (figure (b) ). we also tested the neutralizing activities of these three mabs against two other zikv isolates, mr (the first zikv isolated from the zika forest in uganda) [ ] and prvabc (a zikv isolated from puerto rico). all three antibodies cross-neutralized mr and prvabc (table ) . intriguingly, one of the mabs, c , showed binding to denv ediii protein but not to other denv ediii proteins. the ed of c with denv ediii was . ng/ml, which was comparable to zikv ediii binding ( . ng/ml). we then measured the neutralization activity of these three representative mabs against denv and found that mabs b and a showed no denvi ediii cross-reactivity and failed to neutralize denvi virus at µg/ml, whereas c showed potent neutralizing activity at an ic of . µg/ml (table ) . neutralizing mabs recognized either the ediii or edii lateral ridge (lr) ediii has been recognized as a major target site for nabs that bind to the same or different epitope region. we used a bio-layer interferometry (bli) competition assay to probe whether our e-targeted mabs bind to the same epitope region. a his-tagged e protein, captured to a ni-nta biosensor, was first saturated with one mab, after which competitive binding of a second mab was assessed. the binding competition between mabs b , c , and a and five other zikv e-targeted mabs was measured (figure (a) ). mabs c , d , d , f , and f but not a could block the binding of mab b to e protein, indicating that b and these five mabs share the same or overlapping epitopes on zikv ediii. interestingly, mab c binding to e protein was blocked by mab b and no other etargeted mab, indicating that b may bind to a broader epitope footprint on ediii, whereas c binds to a smaller epitope region that overlaps with the binding region of b . we next mapped the detailed epitope residues for mabs b , c , and a using a shotgun alaninescanning mutagenesis library of zikv prm and e protein variants [ , ] . mab b recognized an epitope region that includes residues t , g , e , n , and k along the lr region of ediii. notably, mab c only recognized k on the lr region as a key residue (figure (b and c) ). amino acid sequence alignment between e proteins of zikv and denv revealed that residue k on zikv e protein corresponds to k on denv , which is not present on the e proteins of denv , denv , or denv ( figure (d) ). mab a is an edii-targeted antibody that recognized residues d , k , and k on the edii. we confirmed that an individual mutation of these key residues reduced binding activity by more than % when compared with that of wt prm-e protein (figure (b) ). analysis of the immunoglobulin heavy chains of b , c , and a revealed that they were derived from germlines hv - * , hv - * , and hv - - * , respectively (figure (a) ). the shm rates of these heavy chains compared with their predicted germline sequences were relatively low, at . % for b h, . % for c h, and . % for a h, which is lower than that of antibodies isolated from annual trivalent inactivated influenza vaccine (tiv) donors [ ] and chronic human immunodeficiency virus (hiv)- patients (> %) [ , ] . compared with their germline sequences, the shms of a h were in the cdr , cdr , and cdr regions, whereas shms of c h and b were found sporadically in the cdr , fr , cdr , fr , and cdr regions ( table ). we paired the heavy chain predicted germline (hgl) of c h ( c hgl) with the light chain (kappa) of c ( c k) and similarly paired a hgl with a k and expressed these heavy chain germline-reverted antibodies in hek- cells. these germline-reverted antibodies showed reduced binding activities to e protein at . µg/ml for a gl, which is about times lower than that of mature a ( . µg/ml), and . µg/ml for c gl, which almost completely lost binding ability compared with that of mature c ( . µg/ml; table ). therefore, shms are necessary for strong binding to e protein, but only a low level of shms is needed to improve binding. to evaluate the protective effects of e-targeted mabs, we developed a zikv lethal infection mouse model. intraperitoneal infection of -day-old scid neonates with . × pfu zikv gz caused % lethality within days; neonatal mice that received no treatment showed disease symptoms, including ruffled fur, trembling and shaking, and body weight loss ( figure (a) ). neonatal mice treated with mab b at either , µg, or µg h after infection survived the lethal infection (figure (b) ). titration of viral load from brains and spleens collected days after infection revealed a dose-dependent decrease in viral rna in mice treated with mab b , whereas mice that received no treatment had much higher viral rna levels in the brain and spleen (figure (c and d) ). treatment with µg mab b resulted in the best protection, with no detectable viral rna in the brain and spleen. we also demonstrated in subsequent experiments that zikvinfected neonatal scid mice survived and did not exhibit weight loss after treatment with µg of either mab c or a (figure (e and f) ). in a separate experiment, an unrelated mab, g , which is specific for h n influenza virus, showed no protective effects on zikv-infected neonatal scid mice (data not shown). therefore, both ediii-targeted and edii-targeted mabs can effectively protect neonatal scid mice from lethal zikv infections. the emergence of zikv in south america has raised a global health concern due to the link between zikv infection and microcephaly in infants and guillain-barré syndrome in adults. the search for and development of vaccines and therapeutics to prevent and control zikv infection are thus necessary. for example, nabs have been found effective in combating emerging viruses, such as the middle east respiratory syndrome coronavirus (mers-cov), ebola virus, and influenza virus [ , [ ] [ ] [ ] . in the present study, we cloned zikv e proteinbinding mabs from the memory b cells of a zikvinfected chinese patient and selected three mabs for further study. we characterized the zikv e protein epitopes recognized by these mabs and analysed the shm pattern. two of the most potent mabs, b and c , are ediii-targeted and showed neutralizing activities against three different zikv strains, including the south american circulating strain (gz ), african strain (mr ), and american strain (prvabc ). mab b recognized several residues in the lr region of ediii, and an individual mutation in these residues led to a > % decrease in binding activity compared with that of wt prm-e protein. a previous study also demonstrated that an e k mutation alone abolished the neutralizing activity of the lr-targeted mab zka [ ] . meanwhile, the heavy and light chains of mab c were derived from the vh - and vk - germlines, respectively. it has been reported that zikv mabs with vh - /vk - paired antibodies are present in five of six people that were sequentially infected with denv and zikv; thus, these mabs were determined as recurrent antibodies that recognize both zikv and denv viruses [ ] . our findings, along with observations by other studies [ ] , suggested that vh - may be preferentially enlisted in response to zikv and denv infections. it is interesting to note that both mab b and c recognized k in the lr region of zikv ediii. mabs reported by others, such as z and z , also recognized k on zikv ediii or k on denv ediii [ ] . although mab c neutralized the african zikv strain mr , another reported mab, zikv- , that also recognized k of the zikv e protein and neutralized the h/pf/ strain, failed to neutralize the mr strain [ ] . this difference may because the current zikv strains gz and prvabc possess e in the ediii region, whereas african strain mr has d in the ediii region. therefore, k in lr region of ediii appears to be a key target residue for ediiitargeted antibodies to exert neutralizing activities. mabs that recognized k showed potent in vitro neutralizing activity, supporting the notion that residue k is a hotspot for effective neutralizing activity of ediii-targeted mabs [ , , , , ] . it is important to note that most reported e-targeted neutralizing mabs are ediii-targeted, whereas only a few neutralizing mabs are edii-targeted. mabs showing broad neutralization activities against flaviviruses have been reported to recognize the edii targeting either the fusion loop epitope (fle) or ede regions [ , ] . antibodies targeting the fle are conserved among flaviviruses and can bind to zikv e protein. it has been reported that fle-targeted antibodies show poor neutralizing activities but have strong infection enhancement in vitro [ ] . meanwhile, antibodies targeting the ede show potent neutralizing activities against zikv and can protect against zikv infection in mouse and rhesus macaque models [ , ] . notably, mab a in our study showed potent neutralizing activities against all three tested zikv strains and is likely an edii-targeted mab. it also recognized residues d , k , and k and is similar to the reported ede-targeted antibody zikv- , which recognizes d , k , and q [ ] . both mabs recognize the same two residues in the edii region. structural analysis indicated that mab zikv- is cross-linked with e monomers within dimers and neighbouring dimers in the zikv particle [ , ] . therefore, mab a may not exhibit the same interaction with the virus particle as zikv- . recently, another ede-targeted mab, zikv- , was reported to neutralize multiple zikv strains and recognize residues d , m , r , and k in the edii region [ ] ; both mab a and zikv- recognize residues d and k . notably, mab a , zikv- , and zikv- were all isolated from memory b cells of zikv table . cdr , fr , cdr , and fr amino acid sequence of b h, c h, and a h with their corresponding vh germline sequences and predicted cdr sequences. convalescent patients. it is possible that residues d , k , and k are hotspots for ede-targeted neutralizing antibodies. future structural biology analysis is required to confirm if mab a is indeed an edetargeted antibody. antigen-specific b cells undergo a process termed shm to increase antigen affinity. interestingly, the neutralizing mabs b , c , and a had relatively low shm rates in their vh genes, which is much lower than the antibodies isolated from annual tiv vaccine donors [ ] and chronic hiv- patients [ , ] . it is possible that when a person is exposed to zikv infection, e-targeted antibodies that require low shm rates to bind and neutralize zikv were generated. even with relatively low shm rates, these limited mutations appeared essential for conferring binding activities. when the mabs c and a heavy chains were reverted to their predicted germline sequence and paired with their respective mature c and a light chains, binding activity to zikv e protein was found almost completely impaired and times lower than that of mature a , respectively. therefore, (d) viral load in the spleen (n = - ). total rna was extracted from the homogenates of the brain and spleen. zikv genomic rna was evaluated using one-step qpcr. viral loads are expressed as the genome copy number per microgram tissue. (e) body weight changes in zikv-infected mice treated with mab a or c at µg/mouse. (f) survival curve of zikv-infected mice treated with mab a or c . data are representative of two independent experiments and presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significance (one-way anova). a few mutations are sufficient but necessary to confer neutralizing activities against zikv infection. in addition to evaluating the neutralizing activities of representative mabs in inhibiting zikv infection in cultured cells, we also tested the protective efficacy of these mabs in a neonatal scid mouse model. zikv infection in wt mice does not result in disease, whereas suckling wt mice are susceptible to infection [ , ] . mice lacking interferon signalling, such as a (type i ifnar ko) [ ] , interferon regulatory factor (irf) / / triple ko [ ] , and ag (type and type ifn ko) [ ] , are susceptible to zikv infection with detectable zikv in the brain, spinal cord, and testes; these mice died within - days post-infection. in this study, we developed a scid beige suckling mouse model for evaluating the protective ability of mabs against zikv infection. scid beige mice are deficient in t, b, and nk cells and are thus more suitable for evaluating the net effect of anti-zikv mabs. zikv infection of scid beige suckling mice led to neurological symptoms and high viral loads in the brain and spleen, which was eventually lethal. all three mabs tested (ediii-targeted mabs b and c and edii-targeted mab a ) showed protective effects in zikv-infected scid mice. therefore, nabs against zikv cloned from convalescent patients have the potential to be further developed for treating zikv infection. there is an opinion that e-targeted mabs may mediate antibody-dependent enhancement (ade) of virus infection. we found that mab b enhanced zikv infection in k cells at a very low concentration of ng/ml. however, we believe that ade is not a critical concern because the concentration of mabs used for treatment are much higher. nevertheless, engineering of the mab fc fragment to minimize fc gamma receptor-mediated infection would be beneficial in improving the practical usage of these neutralizing mabs. overall, our study findings provided insights into the antibody response after zikv infection and demonstrated the potential of mabs in zikv treatment. zika virus: new clinical syndromes and its emergence in the western hemisphere zika virus: a previously slow pandemic spreads rapidly through the americas effect of acute zika virus infection on sperm and virus clearance in body fluids: a prospective observational study the emergence of zika virus and its new clinical syndromes isolations and serological specificity zika virus outside africa evolutionary enhancement of zika virus infectivity in aedes aegypti mosquitoes assessing the global threat from zika virus zika virus: recent advances towards the development of vaccines and therapeutics guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study possible association between zika virus infection and microcephaly -brazil zika virus associated with microcephaly the . Å resolution cryo-em structure of zika virus structure of the thermally stable zika virus conformational changes of the flavivirus e glycoprotein flavivirus cell entry and membrane fusion structural insights into the neutralization mechanism of a higher primate antibody against dengue virus structures of the zika virus envelope protein and its complex with a flavivirus broadly protective antibody the envelope glycoprotein from tick-borne encephalitis virus at Å resolution characterization of a structural intermediate of flavivirus membrane fusion recurrent potent human neutralizing antibodies to zika virus in brazil and mexico neutralizing human antibodies prevent zika virus replication and fetal disease in mice cross-reactivity, and function of antibodies elicited by zika virus infection molecular determinants of human neutralizing antibodies isolated from a patient infected with zika virus delineating antibody recognition against zika virus during natural infection excretion of infectious zika virus in urine maturation and diversity of the vrc -antibody lineage over years of chronic hiv- infection high-throughput isolation of immunoglobulin genes from single human b cells and expression as monoclonal antibodies the highly customized and integrated system for ig and tr standardized v-j and v-d-j sequence analysis rapid generation of human-like neutralizing monoclonal antibodies in urgent preparedness for influenza pandemics and virulent infectious diseases comparison of plaque-and flow cytometry-based methods for measuring dengue virus neutralization a high-throughput shotgun mutagenesis approach to mapping b-cell antibody epitopes a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus rapid cloning of high-affinity human monoclonal antibodies against influenza virus structural repertoire of hiv- -neutralizing antibodies targeting the cd supersite in donors prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies potent neutralizing monoclonal antibodies against ebola virus infection a human bispecific antibody against zika virus with high therapeutic potential neutralizing human monoclonal antibodies prevent zika virus infection in macaques monoclonal antibodies against zika virus: therapeutics and their implications for vaccine design structural basis of zika virus-specific antibody protection a broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of e protein dengue virus sero-cross-reactivity drives antibodydependent enhancement of infection with zika virus therapeutic and protective efficacy of a dengue antibody against zika infection in rhesus monkeys human antibodies to the dengue virus e-dimer epitope have therapeutic activity against zika virus infection a human antibody against zika virus crosslinks the e protein to prevent infection structural basis of a potent human monoclonal antibody against zika virus targeting a quaternary epitope a mouse model of zika virus pathogenesis zika (prvabc ) infection is associated with t cell infiltration and neurodegeneration in cns of immunocompetent neonatal c bl/ mice characterization of lethal zika virus infection in ag mice key: cord- -j i wlak authors: zarai, yoram; zafrir, zohar; siridechadilok, bunpote; suphatrakul, amporn; roopin, modi; julander, justin; tuller, tamir title: evolutionary selection against short nucleotide sequences in viruses and their related hosts date: - - journal: dna res doi: . /dnares/dsaa sha: doc_id: cord_uid: j i wlak viruses are under constant evolutionary pressure to effectively interact with the host intracellular factors, while evading its immune system. understanding how viruses co-evolve with their hosts is a fundamental topic in molecular evolution and may also aid in developing novel viral based applications such as vaccines, oncologic therapies, and anti-bacterial treatments. here, based on a novel statistical framework and a large-scale genomic analysis of , viruses from all classes infecting host organisms from all kingdoms of life, we identify short nucleotide sequences that are under-represented in the coding regions of viruses and their hosts. these sequences cannot be explained by the coding regions’ amino acid content, codon, and dinucleotide frequencies. we specifically show that short homooligonucleotide and palindromic sequences tend to be under-represented in many viruses probably due to their effect on gene expression regulation and the interaction with the host immune system. in addition, we show that more sequences tend to be under-represented in dsdna viruses than in other viral groups. finally, we demonstrate, based on in vitro and in vivo experiments, how under-represented sequences can be used to attenuated zika virus strains. viruses, the most abundant type of biological entity, are small infectious agents that can only replicate inside the living cells of other organisms (hosts). the viral genetic material is composed of either rna or dna molecule, single or double stranded. viral genomes typically encode three types of protein: proteins for replicating the genome, proteins for packing the genome, and proteins for modifying the function of the host's cell to enhance the replication of the virus's material. , viruses are believed to play a central role in evolution, (e.g. via horizontal gene transfer , - ), be responsible for various human diseases (e.g. aids and respiratory diseases , ) , and also have important applications to biotechnology and nanotechnology. for instance, the recent zika virus (zikv) epidemic in the americas have led the world health organization to declare a 'public health emergency of international concern', , and just recently the novel coronavirus ( -ncov) outbreak in china was declared pandemic by the same organization. due to their complete reliance on the host gene expression machinery, viruses are under constant evolutionary pressure to effectively interact with the host intracellular factors, and at the same time effectively evade its immune system. , thus, understanding how viruses co-evolve with their hosts to ensure their fitness may help in developing novel viral based applications such as vaccines, oncologic therapies, and anti-bacterial treatments. it is natural to expect that viruses and hosts co-evolution patterns are also encrypted in the viral genome. for example, it was shown that high correlation of gc content exists between bacteriophage and related hosts, that a pattern of cpg dinucleotides is suppressed in vertebrate hosts and in their related rna viruses, , that the frequency of tpa dinucleotides is suppressed in invertebrate hosts and in their related rna viruses, and that many long sequences are shared between hosts and their related viruses. , identification and analysis of short dna sequences that are under-represented (also referred to as suppressed or avoided) in genomes of different species were analysed in the past. , for example, in, markov chain models were used to analyse short sequences in the dna of two hosts: escherichia coli and bacillus subtilis. markovian models were used in to predict the frequencies of short sequences and applied them to many prokaryotic species, and the authors in introduced an efficient algorithm to identify sequences that are avoided. in this paper, we analyse under-represented nucleotide sequences in the coding regions of all types of viruses and in the coding regions of their corresponding hosts using a novel statistical framework. these sequences are analysed separately in each of the three reading frames. we provide a large database of these sequences, identify unique and interesting patterns within these sequences, and demonstrate how these sequences can be utilized to attenuate the zikv via in vitro and in vivo experiments. in this section, we briefly describe the main steps of our methodology. a detailed description appears in the supplementary document. the general flow of our analysis is depicted in fig. a . the dataset of virus-host associations was retrieved from previously published data. these include , unique viruses and corresponding hosts, where all the corresponding coding sequences were downloaded and processed. randomization models were used to generate many random variants of the host and virus coding sequences. two different randomization models were used, each control for different biases. a dinucleotide randomization model preserves both aminoacid order and content and the distribution of all possible pairs of nucleotides, whereas a synonymous codon randomization model preserves both amino-acid order and content, and the codon usage bias. these were then used to statistically infer short nucleotide sequences that are under-represented within both the original host and virus genome coding regions, in each reading frame, and those that are common to all three reading frames. these under-represented sequences were analysed and compared among different viral groups and viral proteins, revealing some interesting evolutionary patterns that will be discussed later on. based on this analysis, an attenuated variant of the zikv was engineered and its attenuation was demonstrated in cell lines and in mice. the virus and host coding sequences and association information was retrieved from a published database. in brief, the association between viruses and hosts was derived from the genomenet virus-host database. the database contains , unique viruses and corresponding unique hosts from all kingdoms of life (see supplementary table s ). figure b protists), where we specify for each host domain the portion of the corresponding viruses belonging to each virus type. the virus types in the database are reverse-transcribing (retro), double-stranded dna (dsdna), double-stranded rna (dsrna), single-stranded dna (ssdna), single-stranded rna (ssrna, positive and negative sense), and other (unclassified). the question that we must first address is: what constitutes an under-represented sequence in a coding region? to detect sequences that are statistically under-represented in the coding regions, our statistical background model must capture well-understood coding region features, which are known to be under selection. for example, selection for codon usage bias may cause few short sequences to be in low abundance in the coding regions (as opposed, for example, to regions that are not translated). this, however, does not imply that these short sequences were directly selected against by evolutionary forces. our definition of under-represented short nucleotide sequences in the coding region must then be formulated with respect to all known coding region features (i.e. amino-acids content and order, codon usage bias, and dinucleotide distribution), to suggest possibly new evolutionary forces acting on the viral coding regions. to that end, two randomization models were used to evaluate our hypothesis for short, under-represented nucleotide sequences in the coding regions of the viruses and in the coding regions of their corresponding hosts. the first, called dinucleotide randomization, preserves both amino acid order and content (and thus the resulting protein), and the frequencies of the possible pairs of adjacent nucleotides (dinucleotides). the second, called synonymous codon randomization preserves both amino-acids order and content (and thus the resulting protein) and the codon usage bias. figure c depicts a schematic description of both randomization methods. a selection against short nucleotide sequences that cannot be explained by the canonical genomic features that are preserved by both randomization models implies that these sequences will appear more frequently in the random variants (generated by the above randomization models) than in the original genome. empirical p-values were derived from the empirical null model defined by the above two randomization models. the p-value estimates the probability of obtaining a random value (i.e. the number of occurrences of a sequence in the coding regions) that is the same or larger than the observed value in the original genome. this was performed separately in each of the three reading frames. a sequence was declared underrepresented if its p-values corresponding to the two randomization models were both . . note that in the case of synonymous codon randomization, no under-represented sequence of size three nucleotides can be identified in the first reading frame. specifically, when analysing under-represented sequences in the viruses, we compared the original genome to , corresponding randomization variants generated by each of the randomization models described above. under-represented sequences were then identified separately in each reading frame. in addition, common under-represented nucleotide sequences were identified (i.e. sequences that are under-represented in all three reading frames-see supplementary document, section . . ). this may indicate selection against sequences that may 'interfere' with the process of mrna translation. see supplementary document, sections . and . for an additional method of identifying under-represented sequences in the viruses based on the corresponding hosts (i.e. host-based as oppose to random-based analysis). due to the large size of the host genome, the analysis of underrepresented sequences in the hosts was performed differently than in the viruses. instead, the hosts were analysed relative to their corresponding viruses. recall that a host can be infected by several viruses. specifically, for each pair of a host and a corresponding virus (i.e. a virus that infects that host), we randomly sampled the host coding sequences with a sample size equals the total size of the virus coding sequences. twenty host samples were used for each host-virus pair. each sample was compared with , corresponding randomization variants generated by each of the random models. thus, twenty sets of under-represented sequences were identified in the host, for each reading frame, given a corresponding virus. a sequence that is under-represented in at least ten of the twenty samples, per reading frame, is then considered as under-represented in the host, given the corresponding virus. this is referred to as the sampled majority under-represented set of the host given a corresponding virus (see supplementary document, section . . ). the final set of sequences that are under-represented in the host was defined by the intersection over all the corresponding viruses. see more details in supplementary document, section . . the genome of a thai-strain zikv from an infectious-clone plasmid was evaluated to uncover under-represented sequences (see supplementary document, section . for more details on the zikv strain). first, the two randomized models (dinucleotides and synonymous codons) were used on the zikv coding sequence to identify short sequences that are under-represented. next, oligos of five nucleotides ( -mers) that were identified by both models and showed significant p-values were selected and ranked according to their significance level (see the list of oligos detected in supplementary document, section . ). following, the sequence of the thai strain zikv ns protein was systematically scanned at the nucleotide level (according to the significance in the relevant frame) to identify locations that can be modified with each -mer, but without affecting the amino acid sequence of the protein (fig. ). specifically, we were able to identify and introduce synonymous codon changes in the first reading frame, and synonymous codon changes in the second reading frame. figure . a general scheme of engineering a synthetic sequence. specifically, in the case of the synthetic zikv ur sequence, we introduced different under-represented -mer oligo in the first two reading frames (identified using both randomization models), replacing the original nucleotide sequence while verifying that the protein aa sequence remains unchanged. the modified ns sequence (hereafter named ur ) was later synthesized as plasmid dna, amplified by pcr, and used to build zikv-ur strain by gibson assembly. the first-passage stock virus was produced using vero cells. synthetic strain preparation: the infectious-clone plasmid of the thai-strain zikv was constructed from pcr products of viral cdna. the transfection of the plasmid into mammalian cells generated infectious virus with replication kinetics similar to those of the original virus. the sequence of the infectious-clone plasmid was indeed verified. the viral sequence from this infectious-clone plasmid was evaluated to uncover under-represented sequences as discussed above. cell lines: bhk with rtta was used to generate virus from assembled dna. the supernatant from the transfected bhk was then used to infect vero cells to prepare the virus stock for subsequent experiments. replication kinetics of the wild-type (wt) virus and the ur virus were characterized in vero cells with moi ¼ . . the infectious titre was quantitated with vero cells using immunostaining against e protein by g monoclonal antibodies. animals: the male and female ag mice produced by an in-house colony were used. groups of animals of both genders were randomly assigned to experimental groups and individually marked with ear tags. animals were challenged with malaysian zikv, zikv wt synthetic, ur , or vehicle. serum was collected from all mice dpi for assessment of neutralizing antibodies (neutabs) via prnt assay. mice were monitored for mortality and disease signs daily. individual weights were recorded daily throughout the course of the study. virus: wt zikv (malaysian strain, p - ) was prepared by two passages in vero cells. a challenge dose of ccid was administered via s.c. injection in a volume of . ml. the virus was generated from the same infectious-clone plasmid as the designed variants. quantification of neutab: neutab was quantified using a % plaque reduction neutralization titre (prnt ) assay. serum samples were heat inactivated at c for min in a water bath. one half serial dilution, starting at a / dilution of test sera was made. dilutions were then mixed : with an appropriate titre of zikv in mem containing % fetal bovine serum (fbs) and incubated at c overnight. the virus-serum mixture was then added to individual wells of a -well tissue culture plate with vero cells ( e cells/ well). viral adsorption proceeded for h at c and % co , followed by addition of . % ( , cps) methylcellulose overlay medium containing % fbs to each well. plates were incubated for days, and then stained with crystal violet [with % (wt/vol) crystal violet in % (vol/vol) ethanol] for min. the reciprocal of the dilution of test serum that resulted in > % reduction in average plaques from virus control was recorded as the prnt value. to identify short under-represented nucleotide sequences, we compared the number of appearances of each , , and nucleotides sequences in each reading frame of the original genome with many corresponding randomization variants. our randomization models preserve the basic canonical features of the coding sequences, i.e. amino-acids composition, codon usage bias, and dinucleotide distribution (see section . ). thus, an under-represented sequence cannot be explained by these canonical features and may be selected against by other evolutionary forces. to estimate the false discovery rate, we performed two separate evaluations. first, we generated , randomizations (instead of , ) for few randomly selected viruses and verified that underrepresented sequences that were detected using , randomizations were also detected using , randomizations. in the second evaluation, we performed identifications of under-represented sequences in random variants of the viruses (rather than in the original genome). specifically, a random variant of each virus was randomly selected, and the p-value was evaluated relative to this (random) variant (see supplementary document, section . . . ). comparing the number of under-represented sequences identified in the original viruses and the randomized variants of the viruses yields an estimation of a false discovery rate of . % (for m ¼ ), . % (for m ¼ ), and . % (for m ¼ ). the under-represented sequences identified were further processed by analysing different virus and host groups. specifically, we analysed under-represented sequences for each virus group, for each host domain, for all viruses that corresponds to the same host, and for different combinations of host domains and virus groups (see supplementary document, section . ). a complete list of the most abundant under-represented sequences among the different virus groups is available in supplementary table s . in addition, we refined our analysis of under-represented sequences in the viruses by analysing different protein groups. we classified all viral genes into five mutually exclusive functional groups [surface, structural, enzymatic, unknown (unclassified genes), and other (hypothetical genes)] and showed that the selection against short nucleotide sequences depends on the viral protein function. finally, we performed a test study using zikv, where we engineered under-represented sequences into the genome of an asian zikv and studied their effect both in vitro and in vivo. figure a and b depicts the average number of under-represented sequences of size m ¼ , , and nucleotides, identified in few subsets of viruses in both the original and random variants of the virus. see supplementary document, section . for details about the different subsets, and supplementary document, section . . . for generating random variants of viruses. as shown in the figures, the average number does indeed increase with the sequence size. also, many under-represented sequences are found in dsdna viruses that infect bacteria and vertebrate hosts. the average number of underrepresented sequences found in the random variants of the viruses is between and % of the average number found in the original genome, suggesting a false discovery rate < %. since the genome of dsdna viruses tend to be on average larger than the genome of rna viruses, we aimed at evaluating if the larger number of under-represented sequences identified can be simply attributed to a better statistical signal due to the larger nucleotide size of these viruses. a sampling analysis that we performed (see supplementary document, section . ) suggests that the number of under-represented sequences identified in dsdna viruses matches their genomic size, when compared with rna viruses. a complete list of under-represented sequences of sizes m ¼ , , and nucleotides in all viruses in the database is available in supplementary table s (random-based) and in supplementary table s (host-based). our analysis suggests that among the most abundant common underrepresented nucleotide sequences (i.e. sequences that are underrepresented in all three reading frames) are homooligonucleotide repeats, specifically in viruses. these are sequences of the form xx.x, where all x contain the same nucleotide. figure a note that among these, specifically in viruses, are sequences containing the same nucleotide repeated m ¼ , , or times (i.e. sequences that correspond to the same colour repeating m times in the figure) . a finer resolution of these common under-represented sequences is provided in fig. b , where we depict these sequences separately for different subsets of hosts (left figure) and subsets of viruses (right figure) . see supplementary document, section . for more details of the different subsets. table lists the six most abundant common under-represented nucleotide sequences of size m ¼ , , and in dsdna viruses. all homooligonucleotide sequences (shown in red coloured text) are among these most abundant sequences. one possible reason for this general selection against homooligonucleotide (in all three reading frames) in both viruses and hosts is to reduce erroneous frame shifts as ribosomes traverse the mrna while decoding it codon by codon. a sequence containing a repetition of the same nucleotide in the coding sequence may cause the ribosome to miss the codon boundary, resulting in a frame shift and thus a non-functional and most likely deleterious protein. , this must be recognized and degraded by energy-consuming intracellular proteolytic mechanisms. since translation is the most energetically consuming process in the cell, it is believed that transcripts undergo selection to minimize this energy cost. [ ] [ ] [ ] [ ] [ ] selection against sequences of repetitive nucleotides reduces faulty translation, thus minimizing the overall translation cost. it is possible that this selection against homooligonucleotide repeat is indeed more pronounced in viruses than in hosts since viruses are under much stronger evolutionary selection as they have a larger effective population size and thus a stronger effect of these types of mutations on their fitness. another possible reason may be related to different host immune evasion mechanisms used by viruses (see section . ). we also evaluated the sequence overlap between common underrepresented sequences in viruses and transcription factor binding sites and again found a general selection against homooligonucleotide repeats. these are reported in supplementary document, section . . a nucleotide sequence is called palindromic if it is identical to its reverse complement. obviously, palindromic sequences are of even length. our analysis reveals that . % of all common underrepresented sequences of size m ¼ nucleotides in viruses are palindromes. excluding homooligonucleotide repeats this becomes %. note that only . % of all possible sequences of size m ¼ nucleotides are palindromes. we also evaluated the number of palindromes in random variants of the viruses. these random variants preserve basic transcript features such as amino-acid order and content, codon usage bias and dinucleotide distributions. only . % of all common under-represented sequences of size m ¼ in the random variants of the viruses were found to be palindromes. these findings suggest that indeed the coding regions of viruses are selected against short palindrome sequences. figure a and b depicts the percentage of palindromic sequences of size m ¼ nucleotides that are common under-represented sequences in subsets of hosts and viruses. it was found that palindromic sequences are selected against only in one subset of hosts: bacterial hosts that are infected by dsdna viruses. in addition, palindromic sequences were found to be selected against in dsdna viruses that infect either bacteria (i.e. bacteriophage) or vertebrate hosts. , as depicted in fig. a ). figure c and d depicts the total number of occurrences of each palindrome as under-represented sequence in dsdna viruses that infect bacteria and vertebrate hosts, respectively. in these sub-figures we analysed under-represented sequences regardless of reading frames. two cases are shown: the case where the real virus genome is used (shown in blue colour), and the case where a randomized variant of the virus genome is used (shown in red colour). note the scale difference in the y-axis between the real and the randomized results. the results in the figures imply that dsdna viruses undergo selection against short palindrome sequences. it has been proposed that the principal underlying reason for the apparent avoidance of short palindromes in dsdna viruses is because they are targets for many restriction-modification systems and possibly for general recombination systems as well. , , , , restrictionmodification systems protect bacteria and archaea from attacks by bacteriophages and archaeal viruses. a restriction-modification system specifically recognizes short sites in foreign dna and cleaves it, while such sites in the host dna are protected by methylation. to evaluate the hypothesis of palindromes avoidance in viruses due to restriction-modification systems, we downloaded all restriction enzyme patterns from the rebase database (we used version , which contains information for different restriction enzymes) and evaluated the overlap between the common under-represented nucleotide sequences we identified and the restriction sites from rebase. figure e depicts the number of exact matches between the most abundant common under-represented palindrome sequences of size m ¼ nucleotides in dsdna viruses and restriction sites. the figure also depicts the corresponding enzyme name and the p-value for each common under-represented sequence. the p-value was computed by evaluating the match between common under-represented sequences of random variants of the viruses and the restriction sites. figure f depicts the number of restriction sites that are supersets of the most abundant common under-represented palindrome sequences. p-values were computed as in the case of an exact match. to show that the correspondence between selection against short palindromic sequences in viruses and restriction sites cannot be explained by basic coding region features such as amino-acid content and order, codon usage bias and dinucleotide distribution, we also evaluated the overlap between restriction sites and common under-represented sequences of random variants of viruses. this is reported in supplementary document, section . . a complete list of all common under-represented palindromes of size m ¼ is provided in supplementary table s . common under-represented sequences were only identified in two subsets of hosts. on the other hand, common under-represented sequences were identified in all eight subsets of viruses. our analysis reveals that dsdna viruses infecting bacteria and vertebrate hosts have the largest number of common underrepresented sequences among the different virus subsets. this, as suggested above, seems to be due to the size of dsdna viruses when compared with ssdna and rna viruses. on the other hand, bacteria that are infected by dsdna viruses have the largest number of common under-represented sequences among the different host subsets. thus, the stronger selection for under-represented sequences in bacteria may induce stronger selection for under-represented sequences in viruses that utilize this host. in addition, we evaluated the number of under-represented sequences identified in the real genome of the viruses when compared with the randomized genome of the viruses. this is reported in supplementary document, section . . indeed, many more sequences are identified as under-represented in the real genome of the virus. on average over all viruses and the three sequence sizes, there are stds more under-represented sequences in the real genome in comparison to the random genomes, implying that these cannot be explained by basic coding region features, and suggesting possibly new evolutionary forces acting on the viral coding regions. note that since we analyse each pair of a host and a corresponding virus separately, the set of under-represented sequences in a host above is the sampled majority under-represented set. for obvious reasons, sequences that are not under-represented in both host and virus coding regions constitute the majority of the sequences and are thus not reported here. a complete list of all under-represented sequences within the three classes above for all hosts and viruses in our database is available in supplementary table s . in general, an under-represented sequence of m nucleotides may contain sub-sequences that are themselves under-represented. thus, it may be interesting to identify unique under-represented sequences, i.e. sequences that do not contain any sub-sequences that are underrepresented. for each pair of a host and a corresponding virus, a sequence belonging to one of the three classes above is referred to as a unique under-represented sequence if it does not contain any subsequence that is under-represented in that class. specifically, a unique common under-represented sequence of size m ¼ (m ¼ ) nucleotides doesn't contain any sub-sequence of size m ¼ (of size m ¼ and of size m ¼ ) nucleotides that is common under-represented sequences. a complete list of all unique common under-represented sequences within the three classes above for all hosts and viruses in the database is available in supplementary table s . the correspondence of the most abundant under-represented sequences between viruses and their related hosts is depicted in fig. for different host and virus subsets. each panel depicts both the most abundant common under-represented sequences (left) and the most abundant unique common under-represented sequences (right), where the panel names correspond to the class names. our first observation is that many under-represented sequences are indeed unique. for example, comparing the cases of m ¼ and m ¼ of class a (left sub-figure middle and bottom rows, respectively) with the corresponding unique set (right sub-figure top and bottom rows, respectively) reveals that the majority of the most abundant sequences is unique. second, homooligonucleotide repeats are among the most abundant sequences in all three classes. in addition, more sequences were identified in class b over the different subsets than in the other two classes. for example, table lists the most abundant unique sequence of classes b and c in all the different subsets of hosts and viruses. as shown in the table, unique sequences were identified in all subsets in class b, as oppose to class c. the viral genome encodes different types of proteins that are necessary for the life cycle of viruses in their respective hosts. these, in general, include surface proteins that interact with the host receptors and enable attachment and entry to the host cell, structural proteins that serve as the building blocks of the virus, and replicating enzymes, such as rna and dna polymerase, that are required for the replication of the virus. in addition, many other proteins, some of which are uncharacterized, are diversely involved in different regulatory and accessory functions. here, our aim is to refine the analysis of under-represented sequences in viruses by analysing, separately, different protein groups. to that end, and similarly to, we classified all viral genes into five mutually exclusive functional groups (functional sets): surface, structural, enzymatic, unknown (unclassified genes), and other (hypothetical genes). specifically, for each virus in the database, we divided its genome into the five gene sets defined above. each gene set contains all the virus genes of the same functional group. for example, the surface gene set of a virus contains all the genes that encode surface proteins in the virus's genome. a set might be empty for a particular virus if no genes of the corresponding functional group exist in that virus. see supplementary document, table s for a list of the total number of sets and genes of each functional group in the database. the analysis of under-represented sequences was then performed separately in each of the five gene sets for each of the viruses in the database (see more details in supplementary document, section . ). a complete list of all under-represented sequences in each viral functional group over all viruses in the database is available in supplementary table s . we first analysed the average number of under-represented sequences identified in each gene set. to control for the difference in the average gene size and the number of genes in each set, we randomly selected , , , , , , , , and , genes from each of the surface, structural, enzymatic, unknown, and hypothetical functional groups, respectively. this means that the number of identified under-represented sequences is analysed over similar region sizes, and the differences between the different sets cannot be explained by the genes' nucleotide size in each set. figure a depicts the average number of under-represented sequences (over all three reading frames) identified in each of the gene set over the (randomly selected) subset of genes. relatively small number of under-represented sequences were identified in surface genes (that participate in the recognition of the host receptors), when compared with the other gene sets. at least twice as many were identified in many of the enzymatic genes. these proteins interact closely with the host cell machinery, are essential for the viral replication cycle, and thus must use mechanisms that guarantee their function. figure b depicts the most abundant common under-represented sequences within each viral functional group. these differ between the different functional groups; however, homooligonucleotide sequences appear among the most abundant common underrepresented sequences in all groups. we designed an attenuated zikv variant based on the underrepresented analysis we performed. such variants may be useful in the future for generating a live-attenuated vaccine. specifically, we introduced synonymous mutations to the ns nucleotide sequence, which includes under-represented sequences, and named the new variant ur (see details in section ). infection studies in vero cells demonstrated fractional variant attenuation of the ur virus, which was correlative with our model predictions (see foci size in fig. a, right bottom) . in addition, infectious virus collected and evaluated from the ur variant showed substantial attenuation relative to wt zikv (fig. a) . there is evidence that ag mice lacking ifn-a/b and ifn-c (types i and ii interferon) receptors can be valuable for evaluating the efficacy of new vaccines and anti-viral treatments for zikv. , therefore, as these mice are immune compromised, various strains of zikv cause lethal infection and disease, and will typically cause morbidity and mortality. depending on the strain, severe disease is observed between and weeks after virus challenge. , thus, to further test the synthetic vaccine attenuation level in vivo, ag mice were challenged with attenuated zikv preparations as well as synthetic wt zikv. these inoculations were done in parallel with the original virus grown in cell culture. infection with the synthetically attenuated zikv strains was lethal in all inoculated ag mice. however, the mortality curve of mice infected with ur was delayed, when compared with that of wt malaysia and wt synthetic zikv (average of . days in ur vs. and . in wt malaysia/synthetic zikv, respectively; see fig. b ). no mortality was observed in unvaccinated controls, and mice vaccinated with vehicle (fig. b) . weight loss was also observed in all the infected mice ( - %; see fig. c ). normal control mice experienced general weight gain throughout the experimental period (fig. c) . weight loss corresponded well with mortality, and mice typically lost substantial weight, requiring humane euthanasia. neutab is the primary mediator of protection in vaccine studies in this model. , therefore, serum samples were taken to determine the presence of neutab in infected mice. the neutab titre was evaluated in vaccinated mice weeks after vaccination. mice vaccinated with synthetic wt or ur had significantly (p < . ) elevated neutab titres when compared with vehicle controls (see fig. d ). as expected, no neutab was detected in mice vaccinated with vehicle or in normal control groups (see fig. d ). the virulence levels of ur were somewhat lower than the levels of the malaysian and synthetic wt strains, thus demonstrating that under-represented sequences can be potentially used in the design of live attenuated zikv strains. accordingly, additional attenuation of this variant (e.g. by introducing similar changes to other zikv proteins) may further decreases the lethality of the mice infected by it. since ag mice are very susceptible to zikv infection, mouse model might be too stringent to test these live attenuated vaccine candidates, as human infection is generally sub-clinical after natural zikv infection, hence the attenuated strain might be effective in an immunocompetent model. we compared the average number of under-represented sequences identified in each pair of a virus and its corresponding host. see supplementary document, section . for more details. we found that in % of the cases the average number was larger in the hosts. we believe that this is due to the fact that the viral genome is usually populated with many overlapping codes and genes, when compared with cellular organisms. [ ] [ ] [ ] this introduces many constraints along the viral genome, which can decrease the number of under-represented sequences in the virus. for example, a sub-optimal codon within the host coding region may be synonymously replaced by evolution without affecting the host fitness. however, due to overlapping codes, replacing a sub-optimal codon within the viral coding region may affect multiple proteins and genes, and thus be deleterious to the virus. in this study, we analyse sequences of three, four, and five nucleotides long that are under-represented in the coding regions of viruses of all types and in their corresponding host coding regions. this study is based on a novel statistical evaluation that controls for classical coding region features, which is performed separately in each of the three reading frames. we provide various novel discoveries that may shed light on the evolution of viral dna sequences and on the virus co-evolution with its respective hosts. it is important to emphasize that the observed patterns may be related to various variables and their complex interactions, include gene expression optimizations, various mechanisms for escaping the host immune system, and co-evolution with the corresponding hosts. for example, it was reported that suppression of cg dinucleotides in hiv- is due to coevolution with its vertebrate host to avoid the host defence mechanisms. in general, our analysis reveals that under-represented viral sequences are related to different mechanisms such as restriction modification systems and possibly to alternative or unknown immune escape mechanisms, as these sequences cannot be explained by canonical mechanisms that may suggest, for example, classical viral recognition using antibodies. we show that homooligonucleotide repeats are the most abundant under-represented sequences in both viruses and hosts. a possible explanation for this avoidance is to reduce an erroneous ribosomal frame shifts and thus reduce faulty translation and consequentially the overall translation cost. however, as this motif is shown to be shared between hosts and viruses, our analysis also indicates that a stronger selection pressure against these sequences exists in viruses. this again can be attributed to escape mechanisms from the host immune system, as the virus nucleotide composition evolves to be similar to the host, and it is certainly possible that an excess avoidance of homooligonucleotide repeats reduces viral recognition by classical host immune mechanisms. there may be other relevant explanations such as interaction with small rna genes (e.g. mirnas). it is possible, for example, that these sequences may increase the efficiency of mirna and mrna interactions and thus decrease expression levels. this should be studied further. in addition to homooligonucleotide repeats, we show that palindromes are among the most abundant under-represented sequences in viruses. specifically, excluding homooligonucleotide repeats, our analysis reveals that % of all under-represented sequences of four nucleotides long in viruses are palindromes (where only . % of all possible sequences of that size are palindromes). indeed, analysis of palindromes avoidance in viruses was performed previously. it was shown that palindromes are the most under-represented short sequences in a prokaryotic genome. [ ] [ ] [ ] for example, it was reported that short palindromic sequences are avoided at a statistically significant level in the genomes of several bacteria. four and six nucleotides palindromic sequences that are avoided were reported for few viruses and hosts in, and avoidance of palindromes in several dozen phage genomes was reported in these analyses are based on statistical counts of certain sequences in the given dna and thus do not control for canonical coding region features (codon usage bias, amino acid order and content and dinucleotide distribution) as was done in this study. in addition, our analysis is performed over a larger set of viruses of all types and their corresponding hosts, and at a reading frame resolution. thus, we believe that the results reported here may be more accurate, and should provide a better understanding of this phenomenon. one plausible explanation for avoidance of palindromes in viruses is because they are targets for many restriction-modification systems and possibly for general recombination systems as well. we statistically show a high overlap between under-represented palindromes in viruses and restriction enzyme patterns. this overlap cannot be explained by classical coding region features. restriction of recognition sites has been observed in genomes of prokaryotic organisms. , [ ] [ ] [ ] the authors in analysed the avoidance of restriction sites in few bacteriophage, and concentrated on sites containing six nucleotides. rusinov et al. studied most known recognition sites (both palindromic and asymmetric) in thousands of prokaryotic genomes and found factors that influence their avoidance. it was also shown that the recognition site avoidance correlates with the lifespan of restriction-and-modification systems. recently, the authors in the numbers in parenthesis indicate the frequency of occurrences in percentage. x indicates that no corresponding sequence was identified. analysed avoidance of recognition sites of restriction-modification systems in the genomes of prokaryotic viruses and found it to be a widespread but not a universal anti-restriction strategy of these viruses. the method used by the authors is based on a compositional bias calculation, which is the ratio of the observed to the expected frequency of a sequence, where the expected frequency is estimated based on the observed frequencies of all sub-sites of a given sequence. the compositional bias measure was originally used in for analysing over-and under-represented sequences in dna viruses. since the compositional bias measure doesn't account for a statistical background that preserves know evolutionary forces, we believe that a more accurate and comprehensive procedure of identifying underrepresented sequences is the one used here. in addition, we analyse the distribution of these underrepresented sequences among various viral and host groups. we show, for example, that dsdna viruses infecting bacteria or vertebrate hosts contain a larger set of under-represented sequences than other viral types and that this may be related to their larger genome size. furthermore, we show that on average the set of sequences that are under-represented in viruses but are not under-represented in their related hosts is the largest set among different host-virus underrepresented correspondence. we also show that the selection against under-represented sequences in viruses depends upon the protein function. for example, larger number of sequences is shown to be under-represented in enzyme genes than in surface genes. moreover, even larger number of sequences is found to be under-represented in genes with (currently) unknown functionality, prompting further investigation into the nature of these genes. the differences between these groups may also be related to the expression levels of the different proteins. if, for example, surface genes tend to have low expression levels then they may be under weaker selection for features such as under-represented sequences. vaccines are a topic of a singular importance in present day biomedical science. however, the discovery of vaccines has so far been primarily empirical in nature requiring considerable investments of time, efforts, and resources. to overcome the numerous pitfalls attributed to the classical vaccine design strategies, more efficient and robust rational approaches are highly desirable. one direction in designing in silico vaccine candidates may be based on exploiting the synonymous information, encoded in the viral genomes and related to gene expression, for attenuating the viral replication cycle while retaining its genotype and structure. the analysis and results reported here may have important implications in vaccine synthesis. specifically, the outcomes of this study may provide clues and guidance into practical design of efficient and safe viral vaccines via attenuated viral material. furthermore, it may also prove to be beneficial for other biotechnological objectives related to viral based products such as developing oncolytic viruses and engineering phages to fight bacteria. [ ] [ ] [ ] [ ] [ ] [ ] indeed, we demonstrate, both in vitro and in vivo, how under-represented sequences can be utilized to obtain an attenuated zikv. the aim of these experiments is an initial proof of concept. of course, additional experiments with more variants and controls are needed to better understand the effect of these under-represented sequences on the viral growth rate and fitness. for example, it will be helpful to study additional mutants that do not possess underrepresented sequences but include other types of mutation. however, it is important to emphasize an interesting and a non-obvious aspect of these experiments. the introduced mutations are silent and thus did not alter the encoded protein. based on our experience, in many cases silent mutations may not affect the viral fitness, and furthermore, there are cases where they may even improve its growth rate. also, it is important to emphasize that in these experiments both the wild-type and the mutant variants were generated by the same process and from the same infectious-clone plasmid. finally, the randomization models used in this study may not completely preserve the viral rna secondary structure, and thus the selection for under-represented sequences may be partially due to alterations in secondary structures. fields virology tinkering with translation: protein synthesis in virus-infected cells the role played by viruses in the evolution of their hosts: a view based on informational protein phylogenies horizontal gene transfer in prokaryotes: quantification and classification evolution of complexity in the viral world: the dawn of a new vision giant viruses, giant chimeras: the multiple evolutionary histories of mimivirus genes rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults viral infectious disease and natural products with antiviral activity negative-strand rna viruses: applications to biotechnology viruses and their uses in nanotechnology zika virus outbreak rapid spread of emerging zika virus in the pacific area the evolutionary genetics of viral emergence viral adaptation to host: a proteome-based analysis of codon usage and amino acid preferences patterns of evolution and host gene mimicry in influenza and other rna viruses cg dinucleotide suppression enables antiviral defence targeting non-self rna virus-host coevolution: common patterns of nucleotide motif usage in flaviviridae and their hosts evidence of a direct evolutionary selection for strong folding and mutational robustness within hiv coding regions universal evolutionary selection for high dimensional silent patterns of information hidden in the redundancy of viral genetic code exceptional motifs in different markov chain models for a statistical analysis of dna sequences comparison of methods of detection of exceptional sequences in prokaryotic genomes on avoided words, absent words, and their application to biological sequence analysis, algor avoidance of palindromic words in bacterial and archaeal genomes: a close connection with restriction enzymes evolutionary role of restriction/modification systems as revealed by comparative genome analysis computational dna sequence analysis restriction-modification systems interplay causes avoidance of gatc site in prokaryotic genomes molecular evolution of bacteriophages: evidence of selection against the recognition sites of host restriction enzymes the significance of distance and orientation of restriction endonuclease recognition sites in viral dna genomes avoidance of recognition sites of restriction-modification systems is a widespread but not universal anti-restriction strategy of prokaryotic viruses over-and under-representation of short oligonucleotides in dna sequences forbidden penta-peptides linking virus genomes with host taxonomy infectious clone plasmid of a thai-strain zika virus and its fluorescent reporter system for high-throughput assay and vaccine development a simplified positive-sense-rna virus construction approach that enhances analysis throughput multi-color fluorescent reporter dengue viruses with improved stability for analysis of a multi-virus infection ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use translational accuracy and the fitness of bacteria an integrated approach reveals regulatory controls on bacterial translation elongation gradients in nucleotide and codon usage along escherichia coli genes selection for reduced translation costs at the intronic ' end in fungi multiple roles of the coding sequence ' end in gene expression regulation non-ltr retrotransposons encoding a restriction enzyme-like endonuclease in vertebrates predicting rad-seq marker numbers across the eukaryotic tree of life lifespan of restriction-modification systems critically affects avoidance of their recognition sites in host genomes one recognition sequence, seven restriction enzymes, five reaction mechanisms rebase-a database for dna restriction and modification: enzymes, genes and genomes protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive b and t cells characterization of lethal zika virus infection in ag mice characterization of a novel murine model to study zika virus protective efficacy of zika vaccine in ag mouse model a zika vaccine targeting ns protein protects immunocompetent adult mice in a lethal challenge model pacing a small cage: mutation and rna viruses evolution of viral proteins originated de novo by overprinting hidden silent codes in viral genomes statistical analyses of counts and distributions of restriction sites in dna sequences rna virus attenuation by codon pair deoptimisation is an artefact of increases in cpg/upa dinucleotide frequencies live attenuated influenza virus vaccines by computer-aided rational design changes in codon-pair bias of human immunodeficiency virus type have profound effects on virus replication in cell culture bacteriophages and their implications on future biotechnology: a review bacteriophages and biotechnology: vaccines, gene therapy and antibacterials taking aim on bacterial pathogens: from phage therapy to enzybiotics experimental molecular evolution of bacteriophage t we are grateful to the anonymous referees for comments that greatly helped in improving this paper. the work of y.z. was supported by the israeli ministry of science, technology and space and by the edmond j. safra center for bioinformatics at tel-aviv university. the animal research ethics committee at utah state university approved this research. supplementary data are available at dnares online. key: cord- -vql e j authors: wang, xinyi; tai, wanbo; zhang, xiaolu; zhou, yusen; du, lanying; shen, chuanlai title: effects of adjuvants on the immunogenicity and efficacy of a zika virus envelope domain iii subunit vaccine date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: vql e j zika virus (zikv), a mosquito-borne flavivirus, has attracted global attention due to its close association with congenital zika syndrome and neurological diseases, and transmission through additional routes, such as sexual contact. currently there are no vaccines approved for zikv, and thus, there is an urgent need to develop an effective and safe zikv vaccine. domain iii (diii) of the zikv envelope (e) protein is an important vaccine target, and a vaccine developed using a mutant diii of e (ediii) protein protects adult and pregnant mice, and unborn offspring, against zikv infection. here, we have used immunocompetent balb/c mice treated with anti-interferon-α/β receptor (ifnar ) antibodies to investigate whether three adjuvants (aluminum (alum), monophosphoryl lipid a (mpl), and mf ), either alone or in combination, could improve the efficacy of this ediii subunit vaccine. our data show that, although vaccine formulated with a single adjuvant induced a specific antibody and cellular immune response, and reduced viral load in mice challenged with zikv, the combination of alum and mpl adjuvants led to a more robust and balanced immune response, stronger neutralizing activity against three recent zikv human strains, and greater protection against a high-dose zikv challenge. particularly, the combination of alum with mpl significantly reduced viral titers and viral rna copy numbers in sera and tissues, including the male reproductive organs. overall, this study has identified the combination of alum and mpl as the most effective adjuvant for zikv ediii subunit vaccines, and it has important implications for subunit vaccines against other enveloped viruses, including non-zikv flaviviruses. zika virus (zikv) is a mosquito-borne flavivirus first isolated from a rhesus macaque in uganda in [ ] . most zikv infections are asymptomatic or show mild clinical symptoms, such as fever, headache, or rash. however, some adults infected with zikv present with guillain-barré syndrome (gbs), a disorder of the nervous system that results in muscle weakness and/or paralysis [ , ] . in addition, infection during pregnancy may cause congenital zika syndrome (czs), which is associated laboratory animals, national research council committee [ ] . the protocols have been approved by the institutional animal care and use committee (iacuc) of the new york blood center (permit numbers: and ). this protein was prepared as previously described [ ] . briefly, zikv diii of e protein containing m n and e t mutations at residues and of e protein (hereinafter ediii) was constructed using a mutagenesis kit (thermo fisher scientific, waltham, ma, usa) and a zikv wild-type plasmid expressing residues - (diii) of e protein and a c-terminal fc tag of human (hfc) igg [ , ] . the recombinant ediii protein was transiently expressed in the culture supernatant of t cells, and purified by protein a affinity chromatography (ge healthcare, chicago, il, usa). the above purified zikv ediii protein was used to immunize mice in the presence or absence of various adjuvants as previously described [ ] . briefly, mice were intramuscularly (i.m., µl/mouse) immunized with ediii protein ( µg/mouse) and one of the following adjuvant(s): alum (i.e., aluminum hydroxide, µg/mouse, invivogen, san diego, ca, usa), mpl ( µg/mouse, invivogen), alum ( µg/mouse) + mpl ( µg/mouse), or mf ( µl/mouse) [ ] . mice injected with ediii protein or phosphate-buffered saline (pbs) only were included as controls. the immunized mice were boosted once with the same immunogens at three weeks, and sera were collected at days post-last dose to detect antibody responses and neutralizing antibodies, as described below. zikv e, ediii, or hfc-specific antibodies in immunized mouse sera were analyzed by elisa as previously described [ ] . briefly, elisa plates were coated with zikv ediii protein, zikv full-length e protein with a his tag (aviva systems biology, san diego, ca, usa), or a c-terminal hfc-fused control protein containing a receptor-binding domain (i.e., rbd-fc) of middle east respiratory syndrome coronavirus (mers-cov) spike protein [ ] ( µg/ml) overnight at • c, and blocked with % fat-free milk in pbst (pbs containing tween- ) at • c for h. the plates were washed with pbst for times, and sequentially incubated with serial dilutions of mouse sera and horseradish peroxidase (hrp)-conjugated anti-mouse igg ( : ) or igg-fab ( : ) (for anti-zikv-e or anti-hfc antibodies), igg ( : ), or igg a ( : ) antibodies (thermo fisher scientific) at • c for h. the reaction was visualized after addition of , , , -tetramethylbenzidine substrate (sigma, st. louis, mo, usa) and stopped with n h so . absorbance at nm was measured using an elisa plate reader (tecan, morrisville, nc, usa). three recent zikv human strains, including r ( /honduras), pan ( /panama), and prvabc ( /puerto rico), were used in the study. briefly, viruses were grown in vero e cells and detected for viral titers using a plaque-forming assay [ , ] . mouse sera (about µl) and tissues (about mg for eye, and mg for heart, spleen, muscle, and brain) collected days post-challenge were also detected for zikv titers as described above, and the detection limits were about plaque-forming unit (pfu)/ml for sera, pfu/g for eye, or pfu/g for heart, spleen, muscle, and brain tissues. neutralizing antibodies in immunized mouse sera were detected by the prnt as previously described [ , ] . briefly, pfu of zikv was incubated with -fold serial dilutions of mouse sera at • c for . h, which were added to vero e cells and incubated at • c for h. the cells were then overlaid with dmem containing % carboxymethyl cellulose and % fbs, cultured at • c for - days and further stained with . % crystal violet. the neutralizing titer based on the serum neutralizations at a % plaque reduction (prnt ) was calculated using the calcusyn computer program [ , , ] . the immunized mice were challenged with zikv as previously described [ , ] . briefly, nine days post-last immunization of zikv ediii protein with or without respective adjuvant(s), or pbs control, mice were pretreated with a blocking anti-interferon-α/β receptor (ifnar ) antibody ( mg/mouse, leinco technologies, fenton, mo, usa); and h later, they were intraperitoneally (i.p.) challenged with zikv (strain r , × pfu; µl/mouse). mouse splenocytes were isolated at days post-challenge and detected for cellular immune responses using flow cytometry analysis. mouse sera and tissues were collected as described above, and detected for zikv titers and rna copies using the plaque-forming assay and quantitative reverse transcriptase pcr (qrt-pcr), respectively. flow cytometry analysis was performed to detect zikv-specific cellular immune responses as previously described [ ] . briefly, splenocytes ( × cells/well) were isolated from immunized mice days post-zikv challenge, and treated with × red blood cell lysis buffer (biolegend, san diego, ca, usa), followed by incubation with a zikv ediii peptide mixture (final concentration of µg/ml) (table ) in the presence of brefeldin a ( µg/ml, biolegend), and cultured at • c for h. after stimulation, the cells were washed with pbs, and stained for surface markers using percp/cy . anti-mouse cd -positive (cd + ), fitc-anti-mouse cd -positive (cd + ), and af anti-mouse cd antibodies (biolegend). after fixation and permeabilization, the cells were further stained for intracellular markers using pe anti-mouse interferon-gamma (ifn-γ), brilliant violet tm anti-mouse interleukin (il- ), and brilliant violet tm anti-mouse il- antibodies (biolegend), which were analyzed using a flow cytometer (bd lsrfortessa system). this assay was performed to detect zikv rna copies in sera and tissues of challenged mice as previously described [ , ] . briefly, qiaamp minelute virus spin kit and rneasy mini kit (qiagen, germantown, md, usa) were used to extract rnas from sera and tissues, respectively, which were quantified by qrt-pcr using power sybr green pcr master mix, ambion rnase inhibitor, and multiscribe reverse transcriptase (thermo fisher scientific) in viia master cycler pcr system (thermo fisher scientific). the forward primer ( -ttggtcatgatactgctgattgc- ) and reverse primer ( -ccttccacaaagtccctattgc- ) were applied for amplification. a qrt-pcr standard curve was made via serial dilutions of a recombinant plasmid expressing the zikv membrane and e proteins, and a linear standard curve in the range of - rna copies (correlation coefficient: r value > . ; detection limit: rna copies) was selected for calculation of zikv rna copies in the test samples. mouse sera (about µl) and tissues, including eye, heart, spleen (about mg), muscle, and testis (about mg), were collected days post-challenge, and detected for zikv rna, so the detection limit was about rna copies/ml (for sera), × rna copies/g (for eye, heart, and spleen), and . × rna copies/g (for muscle and testis) [ ] . all values were expressed as a mean with a standard error of the mean (s.e.m). statistical significance of all data among different groups was calculated using one-way anova: tukey's multiple comparison test based on the graphpad prism statistical software. *, **, and *** indicate p < . , p < . , and p < . , respectively. comparison of the adjuvanticity of alum, mpl, mf , and in combination, for immunization with zikv ediii to compare the potency of adjuvants, balb/c mice were immunized with a zikv non-neutralizing epitope-masked, ediii subunit vaccine in the presence or absence of alum, mpl, and alum adjuvant in combination with the mpl adjuvant. in addition, mf was included as a control adjuvant. at seven days following the second dose, sera were collected and the antibody titers specific to zikv ediii (containing a c-terminal hfc tag) or e (without fusion with hfc) were determined ( figure ). when used without an adjuvant, ediii was immunogenic and induced ediii-specific igg antibodies (total igg), while inclusion of alum or mf adjuvant significantly improved the antibody response. although mpl did not appear to enhance the antibody response to ediii, when used in combination with alum, the antibody response was significantly higher than for ediii with alum or mf alone ( figure a ). since ediii protein was fused with the hfc tag, igg antibodies targeting hfc were also generated, showing a similar trend as those specific to ediii ( figure b ). nevertheless, when coating the elisa plate with a zikv full-length e protein without hfc, significantly high-titer igg antibodies were induced, particularly in the alum and mpl-adjuvanted ediii ( figure c ), suggesting that fusion of hfc to the ediii subunit vaccine did not affect the generation of zikv-specific igg antibodies. multiple comparison test based on the graphpad prism statistical software. *, **, and *** indicate p < . , p < . , and p < . , respectively. to compare the potency of adjuvants, balb/c mice were immunized with a zikv nonneutralizing epitope-masked, ediii subunit vaccine in the presence or absence of alum, mpl, and alum adjuvant in combination with the mpl adjuvant. in addition, mf was included as a control adjuvant. at seven days following the second dose, sera were collected and the antibody titers specific to zikv ediii (containing a c-terminal hfc tag) or e (without fusion with hfc) were determined ( figure ). when used without an adjuvant, ediii was immunogenic and induced ediiispecific igg antibodies (total igg), while inclusion of alum or mf adjuvant significantly improved the antibody response. although mpl did not appear to enhance the antibody response to ediii, when used in combination with alum, the antibody response was significantly higher than for ediii with alum or mf alone ( figure a ). since ediii protein was fused with the hfc tag, igg antibodies targeting hfc were also generated, showing a similar trend as those specific to ediii ( figure b ). nevertheless, when coating the elisa plate with a zikv full-length e protein without hfc, significantly high-titer igg antibodies were induced, particularly in the alum and mpl-adjuvanted ediii ( figure c ), suggesting that fusion of hfc to the ediii subunit vaccine did not affect the generation of zikv-specific igg antibodies. immunization and challenge schedule. mice were immunized intramuscularly (i.m.) with pbs (background control) or with zikv ediii without an adjuvant, or with aluminum salts (alum), monophosphoryl lipid a (mpl), alum + mpl, or mf adjuvant and boosted at day after the initial dose. sera were collected at seven days after the boost (day ) and used to determine zikv e or ediii-specific antibody, and neutralizing antibody levels. mice were injected with an anti-interferonα/β receptor (ifnar ) antibody (in order to become susceptible to subsequent zikv infection) [ , ] at nine days after the boost (day ) and subsequently challenged with zikv (strain r , × plaque-forming unit (pfu)) the following day (day ). sera and tissue samples were collected three days post-challenge (day ) and used to determine zikv titers and viral load (rna copies). splenocytes were also tested for the zikv ediii-specific cellular immune response. immunization and challenge schedule. mice were immunized intramuscularly (i.m.) with pbs (background control) or with zikv ediii without an adjuvant, or with aluminum salts (alum), monophosphoryl lipid a (mpl), alum + mpl, or mf adjuvant and boosted at day after the initial dose. sera were collected at seven days after the boost (day ) and used to determine zikv e or ediii-specific antibody, and neutralizing antibody levels. mice were injected with an anti-interferon-α/β receptor (ifnar ) antibody (in order to become susceptible to subsequent zikv infection) [ , ] at nine days after the boost (day ) and subsequently challenged with zikv (strain r , × plaque-forming unit (pfu)) the following day (day ). sera and tissue samples were collected three days post-challenge (day ) and used to determine zikv titers and viral load (rna copies). splenocytes were also tested for the zikv ediii-specific cellular immune response. combination of alum with mpl adjuvant induced significantly higher igg and igg a antibody titers than either alum or mpl alone, or when mf was the adjuvant ( figure d ,e). when used in combination, mpl and alum adjuvants induced a more balanced antibody response, indicated by a low igg (th )/igg a (th ) ratio. in contrast, alum generated a th -biased igg antibody response, while ediii alone, or when formulated with mf , showed a slight th bias ( figure f ). the above data demonstrate that the combination of alum with mpl adjuvant enhanced the levels of ediiispecific igg antibodies (igg and igg a) and produced a more balanced igg response. next, we compared the levels of anti-zikv neutralizing antibodies induced by ediii vaccine when formulated with the different adjuvants. to this end, sera collected at seven days after immunization with the second dose of ediii were tested for neutralization against three human zikv we also determined the levels of the igg and igg a antibodies induced by ediii at seven days after the second dose. ediii alone induced a good igg response while the igg a response was noticeably weaker ( figure d ,e). when used alone, alum induced higher levels of igg (but not igg a) antibodies than ediii without an adjuvant, while mpl induced significantly higher levels of igg a (but not igg ) antibodies. when used in combination, alum with mpl significantly improved the ability of ediii to induce the igg and igg a antibody responses ( figure d,e) . moreover, the combination of alum with mpl adjuvant induced significantly higher igg and igg a antibody titers than either alum or mpl alone, or when mf was the adjuvant (figure d ,e). when used in combination, mpl and alum adjuvants induced a more balanced antibody response, indicated by a low igg (th )/igg a (th ) ratio. in contrast, alum generated a th -biased igg antibody response, while ediii alone, or when formulated with mf , showed a slight th bias ( figure f ). the above data demonstrate that the combination of alum with mpl adjuvant enhanced the levels of ediii-specific igg antibodies (igg and igg a) and produced a more balanced igg response. next, we compared the levels of anti-zikv neutralizing antibodies induced by ediii vaccine when formulated with the different adjuvants. to this end, sera collected at seven days after immunization with the second dose of ediii were tested for neutralization against three human zikv strains (r , vaccines , , of pan , and pavabc ). for all three strains, alum and mpl as the adjuvants induced a significantly stronger neutralizing antibody response than ediii alone, while mpl or mf only enhanced the neutralizing antibody response for the r strain ( figure a-c) . importantly, for all three zikv strains, the neutralizing antibody titers induced by ediii were significantly higher when alum was used in combination with mpl than for alum, mpl, or mf as the sole adjuvant ( figure a-c) . in contrast, sera injected with pbs only induced a background-level neutralizing antibody ( figure a-c) . these data show that although single adjuvants increased the anti-zikv neutralizing antibody response, the combination of alum with mpl adjuvant consistently induced the highest level of neutralizing antibodies against all of the zikv strains investigated. vaccines , , x of strains (r , pan , and pavabc ). for all three strains, alum and mpl as the adjuvants induced a significantly stronger neutralizing antibody response than ediii alone, while mpl or mf only enhanced the neutralizing antibody response for the r strain ( figure a-c) . importantly, for all three zikv strains, the neutralizing antibody titers induced by ediii were significantly higher when alum was used in combination with mpl than for alum, mpl, or mf as the sole adjuvant ( figure a-c) . in contrast, sera injected with pbs only induced a background-level neutralizing antibody ( figure a-c) . these data show that although single adjuvants increased the anti-zikv neutralizing antibody response, the combination of alum with mpl adjuvant consistently induced the highest level of neutralizing antibodies against all of the zikv strains investigated. splenocytes isolated from immunized mice three days after zikv challenge (strain r ) were used to investigate if the adjuvants enhanced the zikv ediii-specific cellular immune response. specifically, we determined the levels of secretion of ifn-γ (th ), il- (th ) and il- (th ) by cd -positive (cd + )-cd + t cells, and ifn-γ and il- by cd + -cd + t cells. for both t cell subsets, only low levels of these cytokines were detected when mice were immunized with ediii alone ( figure a -e), suggesting that ediii without an adjuvant did not induce a strong ediii-specific t cell response. when used individually, alum or mf increased the secretion of il- by cd + -cd + t cells, while increased expression of il- was only seen for mpl ( figure b,c) . for cd + -cd + t cells, inclusion of alum significantly increased ifn-γ expression, while il- expression was increased by alum and mf ( figure d,e) . when used in combination, alum with mpl adjuvant significantly enhanced expression of ifn-γ and il- cytokines tested in both cd + -cd + and cd + -cd + t cells over that observed when the adjuvants (alum, mpl, and/or mf ) were used individually ( figure a,b,d,e) . these data indicate that although individual adjuvants enhanced the ediii-specific cellular immune response, the combination of alum with mpl had a greater effect, particularly in cd + -cd + t cells. figure . detection of neutralizing antibody titers in immunized mouse sera. sera collected at seven days following the second dose of ediii were tested by a plaque reduction neutralization test (prnt) assay for neutralizing antibodies against zikv human strains r (a), pan (b), and prvabc (c). pbs shows the background level of neutralizing antibodies. neutralizing activity for the indicated adjuvants is presented as the neutralizing antibody titer based on the serum neutralizations at a % plaque reduction (prnt ). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. splenocytes isolated from immunized mice three days after zikv challenge (strain r ) were used to investigate if the adjuvants enhanced the zikv ediii-specific cellular immune response. specifically, we determined the levels of secretion of ifn-γ (th ), il- (th ) and il- (th ) by cd -positive (cd + )-cd + t cells, and ifn-γ and il- by cd + -cd + t cells. for both t cell subsets, only low levels of these cytokines were detected when mice were immunized with ediii alone ( figure a -e), suggesting that ediii without an adjuvant did not induce a strong ediii-specific t cell response. when used individually, alum or mf increased the secretion of il- by cd + -cd + t cells, while increased expression of il- was only seen for mpl ( figure b,c) . for cd + -cd + t cells, inclusion of alum significantly increased ifn-γ expression, while il- expression was increased by alum and mf ( figure d,e) . when used in combination, alum with mpl adjuvant significantly enhanced expression of ifn-γ and il- cytokines tested in both cd + -cd + and cd + -cd + t cells over that observed when the adjuvants (alum, mpl, and/or mf ) were used individually ( figure a,b,d,e) . these data indicate that although individual adjuvants enhanced the ediii-specific cellular immune response, the combination of alum with mpl had a greater effect, particularly in cd + -cd + t cells. to investigate if the adjuvants increased protection against zikv infection, immunized mice were challenged with a high-dose zikv (strain r , × pfu/mouse) one day after administering an anti-ifnar antibody (figure ). viral load was determined by a plaque-forming assay and a zikv rna copy number determined by qrt-pcr assay using sera and tissues collected three days post-challenge, an optimal time point to detect zikv infection in anti-ifnar antibodytreated immunocompetent mice, including balb/c (our unpublished data) [ ] . similar levels of zikv were seen in sera and most tissues (eye, heart, spleen, and muscle) for mice immunized with ediii without an adjuvant or with pbs ( figure a -e), whereas only in brain tissue, a significant reduction in viral titers was observed for mice immunized with ediii alone ( figure f ). these results suggest that immunization with ediii is unable to elicit adequate protection against zikv challenge. mice immunized with ediii formulated with a single adjuvant (alum, mpl, and/or mf ) had significantly reduced viral titers compared to those immunized with ediii alone (sera, heart, spleen, muscle, and brain) ( figure a ,c-f) and those immunized with pbs (sera, eye, heart, spleen, muscle, and brain) ( figure a-f) , suggesting that inclusion of an adjuvant increased protection. importantly, in sera and all of the tissues tested, the combination of alum with mpl adjuvant resulted in significantly lower, or undetectable, viral titers than other immunization conditions with a single adjuvant (alum, mpl, and/or mf ) ( figure a-f) . these results demonstrate that, although single adjuvants can increase protection, the greatest level of protection against a high-dose zikv challenge is achieved when ediii vaccine is formulated with both alum and mpl adjuvants. to investigate if the adjuvants increased protection against zikv infection, immunized mice were challenged with a high-dose zikv (strain r , × pfu/mouse) one day after administering an anti-ifnar antibody (figure ). viral load was determined by a plaque-forming assay and a zikv rna copy number determined by qrt-pcr assay using sera and tissues collected three days post-challenge, an optimal time point to detect zikv infection in anti-ifnar antibody-treated immunocompetent mice, including balb/c (our unpublished data) [ ] . similar levels of zikv were seen in sera and most tissues (eye, heart, spleen, and muscle) for mice immunized with ediii without an adjuvant or with pbs ( figure a-e) , whereas only in brain tissue, a significant reduction in viral titers was observed for mice immunized with ediii alone ( figure f ). these results suggest that immunization with ediii is unable to elicit adequate protection against zikv challenge. mice immunized with ediii formulated with a single adjuvant (alum, mpl, and/or mf ) had significantly reduced viral titers compared to those immunized with ediii alone (sera, heart, spleen, muscle, and brain) ( figure a ,c-f) and those immunized with pbs (sera, eye, heart, spleen, muscle, and brain) ( figure a-f) , suggesting that inclusion of an adjuvant increased protection. importantly, in sera and all of the tissues tested, the combination of alum with mpl adjuvant resulted in significantly lower, or undetectable, viral titers than other immunization conditions with a single adjuvant (alum, mpl, and/or mf ) ( figure a-f) . these results demonstrate that, although single adjuvants can increase protection, the greatest level of protection against a high-dose zikv challenge is achieved when ediii vaccine is formulated with both alum and mpl adjuvants. , and brain (f). sera and tissues were collected at three days post-challenge with zikv (strain r ), and viral load determined by a plaque-forming assay. mice immunized with pbs served as the control. the detection limits for the samples were ~ pfu/ml (sera), ~ pfu/g (eye), and ~ pfu/g (heart, spleen, muscle, and brain). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. consistent with viral load, mice immunized with ediii alone had only slightly lower levels of zikv rna in sera and tissues (eye and spleen) compared to those immunized with pbs ( figure a ,b,d), confirming that an adjuvant is required to induce protection. similarly, inclusion of a single adjuvant (alum, mpl, and/or mf ) resulted in a significantly lower level of zikv rna in sera and tissues (eye, heart, spleen, or muscle) compared with immunization with ediii alone (figure a-e) . furthermore, the levels of zikv rna in sera and all of the tissues, when alum and mpl adjuvants were used in combination for immunization, were significantly lower than when a single adjuvant (alum, mpl, and/or mf ) was used ( figure a-e) . these results are consistent with the levels of viral load ( figure ) and confirm that protection against zikv is greatest when alum and mpl adjuvants are used in combination. zikv can replicate in the reproductive organs for long periods; therefore, we also determined the amount of zikv rna in testes of immunized and challenged mice. similar to the results in other tissues, inclusion of a single adjuvant reduced the level of viral rna more than immunization with ediii alone or with pbs ( figure f ). moreover, viral rna copies were the lowest in testis of mice receiving ediii formulated with alum and mpl in combination ( figure f) . importantly, our results show that formulation of ediii with a combination of alum and mpl adjuvants can reduce viral rna copies in male reproductive organs. figure . detection of zikv titers in challenged sera (a), eye (b), heart (c), spleen (d), muscle (e), and brain (f). sera and tissues were collected at three days post-challenge with zikv (strain r ), and viral load determined by a plaque-forming assay. mice immunized with pbs served as the control. the detection limits for the samples were~ pfu/ml (sera),~ pfu/g (eye), and~ pfu/g (heart, spleen, muscle, and brain). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. consistent with viral load, mice immunized with ediii alone had only slightly lower levels of zikv rna in sera and tissues (eye and spleen) compared to those immunized with pbs ( figure a ,b,d), confirming that an adjuvant is required to induce protection. similarly, inclusion of a single adjuvant (alum, mpl, and/or mf ) resulted in a significantly lower level of zikv rna in sera and tissues (eye, heart, spleen, or muscle) compared with immunization with ediii alone (figure a-e) . furthermore, the levels of zikv rna in sera and all of the tissues, when alum and mpl adjuvants were used in combination for immunization, were significantly lower than when a single adjuvant (alum, mpl, and/or mf ) was used ( figure a-e) . these results are consistent with the levels of viral load ( figure ) and confirm that protection against zikv is greatest when alum and mpl adjuvants are used in combination. figure . detection of zikv rna in challenged sera (a), eye (b), heart (c), spleen (d), muscle (e), and testis (f). sera and tissues were collected at three days post-challenge with zikv (strain r ) and viral rna copies determined by qrt-pcr. mice immunized with pbs served as the control. the detection limits were ~ rna copies/ml (sera), ~ × rna copies/g (eye, heart, and spleen), and ~ . × rna copies/g (muscle and testis). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. protein-based subunit vaccines are generally safer than vaccines based on live attenuated or inactivated viruses but usually have low immunogenicity and efficacy [ , ] . however, vaccine performance can be improved by structure-based designs via masking non-neutralizing or unfavorable epitopes, or via antigen conjugation [ , , ] . in addition, the choice of adjuvant can greatly influence the immunogenicity and efficacy of vaccines, including subunit vaccines [ , , ] . our previous studies demonstrated that masking of a non-neutralizing epitope (surrounding residue ) of a zikv diii of e protein subunit vaccine (i.e., ediii) improved immunogenicity and protection of adult and pregnant mice, and unborn offspring, against zikv infection [ ] . here, we extended these studies to investigate the influence of licensed adjuvants on the immunogenicity and protection of mice against high-dose zikv challenge following immunization with this ediii subunit vaccine. vaccines prepared with a single adjuvant (alum, mpl, or mf ) significantly improved the antibody response (total igg, igg , and/or igg a) over immunization without an adjuvant. however, the highest igg titers were obtained when alum and mpl were used in combination. as stated above, adjuvants influence the immune response to vaccines. alum is a potent inducer of the th (igg ) immune response [ , ] . similarly, mf generally induces a slightly-biased th response as seen for vaccines for influenza virus and mers-cov [ , ] . in contrast, mpl induces expression of ifn-γ and il- and, therefore, has a more th bias and generates a more balanced (th /th ) immune response [ , ] . moreover, a combination of mpl and alum adjuvants (i.e., as ) may shift the immune responses from th to th and generate a more balanced immune figure . detection of zikv rna in challenged sera (a), eye (b), heart (c), spleen (d), muscle (e), and testis (f). sera and tissues were collected at three days post-challenge with zikv (strain r ) and viral rna copies determined by qrt-pcr. mice immunized with pbs served as the control. the detection limits were~ rna copies/ml (sera),~ × rna copies/g (eye, heart, and spleen), and . × rna copies/g (muscle and testis). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. zikv can replicate in the reproductive organs for long periods; therefore, we also determined the amount of zikv rna in testes of immunized and challenged mice. similar to the results in other tissues, inclusion of a single adjuvant reduced the level of viral rna more than immunization with ediii alone or with pbs ( figure f ). moreover, viral rna copies were the lowest in testis of mice receiving ediii formulated with alum and mpl in combination ( figure f ). importantly, our results show that formulation of ediii with a combination of alum and mpl adjuvants can reduce viral rna copies in male reproductive organs. protein-based subunit vaccines are generally safer than vaccines based on live attenuated or inactivated viruses but usually have low immunogenicity and efficacy [ , ] . however, vaccine performance can be improved by structure-based designs via masking non-neutralizing or unfavorable epitopes, or via antigen conjugation [ , , ] . in addition, the choice of adjuvant can greatly influence the immunogenicity and efficacy of vaccines, including subunit vaccines [ , , ] . our previous studies demonstrated that masking of a non-neutralizing epitope (surrounding residue ) of a zikv diii of e protein subunit vaccine (i.e., ediii) improved immunogenicity and protection of adult and pregnant mice, and unborn offspring, against zikv infection [ ] . here, we extended these studies to investigate the influence of licensed adjuvants on the immunogenicity and protection of mice against high-dose zikv challenge following immunization with this ediii subunit vaccine. vaccines prepared with a single adjuvant (alum, mpl, or mf ) significantly improved the antibody response (total igg, igg , and/or igg a) over immunization without an adjuvant. however, the highest igg titers were obtained when alum and mpl were used in combination. as stated above, adjuvants influence the immune response to vaccines. alum is a potent inducer of the th (igg ) immune response [ , ] . similarly, mf generally induces a slightly-biased th response as seen for vaccines for influenza virus and mers-cov [ , ] . in contrast, mpl induces expression of ifn-γ and il- and, therefore, has a more th bias and generates a more balanced (th /th ) immune response [ , ] . moreover, a combination of mpl and alum adjuvants (i.e., as ) may shift the immune responses from th to th and generate a more balanced immune response [ ] . consistent with these observations, we found that mf and alum, when used individually as the adjuvant for ediii, induced a th -biased immune response, while, when mpl was the sole adjuvant or used in combination with alum, a more balanced (th /th ) immune response was generated. the humoral and cellular immune responses influence zikv pathogenesis and play key roles in controlling zikv replication [ ] . in particular, neutralizing antibodies, induced by zikv vaccines, may block infection [ , ] , while cd + and cd + t cells secrete ifn-γ, il (th ), il- , and il- (th ) cytokines to further promote antibody production or directly kill infected cells [ ] [ ] [ ] [ ] . cd + t cells are critical for protection against zikv infection in mice, non-human primates, and humans [ , , [ ] [ ] [ ] . in the current study, we observed that alum, mpl, and mf adjuvants enhanced production of anti-zikv neutralizing antibodies; however, the combination of alum with mpl generated significantly higher neutralizing antibody titers than the individual use of adjuvants, and these results were consistent for three emerging human zikv strains. in addition, alum and mpl, when used in combination, enhanced secretion of ifn-γ (th ) and il- (th ) by cd + , particularly cd + , t cells. viral vaccines (e.g., against hbv and hpv) formulated with a combination of alum and mpl adjuvants (i.e., as ) stimulate antigen-specific t and b cells, resulting in stronger antibody (including neutralizing antibodies) and cellular responses than vaccines formulated using alum alone [ , ] . using the zikv ediii protein as a model antigen, our study has confirmed these observations by showing that a combination of alum and mpl adjuvants enhanced the immunogenicity of ediii over that observed with single adjuvants (figures - ) , whereas the alum and mpl combination alone without ediii was unable to elicit zikv-specific immune response and protection against zikv infection [ , ] . zikv does not normally infect immunocompetent mice, including balb/c and c bl/ [ , ] , but does infect immunocompromised mice deficient for interferon receptors (interferon-α/β receptor (ifnar) or a , or interferon-α/β/γ receptor, ag ) [ , , ] . to carry out challenge studies, we used balb/c mice treated with anti-ifnar antibodies, which also render mice susceptible to zikv infection [ , ] . formulation of ediii with individual adjuvants improved protection against high-dose zikv challenge, and was consistent with our virus neutralization studies results, that is, the combination of alum with mpl significantly improved protection further. this was confirmed by showing that a combination of alum with mpl as the adjuvant significantly reduced viral titers and viral rna copies in sera and tissues compared to other immunization regimes. zikv can be transmitted through sexual contact, the virus can persist in female or male (testes and seminal vesicles) reproductive organs for a prolonged period, and viral rna can be detected in semen for up to one year after zikv infection [ , [ ] [ ] [ ] [ ] [ ] . thus, vaccines that reduce viral load in reproductive organs will likely be more effective in reducing virus transmission during sexual contact. importantly, our results show that ediii formulation using alum and mpl in combination significantly reduced viral rna in the testes of zikv-challenged mice, thereby confirming that this adjuvant combination is most likely to yield greater vaccine efficacy than single adjuvants alone. in contrast, ediii alone without adjuvant(s) resulted in the highest viral titers and viral rna copies in sera and tissues tested among all vaccination groups, including reproductive organs, which were only slightly lower than those in pbs-injected control mice. it has been shown that protection against zikv infection is positively correlated with serum neutralizing antibody titers [ , ] ; thus, the high-level zikv in the ediii alone-immunized mice identified above may be partially due to the relatively low serum neutralizing antibody titer induced in these mice. it should be noted that the ediii protein used in this study was fused with a c-terminal hfc tag. fc-fusion proteins have been approved by the fda for human therapy, and their safety and pharmacokinetic activity have been evaluated extensively [ ] [ ] [ ] [ ] . fusion of fc tag to recombinant proteins may promote the proteins to form dimeric structures, significantly improving their solubility, stability, and avidity, as well as increasing their immunogenicity and/or efficacy [ ] [ ] [ ] . fc-fused vaccine design has been applied in subunit vaccines against hiv, influenza virus, ebola virus, and other emerging or reemerging viruses [ ] [ ] [ ] [ ] [ ] . in terms of zikv vaccines, a fc-fused e protein dimer (ze-fc) has been shown to mimic native dimeric status of e protein and induce higher neutralizing antibodies than a monomeric e protein without fc (monze) [ ] . we have also found that hfc-fused zikv ediii proteins form dimeric structures capable of eliciting high-titer antibody responses, neutralizing antibodies, and protection against high-dose zikv infection (figures , , and ) [ ] , consistent with previous findings. in conclusion, using immunocompetent balb/c mice, we compared the immunogenicity, neutralizing activity, and protective efficacy (after injection with anti-ifnar antibodies) of a zikv ediii subunit vaccine when formulated with different adjuvants (individually or in combination). although ediii formulated with alum, mpl, or mf induced a specific antibody and cellular immune response, and reduced viral load, the greatest vaccine efficacy was achieved when alum and mpl adjuvants were combined in a single vaccine. overall, this study has identified alum combined with mpl as the adjuvant of choice for zikv subunit vaccines, and has important implications for subunit vaccines against other enveloped viruses, including non-zikv flaviviruses. author contributions: l.d. and c.s. initiated and designed the study. x.w., w.t., and x.z. performed the experiments and analyzed the data. y.z., l.d., and c.s. wrote, edited, and approved the manuscript. funding: this research was funded by the national institutes of health (nih) grant (r ai ) and intramural funds of the new york blood center (nyb ). zikv strains (r , pan , and pavabc ) were obtained from bei resources. the authors declare no conflict of interest. zika virus. i. isolations and serological specificity zika virus infection as a cause of congenital brain abnormalities and guillain-barre syndrome: systematic review zika virus-associated guillain-barre syndrome in a returning us traveler congenital zika virus syndrome in infants: repercussions for the promotion of families' mental health congenital zika syndrome and extra-central nervous system detection of zika virus in a pre-term newborn in mexico association between microcephaly, zika virus infection, and other risk factors in brazil: final report of a case-control study structural basis of zika virus-specific antibody protection structures of the zika virus envelope protein and its complex with a flavivirus broadly protective antibody the . a resolution cryo-em structure of zika virus yeast-produced subunit protein vaccine elicits broadly neutralizing antibodies that protect mice against zika virus lethal infection zika virus-derived e-diii protein displayed on immunologically optimized vlps induces neutralizing antibodies without causing enhancement of dengue virus infection. vaccines (basel) , , cross-reactivity, and function of antibodies elicited by zika virus infection recent advances in zika virus vaccines protective efficacy of nucleic acid vaccines against transmission of zika virus during pregnancy in mice current status of zika vaccine development: zika vaccines advance into clinical evaluation zika virus vaccine: progress and challenges safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials zika virus vaccines zika virus vaccines: immune response, current status, and future challenges current advancements and potential strategies in the development of mers-cov vaccines the latest advancements in zika virus vaccine development recent advances of vaccine adjuvants for infectious diseases correlates of adjuvanticity: a review on adjuvants in licensed vaccines optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want putting endotoxin to work for us: monophosphoryl lipid a as a safe and effective vaccine adjuvant critical neutralizing fragment of zika virus ediii elicits cross-neutralization and protection against divergent zika viruses rational design of zika virus subunit vaccine with enhanced efficacy guide for the care and use of laboratory animals identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design transfusion-transmitted zika virus infection in pregnant mice leads to broad tissue tropism with severe placental damage and fetal demise peptide-based membrane fusion inhibitors targeting hcov- e spike protein hr and hr domains theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies an immunocompetent mouse model of zika virus infection animal models of zika virus infection, pathogenesis, and immunity a mouse model of zika virus pathogenesis introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines a proof of concept for structure-based vaccine design targeting rsv in humans adjuvants and the vaccine response to the ds-cav -stabilized fusion glycoprotein of respiratory syncytial virus recombinant hemagglutinin produced from chinese hamster ovary (cho) stable cell clones and a pelc/cpg combination adjuvant for h n subunit vaccine development how) do aluminium adjuvants work? adjuvanted influenza vaccines no pain no gain? adjuvant effects of alum and monophosphoryl lipid a in pertussis and hpv vaccines immune response to dengue and zika broadly neutralizing activity of zika virus-immune sera identifies a single viral serotype efficacy of a t cell-biased adenovirus vector as a zika virus vaccine t cell immunity to zika and dengue viral infections cd + t cells promote humoral immunity and viral control during zika virus infection protective to a t: the role of t cells during zika virus infection mapping and role of the cd + t cell response during primary zika virus infection in mice zika virus infection of rhesus macaques leads to viral persistence in multiple tissues animal models of zika virus infection during pregnancy zika virus pathogenesis and tissue tropism comparative histopathologic lesions of the male reproductive tract during acute infection of zika virus in ag and ifnar -/-mice a new threat to human reproduction system posed by zika virus (zikv): from clinical investigations to experimental studies zika virus causes testis damage and leads to male infertility in mice prolonged zika virus rna detection in semen of immunosuppressed patient persistence and clinical relevance of zika virus in the male genital tract fc-fusion proteins in therapy: an updated view use of population pharmacokinetic analyses among fda-approved biologics fc fusion as a platform technology: potential for modulating immunogenicity validation and life-cycle management of a quantitative ligand-binding assay for the measurement of nulojix, a ctla- -fc fusion protein, in renal and liver transplant patients fc-fusion proteins: new developments and future perspectives fcγ fragment of igg as a powerful affinity tag in recombinant fc-fusion proteins: immunological, biochemical and therapeutic properties a recombinant vaccine of h n ha fused with foldon and human igg fc induced complete cross-clade protection against divergent h n viruses a neonatal fc receptor-targeted mucosal vaccine strategy effectively induces hiv- antigen-specific immunity to genital infection adjuvant-free immunization with hemagglutinin-fc fusion proteins as an approach to influenza vaccines ebola virus glycoprotein fc fusion protein confers protection against lethal challenge in vaccinated mice key: cord- -t x gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober ‐ , date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: t x gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks , , , and following index donations from donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by months. urine and saliva detection decreased significantly after weeks and was undetectable by months. of donors who were enrolled in the acute pre-seroconversion stage of infection % ( / ) developed multiple zikv related symptoms week post index donation, compared to only % ( / ) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* , yuelong guo , ritchard g. cable , joseph e. kiss , michael paul busch , grier page , stacy endres-dighe , steve kleinman , simone glynn , alan mast and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) . american red cross, rti international, american red cross blood services, blood systems inc., blood systems research institute, university of british columbia, nih/ nhlbi, blood research institute, nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over , donors were enrolled from us blood centers for ferritin testing. the study population was enriched for racial minorities [ african-american (aa), asian (as), hispanic (hisp)] and for "super donors" ( , who had completed donations in two years without low hemoglobin deferral). the minority donors and the remaining non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml). results/findings: across all subjects, % had ais and % had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed % % decreased risk for ais compared to nhw, while hisp donors had % higher risk. daily use of exogenous iron reduced risk for lf and ais by to %, respectively, while the estimated benefit from less-than-daily use was lower ( to % protection). regular use of antacids was associated with a % or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by % - %, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean ( sd) *p < . compared to batf / , ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to % of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d and d of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration ( % on d vs % on d of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized ( %) and macrophage-depleted ( %) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than % at h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at h posttransfusion. at h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* , amanda l richards and krystalyn e hudson . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod otii mice are predisposed to have autoreactive cd t cells. study design/method: four cohorts of hodxotii f mice ( - mice/ cohort) were bled monthly for months to assess for autoab production. peripheral rbcs were stained with anti-complement (c ) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd and ter to assess for the presence of rbc progenitors. statistical analysis between hod otii autoab vs. hod otii autoabvs. hod -otii was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd t cells were not deleted in the thymus of hod otii mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod otii . however, as they aged, - % of hod otii were positive for rbc autoab by months. thereafter, $ % of the autoab mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in of the cohorts, - % of autoab mice were female. hod otii autoab mice also had enlarged spleens compared to hod otii autoaband hod -otii mice ( . g vs. . g and . g, resp., p< . ). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd hi ter inter ) were observed in the spleens of hod otii autoab mice but not in hod otii autoaband hod -otii ( . % vs. . % and . % resp., p< . ). moreover, autoab and c deposition were found ( . - % and - %, resp.) on ter rbcs in all of the hod otii autoab mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b -a a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards , christopher a tormey and krystalyn e hudson* . background/case studies: red blood cell (rbc) alloimmunization occurs in up to % of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c bl/ (b ) mice were treated with pbs, or anti-ly g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at - hours post-transfusion. anti-hod alloantibody generation was assessed days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n ), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n ). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls ( / experiments, p< . ). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l ), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p< . ); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b -a b cxcr pd and ccr expressions characterize responders to rbc immunization benoît vingert* , , , marie tamagne , , , sadaf pakdaman , , , anoosha habibi , , , philippe bierling , , , , , rachid djoudi and france pirenne , , , . efs ile de france, laboratory of excellence gr-ex, imrb u -eq , ap-hp, universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd t cells have a th profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd t cells with a cxcr pd phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr pd hi profile, with a differentiated expression of ccr . ccr is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr and cxcr can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr pd lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr pd cells were compared in groups of transfused sickle cell patients : alloimmunized (n ) and non-alloimmunized patients (n ). the analysis was also performed in non-transfused healthy controls (n ). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr pd hi subpopulation expression was identical between transfused groups and controls. ccr and cxcr expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr expression was very strong independently of the expression of pd . in the aim to determine the help of the circulating cxcr pd cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr pd subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr pd cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr on circulating cxcr pd cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr pd profile and the ccr expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* , ashley bennett , kathryn girard-pierce , connie arthur , amanda mener , patty zerra , christopher a tormey , jeanne hendrickson and sean stowell . emory university, yale-new haven hospital, yale university, emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod kel). to examine the role of cd t cells, pic/kel primed b recipients were cd t cell depleted prior to transfusion. in addition, b recipients were adoptively transferred with cd t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < . ); pic/kel primed recipients transfused with (hod kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < . ) and transfer of cd t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < . ). conclusion: these results demonstrate that cd t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c bl/ ) mice or oti mice, whose cd t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after , , , , and hours and on days - . results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after hours and peaking at hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is - % versus > % in wt recipients at hours (p< . ), whereas transfusion of wt platelets into either oti or wt recipients is approximately % at hours after transfusion. adoptive transfer of oti cd t cells into wt mice recapitulates the effect, with significant mova platelet clearance at hours compared to wt platelet clearance (p< . ). conclusion: this work extends the ability of cd t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c bl/ mouse blood and treated with uv r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv r treated wbc-rich prp, or uv r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c bl/ donor blood. a second transfusion of untreated wbc-rich c bl/ prp was given weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p< . ) or necrotic (p< . ) wbcs, but not those given uv r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c bl/ wbcs were reduced in recipients of either uv r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so ) of venous blood is generally assumed to be around - % as measured from a central venous line. however, a recent investigation of so levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so distribution (mean . % . % [yoshida et al. ; blood transfusion , ] . the present study was undertaken to determine the distribution of so in lr-rcc produced at a medium-size blood center using a novel non-invasive so probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so levels. study design/method: the so from units of lr-rcc were examined on five consecutive days representing % of the collected units during the period at a regional blood center where all the units were processed at room temperature within hours of blood collection. so was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a u ; pendar technologies, cambridge ma). in addition to so , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n ) from human volunteers were stored in as- under normoxic, hyperoxic, or hypoxic conditions for up to days (so ranging from < to > %) prior to uhplc-ms metabolomics analyses in presence of c, n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so carried out non-invasively at a blood center yielded a similar wide distribution as previous study from units of lr-rcc procured and sampled invasively within hours after blood collection [yoshida ibid]. the shape of the so distribution appeared near normal with the mean of . % . %, median . %, range < % to > % and inter-quartile range (iqr) of . %- . %. male donors showed higher so compared to female donors (p< . ). no correlations were observed between so levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so levels ameliorate the energy and oxidative metabolic lesion. lower so levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so levels was observed from lr-rcc manufactured at a blood center using -hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a -fold increase in the absolute lymphocyte count (ko . . x /l vs. wt . . x /l, p . ), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko . . fl vs. wt . . fl, p . ). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd foxp regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd foxp tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd foxp tregs derived exosomes and their functions involved in cd tregs mediated immune-modulation were seldom reported. study design/method: cd t cells were freshly purified from pbmcs, cultured with anti-cd /cd antibody packaged beads and il- , and then polarized with tgf-b and rapamycin into cd foxp treg cells. the harvest cells were co-cultured with cd /cd beads stimulating cd cd effector cells in the transwell plate. the supernatant derived from cd tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd , cd , tsg and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir- , let- b, let- d were measured by qpcr. the precipitated exosomes were further purified by cd immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd treg cells could suppress the proliferation of effector cells with a small decline (p> . ), which means some non-contact factors involved in the cd treg mediated immune modulation. a total number of . . / cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle - nm in diameter ( . . nm by nta). cd and cd were expressed on these background/case studies: regulatory t cells (tregs), containing cd and cd subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd foxp regulatory t cells (ntregs) in inflammation conditions (including instability of foxp , conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd regulatory t cells in inflammation and transfusion. study design/method: human cd tregs were induced with tgf-b and rapamycin from cd t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd tregs when encountering with inflammation were test by foxp expression, th and th cells conversion in inflammations conditions (il tgf-b il il and il tgf-b il b il ) on day , day and day . in vivo, cd tregs were transfused into cia mice and then their survival in mice and foxp express were evaluated to reveal the stability of cd tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd tregs when induced factor tgf-b and rapamycin were removed by testing the foxp expression on day , day and day . results/finding: ex vivo induced human cd treg were foxp ( . . %) and did not secret il a (both in supernatant and % of cells). foxp express in cd tregs were maintained after induced factor tgf-b and rapamycin were removed on day , day and day . in vitro, foxp , il and ifn-c expression has no significant difference when compared with controlled tregs on day , day and day and did not secret il a when encounter with inflammation conditions (il tgf-b il il and il tgf-b il b il ). in vivo, cd treg cells were transfused into cia mice on the peak of disease onset ( days after the first collagen immunization, has inflammation condition in vivo) to test cd tregs survival. cd tregs were found in cia mice foot ( . . %), blood ( . . %) and spleen cells ( . . %) hours after transfusion and their % of foxp were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd tregs are stable in inflammation and transfusion and can maintain foxp expression when induced factor were removed, these make cd treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd /cd /cd expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p s k and its downstreanm proteins, especially the protein s , which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s related protein translation inhibition. xiaoyun fu* , , mikayla anderson and james c zimring , . bloodworksnw research institute, university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day (one day past their expiration). bioactive lipids including common fatty acids, oxylipins, and lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in stored rbc units. for example, arachidonic acid (aa) ranges from . - . mm, linoleic acid (la) ( . - mm), dihomo-c-linolenic acid (dgla) ( . - . mm), eicosapentaenoic acid (epa) ( . - . mm), docosahexaenoic acid (dha) ( . - . mm), and alpha-linolenic acid (ala) ( . - . mm). ten oxylipins including hetes, hodes, and dihomes, and lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of analytes quantified, showed a significant difference in concentration among different blood types by one-way anova testing (fdr< . ). the ab rh blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh ). the fold increase of o rh-/o rh among pufas ranges from . to . , suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna- was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna- targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps ( nm) surrounded by a thick fluorescent silica shell ( nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r rnaprobe . and r dnaprobe . ) with mirna- concentration, down to a -nm limit of detection. hybridization assays in % human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in % human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. . . a . . . . a . . . . a . . total atp,lm/ghb . . a . . . . a . . . . a . . extracellular lactate,mm a extracellular glucose,mm a a a extracellular na ,mm a extracellular k ,mm a a a a p< . , paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n ) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by % ( / , ) compared to the previous year without pathogen inactivation ( / , , p . , chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased ( / , vs / , ). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count ( . x ) than unaffected controls ( . x ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above . x . in vitro quality of single dose amotosalen/uva treated platelets in % plasma/ % pas- after days of storage crystal stanley , marguerite kelher , nero evero , melissa vongoetz , betsy donnelly and anna erickson* . belle bonfils memorial blood center, university of colorado, cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas- to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct ) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas- and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in % plasma/ % pas- , collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, . . platelets in ml, were collected on the trima apheresis platform in % plasma/ % pas- . a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n , were ml (t) and ml (c) with doses of . . (t) and . . (c). all pc were stored under the same conditions and evaluated on day and day for physical/metabolic characteristics. results/finding: on days and all t and c pc had ph c ! . . the dose recovery for t was % %. on day , t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table ) . conclusion: trima pc in % plasma/ % pas- treated with the inter-cept blood system for platelets using the sv set and stored for days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan , , yang yu , li-ping sun , shu-fang wang , rui wang , lei-ying zhang and deqing wang* . the department of blood transfusion, the pla general hospital, the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d , d and d . we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in % final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. levels of k and lactic acid (la) were tested using automatic biochemical analyzer. k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. treated hips-cms with d ssrbcs, d k and cell culture media for h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: d ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d ssrbcs stop beating, but beating patterns restored at h. hips-cms treated with d ssrbcs stop beating, and beating patterns did not restored at h. levels of k and la in ssrbcs changed most obviously. only d k solution made hips-cms stop beating and can restore in h; d k, d k and la solution did not influence the beating pattern in at the end of the treatment for h, hips-cms treated with d ssrbcs show obvious shrinkage. at the end of the treatments for h, cells treated with d k and d ssrbcs both show obvious shrinkage, the shrinkage in d ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. gene expression array results show a total of genes were differentially expressed in d ssrbcs group compared with naive group. there was no consistent separation within the d k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. further study should be applied to signal pathways on ssrbcs induced cytotoxicity. large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as- as compared to sagm. the presence of citrate in as- seems to be necessary to prevent hemolysis of thawed cells. during storage in as- , atp and , -dpg levels rapidly decline. recently developed additive solutions like pag m and as- have shown to better maintain , -dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag c in which the mannitol of pag m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at - c in pag c. study design/method: leukoreduced rcc (n ) in pag c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at - c. on day , rccs were glycerolized using acp (haemonetics v r , braintree, ma) to a final concentration of % (w/v), frozen and stored for at least two weeks at - c. after thawing and deglycerolization using acp , rcc were resuspended in pag c. during storage at - c, stability (hemolysis), atp and , -dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n ) resuspended in or sagm (n ). results/finding: pre-freeze storage in pag c resulted in increased , -dpg levels at day as compared to storage in sagm, resp. . . mmol/g hb and . . mmol/g hb. hemolysis during post-thaw storage in pag c remained below . % for days and was comparable with storage in as- . in sagm, hemolysis remained below . % for days. during the first weeks of post-thaw storage in pag c, both atp and , -dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag c showed significantly higher atp and , -dpg levels compared to as- or sagm. while in sagm and as- , , -dpg levels were undetectable after days post-thaw storage, in pag c, , -dpg levels only decreased to . lmol/g hb after days of storage. conclusion: pre-freeze storage in pag c resulted in increased , -dpg levels. as compared to as- , post-thaw storage in pag c showed comparable hemolysis while atp and , -dpg levels were much better maintained. based on a maximum allowed hemolysis of . % and an atp content of > . mmol/g hb, thawed rcc can be stored at - c for days in pag c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold ( c, c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n - ) in % iso using a trima or in % int using an amicus and stored for days at rt and c. samples were tested on day (baseline, bl), , , and of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at % hct. labeled blood was perfused through microfluidic channels (fluxion) coated with ug/ml type- collagen at s - shear rate. images were acquired every sec for min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p< . . results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day of storage compared to bl (bl: . . %, rt: . . %; p< . ). c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day (p . ) and compared to c-int by day (p< . ). conclusion: our work suggests that c storage of plts collected with a trima ap system in iso for up to days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at c. these results are surprising since both c-int and c-iso have been shown to express similar levels of cd p, pac- , and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table . comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after -week storage between unirradiated and irradiated groups (n ) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone ( mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin ( mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th cells and increase in cd regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th ,th ,tfh and tfr cd subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* , sarah kloss , sara crew and sandra j nance . american red cross, american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from plasma and serum clinical specimens. group contained a single hpa alloantibody specificity with or without hla antibodies (n ). group included specimens with hla antibodies alone and group consisted of patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa- , hpa- , hpa- , hpa- , hpa- , gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is % concordance observed for hpa- a, hpa- b and hpa- b antibodies. the pak-plus assay had difficulty discriminating hpa- b from hpa- a antibody when hpa- a antibody was present ( false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa- a when compared to hpa- b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within % of the cut-off for pakplus and < . adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut overexpression on cell motility. results/findings: fut overexpression increases both ley expression and cell migration, while fut knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac . as tumor promoter, hdac becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n ) and stored at c for up to days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means sem, and paired student's t-tests were used to determine statistical significance (p< . ). results/finding: on day , p-selectin levels were significantly higher in pre than bl (p . ). mirasol treatment caused a significant increase in pac- expression compared to pre (pre: . . %, post: . . %; p . ), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post- samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after day storage at c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day c-stored pas plts followed by incubation ( minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* , arthur p. bode , anne s hale , michael stanton , mark johnson and g. michael fitzpatrick . cellphire, bodevet, inc, cellphire, background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to , , and . % of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control ( -day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to % of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of % and % of the tcpc produced a significant decrease in blood loss. the lcps at % and % tcpc were as effective in mitigating blood loss as -day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to % of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of % and % of the tcpc reduced blood loss. these results suggest a starting dose above . % tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as % tcpc had similar efficacy signals as % tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso c. the study on pcr-ssp technique for the genotyping of cd - del.ac mutation and the genetic polymorphism of cd - del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd (platelet glycoprotein iv, scarb ) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd deficiency in china. cd gene mutation is the main reason that leads to cd deficiency. cd - del.ac (frameshift at aa ) mutation is one of the cd mutations that causes cd deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in pediatric patients who received both doses of the mmr vaccine at and months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are ! % for all mmr components. results/finding: table shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged . years ( . to . years). thirteen patients ( %) were chronically transfused at the time of serology. twenty-three patients ( %) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper , , franklyn cladis , richard saladino , darrell triulzi , barbara a gaines and mark yazer* . university of pittsburgh, children's hospital of pittsburgh of upmc, institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients ! years old and ! kg with evidence of hemorrhagic shock were eligible to receive up to cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (< ) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately months, trauma patients received wb: group o and group a recipients, % male, median (iqr) age was ( . - ) and % blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of ( - ) and % mortality rate. the median (iqr) quantity of wb transfused to group o recipients was . ( . - . ) ml/kg versus . ( - ) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean standard deviation haptoglobin concentrations for non-group o recipients was . . mg/dl on day , . . mg/dl on day , and . . mg/dl on day ; the corresponding haptoglobin concentrations for group o recipients were . . mg/dl, . . mg/dl, and . . mg/dl, respectively (p> . for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n ) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n ). the mean standard deviation platelet volume administered was cc for whole blood recipients versus cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) with anti-human igg only, and a positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a minute c incubation, followed by automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer and the breast milk showed anti-d with a titer between and . the patient had a consistent plasma anti-d titer of . the patient's mother chose to stop breast feeding after weeks, and the patient's hemoglobin was improved at and weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that % of c scd patients from the west indies and west and central africa are partial c and at ( %) risk for alloimmunization to the c antigen through transfusion of c rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce( g, t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce( - )-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of patients with genotype/rh phenotype data available, ( . %) were c antigen positive serologically. the allele frequency of rhce*ce( g, t) was . . in total, ( . %) patients possessed rhce*ce( g, t) in the absence of conventional c gene in trans. of the c antigen positive patients, individuals ( . %) were predicted to be partial c based on four molecular profiles [rhce*ce( g, t)/rhce*ce: ; rhce*ce( g, t)/rh*ce: ; rhce*ce( g, t)/rh*ce( g): ; rhce*ce( g, t)/rh*ce( g, t): ]. in these partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after transfusion exposures ( c-antigen negative units; mean: , range: - ), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce( g, t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* , megan l nguyen , melanie c proctor , david e krysztof , gregory a foster , erin k sash , sandy s dickson , joua yang , jeffrey m linnen , kui gao , jaye p brodsky and susan l stramer . american red cross, grifols diagnostic solutions inc., grifols diagnostic solutions, inc, quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on / / (fl, ga, sc, ms, al). following revised guidance on / / , testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on / / . travel history questions were discontinued on / / . confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of / / , , , donations were tested including , ( %) in , mps. no reactive donations were identified by mp-nat. of the , , id-nat donations, were initial reactive (ir) of which ( %) confirmed positive (cp) by subsequent testing (cp rate of : , ; positive predictive value of %; specificity of . %). five ( %) cp donations were id-nat repeat reactive (rr); ( %) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and in fl, of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from to days prior to donation. two donors with a travel risk reported clinical symptoms; cp donors ( %) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than copies (c)/ml to about Ê c/ml. at the time of writing, the longest period of detection in rbcs was days vs. days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from ir and all rr donors, ranging from to c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of were prepared by diluting nat plasma : and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n ) were sorted into categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm /high vl; igm /low vl. results/finding: of , donations collected april -december , were reactive for zikv rna. igm-index donations had higher vls (mean . x vs . x iu/ml) and higher proportions of simulated mp-detectable results ( % vs %) than igm donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm donations increased (table ) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r / systems lisa lee pate* , phillip c williamson , michael paul busch , susan rossmann , scott jones , ann butcher , john duncan , jean stanley and susan a galel . roche molecular systems, inc., creative testing solutions, blood systems research institute, gulf coast regional blood center -sugar land, qualtex laboratories background/case studies: in february , the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march , and testing of puerto rico donations began on april , . as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august , the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april , -february , using the investigational cobasv r zika for use on the cobas v r / systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a : dilution to simulate mini-pool testing. results/findings: a total of , , blood donations were screened using cobasv r zika. of ir donations, were repeat reactive (rr), non-rr and had no repeat testing. of the rr donations, were positive by altnat; of these were igm positive. all altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the rr donors that were not igm positive on index, enrolled in follow-up and all seroconverted. of non-rr donations, were altnat negative and is pending supplemental testing. / donors were igm positive on index. donors were igm negative on index; / enrolled in follow-up; remained igm negative and was gm inconclusive. of donations without repeat testing results, met criteria for positive ( was altnat positive, igm negative and altnat negative, igm positive). donation is pending additional testing. altogether, / ir donations met the criteria for true positive on the index donation. / ( %) true positive donations were reactive when retested in a simulated minipool. / were igm positive. conclusion: . % of the , , donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* , marc germain , gilles delage , maria esther lopes and yves gr egoire . hemorio, hemaquebec, h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia ( ) , and in brazil ( / ), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since % of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january st , through november, th , , from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: days, with % of the values lower than days); % of infected donors with symptoms lasting days; . donation/donor/year for wb and . for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x ( -proportion of refused donors) x ( proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain : , , for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from donations). the residual volume of mp plasma, . - . ml, is routinely discarded. beginning in april each blood center saved $ mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april - ) mp were combined into pools of donations; thereafter mp were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and % confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity ( % limit of detection < copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first months of samples is complete for , mp, comprised of , donations collected from april to october , . a total of pools were positive, with detected between april-june . the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over . % of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over . % of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in , zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* , sze sze chua , mars stone , michael paul busch and ai leen ang . health sciences authority, blood services group, blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on august . the numbers rose rapidly to cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since october . zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using blinded frozen samples consisting of replicates of half log dilutions of the who international standard for zikv and replicates of negative controls prepared by bsri. probit analysis was performed to determine the % and % limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a member blinded zikv reference panel from the usa-fda. results/finding: a total of , donations were screened from october to march , with false positive case and zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of . x copies/ml. zika igm was negative in the index donation sample but present in the -day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be . copies/ml at % lod and . copies/ml at % lod. the procleix zikv assay detected rna in out of patient samples and provided . % agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of in , donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types , and express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to reactivity on initial gel testing. if genotyping demonstrated weak d types , or , the intent was to manage the patient as rhd-positive. if weak d types , or were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in to weeks. results/finding: rhdgenotyping was performed on patient samples over months. of these patient samples, ( %) were weak d types or . the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types and patients have not received transfusions at this institution since they were genotyped. four of obstetric weak d types and patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this month study period serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using fda approved anti-d reagents. when reactivity with all reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of months we tested rhd-negative blood donors. there were ( . %) partial-d, weak d ( . %), and ( . %) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs - _ deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these donors showed that rhd-negative recipients received rbcs from of these donors. five of these recipients underwent antibody screening after an average follow-up period of months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which % of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d " with reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october to march , we performed routine d typing (neo, immucor) on obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of using at least antibody. solid phase and manual testing used the series and series reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh ) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all samples. two of ( . %) were d with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs - c, rs ; ivs c, rs ; and ivs a, rs ). two ( . %) were d and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four ( . %) patients had rhd alleles with known potential to make anti-d (rhd*dol , rhd*dar . , and with weak d type . ). one had weak d type , which has uncertain susceptibility to alloimmunization and one was weak d type , which has not yet been associated with anti-d. interestingly, two ( . %) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type is a variant of the rhd protein that comprises an amino acid substitution located in the th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c. g>c which is the first nucleotide of the exon of the rhd gene and thus could be implicated in exon skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v (agilent) and the nextseq platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of patients previously characterized by beadchip technology. interestingly, out of carry the c. - c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last patients, one has been previously characterized as rhd weak type carrying the c. g>c (p.gly ala). independently, sanger sequencing on unrelated rhd weak type samples pinpoint to a linkage disequilibrium between c. g>c (exac, maf . ) and the c. - c>t (exac, maf . ). in silico analysis of both mutation located close to the splice acceptor site of the exon does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon , mutant rhd c. g>c, mutant rhd c. - c>t and double rhd mutants c. g>c plus c. - c>t, we showed no influence on skipping of exon due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type between ala (transmembrane helix ) and val (transmembrane helix ) hampering membrane insertion. conclusion: the c. - c>t variation is always associated in cis with the missense mutation c. g>c on the allele rhd weak type . the c. - c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type red blood cells is due to the substitution of alanine at amino acid position to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has transmembrane (tm) and tilted ureapore a-helices, a long extracellular connector segment, and cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p. . we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and - aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, ) . results/finding: seven snmvs located within amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all at the exofacial ends (p.a t, p.w r, p.v d) are jk-weak; the two jkneg exceptions p.g e and p.g e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v m, p.e k, p.v i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the jk-neg variants are within aa (p. -p. ) of jk a/b at p. . none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n s and p.s p are adjacent to p. f and p. l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc a gene, which encodes the urea transporter ut-b . the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in . in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc a gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within aa from tm a-helix end v i, a t, w r, w r, g e, g e, v d* cytoplasmic n-terminal v m, g s, e k, l p in membrane tm and urea-pore a-helices r w, r q, g d, i t, a v, l r, a t ‡, a a §, l f, n s, s p, t m / * second nucleotide variant in this allele is synonymous (p.p p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a mbp region in q . - . with an lod score of . . using deep sequencing, we identified a potential deleterious mutation in the znf gene, which deletes bp resulting in loss of an entire zing finger domain. the identical del -znf mutation is present in all affected individuals, and is absent from all controls tested (n> ). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf del . none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc a gene, is a urea transporter that has been associated with renal function, we found that people with the znf del in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf , prevalent in southern spain due to a founder mutation, leads to ut-b dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-( -ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis( -ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at days and year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the pools included group a, group o and group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than - c within hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day (pool), day , and year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day , day , and year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp . ppm; mehp . ppm; deht . ppm; and meht . ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than % of its initial value. plasma stored in deht bags had an average plasticizer content % lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at g to separate rbcs from platelet-rich plasma (prp). prp was diluted -fold in pipes-saline with . mm pge and centrifuged at g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of - % and , - , platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted : (spdp %) with plasma from a patient with type vw disease (t vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of s - for seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was % (spdp/ffp > . ). results/finding: six batches of spdp/ffp were evaluated using subjects. there was no statistical difference between the spdp/ffp pairs (p . ). the mean ratio of spdp/ffp was . with a % ci of . - . . comparing spdp vs. spdp %, there was no difference (median ratio . , range: . - . ) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was % greater than in samples reconstituted with ffp. the lower limit of the th % ci is a difference of %, which is less than the a priori determined margin of noninferiority of %. even with % dilution with t vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* , qiyong peter liu , grantham c. peltier , ryan c. carney , ashley s. taylor , colby s. mcintosh , james a. bynum and andrew p cap . u.s. army institute of surgical research, velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: ( ) ffp; ( ) ffp with mm glycine; ( ) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; ( ) spdp pretreated with glycine-hcl ( mm); and ( ) spdp pretreated with glycine-hcl:glycine ( mm: mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood ( % hct with platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < . ). fibrin polymerization density was slightly diminished in rspdp vs. ffp ( . vs. . o.d., p < . ), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < . ). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < . ) and an additional twofold in pretreated spdps vs. rspdp (p < . ). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < . ). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples ( . % surface coverage vs. . - . %, p < . ). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* , anita tuip-de boer , ruqayyah almizraq , jason p. acker , philip j. norris , jennifer a muszynski and nicole juffermans . academic medical center, university of alberta, canadian blood services, blood systems research institute, nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from donors (blood type a or b). supernatants were prepared after - (fresh) and - days of storage (stored) for measurement of thrombin generation and ev analysis. a type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to % stretch using a cellstretcher. control cells were not stretched. after hours, il- and il- production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il- and il- production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p< . ) . incubation of stretched cells with stored wbf products resulted in higher il- production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by different methods from individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after - days (fresh) and - days (expiry). monocytes were co-cultured in media plus % rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table ) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin- production was higher after exposure to fresh wbf ( % control, p . ) or wbd at expiry ( % control, p . ). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* , alessandro tocchio , anita howell , kaushik sridhar , jason p. acker and utkan demirci . stanford university, canadian blood services, centre for innovation, background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of - g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at , , , , and days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from volunteers with four different age and sex categories (male, - years; male, > years; female, - years; female, > years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young ( . g/ml) and older female donors ( . g/ml) (p < . ). moreover, rbcs from young males ( . g/ml) were significantly less dense compared to rbcs profiled from older female donors ( . g/ml) (p < . ). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il- (pg/ml) il- (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p< . ). substantial correlations were also found between orp and free hemoglobin (p< . ) and orp and free heme (p< . ). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections vs (p< . ). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than days compared to rbcs stored for days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of to days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to days storage duration -reference group), medium age (at least rbc of - days storage), and oldest (at least rbc greater than days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every days, and b) a finer partition using cut-points every days. results/finding: , patients receiving , rbcs were included in the analysis. exposure to rbcs stored for more than days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for days or less) after adjusting for several fixed and time-dependent potential confounders (hr . ; % ci: . , . ; p . ). exposure to blood stored for at most - days yielded a similar hazard ratio (hr . ; % ci: . , . ; p . ). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than days compared to exclusive exposure to rbcs stored days or less was not significant (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . similar findings were obtained with partitioning exposure data into day intervals where exposure to rbcs stored for - days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for - days (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . conclusion: individuals exposed to rbcs stored for more than days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* , cynthia walser , tatsuro yoshida , andrew dunham and pedro cabrales . university of california san diego, new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o saturation < %) stored rbcs, or anaerobic/hypercapnic (o saturation < % and pco (@ c) $ mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as- after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either ) conventional; ) anaerobic; or ) anaerobic/hypercapnic conditions. rats ( - g) were hemorrhaged to % of blood volume, held in hypovolemia for minutes, and resuscitated to recover blood pressure to % pre-hemorrhage with prbc stored for either or weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. ( %) neg patient showed short term response and ( %) patients showed progressive disease. at the neg group standard eval ( %) patient showed response and ( %) had progressive disease. ( %) neg patient had long term response compared to ( %) pos patients. at the pos short term eval ( %) patients showed response and ( %) patients had progressive disease. at the pos group standard eval, ( %) patients showed response and ( %) patients had progressive disease. overall, ( %) pos patients responded compared to ( %) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd neutralizing substance could play a role in treatment response. alternatively, reduced cd expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a -hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate % replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of . m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* , laurie sutor , , germ an leparc , marjorie doty and william crews . carter bloodcare, ut southwestern medical center, oneblood background/case studies: anti-cd drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with . m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and . m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual ( th edition). each of the plasma aliquots was further separated into aliquots and stored at - c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. ( ) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than with the untreated or dtt-treated cells during the study. conclusion: long term storage of . m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque- ), pooled, suspended in cryopreservation media ( % dmso; : ) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips ( c, % co , h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o ) rbcs sensitized with either anti-d (positive control), anti-scianna- (sc ) or anti-anwj or lipopolysaccharide stimulated for h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/ monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed . % viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il- b, il- , il- , mip-a (p < . ), mip-b and gro (p < . ) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc -and anwj-sensitised rbcs resulted in a pi of . % and . . % respectively vs anti-d sensitized rbcs (pi: . %). a weak ( ) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi> %). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in patients, involving antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all patients had - positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients and typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c by hea precise-type. ega-treated rbcs gave reactions with the same anti-c reagent. patient rbcs gave variable reactivity (vw- ) with bio-rad seraclone and ortho bioclone anti-c. patient rbcs gave reactivity with all anti-c reagents when incubated for the maximum incubation time allowed. patient rbcs were jk(a ) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients and tested s with bio-rad seraclone anti-s ( - ), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat rbcs but not all manufacturers include reagent limitations regarding testing of dat rbcs. we describe cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and cases with false positive tests with anti-s (n ) and anti-jk a (n ) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with - positive dat and supports testing to dissociate igg from rbcs strongly dat before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/ opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r : prob(ab|al-loexp), so that prob(ab) prob(ab|alloexp)*prob(alloexp) and r ; rewriting prob(alloexp) prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar months before ( / - / ) and months after ( / - / ) was selected; for state b, a similar months before ( / - / ) and months after ( / - / ) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (< months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a -and -fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from to (state a) and to , (state b), which annualized, represents a potential gain of , (state a) and , (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from - , there were donors identified who had changed their gender from their birth gender; female donors changed their gender to male and male donors changed their gender to female. there were ( %) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to . ml/kg or % of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the % limit. variable volume scales [vvs] can be programmed to vary unit volume (up to ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by ml at ebvs < . l in donors ! yo, but increase by - ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the mos. before a mo. phased implementation of the vvs, and the subsequent mos. multivariable analysis [mva] by -mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods & , continued during impl and post-impl periods & , returning to the baseline rate in post-impl periods & (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods & . the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of . ml during post-impl periods & from the temporally matched baseline & pre-impl period . conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* and yves gr egoire . hema-quebec, h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, . the vaccine is produced with live and attenuated yfv, which can circulate for at least weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received , blood donors candidates; from those, , were accepted as a blood donor, after medical interview. the deferral rate was . %. at the same period of the year , there were , prospective donors, and blood donations. the deferral rate was . %. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, , additional donors, compared to same dates. that represents a . % increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from . % in to . % in . conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of ! . x is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: , apheresis collections from centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of . x for single (s), . x for double (d), and . x for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or % plasma) assuming i) a minimum dose (allowing for production loss) of . x for s and . x for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as %) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc rbc plt/p plt plt/rbc/p plt/rbc plt plt/rbc plt/p #donations citrate exposure (mls) - study design/method: a randomized ( : ), placebo-controlled, single blind, subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into cohorts, receiving increasing doses, ranging from / , - / of the lowest effective dose found in the above rabbit model. cohorts and received the / th dose, but cohort received two / th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for hrs post infusion and followed for up to days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of aes: were treatment emergent (teae), of which were treatment-related ( thrombosomes and control). all teaes were mild or moderate in severity. in cohorts and , / thrombosomes subjects had treatment related adverse events. one cohort subject developed an upper respiratory infection and elevated wbcs within hours post infusion, which resolved by hours, and an elevated d-dimer at hours post infusion, which resolved by day . this subject also had an elevation of prothrombin fragment at baseline, which increased post transfusion and peaked at hours with resolution by day . one cohort subject developed non-specific t-wave changes at and hours following her nd infusion that resolved by day without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort subject developed an igg platelet autoantibody on days - , which was undetectable on days - ; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days - , and negative on days - . background/case studies: cryopreservation of platelets (plts) could extend the shelf life from - days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with % dmso and stored at c. after thawing, the unit was reconstituted in thawed ffp spiked with either lm puromycin (pm) or nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after , and hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x- containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by -fold during hour storage. immunoblot analyses of the plts showed a -and -fold increase in pm incorporation after and hours of storage, respectively. massspectrometry revealed unique proteins that were synthesized after hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac , rap and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac , rap and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in , the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r - cooler with units of ffp, units of rbcs, and unit of whole blood. three to ml of platelets were collected via syringe from each unit at min (before storage in cooler or refrigerator) and after . , , , , and hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac- binding) were measured by coulter counter, channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p< . deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c -a h molecular sieving: beyond genotyping ghazala hashmi , reinhard klemm and michael seul* , . biomolecular analytics, immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi http://bit.ly/ ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, * * samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for sensitized sickle cell anemia ("sca") patients (tb in cas-tro , http://bit.ly/ oplxhr, excluding le and e(variant) and assuming request per patient), presenting with up to allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only = plate holding * candidate units from actual black donors, followed by profiling of samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for of requests ( . %), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining pooled samples produced additional assignments to a second set of requests, for a total of assignments from only wells. in another scenario, sieving of a full plate of * samples, produced $ assignments for two successive batches of requests from sca patients, a yield exceeding . x. sieving alone typically fills - % of requests of moderate complexity ( ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez , monica kalvelage , ghazala hashmi* and michael seul . biomolecular analytics, lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou http://bit. ly/ ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including at the rhce locus) that encode mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag " (e.g. e ,c ,e ,c ) to specific combinations of "ag " (e.g. c e k fya and c e jsa ) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the c samples, that were also v and vs and, among the e samples, that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select specific "ee" pools of which were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b ( pools), co a|b ( ) and others. conclusion: molecularsieving of a single -well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c , e and jsa . these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam (landsteiner-wiener) and ackr (duffy). for longer genes, such as abo of > kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of > kb each was used for all physically confirmed ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v . . was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least clades representing clusters of to alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors ) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons - , and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors . a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos (b galnt ) cells, and cell-surface expression of fors antigen was immunologically monitored with a monoclonal anti-fors antibody. results/findings: we found that met thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon or of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met thr/ ser or exon / deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt genes is reminiscent of common ancestral origin of alpha , -gal(nac) transferase genes. the finding that at can synthesize fors implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of , cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period / / - / / . abo genotyping targeting specific snps for groups a, a , b, o , and o and, if needed, gene sequencing was conducted in cases with indeterminate results, and in cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two ( . %) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in % the predicted abo phenotype was a rh neg (table a ). the predominant donor race was caucasian ( %). four cbu with abo discrepancy were also evaluated by genotyping (table b) . in of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r / systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: , fresh and , frozen edta plasma samples from american red cross donors, collected from february - , were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of , valid results, a total of donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a -year old male in indiana, a -year old male in california, and a -year old female in kentucky. all donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna ( iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as a, the california donation genotype b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was % ( % exact ci: . % to %). conclusion: based on the confirmed-positive donations of , tested, the hev prevalence was . % ( % exact ci: . % to . %) with a detection rate of : , ( % ci, : - : , ). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than % of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using % confidence intervals. this analysis contains data from / / - / / . results/findings: among , , donations reported ( . % from firsttime and . % from repeat donors), there were respectively , and cp results for hbv, hcv and hiv with corresponding rates of . , . and . per , (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of : , : and . : for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv . vs . ; hcv . vs . ; hiv . vs . ). in general, higher rates for all markers were seen among minority donors, those in the - -year age group (also - year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when -month periods were compared. conclusion: data from major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* , melanie c proctor , deanna self , monique portugal , adrian gurrola , laura tonnetti , sonia bakkour , cheryl lobo , michael paul busch , susan l stramer and jeffrey m linnen . grifols diagnostic solutions inc., american red cross, blood systems research institute, new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening , unlinked whole blood donations collected from august th to april th in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of . results/finding: the procleix babesia assay detected all four babesia species with a % lod ranging from . - . copies/ml. the preliminary % lod in parasites/ml ranged from . - . p/ml for b. microti (n ), from . - . p/ml for b. duncani (n ), and from . - . p/ml for b. divergens (n ). of the , donations screened, initial reactive and confirmed positive donations were identified for specificity of . % ( %ci: . - . %). of the confirmed positive specimens, were reactive by both ifa and pcr, by ifa only and by pcr only. all confirmed positive samples were reactive in lysate pools of . donors of reactive donations resided in ct ( ), nj ( ), nh ( ) and me ( ) for an overall incidence of : , , and : , in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* , whitney r steele , ed p notari , james haynes , roger y dodd and susan l stramer . american red cross, american red cross (retired) background/case studies: from - , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from - . study design/methods: prevalence was calculated in -year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of . , . and . days for hbv, hcv and hiv, respectively. linear regressions were calculated with p< . (*) as significant. results/findings: from / / - / / , there were more than million donations from , , donors ( . % female, % first-time (ft), . % caucasian). there were significant decreases in donation prevalence for hbv and hcv (p . and . ), but no significant decrease in hiv during the years (see table for f and r values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p . and . ). prevalent ft donors were significantly more likely to be male ( . % -hbv, . % -hcv, . % -hiv; p< . ). incidence for all agents declined (significant only for hbv; p . ). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last -year period ( in - vs. in - ) . hcv incident donors in - were more likely to be male ( . % vs . % in - , p< . ) and were younger ( . % vs. . % in - < years, p . ). overall, incident donors were more likely to be caucasian males (p< . ). rrs for all agents decreased over time with rrs in - of in , , ; in , , ; and in , , for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the -year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in , mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as- rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with lm amustaline, and incubated for hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, > . log , or > . log pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was > . log , or > . log pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts- inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts activity of < % and high inhibitor ( . - ). mean age of cohort . years (range - ). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean . x /l, range - x /l) and low a-ipc (mean . x /l, range . - . x /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab mg/m ( patients) and cyclophosphamide mg/m (one patient). tpe continued until platelet count reached x /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of . days [range - days]) when they achieved a three-fold increase in a-ipc from baseline (mean . x /l, range . - . x /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean . x /l, range - x /l) and a-ipc (mean . x /l, range - . x /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of . days (range - days) mean platelet count was . x /l (range x /l) and mean a-ipc . x /l (range . - . x /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of . days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* , michelle n stram , joan sevcik , alesia kaplan , and joseph e. kiss , . department of pathology, university of pittsburgh medical center, blood systems inc., university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within - hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january , to november , was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts- activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the ttp patients identified, were excluded due to missing temporal data for important variables. the majority ( %) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients ( %) had a prior history of ttp and % had severe adamts deficiency on admission. the median time from tpe request to initiation was . hours (interquartile range: . - . hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table ) . treatment was not started within an -hour window in patients; the median time to cv access was significantly longer in these patients ( . vs . hours, p< . ). two of these patients had a prior history of ttp and only four patients had severe adamts- deficiency. the majority (more than %) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table ) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus - hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi- a transfusion vol. supplement s hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, through january, , we performed cytapheresis (cy) treatments (txs) for pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. pts ( %) had cml-at and received leukapheresis (lp) txs; pts ( %) had et and received thrombocytapheresis (tc) txs. cml-at pts presented with median wbc x /l (range - x /l), of which % had blast percent > % or blast count > x /l. median age was years ( - years); % were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. % of cml-at pts had no sxs of lks; % pts had sxs of either cns or pulm lks ( sxs), and % pts had sxs of both cns and pulm lks ( sxs). et pts presented with median platelet (plt) count of: x /l ( - x /l)and % pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was years ( - years); % pts were male. results/finding: all pts received a course of cy tx with following objectives: ) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and ) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) < x /l for cml-at pts, and plt ct < x /l for symptomatic et pts and < x /l for asymptomatic et pts. cml-at pts received median of lp txs (mean . txs/pt; range - txs). et pts underwent median of tc txs (mean . txs/pt; - txs). outcomes were evaluated by percentage of pts who: ) reached wbc (or plt ct) tx goal, and ) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved > % reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, % pts improved, % pts stabilized; and % pts worsened. in et cohort, % improved, % stabilized, and % were unchanged. for cml-at pts, median final wbc ct x /l (range - x /l); % pts received ind chemo. for et pts, median final plt ct x /l ( - x /l); % pts had resolution of thrombotic a transfusion vol. supplement s symptoms. % of cml-at pts and % of et pts expired within - days after course of cy tx. of expired pts, pts had both blast crisis and sxs of cns/ pulm lks; pt had intracranial hemorrhage or cva; and pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median - txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used % albumin or % albumin- % normal saline ( / ) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered ( % albumin vs / ), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where % albumin was used versus those that used / . covariates included were fluid types, age and gender. odds ratios (or) and % confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < . . results/finding: during the study period, procedures were documented for subjects ( % female), age range - years, of which , ( . %) received / . the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with % albumin had a significantly lower risk of having either event than by using / , [p . , or (ci): . ( . , . )] , and also had a significantly lower risk of causing hypotension [p . , or (ci): . ( . , . )] in addition to a lower risk of causing citrate toxicity [p . , or (ci): . ( . , . )]. age had a significant effect on having a hypotensive event [p . , or (ci): . ( . , . )] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a year period and compared it to published literature. study design/method: we conducted a -year retrospective study of ta procedures performed and aes were classified according to criteria described in table . during the study period, ta were performed using cobe spectra (software versions . and . ) and since the spectra optia apheresis system (version . ). literature search was conducted for data published on aes associated with ta. four studies from us and non-us studies (canada, europe and japan) were analyzed. trend for ae rates from - was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was . % ( of , procedures) during year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher ( . %, p< . ) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho . , p . ) over the years and significant down trend of moderate and severe aes with a spearman rho of - . (p . ) and - . (p . ) respectively. there were no fatalities during the study period. majority of aes were grade i ( %) and grade ii ( %): / ( . %) procedures were not completed due to aes. comparison of aes [ . % ( / , )] to both european [ . % (n , , / , ) ] and other us studies [ . % (n , / , )] showed a statistically significant difference (p< . ). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table ) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether platelet donors with a donation activity of up to platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* , jordy jurgens , jacoline buchner-doeven , joris roelofs , philip spinella , jennifer a muszynski , carel goslings and nicole juffermans . academic medical center, washington university school of medicine, nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells ( days old) and platelets ( days old) by washing. plasma was filtered through a . um filter. rats ($ grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $ % of their estimated blood volume, which was calculated to be ml/kg. hemorrhage continued until a mean arterial pressure of mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of ml/kg of blood products in a : : ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april , which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and -day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january and july (n ). because of missing data on patient characteristics, patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january to march , n ) and after (april to july , n ) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of baseline variable, generating pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common ( . % vs . %, p< . ) and ventilation time was longer ( h vs h, p . ) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group ( ml vs ml, p . ; and ml vs ml, p< . , respectively). however, -day mortality was not statistically different between the groups ( . % vs . %, p . ). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april , www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over , joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy . length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of -factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: -factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in . marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p . ) and ptt (p . ) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p . ) and plasma (p . ) after off-label use was significantly greater than on-label use. cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were . times (p . ) more with cell saver or anh, and . (p . ) times more with cpb. post-pcc thromboses were identified in cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to days. the transfusion service medical director reviews the case and gives final approval. we observed only patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight ( ) patients were in-patients continuously until delivery. five patients were discharged prior to delivery- moved to another state, was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was days (range - ). six ( ) patients delivered within days of approval. after approval, the mean number of additional specimens per patient was . (range, - ). no patient required transfusion prior to delivery. five patients received transfusion of at least rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only patients delivering within days of approval for extended specimens, patients avoided collection of at least specimen each, and patients avoided at least collections each. since new antibodies are not detectable for at least days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* , , jan m. binnekade , benjamin nota , pieter r tuinman , kirsten van de groep , olaf l cremer , janneke horn , marcus j schultz , robin van bruggen and nicole p juffermans . academic medical center, sanquin research and landsteiner laboratory, vu university medical center, university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in tertiary icus in the netherlands comparing patients who developed ai during icu stay with control groups: non-anemic patients with sepsis, non-anemic patients without sepsis, and patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron ( . vs. . mmol/l, p< . ) and transferrin saturation ( vs. %, p< . ), and low ferritin ( vs. mg/l, p< . ). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately . x cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to %, whilst leaving % of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than % of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately % of the cd fraction post separation and freeze thaw (table ) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table ) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd cd ) and early projenitor cells expressing oct and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: . routine recovery of the wcf at levels higher than current methods, independent of volume. . higher percentage recoveries of all cell types tested than can be achieved with existing methods. . markedly higher post-thaw recovery of viable nucleated cells than any current methodology. . almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving > . x lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd cell target of . x /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf plerixafor (g pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< . considered significant. results/finding: no alc and alc collections occurred among the patients. fenwal amicus was used for % of the no alc and % of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was hodgkin's and non-hodgkin's lymphoma (no alc); hodgkin's and non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc . , lymph . x /l) than alc (wbc . , lymph . x /l). equivalent whole blood (corrected for ac) was processed for no alc ( . l) and alc ( . l). for alc group, extra collections beyond cd target were: days: %, day: %, days: %, days: %, and days: %. significantly more patients were mobilized with g pl in no alc group (n ) than alc group (n ) and collections in alc group had mobilization discontinued after cd cell target reached. there was no significant difference in g ( . x lymph) compared to g pl mobilized collections ( . x lymph); both were significantly higher than the collections where mobilization had been discontinued ( . x lymph). days to wbc engraftment ( . no alc vs . alc) and platelet engraftment ( . no alc vs . alc) were not significantly different. median number of collections for no alc ( ) and alc ( ) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the . x lymph/kg or even the . x lymph/kg targets. implementation of a lymph target increased patients obtaining . x lymph/kg from % to %. only % had < . x lymph/ kg. discontinuation of mobilization once cd cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* , and nicolas pineault , . canadian blood services, university ottawa, canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost hours at room temperature (rt) as long as units are cryopreserved by -hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n ) were split with one half processed immediately (baseline - hours) and the second after hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd cells and cd cell (n ). primary nsg mice were transplanted with a ucb cell dose that contained a total of , annexinv neg viable cd cells. the latter was done to avoid any bias towards one group or another. short term platelets ( vs. hplt/ml, p . ) and leucocytes ( . % vs. . % hcd , p< . ) engraftment at -weeks were significantly reduced in stored mice vs. baseline (n ), and similar results were observed long-term at -weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated months post-transplant. strikingly, the frequency of human cd bm cells was -fold greater in baseline vs. stored mice (p< . , n ). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured -weeks post-transplants were reduced by % in unit , and by % in unit . conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($ mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to %, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of sec). total lymphocyte recovery was % and monocyte concentration was reduced %. furthermore, in a two-pass process platelets were reduced by %. in a -fold parallel system we tested rbc separation from plasma and achieved % separation at ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from clinical protocols from - . an infusion reaction was defined as any symptom from the time of nk cell infusion up to hours afterwards. a severe reaction was defined as any symptom with grade or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r . . . two major endpoints of interest were: ) infusion reaction with any symptom and ) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of nk cell infusions. there were ( %) patients with an infusion reaction of any symptom and there were ( %) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . ). infusion rate (ml/min/kg) was also similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . respectively). incubation of nk cell product overnight in il- vs il- had similar reaction rates for those with any symptom ( % had reaction with il- , % had reaction with il- , p . ) and those with severe reaction ( % had severe reaction with il- , % had severe reaction with il- , p . ). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median . x ) versus those without (median . x , p . ). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan , maryanne c herzig* , barbara a christy , james a. bynum and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at - . mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at - , cells/ well and cultured in well plates for - h in their respective medias. on day , mscs were washed, resuspended in pbmc media and incubated with or without , freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, - lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by h, with > fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was . %. inter-assay variation of msc preps run under identical conditions was . %. inhibition of pbmc proliferation was graded from - % over the range msc concentrations therefore an ec of msc cell number resulting in % suppression of pbmc could be determined for each msc prep. this ec however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute % or more of the us blood supply. differences between donors - years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged - were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the / academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml) were estimated for , , and - yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of donors contributed donations. donors were evenly split by gender, % were ft donors, and % were - yo. ft and rpt - yo donors had on average lower ferritin values at enrollment (p<. ), and a greater percentage were iron-depleted than donors - yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged - have sharply higher risk for iron depletion than donors - yo. odds for lf were to times greater in the younger donors, and for ais were -to fold higher. preliminary statistical models indicate yo donors may have greater risk for lf than or yo by to percentage points, controlling for other factors (p . ). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in - yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on / / by a large blood collector. testing was performed on successful - y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < ng/ml in females (f) and < ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations ( months for f and months for m) and counseled to take - mg of elemental iron daily for days. for m and f, a ferritin < ng/ml indicated absent iron stores (ais) and < ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! ng/ml in f and ! ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior months. an appreciable number of donors with no rbc donations in the prior months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* , joan williams , michelle humphries , nancy haubert , ben reynolds , michael phillips , randall spizman , ralph r vassallo , hany kamel , sally caglioti , german leparc , and phillip c williamson . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. study design/methods: over , serum samples from donors aged , and years were analyzed for ferritin levels using the beckman coulter au instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among , adolescents (ages - ; . % female) who donated blood from to . study design/method: abnormal risk factor levels were defined as hba c ! . %, sbp/dbp ! / mm hg and tc ! mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, , ( . %) adolescents had at least one abnormal risk factor ( . % of males, . % of females). of these, , adolescents had isolated abnormal risk factors, and , adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as ! . g/dl for men and ! . g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from blood services worldwide and complete data were available for blood services. deferral percentages for low hb varied from . % to . % among male donors and . % to . % among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with % lower hb deferral rates in men ( % confidence interval [ci] % to %) and % lower rates in women ( %ci % to %). iron supplementation was associated to % lower hb deferral rates among women ( %ci % to %) but there was no evidence of such an effect among men (p . ). each one-week increase in minimum donation intervals resulted in % lower hb deferral rates among women ( %ci % to %) but not among men (p . ). at the % level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: . % of all candidates for wb donation were deferred in continental france in . deferral was significantly more frequent in women ( . %) than in men ( . %), due to anemia in . % of deferred women and . % of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from to weeks. analysis (table) identified main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the previous years. conclusion: the main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, days stored or days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, ng/kg). blood was sampled every hours up to hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from . e (iqr . e - . e ) /ml in the fresh product to . e (iqr . e - . e /ml; p< . ) in the stored product (p < . ), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: cases of taco and matched controls were enrolled from , transfused patients who received , blood components from may until july . taco incidence was case per patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation ( % vs. %; p < . ), experienced longer intensive care ( vs. days; p . ) and hospital length of stay following transfusion ( vs. days; p< . ), and had higher mortality ( % vs. %; p . ). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from to days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july and to routinely extend ap outdate to day since february . this study reports a month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july -january , ap underwent rt on day . day and units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day had a second rt performed. from february -january , ap underwent rt on day with routine outdate extension to days by performing a second rt on day and a third rt on day , as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type ) or repeat rt positive with negative confirmatory culture (type ). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july , , ap were entered into inventory. of these, , ( %) were transfused prior to rt testing. the remaining ( %) underwent rt on day or day . of these ( . %) were rt positive ( type fp, returned to inventory; type fp, discarded), leaving a total available inventory of units tested by rt. of these, ( % of original inventory) were transfused before the end of day and the remaining ( % of original inventory) reached a day outdate. a total of ( % of original inventory) were transfused on day or day . of these, underwent a second rt on day ( rt positives; fp type one and fp type ) and underwent a third rt on day (no positive results). a total of ( % of original inventory) outdated on day . of these, underwent a second rt on day (no positive results). conclusion: to date we have performed rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted % to only %. a total of ap have been tested twice by rt ( on day and ; on day and ) with ( . %) positive results, both of which were deemed fp by repeat testing or culture. a total of units have been tested times (day , day and day ) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with to million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in . contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between - , parasites/ml. each parasite concentration in wb was tested x . an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at c for up to days; platelets were stored at c (rt) under agitation for days and plasma was frozen at - c. aliquots for culture were removed weekly from rbcs, daily from platelets and after days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at c for detection of live parasites for up to weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at c, rbcs from all units spiked with , parasites/ml were positive for up to days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with parasites/ml were positive for up to days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with , and parasites/ml were positive up to days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at c for up to weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* , marion lanteri and larry corash . cerus corporation, scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom ( - ), french ( - , swiss ( - ), and belgium( - hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately . million dlvbc-screened were issued with a day outdate after release into inventory days after collection, and $ . million amotosalen/uva-treated pc were released into inventory on day or , with a to day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored > . million conventional, non-dlvbc-screened pc and recorded str and fatalities. concurrently, zero definite and possible str were reported with , amotosalen/uva-treated pc, significantly fewer than with conventional pc (table ) ( . str per million vs. . per million, p< . ). one definite, possible, undetermined/indeterminate non-fatal str and contaminated "near miss" pc were reported with . million dlvbc-screened pc between and , for a reduced falsenegative rate compared with the prior five years ( . str per million vs. . per million, p < . ). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october for all platelets received at our institution. at time of receipt at the blood bank (day post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at c for three days. results/finding: a total of / , ( . %) platelet products were successfully cultured ( / [ . %] and / [ . %] in october and march respectively). over the -month period, two true positive cultures were obtained (incidence of in platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us . per product tested. the cost per averted case was $us , . conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january and december . the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the -year study period, a total of transfusions reactions were reported, of which were bcptr ( . % of transfusion reactions). of the bcptr, ( %) were associated with apheresis platelets, ( %) with red blood cells, and ( %) with plasma. recipient diagnoses spanned hematologic/oncology (n ), renal (n ), cardiac (n ), autoimmune (n ), and obstetrics (n ). an organism was identified in both the blood product and recipient in ( %) cases; in ( %) cases an organism was grown in the blood product but not the recipient; and in ( %) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in of the cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever ( %), chills ( %), nausea and vomiting ( %), pain ( %) and dyspnea ( %). blood pressure (bp) decreased in %, increased in %; % of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie , jenna lebedev , linda kapp , xiaohong wang , meghan delaney , lay see er and james c zimring* . bloodworksnw research institute, bloodworks nw, university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg -igg ), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all known variants. study design/methods: the heavy and light chain variable regions of an anti-k monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg , igg , and igg had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table ). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg or for any of the monoclonal ahgs tested. monoclonal anti-igg had a blindspot for igg - , due to the shorter hinge region on igg - . no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* , yves dominique pastore and maryse st-louis . chu sainte-justine, hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since , our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of , scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september , ( %) patients had been transfused and had antibodies with known blood group antigen specificity: anti-c, anti-e ( ), anti-hrb, anti-fya, anti-jka, anti-jkb ( ), anti-s, anti-m, anti-sc , anti-leb ( ). seventeen patients ( . %) were either d or partial d. rhce results showed that patients expressed a normal c antigen and expressed partial c. as for e antigen, had a normal antigen, bore a partial antigen and were weakly expressed. fy(a b ) phenotype was found in ( %) patients. a total of genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos , emilia sippert , mayra dorigan de macedo , sheila fatima perecin menegati and lilian castilho* , . hemocentro unicamp, university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa- a, il b- t cytokine polymorphisms, rhag g>a and hla-drb * alleles may predict a good responder phenotype (sippert et al, transfusion ) and that rhag a and hla-drb* alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included non-alloimmunized patients with scd, homozygous for hbs, receiving a range of - rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa- g>a, il b- c>t) and the rhag g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among non-alloimmunized patients, were homozygous or compound heterozygous for rh variant alleles. from those, had rhag a and/or hla-drb* alleles and at least one cytokine polymorphism (tnfa- a or ilb - t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and were confirmed by sequencing. the third sample was found to be rhce*cevs. ,rhce*cebi on sequencing (predicted phenotype v ,vs ). the samples were typed as v (or ce s ) and vs (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[ g]in samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising % of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies ( % of patients) bound young and old rbcs with no apparent prejudice. band- is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band- is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band- to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band- aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band- tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing type i and type ii patients, we found that type i is characterized by percollv r fractions (similar to healthy storage-matched controls) but increased band- tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by - percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band- tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band- suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* , burak bahar , jeanne hendrickson , krystalyn e hudson and christopher a tormey . yale-new haven hospital, yale university, background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam abstract algorithm, e value x - , word size , gap costs: existence exten-sion ). search results were restricted to bacteria and fungi, with a selective threshold of > % identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b - . , r . & p . ); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of alloimmunized patients reviewed, were culture-positive. of these, % of the anti-c/c group ( of patients) and % of the anti-k group ( of patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from - . %. overall, . % ( of patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of > % sequence identity. while . % ( of ) patients reviewed had positive cultures for klebsiella species, . % of these ( of patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed tegs performed on patients undergoing cv surgeries at our institution from jan to dec , . no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at minutes (min) all within reference range), "hypocoagulable" (r> min, k> min, a< degrees, ma< mm) and "hypercoagulable" (r< min, k< min, a> degrees, ma> mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of tegs analyzed, patients ( %) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients ( % vs. %, p . ). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused ( % vs. %, p . ). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused ( % versus %, p . ). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from liver transplants, performed from to in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, olts were performed. a total of patients was submitted to cs. the median age was years (range - yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in , % of the patients. the average meld score was , , and it was slightly higher in the cs group ( , vs , , p< , ) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was ml and mean reinfused blood volume was ml. allogeneic blood transfusion was required in , % patients in the cs group, compared to , % patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group ( , units vs , units, p< , background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july -december was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp ( and products, respectively) however, obp wastage occurred more frequently in the month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations ( versus products). this is skewed by one month when products were wasted due to expiration of product on the floor. cooler-related issues ( ) and products dwelling too long out of a controlled environment ( ) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were . %, . %, and . %, respectively, with a total exsanguination protocol waste rate of . %. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p . ). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d ) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d- (table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* , edward smith , thomas brown , foeks jeremy , metcalf suzanne , james johnson , peter davis , karafa sw badjie and abba zubair . department of laboratory medicine and pathology, transfusion medicine, mayo clinic, department of anesthesia, mayo clinic background/case studies: our institution performs an average of solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused o( ) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the o( ) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in % of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a y.o. female patient should not have received o( ) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with admissions during the hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight ( kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o( ) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o( ) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately , surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from - %, whereas transfusion rates for vaginal and robotic pfd surgeries range from . - . % and . - . %, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately % of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may -may in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p< . . results/finding: we identified patients for analysis, of whom ( . %) had a preoperative t&s ordered. two ( . %) of these patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine ( . %) of the patients required a second abo/rh specimen per hospital protocol; ( . %) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table ) . no abo/rh discrepancies were identified. one patient received unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution ( . % vs. %, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n ) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of . g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs- i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), units were subjected to the volume reduction while recording the time needed to process all units. this was performed twice for a total of units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of % (range %- %). in units between and days (n ), the estimated mean residual k was . meq (range . to . ). in the two mock mtp trials, the time to complete the procedure was approximately minutes and we estimate an additional - minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal ( ) and caesarian ( ) births. uc collections were divided into segments to test conditions. segment explants were placed on . % gelatin-coated gridded tissue culture plates ( explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the remaining tissue segments were soaked in (ab/am) saline solution for hr and hrs at c, respectively. tissue segments were frozen in cryo bags with a proprietary % dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process or times the total blood volume (bv) of the patient, up to a maximum of liters (l) per procedure, to obtain peripheral blood cd stem cells. as a consequence, a patient often would need to spend hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd pre-count and cd yield, normalized per liter of blood processed, was derived utilizing the patient's cd pre-count, the patient's weight in kilograms (kg), and the target cd dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd stem cells. the initial equation was modified to add an additional % to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in patients, representing both allogeneic and autologous donors, the average blood volume processed was . l. the range was . l - . l. the target dose was achieved in all patients. our previous practice for these patients would have required, assuming a standard bv procedure, processing an average of up to l per patient, with a range of - l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd yield. the result was a high correlation between these two ratios (r . ), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r . , confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses ( - ) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june and march . patients (n ) evaluated were diagnosed with malignant lymphoma (n ), multiple myeloma (n ) and primary amyloidosis (n ) and were mobilized according to standard protocols. collection cd cellularity target was established ! x e /kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by > years old, previous fludarabine, lenalidomide, or bendamustine treatments or ! previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v . . results/finding: the media (range) general collection parameters were: cd (day ) . /ml ( . - . /ml), blood volume processed ml ( - ml) and . ( - . ) exchanged volemias. seventeen patients were considered bad mobilizers, needed plerixafor and had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p . ]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n ), fever (n ) and flu syndrome; all grade ]. two patients could not undergo hematopoietic stem cell transplantation due low cd cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd ) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with % fetal bovine serum. bmsc and amsc at passage - were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses ( . /ml, /ml and . /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par ) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within min and hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: seconds, versus low, medium and high doses of amsc ( , , and seconds), and bmsc ( , , . seconds), p< . ), clot formation time (cft, p< . ) and increased alpha angle (p< . ) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par . no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* , neil bagamasbad , reynold dilag , melissa nasser , nicole bauer , jennifer wheeler and mary berg . department of pathology, university of colorado -anschutz medical campus, department of medicine, division of hematology, university of colorado hospital, scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from collection procedures using the mnc protocol and collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd )-positive (cd ) throughput, cd collection efficiency (ce%), platelet loss a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included and allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd throughput was significantly higher in the cmnc group than the mnc group. the cd ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient # , originally typed as an a , had bone marrow donor and cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient # is now typing as type o. patient # was originally typed as a and received a bone marrow transplant from a type b donor. patient # is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient # and patient # indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with % fetal bovine serum under either normoxia ( % o ) or hypoxia ( . % o ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd /cd and cd were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc ( . /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by %, but depressed the growth of amsc by % at day in comparison to normoxia. both bmsc and amsc equally expressed cd and cd but not cd under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: (control), versus (bmsc), and (amsc) seconds) by natem. hypoxia also significantly shortened ct ( (bmsc), (amsc) seconds, p< . as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd cells. study design/method: cryopreserved cd cells from healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef -alpha-yfp lentivirus ( . % concentration) and media (x-vivo- , human serum albumin(hsa), ng/ml each of cytokines (scf, tpo and flt -l) over days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of % dmso, % pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd cd -cd ra -cd cd f cells) phenotyping and cfu assays were done following first thaw (pt ), post-transduction (ptxn) and second cryopreservation-thaw (pt ). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd %, cfus were similar before and after pt . hscs ranged from to cells/ cd cells in the pt -tr arm compared to a range of to / cd cells after pt . viability, % cd and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt (table) . conclusion: dec of mpb human cd cells decreases tnc recovery, but has minimal effects on cd cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd % in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of % hydroxyethyl starch, % human serum albumin and % dmso at final concentration. pbsc were cryopreserved by direct immersion on - c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of consecutive days of neutrophil count > . x /l and platelet count > x /l after days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd was below x cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd on the day of the collection versus collected cd per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd was calculated. final laboratory count of cd per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss software. results/findings: among patients collecting hpc for autologous transplantation, , % needed only one day of hpc harvesting, while , % needed two days and , % needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was - , %. after comparing predicted values with cd collected in the final product, we found a very strong correlation of . (p< . ) for patients and a strong correlation . for healthy donors (p< . ). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; ! % cd , cd , cd and % cd , cd , cd , cd , hla-dr) study design/method: umbilical cord tissue (n ) was washed, blood vessels removed, cut into . - mm pieces, and washed twice in saline. fresh tissue was immersed in . % saline for same day culture, while frozen tissue was cryopreserved for at least hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a cm tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures > % confluence. all cells were tested on an msc flow panel at passage just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of days (fresh . , frozen . ), and days (total) for the msc's to reach passage (fresh . , frozen . ). all cells were ready for flow analysis in approximately weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p . ), or their growth rates (p > . for all). flow cytometry showed average ! % for positive markers and % negative markers. there was no statistical difference between fresh and frozen flow result (p > . ). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of up to years ( to ) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with % concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a c water bath and . ml aliquots were diluted at a : proportion with % human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using -aad marker through flow cytometric analysis. results/finding: ucb storage period was . years (mean) and cell recovery was . % (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p . ). post-thaw cell viability of . % (mean) showed no statistically significant correlation with unit storage period (p . ). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln vapor in a dmso-based cryoprotectant for yrs. ( . . ; n ). units were rapidly thawed and rinsed in dpbs, then pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a x grid pattern in msc-supportive medium and incubated for days, after which the tissue was discarded and media exchanged. cells were isolated on the th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a % success rate. cells were positive for the msc markers cd , cd , and cd ( . . %, . . %, and . . %, respectively) and negative for the hematopoietic markers cd / ( . . %). passage and passage doubling times were . . days and . . days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical a transfusion vol. supplement s research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd yield prediction algorithm ines bojanic* , nelly besson , ivana vidovic and branka golubic cepulic . department of transfusion medicine and transplantation biology, university hospital centre zagreb, terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version ) in adult and pediatric lvl. a prediction algorithm for cd cell yield was also tested. study design/method: we evaluated retrospectively lvl performed in adult patients, and lvl in pediatric patients treated in uhc zagreb from march till september . mobilization regimen combined chemotherapy and filgrastim; poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio : ). in patients weighting kg (n ), a rbc prime was performed. cd , lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd cell count and cd cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd values to real cd yield. results are presented as median (iqr). results/finding: in both groups, cd , ly and mo ces were high. target cd dose was successfully reached in procedure in ( , %)adults and in ( . %) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in ( . %) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd cells and cd cells collected/ blood volume was observed in both groups (r . and . in adults and children respectively, p< . ) suggesting cd yield could be predicted based on precd cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd yield and observed cd yield (r . and . in adults and children respectively, p< . ) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of . ( . - . )l of blood in adult procedures, and . ( . - . )l in pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd , ly and mo ce were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* , aniko barta , arpad batai , zoltan csukly , zita farkas , laszlo gopcsa , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) times per case weekly at a dose of million cells/kg. clinical response was assessed days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all patients had received cycles of msctreatment ( dose per cycle). the median age was years old ( - ) with a male/female ratio of : . distribution of the original malignancies (n): acute myeloid leukemia: ; acute lymphoblastic leukemia: ; myelofibrosis: ; myelodysplastic syndrome: ; multiple myeloma: ; t-cell lymphoma: . nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median rd day ( - ). the involved organs were skin ( ), gut ( ), skin and gut combined ( ) and even lung in cases. the median time of msc's first infusion was days after the stem cell transplantation (hsct) and ( - ) days after the first episode of gvhd. of the cycles of msc-treatment led to complete remission ( . %) and resulted inpartial remission ( . %). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with % overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is million cd cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the million cells/kg goal. the ideal minimum cd cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from patients to evaluate the predictive value of the cd cells/ml level. data was collected over months from every patient who underwent a stem cell collection. four patients were allogenic donors and were autologous donors. the patients' weight, diagnosis, and pre-procedure cd cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd cells collected were recorded. the collection efficiency and the cd cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd cells/ml and post-procedure cd cells/kg (r . ). any patient who had a pre-procedure cd cells/ml count of or greater had a collection of at least million cells/kg. any patient who had a pre-procedure cd cells/ml count of or less collected less than conclusion: the pre-procedure cd cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd cells/kg level. to confidently know that a patient will be able to produce the desired million cells/kg, a pre-procedure cd cells/ml count of at least should be obtained. for any patient with a count below , they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between and cd cells/ml should be conducted. heidi elmoazzen , antonio giulivi , michael halpenny* , lisa martin , donna perron , chris bredeson , lin yang , locksley mcgann , paul birch and jason p. acker . canadian blood services, ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso ( % final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd , viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in % dmso and . % hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of . days for anc with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current % dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using % dmso and . % hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from % to %. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is . mg/kg, therefore patients weighing > kg would require a second vial, thus doubling the drug cost. in we implemented a policy of capping plerixafor at mg for patients weighing > kg. this retrospective study compares the mobilization of patients > kg who received capped doses ( ) ( ) ( ) ( ) , with historical control patients ( - ) who received full or uncapped doses. study design/method: patients weighing > kg with crcl > ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of and consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd /cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at mg for patients > kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (< yrs) have a higher pre-apheresis %cd level than any of the other groups, reaching statistical significance when comparing the %cd pre-apheresis between the youngest group (< yrs) and the oldest group (> yrs). hispanic donors show statistically similar %cd pre-apheresis levels over all age groups. moreover, the hispanic older age group (> yrs) had a statistically higher %cd pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last years was analyzed to determine the th percentile, median and th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc ( x cell / ml) and three low wbc ( x cells / ml) concentrations, each at high ( ml), low ( ml) and median ( ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax (pericell protocol, cs. . kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and -aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting -aad viability of % [range - ]% and a hematocrit of % [ - ] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was % [ - ] with a % [- - ] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of [ , ] % and a change in -aad viability of [ , ] % from the input product. the method was found to have a cv of . %. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating a transfusion clinical assessment. in the first phase, cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd -aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to hours post thaw. the second in vivo phase included use of an infusion pump for consecutive autologous patients, with comparison of infusion and transplant outcomes to previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than % within hour, while cd cell viability remained stable up to hours post thaw. small aggregates appeared after hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were . . and . . , respectively (p-value . ). platelet days to engraftment for pump and drip were . . and . . , respectively (p-value . ). infusion rates were slightly higher for the pump group. for control patients, required transfer of products to syringes due to slow infusion rate and others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the zikv ineligible cbus was: caucasian %, asian %, black/aa %, and multi-race %. racial distribution of all clinical cbu donors was caucasian %, asian %, black/aa %, and multi-race %, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: % of all ineligible cbus and % of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete , -diphosphoglycerate ( , -dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring , -dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n ), which were stored in sagm for days, to act as untreated controls. the remainder of each unit ($ ml) underwent treatment with the rejuvenation solution ( ml, minutes at o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros . pc were centrifuged at g in a sorvall rc c centrifuge (sorvall, usa) for min. the combination cryoprotectant dmso dextran (cryosure dex , germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at degrees c (barkey plasmatherm) for min. cpc osmolality was measured with an osmomat osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru u ). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso dextran (cryosure dex ) , as a cryoprotectant, to obtain a final concentration of % dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m were instrumental in automating that phase. pc to be frozen had an osmolality of no less than mosm/l. prp and ppp were frozen at a cooling rate of - c/min and stored at - in the chest freezer for up to months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru u ). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to mosm/l. freeze-thaw recovery of platelets was % or more of the original population. defrosted pc were stored at - with continuous gentle stirring from a helmer platelet agitator for no longer than hours before transfusion. it took no more than min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day and day evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day . a total of lrap units were tested over a -month period: were cultured and rapid tested on day ; were rapid tested on day . the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were true negatives (tn) and false positive (fp) on day when tested by bact/alert, with tns on day . bactx testing results showed tns on day and tns on day . testing using the pgd kit showed tns on day ; and tns and fps on day . fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in % plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day and day during the night shift to be accomplished without additional staffing and allows to extend outdate to day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for year jos lorinser , pieter f van der meer , hans van der heiden and dirk de korte* . department of product and process development, sanquin blood bank, mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers ( ml) is unknown. if these products can be stored at - c it will be feasible to store this product in -star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at - c or <- to - c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <- c for - months and controlled thawing, six different sera were used to fill a large number of mini ( ll) containers, which were refrozen and stored at either - c or <- c. during storage at months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <- c. growth factors tested were pdgf-aa&ab/bb, tgf-ß / / , vegf, a transfusion vol. supplement s egf, fgf . the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß were the most abundant growth factors, on average , resp. ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average ng/ml. tgf-ß , egf and vegf were detected at relatively low values, resp. ng/ml, . ng/ml and . ng/ml. average levels of fgf and tgf-ß were close to detection limit (< . ng/ml). the controls stored at <- c showed for all growth factors close to % of the initial values in samples at t (moment of filling mini containers). for serum stored at <- c for up to months, most factors showed less than % decrease, except for pdgf-aa and tgf-ß , showing % resp. % lower values. for serum stored at - c the values for tgf-ß , egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß showed a decrease of resp. , and %. conclusion: human serum eye drops can be stored in the new micro dose device at - c ( -star household freezers) or <- c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at - c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond year. ruqayyah almizraq* , heather inglis , phillip norris , , jennifer a muszynski , nicole juffermans , jelena holovati and jason p. acker , . university of alberta, blood systems research institute, university of california, san francisco, nationwide children's hospital, academic medical center, canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n per method). residual platelets and white blood cells (wbcs) were measured on day using flow cytometer (fc). on storage day and , number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day , apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p< . , wbd p< . ) and wbf (vs: apheresis p< . , wbd p< . ) methods. while rcf units yielded the lowest count of platelet-evs (cd a ) on day and , the highest number of platelet-evs were in apheresis (day ) and in wbd (day ). similarly, there was significant difference among methods in the number of wbc-evs (cd , cd , cd , cd , cd b ) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day vs day in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< nm) was greater than large evs (! nm) in all of the products on day and , and the highest level of evs < nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p< . ). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at centigrade maryanne c herzig* , crystal lafleur , chriselda g fedyk , sherrill j. slichter and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research, university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least weeks of storage at c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for , , , or days after collection. units were stored for days without agitation. units stored for , or days were agitated during storage with a model hybridization incubator at c set for end over end rotation at - rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at - c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai- ) as another fibrinolytic measure; and complement activation markers c a, c d, c a and c b- . data was analyzed by one way repeated measure anova. results/finding: only % of the platelets were recovered in units stored for days without agitation. these levels did not meet fda requirements of . x platelets per wb unit. subsequently, wb was agitated and platelet recovery was - %. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t (day of collection) and t , , , or measurements. significant elevations of pai- and scd l indicate activation of platelets and inhibition of fibrinolysis (p< . ). activated complement peptides c a, c a, and c d were all elevated over time (p< . ) while sc d- was not. however, only c a and c d levels at t were above normal reference ranges at . and . times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at c for - days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc d- reported, wb showed elevation of c a, a and c d and not sc d- . complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* and christian todd . cerus corporation, community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly . x . x . x . x rcf . x . x . x . x apheresis . x . x . x . x wbd . x . x . x . x platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following collection targets: . x in ml, . x in ml, . x in ml, and . x in ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of . x or . x was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: % of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was . . conclusion: it is possible to treat % of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward % while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* , andrey skripchenko , fei xu , ying li , stephen j wagner , pamela h whitley and jaroslav g vostal . fda/cber/ obrr/dbcd/lch, american red cross holland laboratory, american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature ( - o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts ( hrs ct: hr o c [tc]). autologous apheresis plts stored for -days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n ) and the same non-labeled plts were also infused into mice (n ). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts < % were considered background. results/finding: the mean recoveries of infused plts were . . % for rt, . . % for tc and . . % for ct in humans. in mice, mean recoveries of the same plts were . . % for rt, . . % for ct and . . for ct (mean sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $ % and ct was $ % of rt. in mice tc was $ % and ct was $ % of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to % for rt plts. human tc plts had % auc while ct plts had % auc compared to rt plts in humans. in comparison, the same tc plts had % auc and ct plts had % auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are . and . , respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* and geeta paranjape , . coastal bend blood center, carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g with the compomaster net software for data management. implementation was planned for a november go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g s and compomaster in june . training and validations were successfully completed and a full launch occurred mid-march . device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december . validation was completed and signed off in march of . manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g system. data points were collected from units bi and units ai. results/finding: upon initial implementation, staff training and use, the compomat g was found to be easy. plt weight spread was reduced from an average of gm to an average of gm. actual plt weights were reduced from an average of gm to gm, resulting in an average increase in recovered plasma of . ml per unit. plt count on average increased from a count of to ( /mm ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by . % after implementation of the compomat g and our plt concentrations increased on average by %. we were able to consistently produce a smaller volume plt (average gm), which gave us . ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell , angela hill , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: abo/rh matched lr sagm rccs were pooled and split to produce large ( ml) and small ( ml) rccs. the rccs were stored to d and glycerolized manually by mixing ml of glycerol with the rcc in a ml freezing bag. units were frozen at - c for ! h before being removed from frozen storage and thawed in a c water bath. large rccs and small rccs were deglycerolized using the organization's current procedure on the cobe cell processor prior to re-suspension in . % saline, . % dextrose. the remaining rccs were transferred into a l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of %, and deglycerolized in a ml centrifuge bowl on the acp- with re-suspension in as- . rbc quality was tested at h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p . , acp : p . ) and lower cell recovery (cobe: p . , acp : p< . ) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe had higher hemolysis (p< . ) and supernatant potassium (p . ) than did small volume rccs. large cobe rccs had higher hematocrits (p . ), hemoglobin (p . ), and recovery (p . ) than did large acp- rccs. however, all cobe rccs had higher (p< . ) hemolysis ( . . %) levels than did acp- rccs ( . . %). cobe rccs failed to meet regulatory hemolysis standards of . %. conclusion: addition of a ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as- and storage for h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp- cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using . % saline and centrifugation and the semi-automated washing method (sam) using the cobe blood cell processor. study design/method: in this study, units of single donor platelets were evaluated ( washed using the mm and washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas . results/finding: table shows that the average platelet recovery for the sam ( %) was significantly higher compared to the mm ( %). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took - minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas- ) for input platelet doses of . to . platelets in to ml of to % plasma and - % pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas- containing doses of . to . platelets in a volume of to ml. study design/methods: apheresis pcs (amicus v r ) were collected in % plasma and % pas- . one study was performed at the nominal dose ( . - . x platelets), volume ( - ml) in % pas/ % plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition ( . - . x platelets in - ml) using either single or pooled donations. input pcs (n ) were treated with the intercept ts set by the end of day post collection; the incubation time in the compound adsorption device (cad) container ranged from to hours and the intercept treated pcs were stored in containers (n ). day and post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas- treated in the intercept ts set demonstrated acceptable in vitro function (table ). all intercept treated pcs had ph( c) ! . . platelet dose and volume recovery post-treatment ranged from % to % and % to %, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing % plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n ). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n ). numerous in-vitro quality markers (plt concentration, atp, po , pco , ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days , , and for apheresis pcs, and on days , , and for wb-derived pcs. two flow cytometry assays were used to evaluate cd p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion vol. supplement s results/finding: platelet recovery was % and % for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells ( % (test), vs. . % . (ctl) on day ) and a higher rate of cd p expression than control pc units ( % (test), vs. % (ctl)) on day ). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* , lorraine blagg , christi e marshall , herman woodson , sean erony , krishna patel and eric gehrie . the johns hopkins hospital, johns hopkins hospital transfusion medicine dept, johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day . as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day . the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of lrap were tested. lraps initially tested negative by bactx, while lraps initially tested positive by bactx. all initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was . %. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only platelet units. the expected rate of bacterial contamination of platelets is less than per units. the . % initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as- and cp d/as- rbc alan d. gray* , matt landrigan , pamela whitley , michael wellington , sherrie sawyer , shalene hanley , emily rondeau , louise herschel , neeta rugg , patricia a.r. brunker , shawnagay nestheide , jose cancelas-perez , larry dumont and zbigniew m. szczepiorkowski . and , -dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for > hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood ( - ml) was collected and processed at sites into leukocyte-reduced rbc (a total of n cpd/ as- and n cp d/as- ). ml of rejuvenation solution (citra labs) was added to each rbc on day (d- ), incubated for minutes with agitation at c water bath (helmer dh ), washed (haemonetics acp ), and stored in as- at - oc for days (d- through d- ). in vitro recovery (%) was calculated and hemolysis, atp, and , -dpg were determined on day , d- , d- after rejuvenation and washing (postrjv), d- , d- , d- , and d- . all units were cultured on d- postrjv and on d- , and then concentrated by centrifugation on d- . results/finding: in vitro rbc recoveries were . % and . % (as- and as- , respectively) and no bacterial growth was observed. hemolysis on d- was maintained < % in / ( %) as- units and / ( . %) as- units. all as- and as- units ( %) had hemolysis < % following concentration by centrifugation. morphology score was reduced to % (as- ) and % (as- ) by d- , restored after rejuvenation ( %, %, respectively) and maintained through d- (> %). atp was restored and maintained above fresh levels after rejuvenation. , -dpg was restored above fresh levels and was maintained ! % of fresh levels through d- . all values were significantly different compared to d- except as noted (p< . , paired ttest) ( table ) . conclusion: rejuvenation of stored rbc restores atp and , -dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d- when compared to nonrejuvenated rbc on d- . this study is funded by zimmer biomet. storage > hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante , jason p. acker* , and jelena holovati . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during -day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and , -dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome rejuvesol-treated (l r). the prbcs were incubated for h at c with hepes-nacl (sham), liposomes (dopc:chol, : mol%, mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and , -dpg at day hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control ( . . %): l ( . . %, p . ), r ( . . %, p . ), l r ( . . %, p . ). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r ( . . , p . ) and l r ( . . , p . ) treatments compared to s ( . . ) but not l ( . . , p . ). rbc rigidity (kei) increased in all treatments compared to sham ( . . ): l ( . . , p . ), r ( . . , p . ) and r l ( . . , p . ). aggregation amplitude was significantly increased by r treatment only ( . . au vs. . . au, p . ). atp levels were significantly higher in all treatments compared to sham ( . . mmol/g hb): l ( . . mmol/g hb, p . ), r ( . . mmol/g hb, p . ), l r ( . . mmol/g hb, p . ). the levels of , -dpg were no longer detectable in s and l treatments at day . the combined treatment was comparable to r ( . . mmol/g hb vs. . . mmol/g hb, p . ). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer , eric ducas , patricia landry , nathalie dussault , jacques bernier , danny brouard* and anne maltais . h ema-qu ebec, institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the -ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < c) of one to six -ml whole blood units (wbu) within h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between c and c for h under extreme external conditions (- c to c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for h at - c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, -ml wb bags were filled with ml saline . % at t c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (- c) and summer ( c) conditions. shipping boxes were filled with either one or six bags (n ). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under c in . . h and maintain their internal temperature between c and c for h with final values ranging between . c and . c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the c threshold value in . . h and the bags' internal temperatures were within the acceptable range for h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than - c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* , christopher c. c silliman , beth shaz , marguerite kelher and claudia s. cohn . new york blood center, bonfils blood center, department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either % donor plasma (n ) or % pas- / % donor plasma (n ). within hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd ligand (scd l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c ( / products) compared to plasma platelets ( / products); however, the hla-antibody screen-positive supernatants of pas- a transfusion vol. supplement s abstract c platelets had fewer hla specificities ( specificities) compared to those of the plasma platelets ( specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd l levels were increased in the supernatant of pas-c compared to plasma platelets (table ) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd l and not bioactive lipids. although scd l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below x wbc in us and x wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n ) and leukoreduced (lr) rbc units (n ) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of wbc/ul. the spiked samples of , . , , , and wbc/ul were prepared from the source sample of wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations ( , . , , , , wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples ( , , , wbc/ul). tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of samples (plt and rbc) were run on both analyzers, repeated for days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt . , slope . ), (r-rbc . , slope . ). the %diff-plt at , , wbc/ul were . , . and , respectively. the %diff-rbc at , , wbc/ul were . , . and . , respectively. the average total testing time was similar on both instruments; min for the facsvia and min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated % ( of min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* and mickey koh . department of laboratory medicine and pathology, university of minnesota, st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of completed and partially completed surveys were received. results/findings: responses came from countries, but the majority of responses came from the united states (us). of the respondents, % reported aprp use in their hospital. aprp was used predominantly for outpatients, though > % of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by - mds; however, hospitals had > mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures ( %); however, respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, ) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n ) under blood bank conditions at day (d ), at day (d ), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from . - . % at d to . - . % at d . rejuvenation markedly reduced this storage-induced spherocytic shift ( . - . %) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell , angela hill , tracey turner , april xu , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units ( ) than in rcf units ( ), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as- additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: small rcf ( - ml) and large wbf ( - ml) rccs were stored for d before being glycerolized and frozen at - c for ! h. large rccs whose red cell mass exceeded the capacity of the ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a c water bath, deglycerolized and re-suspended in as- . rccs were stored d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p< . ) hematocrit, specific gravity, hemoglobin per unit, supernatant k and na concentration, deformability (ei max ), and higher (p< . ) recovery than did large wbf units. no significant differences in hemolysis, atp, , -dpg, p , rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table ) , however of large wbf units had rbc recoveries < % due to pre-glycerolization volume reduction, and of the small rcf units had hemoglobin values < g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above % and the hemoglobin failure rate would be below % of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. . . . . . . . . elongation index ( pa) . . . . . . . . this study is funded by zimmer biomet. (hasan ) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two ( ) rbc units (leukocyte-reduced), cpd/as- or cp d/as- , on day , day , and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o /g hb) and total releasable oxygen (tro) of the unit (ml o ). orc was determined by assessing the change in % o saturation from mm hg po (e.g., lung) to mm hg po (e.g., venous blood) multiplied by . ml o /g hb (li ). a simulated baseline pretransfusion vo of ml o /min was estimated using the day orc and assuming a g/dl transfusion trigger with a cardiac output of l/min and l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day restored orc and tro to levels greater than day ( table ) . orc of the rejuvenated unit was . . times and . . times greater than rbc on day and day , respectively (p< . ). vo increased after a simulated single unit transfusion of rbc (day , day , and pw) by . %, . %, and . % over the pre transfusion vo , respectively (p< . ). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release . times the volume of o compared to standard, untreated rbcs stored for days. inferior oxygen delivery to tissues (vo max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for days vs days which seem dependent on genetic variability and storage time (bennett-guerrero ). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* , jay srinivasan , gustaaf de ridder , alan d. gray , matt landrigan , keaton charles stoner , angela crabtree , jessica poisson and ian welsby . duke university school of medicine, duke health pathology, citra labs, a zimmer biomet company, zimmer biomet, duke university, department of pathology, durham veterans affairs medical center, duke university hospital, duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted , -diphosphoglycerate ( , -dpg). the loss of , -dpg increases the oxygen affinity of hemoglobin, resulting in lower p (partial pressure of oxygen at % hemoglobin saturation). decreased p may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and , -dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a , leukoreduced prbc stored in as- were obtained from our local blood center. after days of storage, units were divided into separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution ( ml) was added to the cr group, and all groups were then stored for another days at - c. on day of storage, the sr group was incubated for hour at c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps ) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a . . results/finding: significant differences in p were noticed between all groups (table ) . ei, ps , and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p ) seen over days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc ( ml) units stored in as- , obtained from a regional blood donor center at expiration ( days), were passed by gravity through sorbent-devices containing ml of multifunctional polymer bead, at a flow rate of ml/min. supernatants were analyzed for k removal as well as free hb, antibodies and cytokines ( -plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k ] from . . to . . meq/l; equivalent to an . % reduction. free hb was reduced by . % from . . to . . mg/ml. antibodies, specifically igg, iga, and igm decreased from . . to . . mg/ml ( . %), . . to . . mg/ml ( . %), and . . to . . mg/ml ( . %), respectively. inflammatory cytokines were significantly reduced, specifically: ip- from . . to . . pg/ml ( . %), mip- b from . . to . . pg/ml ( . %), and pdgf from . . to . pg/ml ( . %). filtration had no significant impact on cell surface markers of rbc viability (< . % decrease) or sensitivity to osmotic changes. values listed represent mean sem (p < . for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency ( . %) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in apheresis and whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and , -dpg profiles to fresh levels. the objective was to compare % hemoglobin-oxygen saturation (p ) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was . . %. hemolysis (%) was similar on day before and after dry-air incubation with the rejuvenation solution ( . . % vs . . %). percent hemolysis (%) decreased after washing ( . . %) and was maintained below < % for all units during storage for hr ( . . %). average atp and , -dpg were restored above the average fresh values. the morphology score decreased $ % by day , which was restored to near fresh values following rejuvenation and washing and storage hr ( . % and . %, respectively). rbc oxygen affinity, as assessed by p , was restored above fresh values. all values were significantly different compared to day (p< . , paired t-test) ( table ) . conclusion: rbc morphology was restored to near fresh and average atp, , -dpg, and p were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and , -dpg were maintained during storage hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d tetrahydrocannabinol (thc) and -oh-d -tetrahydrocannabinol ( -oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and -oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and -oh-thc to produce samples for lc-ms/ms analysis. lc used a c column. post-column detection by ms/ms used positive ion electrospray with q :q ion pairs of m/z . : . (internal standard (is), d -thc), m/ z . : . (thc), and m/z . : . ( -oh-thc). quantitative results for thc and -oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from - ng/ ml for both thc and -oh-thc. limits of quantitation, defined as standard deviations above background, were . ng/ml for thc and ng/ml for -oh-thc. results/finding: a total of donor plasma samples were tested for thc and -oh-thc. no samples tested positive for either thc or -oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a % probability of one or more positives at a prevalence of . % positive samples, and a % probability of one or more positives at a prevalence of . % positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than % among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than hours for post-exposure detection of thc and/or -oh-thc in plasma. conclusion: testing of donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than %. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* , ruqayyah almizraq , daniel millar and jason p. acker . university of british columbia, university of alberta, lightintegra technology inc., canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days , , and of storage. one rcf rcc was tested on days , , , , and and six ml aliquots were stored in parallel and tested on days , , and . all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days , and (p< . ) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the aliquots were consistent at each time point but statistically higher than in the original rcc on and after day of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow % screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n ) and paired ffp aliquots were stored for - days at - c and - c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! % levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c esterase and alpha -proteniase inhibitors). the level of factor xiii in odp was slightly lower, about % of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i ii and ddimer) and complement (c a and c a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion vol. supplement s abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to % in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to minutes. in addition, donations with collection times between and minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from - minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n ) were prepared from one - minutes bc and ml of autologous plasma in a ml pvc-dehp container. as a reference, spc from donations with collection times of < minutes were prepared (n ). in addition, pc were prepared from bc, of which at least bc were from - minutes donations (n ). after pooling of the bc, ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for days at c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume ( vs. ml) and platelet content ( vs. x ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day , ph( c): . . vs . . , other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day and/or in / pc (possibly because sometimes ab incompatibility was accepted). on day , plt showed low cd p expression ( . . %) and phosphatidylserine exposure (annexin v binding, . . %). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from - minutes whole blood donations had a normal composition and showed good in vitro quality during day storage. to substantiate that the exclusion of - minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* , jessie miller , ranee marie wannarka-farlinger , sandra bryant , scott a hammel , sherry stern and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as- rbcs, irradiated and non-irradiated, were selected for the study. the units varied in age, ranging from to days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< . ) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p . ). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after days of storage pei lun karen lim* , erma sofia sumardi , isamar eduardo ancheta , susan lim , christina yip , lip kun tan and shir ying lee . national university hospital singapore, national university hospital, national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within hours of processing and stored at - c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for days and kept at to c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than iu/dl. study design/method: randomly selected units of ffp (n ) were measured for fviii concentration based on clotting assay (stav r -deficient a transfusion vol. supplement s viii diagnostica stago). fviii levels were measured at five time points: prefreezing, , , and hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at to c for minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration ( hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at to c for days for subsequent testing. results/finding: results obtained were listed in table . units to were not tested for fviii at post thaw- hour due to operational issues. the overall fviii concentration decreased at an average of % from pre-freezing to post thaw hour. after further storage of tp post thaw- hour and - hour, residual fviii level remain to be above iu/dl except unit which had a lower initial fviii concentration. at post thaw- hour, out of units tested had residual fviii activity within the pre-set standard of iu/ dl. the average decline from -hour post-thaw to -hour, -hour and hour post-thaw was . %, . % and . % respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least iu/dl of fviii. typically patients with factor levels below iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi , ayman mohamad sabri , ali abdullah alajeafi , ashwaq hasan alhekri , saleem bin mahfouz , ali hasan alkhodari , rawya saeed shealy , marcus picard-maureau* and hussain bana almalki . king abdulaziz hospital and oncology center, cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in % plasma over a day storage period and the new "test" pathogen-reduced, pooled (pools of ) prp pc in % plasma over a day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of leucoreduced test pcs were assessed at day of storage and compared to leucoreduced control pc at day of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day ; the process was completed by day post-collection. samples were taken daily for quality analysis from test and control pc until day and day , respectively. for bacterial spiking, additional pc were spiked with each receiving ml of mcfarland ($ . x cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was . % . , the total average platelet loss at day was . % . . the average platelet loss in the control units at day was . % . . the average ph of the test units at day was . . and in the same range as the control pc, ph . . . glucose concentration in test pc at day ( . . mmol/l) was lower than in the day control units ( . . mmol/l). lactate levels increased during the course of storage; lactate levels at days and were outside the range of the assay (> mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* , jay srinivasan , jessica poisson and ian welsby . duke university, duke university school of medicine, duke university hospital, duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified proteins in cryo; of the most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf ( . mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial ( Á lm) filtration. cryoprecipitate mini-pools ( ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di( -ethylhexyl) phthalate (dehp)] adsorption device and a Á lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table ) . kit ensured bacterial sterility (table ) and most importantly, final product was free of hbv, hcv and hiv (table ) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase- , thereby blocking synthesis of thromboxane a from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n ) were prepared from a nsaid-bc and ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for days at c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n ) were investigated as a reference. values are expressed as mean sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume ( vs. ml) and plt content ( vs. x ) were similar in both groups. on day , both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day was significant higher in a subset of donors who had used ibuprofen (n ). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin ( , - , p< . ), diclofenac ( , - ) and naproxen ( , - , p< . ), compared to normal controls ( , . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (< hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, ] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type diabetes (t d). because of the strong rise of people with t d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t d, but accepted as donor. study design/method: twelve whole blood donors with t d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for days at c and sampled on day , or and . the diabetic marker hba c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 'good' (ph day > . ) and 'poor' (ph day < . ) storing spc were selected and analysed in more detail. results/finding: donors were of age year and primarily men ( %). donors with t d had a higher mean bmi ( . . vs. . . kg/m ) and higher hba c than controls. the spc of both groups had the same volume ( vs ml) and plt content ( vs x ) but on day glucose concentration was higher in the diabetic group ( . . vs . . mm, p< . ). on day , the average in vitro quality was comparable in both groups (data not shown). when combining a transfusion vol. supplement s the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed ( . . vs . . mmol/ day/ plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc- ) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day ('poor': . . vs 'good': . . , p< . ). conclusion: bc from donors with t d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table ). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* , , katharine a downes , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy month old unvaccinated girl presented with history of diarrhea for days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: x /l, platelets: x /l, bun: mg/dl, creatinine: . mg/dl, lactate dehydrogenase u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to x /l, adamts sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation ( x /l and a-ipc of . x /l). two consecutive tpe were completed which resulted in a platelet count decrease to x /l and a-ipc of . x /l. a-ipc ratio was . below the ratio of which has been reported for ttp patients. similarly a-ipc count was not below x /l threshold reported in setting of ttp with severe adamts deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o :h toxin. testing of c , c , factor h, factor h autoantibody, factor i and factor b were normal. adamts activity was %. patient was treated for the infection and platelet count improved within days to x /l, with resolution of her renal failure: bun: mg/dl, creatinine: . mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts activity. many patients with severe autoantibody-mediated adamts deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts activity < %) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value - days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column ), increased peak a-ipc value (results shown as percent increase, column ), delayed a-ipc peak, and delayed plt recovery (table ) . moreover, recurrent episodes required more procedures compared to initial presentation (table ) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february to march on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version . after taking consent from the donors. the target collection of each procedure was a dose of x platelets in - ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at consecutive donations within days were considered. data was analyzed by stata . within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the donors, repeated the plateletpheresis within a week (group i) and underwent nd plateletpheresis within - days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the groups (p . ). though above the eligibility cutoff of . lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at nd plateletpheresis in group ii donors. there were donors who presented to us for the rd time for plateletpheresis with a mean gap between st and rd plateletpheresis being days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p . ). plateletpheresis through all the cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type- receptor antibody (at rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at r ab are reviewed. results/findings: case : the patient is a currently -year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age , and a second deceased donor transplant due to a rejection of the transplanted kidney at age . three years post-transplant, her creatinine (cr) started to rise from . to . mg/dl and a biopsy showed banff criteria grade amr, grade a t-cell mediated rejection (tcmr) and grade interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at rab was identified at > u/ ml (high: > u/ml, intermediate: - u/ml, negative: < u/ml). she received tpe treatments every other day and started losartan. after a course of tpe, at rab decreased to u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to . ml/dl. in one month, her at rab increased again to > u/ml, therefore, she received more tpe treatments with a decrease in her at rab to u/ml. although at rab level increased slightly to u/ml after months, her cr has been stable at . - . ml/dl. case : the patient is a -year-old mean /-se - . /- . % * . % /- . %* * p< . a female with malignant hypertension who received a deceased donor kidney transplant at age . her cr started to rise weeks post-transplant from . to . mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at rab level at u/ml. she received tpe procedures every other day and at rab decreased to u/ml with a decrease of cr to . mg/dl and improved arteriopathy in histology. because her at rab level slightly increased to u/ml over the next weeks, she started weekly tpe treatment. after weekly tpe, tpe treatment was stopped because her at rab level remained relatively unchanged. her cr has been stable at around . ml/dl to date. conclusion: we present kidney transplant recipients who received tpe treatments for high at rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at rab levels; however, weekly tpe had no effect on reducing at rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < %], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a year old man (patient b) with a medical history of hypothyroidism (on synthroid for years), end stage renal disease and non-ischemic cardiomyopathy (ef of - %) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a - . plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within hours of the procedure completion. their total t , t and free t levels trended to normal or near normal range within hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy - weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* , laura martínez molina , cristina muniesa montserrat , octavio servitje bedate , silvia cosano navarro and maria isabel gonz alez medina . banc de sang i teixits, dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) , as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category . since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of patients diagnosed with ss and compare them in their first evaluation once the th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( -methoxypsoralen, -mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from . to total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in / patients) and with online system (therakos) just in . main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is ' % (partial remission . % and complete remission . % with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : u/l) requiring transfusions, mild thrombocytopenia ( x /l), acute kidney injury (bun mg/dl, creatinine . mg/dl). by the third hospitalization day hgb improved to g/dl, however with worsening thrombocytopenia ( x /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days - ). platelet count and a-ipc improved to x /l and . x /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count ( x /l) and a-ipc . x /l. these dynamics did not resemble those which had been described for ttp patients with adamts deficiency. adamts obtained prior to tpe initiation was resulted at this time and was %. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc ( . x /l) that preceded platelet count increase to x /l three days later when patient was discharged. other laboratory values at this time were ldh of u/l, hgb: . g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts deficiency eiman hussein* and jun teruya . department of clinical pathology, cairo university, texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts . since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts , the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts . the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january through march were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts activity of less than %. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts deficiency. eight patients ( %) were associated with suspected bacterial infection. four of the patients ( %) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than kilograms using a single apheresis procedure. study design/method: in october and june , two children with possible leukemia were submitted to tl procedure. they were and months old, and weighted , and , kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, % hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin ( ml of acd-a and , units of heparin), at a blood to anticoagulant ratio of : . a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were . and . / mm . the formula "collection pump flow , x inlet flow x preapheresis wbc count" was used with the goal of removing up to x leukocytes/ml. a single leukapheresis procedure was performed with total blood volume processed per patient. immediately after the -hour procedures, wbc count were . and . wbc/mm , and -hour post tl, wbc count were respectively . and . /mm . net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since . we report the data from the year . study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of , apheresis procedures were performed at hospitals. therapeutic plasmapheresis was the most frequent procedure ( . %) followed by autologous peripheral blood stem cell (pbsc) collection ( . %), allogeneic pbsc collection ( . %), donor leukapheresis ( . %), and therapeutic leukapheresis ( . %). cobe spectra ( . %) and amicus ( . %) were the most widely distributed instruments. centrifugation was the dominant technique ( . %) for therapeutic plasmapheresis. detailed information was given for , therapeutic plasmapheresis procedures performed on patients (some items were not completely filled out). spectra optia ( . %) and cobe spectra ( . %) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently ( . %) as the replacement fluid followed by % albumin ( . %), % albumin ( . %), and % albumin ffp ( . %). most of the procedures were performed for plasma volume ( . %). acd ( . %) and heparin ( . %) were used for anticoagulation. central venous catheter ( . %) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation ( . %), antibody mediated rejection in renal transplantation ( . %), thrombotic microangiopathy ( . %), desensitization for abo compatible renal transplantation ( . %), neuromyelitis optica spectrum disorders ( . %), and hyperviscosity in monoclonal gammopathies ( . %). adverse reactions were observed in . % of the procedures. allergic reaction ( . %), hypocalcemic symptom ( . %), and hypotension ( . %) were frequently reported. therapeutic effect was achieved in . % of the patients. our apheresis registry has been well run for years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride > - mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $ deaths/ , cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of pts who were diagnosed with hp from january, through january, , and referred for immunotherapy evaluation. / ( %) pts received conventional therapy (ct) and pe (pe group), and / ( %) pts received ct alone (ct group). mean age was years (range - ), and % were female. baseline mean triglyceride level (normal < mg/dl) for pe group was , mg/dl ( , - , ) versus , mg/dl ( , - , ) for ct group. baseline mean lipase level (normal < u/l) for pe group was , u/l ( - , ) versus u/l ( - , ) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving - medications. / ( %) of pe group and / ( %) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. / ( %) of pe group and / ( %) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of . pe treatments (txs) (median of , range - daily txs) using % albumin; / ( %) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were < - mg/dl and lipase < - u/l ( . - . x upper limit of normal). mean triglyceride levels after pe txs were , mg/dl ( - , ) for pe group (mean decrease %); mean triglyceride levels after additional hours of ongoing ct were , mg/dl ( - , ) for ct group (mean decrease %). while the pe group achieved a greater mean decrease in triglyceride levels after pe txs (compared to the ct group after hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p> . ). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a -year-old man with a chronic history of hypertriglyceridemia > mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa guidelines, in a patient unresponsive to optimal medical management. asfa guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely % albumin for exchange fluid ( % albumin procedures) or partial plasma replacement ( - units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while were performed with % albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* , , metha apiwattanakul , sompis santipong , jutaluk jaipian , jettawan siriaksorn and ponlapat rojnuckarin . chulalongkorn university, king chulalongkorn memorial hospital, prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june through february were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a % solution. before using, it was diluted to a % albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of tpes in patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was , (range , - , ) ml. although the corrected calcium level was low (< mg/dl) in . % ( / ) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in patients. the first patient had events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was . % ( / ). in , the incidence of tpe adverse effects was . % ( / ) when commercial albumin was used. the difference was not statistically different (p . ). median serum albumin levels pre-tpe and post-tpe were . ( . - . ) and . ( . - . ) g/dl. the increase in serum albumin after tpe was statistically significant (p< . ). eighty-two percent of pre-tpe serum albumin levels were lower than . g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether outpatients who underwent a total of la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer ( %) and ovarian cancer ( %). based on differences in the study protocols la was performed either one-time ( %), two-times ( %) or three-times ( %), with an interval of at least weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, out of patients ( %) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in % of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was . x wbc consisting of . x mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was % lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg -related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to , iu/mls (ref. range < ) and anti-cyclic citrulline peptide antibody was elevated to , units (ref. range < ) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were , and mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was . . peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be . centipoise (cp) at admission (ref. range . - . ). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated - total plasma volume; replacement fluid - % albumin and normal saline in a : ratio; replacement fluid volume: % of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to . cp. serum igg, igm and iga levels decreased to , and mg/dl respectively. her rf had decreased to , iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan -dec ). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with % human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between to years age (m: f; : ) underwent tpe procedures with an average of . per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three ( %) patients had only visual symptoms, ( %) had both visual as well as muscular symptoms whereas ( %) patients had muscular symptoms only. three ( %) out of the seven tested, were positive for aqp -igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in patients with grade- , in patient, and by grade- , in seven. adverse events were observed in % ( / ) of the procedures with allergic reactions to replacement fluid as most common event (n- ) followed by hypotension (n- ). follow up was available in % ( / ) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from . to . for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version . as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version . . the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to / / , plt collections were performed on nine trima accel machines operating with version . . upgrading and validating all nine machines to version . occurred from / / to / / . the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version . ( / / to / / ) was compared to version . ( / / to / / ). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version . and . , adjusting for multiple visits per donor, with significance defined as p-value < . . results/findings: following the upgrade to version . , staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version . of the trima accel showed a statistically significant increase in possible leukocyte contamination from % to % of collections as compared with version . . both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version . . conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version . software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version . currently does not provide added value over version . for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of ml % albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with acute episodes requiring prolong hospital admission of approximately months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of mg/dl, lipase u/l, glucose mg/dl, bicarbonate mmol/l, anion gap . ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day after admission. results/finding: the patient tg decreased by % ( mg/dl) with medical therapy, followed by additional % ( mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below mg/dl at days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from to , a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected , cases whose ferritin levels have been monitored more than twice with an interval of detection in - days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over lg/l. and the upper limit was set to be lg/ l in male and lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: times, to times, to times, to times and more than times. the high frequency (more than times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were , donors included in the study, of which , were male ( . %) and were female ( . %). the mean ferritin was . lg/l in male ( % ci: . - . lg/l) and . lg/l in female ( % ci: . - . lg/l). the result of anova indicates that the group with the highest frequency (more than times) has the significant lowest ferritin level (p< . ). the average change of ferritin if donation over times would up to . and . lg/l in younger and elder y/o male and and lg/l in female. and then for high frequency (half a year more than times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over times in $ days) was reduced from . lg/l in the first period to . lg/l in the third period ( period $ days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* , mary townsend and lizabeth rosenbaum . university of new mexico hospital, blood systems, inc., blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* , william korzun , teresa nadder , susan roseff and elizabeth ripley . virginia commonwealth university, virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for % of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a -hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from to could be a result of a change in the blood drive timing of the training schedule of that location. in , basic trainees at site a were scheduled at day of . in january , the blood drive date changed to day of . the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from to of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in . these observations support the hypothesis that the increase in hemoglobin deferrals in resulted from the implementation of the male hemoglobin standard change from . to . g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional % of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* , eva alonso , oscar bascuñana , monica romero , teresa vich , elena castaño , laura carbonell , eva palomas , saray almerge , francesc carpio and xavier curia . banc de sang i teixits, institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january to december we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, donors came to give blood, of these, ( %) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t but his hemoglobin levels were lower than our selection criteria. of the donors selected for donation ( . %) had sci lower than t and t . adverse reactions to donation ( . %) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of , automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an month period from january to november . the same information was assembled for the automated bp process for the month period of january to november . the automated bp process implemented in mid-december ; so the december data for both and has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < . . results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p . ). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < . ) with the automated bp while and reactions remained non-significantly lower (p . ). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below . g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < . . results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under . . statistically more visits with hgb less than . g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under . was . % higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from january to march . after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of donors who has donated blood in the blood bank's main branch were used as the baseline for this study. % of donors (n ) accepted automatic appointment booking, whereas some donors (n ) were not comfortable with it. % of those who declined still preferred walk-ins (n ) based on their own time schedule, the rest decided that variable situations (n ), donation frequency (n ) and choice of preferred donation locations (n ) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was %. a comparison was made and found that this study shown a significant increase of acceptance rate by %. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. ) , . . poisson distribution, normal distribution, logistic distribution, lognormal distribution a transfusion (p> . ) in donor and reference populations except in younger ( - yrs) male donors (p< . ; donor . %, reference . %). mean donor sbp, dbp, and pulse were . mmhg, . . mmhg, and . . bpm, respectively. screening blood pressure levels consistent with hypertension ( . % male; . % female) in the - year donor group, significantly (p< . ) higher than the reference population ( . % male; . % female). no differences were observed in the - year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in - year old females. developing blood donor educational materials gay wehrli* , susan rossmann , louis m. katz and dan a waxman . university of virginia health system, gulf coast regional blood center -sugar land, americas blood centers, indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, . page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table . results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level ( th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from to after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from . % to . % in and , respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from . % in to % in . moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately . million doses of ap transfused within . billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since . firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than times and had not donated for more than days or less than times with an interval of more than days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by . times from to and the doses of ap increased by . times from to within years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into groups: those who donated ap once, those who donated - times, - times, - times, and those who donated more than times, respectively. it was found that the number of permanent ap donors who donated ap more than times was only ( . %), but they denoted a total of doses of ap ( . %) from to . conclusion: aps increased at a rapid and steady pace in wuhan blood center from to , which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at sites on consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer < ng/ml and zpp levels > umol/mol heme) at hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp . % (r . ) at first and . % (r . ) at second donations. at first donation when compared to fs hb, only . % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. at second donation, when compared to fs hb, only % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p< . ) suggesting strong evidence against correlation. % ( ) responded to the survey of which % ( ) reported not feeling well after donation. it should be noted that noted that % ( ) female study participants reported feeling unwell after the first donation and had ferritin levels below ng/ml but the zpp levels were less than umol/mol heme. of the % ( ) male participants that reported not feeling well none had ferritin levels below ng/ml nor ven or fs zpp levels above umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from . to . gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from blood centers over two intervals, july-dec. and july-dec. (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male m, female f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab . , chicago il). p <. was considered significant. results/findings: data were provided by of centers invited, representing , , and , , wb donations and , and , ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from . % to . % in the two intervals among aggregated donation attempts (p<. ), and for m ap from . to . % (p<. ). the mean "by center" deferral rates (table) were similar to that and significant (p<. ). mean by center hb deferral rates among f donations during the two intervals were . and . % (p . ) for wb, . and . % (p . ) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only centers could provide specific high vs. low vs. irregular pulse deferrals; provided only a summary (i.e total pulse deferrals), and could provide none. for bp, provided detail (high vs. low), summary and none. p deferrals increased in the successive intervals among f wb donors from a center mean of . to . % (p . ) and for m wb donors from . to . % (p . ). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba c levels among those with rbs > mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from st march to st march . total of , blood donors were tested for rbs. those with rbs > mg/dl were further tested for hba c by gold standard hplc method using variant ii biorad. blood donors with > mg/dl rbs and hba c > . % were advised to consult a physician for further evaluation. results/findings: of the , donors tested, ( . %) donors showed a rbs of > mg/dl. forty two ( . %) were males and ( . %) females with a mean age of . years ( - years). of these, ( . %) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining , ( . %) of them had a family history of dm. of these donors, donors did not give a consent for testing for hba c. among the donors tested for hba c levels, ( . %) had hba c > . %. all the donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is . % ( of donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* , hwee huang tan and ai leen ang . health sciences authority blood services group, health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from . to . g/dl last may . the current minimum acceptable hemoglobin for male donors in singapore is . g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels ( . - . ) and in donors with hemoglobin g/dl and above. study design/method: during a month period, serum ferritin testing was performed on regular male whole blood and regular male apheresis donors who made at least donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin . - . ) group b (whole blood with hemoglobin ! , group c (apheresis with hemoglobin . - . ) and group d (apheresis with hemoglobin ! ). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below ug/l is considered low and levels below < ug/l are considered having absent iron stores. results/findings: . % of donors in the study have ferritin levels below ug/l. there were more donors with low ferritin in group a compared to group b, % and % respectively (p< . ). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, % and % respectively (p . ). ferritin results for the groups can be seen in table . conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or . % have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin . - . g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to . g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of % male and % female students in the age group of - years. only % of the students have heard about voluntary blood donation and % of the students have given blood once in their lifetime and among them % are blood donors at the moment. % of the participants believed that there is a specific reason why they don't donate blood and % believed that there is a risk involved for the donors, when donating blood. % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. ( ); miscellaneous effects were reported in courses. side effects led to interruption of supplementation in instances. ferritin levels (mgt sd) at entry into the program and at the last visit were . and . . mg/l in participants, vs . . and . . mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took ! % of the tablets. ferritin levels< mg/l were found in , % of participants and . % of controls. deferral for low hemoglobin was below % in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only % of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under cfr . and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § . to make a collection under this provision if the requirements set forth in § . (a)( ) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in , an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of - ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached , of whom ( %) are c y homozygotes. without active recruitment, accrual rate is about per quarter, with % of subjects qualifying as allogeneic donors. the mean current age is . years, % male, % caucasian. the majority of hh donors ( of an active cohort of ) are in the maintenance phase of therapy with an average of . donations/year and a % deferral rate. over the last years, hh donors contributed approximately - % of the hospital's allogeneic blood supply, averaging whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided - % of blood for in vitro research at our institution with an average of wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over years. since / / , with an increase in male hgb deferral threshold to g/dl, there has been only hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm- non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm- non-invasive occlusion spectroscopy device. over a span of days, eligible blood donors, both male and female, were first screened by the nbm- non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the blood donors for the performance of hb measurement on the sysmex hematology analyzer within - hours of collecting the venous samples. results/finding: the sd of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed . g/dl. the hb measurements obtained from the nbm- and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be . g/dl. the precision of the nbm- yielded a co-efficient of variation of . g/dl and a standard deviation of . g/dl. conclusion: the operators found the nbm- easy to install, maintain, and operate with minimal training. the nbm- non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder , ravi reddy , dhuly chowdhury , don brambilla and edward l. murphy* . sanbs, rti international, ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in and followed them for one year. within days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used -point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. ) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included , first-time black donors with median age and female predominance ( %). within one year, , donors ( %) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable ( strongly agree to strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) . , % ci . - . ), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or . , % ci . - . ). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or . , % ci . - . ) and "i wasn't treated well by the <blood center> staff" (or . , % ci . - . ). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or . , % ci . - . ). a secondary analysis treating the likert scales as -level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard , ramya ghantasala , obhijit d hazarika , nicole leonard , cori a polonski , zachary b wunrow , michelle heleba , jan k carney and mark k fung* . university of vermont larner college of medicine, american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" ( . %) and "traveling is a time for me to relax." ( . %). of the respondents who travel in the summer, very few reported donating while traveling ( . %). summer donation rates between summertime travelers ( . %) and non-travelers ( . %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website ( . %) and phone ( . %). willingness to use a regional blood donation smartphone app was highest among respondents ages of to ( - %) and lowest among ages and older ( - %). of respondents with no prior knowledge of summer seasonal shortages ( %), / rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv , and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin- (il- ) and il . others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent hard spins at rpm for minutes with separation after each spin on a compomatev r g . plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers ( m [ %]: [ %] f); median age years (range - ) donated a unit ( ml) blood from which buffy coats (average volume ml) were processed. the buffy coat process was previously validated on wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures and . all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by % within months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for days. results/findings: there were completed responses, of which ( %) indicated that their hospital had an msbos and ( %) did not. the majority of hospitals without an msbos were academic centers ( / , %) from oceania ( / , %) or europe ( / , %), had between - beds ( / , %); the majority of these hospitals transfused between , - , rbcs ( / , %) per year. / ( %) are going to implement an msbos in . of those with an msbos, the majority / ( %) were from north america. the majority were academic hospitals ( / , %) with - beds ( / , %) that transfused ! , rbc units per year ( / , %) offering a wide range of surgical services. on average there were procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of % of the procedures listed, a pre-operative type and screen for %, crossmatching rbc units for %, and for % of procedures a different recommendation was made. most ( / , %) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only / ( %) of msbos' were created solely by using procedure-specific data, and most ( / , %) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually ( / , %), and the hospital transfusion committee is often ( / , %) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents ( %) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, % of respondents felt that it was regularly used by all surgeons and anesthesiologists; % felt that it was not used at all at their hospital, % did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, % of the hospitals currently without one indicated that it would be implemented in suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a -bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see , hospital admissions and nearly , emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in was approximately $ . m. in nov. , an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $ . m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns ( and ), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee . development of evidence based transfusion triggers. . education on evidence based transfusion triggers across multiple campuses, specialties and resident programs . clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to " " unit instead of " " units. . updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in , we were still able to reduced blood product expenditures by $ , when compared to . conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* , anna w rains and christopher t clark . university of tennessee graduate school of medicine, univeristy of tennessee medical center, univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for week post-delivery, with cost of $ . per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc system. by using the blood volume values, and assigning a value of . ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in , there was a total of , infants delivered at our facility. out of all the deliveries, ( %) infants were transferred to the nicu. of those infants, % received at least one red blood cell transfusion and % received at least one platelet transfusion. of the infants transferred to the nicu, ( %) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to % (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from . % all the way up to . %. in those infants, the birth weight ranged from - grams, and the gestational age ranged from weeks to weeks and days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* , shannon davis , suzan new , vaishali patel and oren guttman . university of texas southwestern medical center, ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable : communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to % of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july ; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of randomized controlled trials ( , conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* and stephanie rogers . dignity health st joseph's medical center, dignity health background/case studies: over million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels > . g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of . g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of . g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to hospitals including cme presentations, online physician and nursing educational videos, communication tools including infographics and " is the new " buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb > . g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy to fytd , there was a % reduction in prbc units transfused to patients with hgb > . g/dl, starting at a baseline of % down to %. this represents an fy annualized savings of $ . m, from a baseline of units per , patients days down to an average units and approximately , fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels > . g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm - . ) reduced rbc units and two studies decreased the percentage of patients transfused (or . ). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or . ) and rbc units transfused (sdm - . ). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a- method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was g/dl/ % or g/dl/ %. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately % of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was units/ month. after the first months of buc activation the mean number of units was units/month a reduction of units/month or % of nonsurgical blood use (p . by t-test). non-surgical rbc use now represents approximately % of the total rbc use hospital wide a % reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital - month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital ( beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance ( q vs. q ): present and completed consents ( vs. %), present and completed nursing flow sheets ( vs. %), transfusion thresholds supported ( vs. %), discharge instructions provided ( vs. %); ( q vs. q ) vital sign compliance ( % vs. %). jwp increased from to ( / - / ). cost savings were realized by decreased utilization and implementation of bpa. (table - q ) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately - trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only percent of coolers fully used. it also consumed valuable staff time as technologists typically made - trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated - hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains - units of group o rhd negative, units of o rhd positive, and units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in , the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june -october were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a -month period, , cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table below. overall, ( %) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." ( ), "active bleeding" ( ), "platelet count of . . ." ( ), and "downtrend" ( ), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, ( %) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years - . study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of , g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey ( questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of complete responses ( %). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, % with physicians, and of these, % reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only % responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of - g/dl ( %), platelet count of - , ( %), and inr of greater than . ( %). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per , inhabitants may vary folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies ( . % and . %), for the remaining strategies weights varied between . % and . %. we estimate that , patients would be eligible for pbm strategies in one year time horizon, resulting in premature death avoided ( . % reduction) corresponding to a gain of approximately , life years and a reduction of , ( . %) disability adjusted life years (daly) relative to the current clinical practice. a decrease of , in-hospital days is expected mainly due to a . % reduction in hospital length of stay and a . % reduction in -day readmission rate. in this population the overall transfusion rate could decrease to . % from the current . % ( . % reduction) implying , blood transfusion avoided and , red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted < ml of rbcs, all cases with ml transfused were captured and only . % of the time > ml were used. if - ml rbcs were predicted to be transfused, > ml were used % of the time. if predicted usage was > ml, % of the time it exceeded ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for > ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than . is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a month period (jan to mar ). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the month period, there were patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of units of blood were requested. units were crossmatched, of which units were sent to the operating theatre (ot). only . % of blood issued to ot were transfused (n ) while the rest were unutilized. the observed ct ratio was . . conclusion: although only % of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of ! . , with almost % of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study patients who received tranexamic acid (txa) (study group) and patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < . g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were unique alert encounters. of these, ( . %) led to a crossmatch being ordered while ( . %) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds ( . g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds ( . or . g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at academic hospitals with data-derived msbos. study design/method: the hospitals were in groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if % of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if - % of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if ! % of the patients had been transfused. data were collected at each center over a month period between january to march and included a maximum of cases per hospital during that one month to ensure equal representation between centers results/finding: between these centers there were a total of cases analyzed. some of the more frequently performed surgeries included orthopedics ( % of cases), general surgery ( %) and cardiac surgery ( %). there were t&s ordered for these cases, of which were positive for antibodies on the day of surgery. of all the t&s ordered, % were ordered in accord with the msbos recommendation, % were ordered when the msbos did not recommend one, and in . % a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio . for prenatal hemoglobin (hgb) - . ). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of % among obstetric patients. study design/method: we studied a sample of anemic (hgb< . g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of women were enrolled, with median age (interquartile range - ) years, median gravida / para and median gestational age weeks. mean hgb before referral was . g/dl and most were already taking oral iron therapy. a total of women were hiv positive with mean cd lymphocytes counts of cells/ul; ( %) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and ( %) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in ( %) of women. there was concurrent chronic disease (n ), infection (n ), vitamin b deficiency (n ) and antenatal hemorrhage (n ); had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a month old boy presented to our institution after a month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige ( ku/l; rr: - . ). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp , while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp cd lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $ . %. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of . % over the last years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from to , with a peak of . % (range . %- . %). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for mtps. results/finding: in , mtp was activated times. in cases the patient did not receive any blood product and in cases plasma was already available at the time of rbc allocation/issue. this left cases to evaluate. the median time to plasma availability was minutes (range minutes - minutes). the mean plasma wastage for mtp activations was % (range - %). of the cha replies, were using thawed plasma and their wastage was </ %. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of minutes. our implementation began with thawing a single unit of plasma for "level neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are . %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated cases of gtx in pediatric patients with age from months to years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of granulocytes donations from to were collected from health donors ( male/ female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, mcg/kg/dose (donors up to kg, maximum of mcg) and oral dexamethasone ( mg), h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with gy. results/finding s: granulocytes number increased times from basal levels and the median of cells collected were . x (range . - . ). in general donors reported no significant adverse reactions. from donations, only in was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table . adverse reactions for patients were minor and not frequent ( in gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* , elena kurnikova and pavel trakhtman . russian institute for paediatric haematology, oncology and immunology, instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period / - / . donors underwent granulocyte mobilization with g-csf in a dose - mg/kg s.c. - hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed granulocytes collections in donors ( f, m), ages to . the mean increment of wbc after stimulation was , x /l ( , - , x /l; f- , x /l; m- . x /l); in the age groups - years (n ) and over years (n ), the mean increment of wbc was respectively , x /l ( , - , x /l) and , x /l ( , - x /l). the mean volume of treated blood was , a , ) . the mean number of total wbc in the product was , x /l ( . - x / l; f- , x /l, m- , x /l). only three donors ( , %) had apheresisrelated adverse effects the analysis included granulocyte transfusions in cases of infectious complications in patients. the mean number of transfusions was . ( - ). each patient received transfusions from mean donors ( - ); the granulocyte transfusions started at the mean days ( - ) after infection; the mean dose of wbc on the transfusion was . x /kg ( - . x /kg). wbc increment was able to estimate after transfusions. wbc increment was recorded after transfusions ( . %); the mean increment was , x /l ( , - , x /l). efficacy was determined by the number of patients who survived an infection episode and amounted to . %. in the cases of severe sepsis, the efficacy of transfusions was . %. the frequency of post-transfusion reactions was %, (n ): febrile nonhemolytic reactions , % (n ); pulmonary complications , % (n ); the anti-hla antibodies formation , % (n ) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp hemolytic disease of the newborn due to anti-rh keegan barry-holson* and jennifer andrews . stanford university, dept of pathology, stanford university, dept of pathology & pediatrics background/case studies: anti-rh is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g p > mother with a negative st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at hours of life because he was found to have anemia (hemoglobin . g/dl), severe hyperbilirubinemia (total bilirubin (t bili) . mg/dl), reticulocytosis ( %) and a positive direct antiglobulin test (igg ). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of . mg/dl on day of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin . g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* , cyril jacquot , valli criss , philippe p pary , jay greenberg , naomi lc luban and edward cc wong . children's national medical center, quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as - %. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of . g/dl/ . %. wbc counts ( x /l) were mildly elevated and platelet counts ( x /l) were within normal limits. her history was notable for upper respiratory tract infection days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total . mg/dl, direct . mg/dl), normal ldh ( u/l), and undetectable haptoglobin (< mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin ( - reactivity) with positive autocontrol. dat was positive for anti-igg and negative for c despite a positive cold antibody screen. the patient weighed . kg with an estimated total blood volume of ml. she initially received simple transfusions totaling ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of %, utilizing the central venous catheter. no adverse events took place over the course of the hour exchange. her one hour post-exchange hb was . g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin ( ). after initiation of steroid therapy (methylprednisolone, mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels ( mg/dl). at a subsequent follow-up visit months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a or gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, . /- . ml/ second) or with a mechanical syringe pump (slowly, ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the g catheter, the mean change in hct was - . /- . % with the one-way valve and . /- . % without (p< . ). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p< . ). during rapid manual transfusion with a g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse . ml when using a one-way valve (change in hct versus time: r - . , p< . ) compared to a significantly different (p . ) slight increase in hemolysis for samples that took less time to transfuse . ml when not using a one-way valve (change in hct versus time: r . , p . ). correlations between time and hemolysis were similar, but insignificant using g with washed rbcs and the g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in . % of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge . study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at weeks gestation with passive anti-d and an anti-ge titer of . she was d-and ge:- ,- , by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge . the birth hemoglobin (hgb) was . g/dl, reticulocyte (retic) was . %, bilirubin (bili) was . mg/dl; the infant was discharged. on day of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb . g/dl, retic . %, and bili . mg/dl. ge -blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh . obstetrics had to authorize maternal blood donation due to her hgb of . g/dl. maternal blood collection and rbc washing was expedited and the infant received ml of maternal rbcs within hours, at which time his hgb was . g/dl. post-transfusion hgb was . g/dl. one week later, the infant was symptomatic with hgb . g/dl, retic . %, bili . mg/dl. a nd aliquot of ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb . g/dl, retic . %, anti-ge titer , and needed another transfusion. the maternal blood stored for just weeks had hemolyzed necessitating a nd maternal donation for baby's rd transfusion. at weeks, the infant's anti-ge titer was , hgb . g/dl, retic . %; no transfusion was necessary. at weeks of life, hgb was . g/dl, retic was . %, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge hdfn. molecular analysis revealed that the mother was homozygous ge -negative ge* .- , the father had homozygous wild type ge* , and the infant was heterozygous ge* /ge* .- . conclusion: the infant had hdfn due to antibodies to the high prevalence ge antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge . hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results ( ) supporting a hemoglobin trigger of g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in , , rbc orders occurred and the top three patient groups were: % in congenital heart disease patients, % in hematology/oncology patients and % in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was . g/dl as measured in the hours prior to rbc order placement. in , ffp orders occurred and the top three patient groups were: % in neonates in the nicu, % in congenital heart disease patients and % in pediatric intensive care patients. average inr of every patient was . as measured in the hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates ( - months of age), infants (> - months of age) and children (> months- years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after months, pr-plt represented % of our platelet inventory (average daily plt inventory: units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among transfused neonates ( - m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased , dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of pediatric patients receiving prbc transfusion over a month period were retrospectively reviewed. a total of patients were identified as receiving allogeneic prbc transfusion. patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was . g/dl with post-transfusion hgb rising to . g/dl. the mean prbc volume transfused was . ml using a dose of ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of - ml/kg in a kg patient, for instance, would translate into full prbc units (about ml) in an average size adult. the current standard dose of - ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a month-old, previously healthy female infant who presented to the hospital with a -week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of . g/dl and . %, respectively, platelets of , , and a reticulocyte count of . %. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of . mg/dl with a direct fraction of . mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens ( total) were non-detectable. the patient was started on a -day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c . an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her -day hospital course, the patient received rbc transfusions on the day of admission and several rbc transfusions thereafter (see table ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day . her hemoglobin rose to . g/dl on hospital day and increased to . g/dl on hospital day . at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to . g/dl on day after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is or . platelet and leukocyte immunohematology, testing and genetics table . of pairs, pairs were complete match ( / ), pairs were partial match ( / ), pairs were complete mismatch ( / ). the matching rate of hla-dpb in our study is %. conclusion: the matching rate of hla-dpb in / hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china ( ) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts . therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a year old male with a -year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts < % and inhibitor of . . on day of admission platelet count was x /l which decreased within five days to x /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were x /l and x /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to . x /l achieving the ratio of previously shown to be diagnostic of ttp. on day his a-ipc and platelet counts had improved to . x /l and x /l respectively. absence of anti-pf antibodies ruled out heparin-induced thrombocytopenia at this time. on day he had an unexpected decrease in both a-ipc and platelet count to . x /l and x /l respectively, worsening by day to . x /l and x /l respectively despite daily tpe. patient received additional tpes that failed to improve a-ipc or platelets which on day were . x /l and x /l respectively. a-ipc had remained at this level for days suggesting that the observed decrease was irreversible. adamts activity remained < % low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa- a, hpa- a, and hpa- a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa- a, hpa- a, and hpa- a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p and gi , specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa- aa, hpa- ab and hpa- aa) were collected and reacted with anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex . the hpa- a serum was diluted to serial dilutions (from neat to / ) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa- a, hpa- a, hpa- a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( . vs . ), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa- a, anti-hpa- a, anti-hpa- a. furthermore, because the platelet was hpa- aa, the hpa- b serum did not react with the coupled beads with mfi was comparable to negative control ( . vs . ). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa- a was / ( . iu/ml) and / ( . iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa- a, hpa- a and hpa- a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in % of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from january to december . clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c , c bp, thbd, dgke, cfhr , cfhr , cfhr and cfhr ) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of patients tested, pathogenic mutations were detected in % ( / ) and vus in % ( / ). % ( / ) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and ( %) had vus. % ( / ) of patients had primary ahus; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. % ( / ) of patients had secondary tma; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. in patients with pathogenic mutations, % ( / ) were children, . % ( / ) had a positive family history of ahus and % ( / ) had recurrent disease. patients with primary ahus had a significantly lower age at presentation ( vs. yrs; p-value: . ) and a higher proportion of pathogenic mutations ( % vs. % p-value: . ) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl and sysmex xn compared with a flow cytometric method farshid ezligini , kjersti roen eriksen , annette vetlesen , thomas larsen titze and geir hetland* , . oslo university hospital, university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion ). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl (horiba abx, montpelier, france) and xn sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x /l sd were , (<) , (<) and for counting by sysmex toa, pentraxl , and the gallios flow cytometer, respectively. sysmex count was the very lowest a transfusion vol. supplement s abstract ( . % less than for flow cytometry), but all plt counts were significantly different (p< . ), although least so ( . %) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing , arishma lata , roland russnak , zachary antovich , heather dunckley and thierry viard* . new zealand blood service, linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of reactions that identify both variants of relevant snps located within hpa genes (hpa- through hpa- , and hpa ). genomic dna purified from blood samples, previously genotyped for hpa- ,- ,- ,- ,- and - by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were % concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late and to date has tested dna samples from blood donors ( donors were tested in duplicate). concordance between the sample replicates was %. there were occasions where the assay had to be repeated, giving a repeat rate of . %. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa- ( . %) and hpa- ( . %) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd antigen deficiency expression in jiangsu chinese han population qing chen* , jianyu xiao and chengyin huang . jiangsu province blood center, jiangsu province blood center background/case studies: cd has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd deficiency varies widely among different ethnic populations, with the frequency of - % in asians and . % of african americans, respectively. however, there is little information on the molecular basis of individuals with cd deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd expression levels and to determine the molecular basis of cd deficiency on the platelet surface of the han population in jiangsu region. cd expression levels on platelets were detected by flow cytometry among blood donors in jiangsu region. donors without cd antigen expression on their platelet surface were further to be determined the expression of cd antigen on their peripheral blood monocyte cells. the coding exons of cd gene and adjacent introns were amplified and sequenced in cd deficient individuals. results/finding: among these blood donors, cd -deficient and cd -expression individuals were . % ( / ) and . % ( / ), respectively. the frequencies of type i and type ii cd deficiency among the study population were . % ( / ) and . % ( / ), respectively. among individual with platelet cd expression, according to mean fluorescence intensity (mfi) value, , and individuals showed low, moderate and high expression levels of cd , respectively, and their mfis were . . , . . and . . (p< . ), respectively. the type i cd deficiency individual were heterozygous for - a>g and - c>g, respectively. among type ii cd deficiency individuals, two harbored a t insertion at position in exon which caused frameshift at codon ; one has a t>c exchange at position in exon which resulted in a tryptophan to arginine substitution at codon ; one has a a insertion before the th bp of the start codon atg in the promoter region; one were heterozygous for t>c and t>g, respectively. conclusion: platelet cd surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd deficiency was higher than that in type i. the study findings indicated that the frequency of cd deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd -deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd -deficient individuals widely varies among ethnic groups, with % to % in japanese, % in sub-saharan africans, . % in african americans, and . % in caucasians. although some studies of cd deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from unrelated platelet-apheresis donors in the eastern china. the expression of cd antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd and peanti-cd ). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd -nagtive phenotype. for those donors with cd -negative platelets, cd antigen expression on monocytes was analyzed further to distinguish between cd type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china ( ) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd in all samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd phenotypes using the (mean sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd deficiency on platelet, in which one sample was cd negative both on platelet and monocyte. the frequency of cd type i and type ii deficiency in the eastern chinese donors was . % and . %, respectively. the average mfi of cd deficiency samples was significantly lower than cd positive samples ( . . vs . . , p< . ). conclusion: the frequency of platelet cd deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd -deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd antibody. background/case studies: cd (gpiv, chromosome q . ) is an kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd deficiency (cd -n) is observed in - % of africans (t g) & is classified as either type i (cd -n plt, cd -n mono) or type ii (cd -n plt, cd mono). an acquired type ii cd -n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type cd -n individuals can develop anti-cd alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an year-old, group o african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin . g/dl & plt count k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, - % blasts & a complex karyotype with del( )(q q ) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < . hla antibody testing was negative (class i panel reactive antibody (pra) %). the patient was plt-xm-incompatible with most donors ( / ). a trial of group o, plt-xm-compatible plts was unsuccessful (cci ). subsequent testing for plt-specific alloantibodies identified anti-cd . fc-phenotyping showed no cd on patient's mono or plt, consistent with type i cd -n. preliminary dna results show that the patient is heterozygous for t g. because cd -n apheresis plt were unavailable from blood suppliers, the patient's children & grandson were screened as possible donors: all showed normal cd expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day ) demonstrated new class i alloantibodies (pra %) in response to transfusion ( apheresis plts, rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd -n & severe plt refractoriness in the setting of new mds, and q-chromosomal abnormalities. the absence of cd on plt & mono support congenital type cd -n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel , kristopher fernandez* , eric senaldi , pascal george , michael seul and ghazala hashmi . biomolecular analytics, central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur http://bit.ly/ q heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $ adult donors who had made ! donations in the previous months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for , , , , , , , , , and for hla class (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/ pdplf ) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a * : chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are , hla alleles documented according to the imgt / hla sequence database in janury , and more than % of them were identified in the last years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a * : allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a * : : : , but nucleotide substitution in exon , by nt c-a (codon acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a year-old pregnant woman typed as ab , who delivered a baby affected by severe hdfn. the newborn was typed as b and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted with the four monoclonal anti-d used (igm clones p x and rum and the blends clones th ms and d d ) and were typed as c-c e-e . an anti-d was identified in her serum. molecular analysis showed the c>t and a>c in exon , the snp t>c changes in exon and the t>g nucleotide change in exon . the set of snps found is similar to the molecular background of dol , except for a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of immuno hematological tests ( abo/d grouping (including newborn samples), extended erythrocytic phenotype, antibody screening, antibody identification, dat) and crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in . % of the abo/d tests (n ), , % of the antibody screening tests (n ), , % of the antibody identification tests (n ) and % of the dat tests (n ). there were discrepancies ( abo/d for the same patient, for antibody screening and antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than min). detection threshold of the d antibody was assessed at . ng/ml ( . ui/ml) whereas the french recommendations are ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a year old g p presented during her fifth pregnancy with anti-kpb with an initial titer measured of . by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams ( . moms) peaking at weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was . mg/dl with . g/dl hemoglobin. the baby typed as o positive, kp (b ) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by weeks of age. his hemoglobin recovered to . g/dl with an indirect bilirubin of . mg/dl at weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c d-specific dat may be too insensitive to detect low, but significant levels of c d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a " " reaction corresponds to about molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c d have been published. however, these are mainly designed to quantify the fraction of rbcs with c d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c d. study design/method: ten microliters (ul) of : (after documenting experimentally that this amount ensured maximum binding of anti-c d) mouse monoclonal anti-human anti-c d (abcam, clone c ) were added to ul of a . % rbc suspension. after incubation for minutes at c, samples were washed x , and ul of : diluted anti-mouse-f(ab) -pe (ro , dako) were added. after incubation at c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a rbcs stained with levels ( fold dilution, : - : ) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c d, edta-stabilized samples from healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c d (values ranging from to , abc) with level of -serum dilution (used to sensitize a rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r . , p < . ). compared with dc-screening , the sensitivity of the flow cytometric assay was superior. it detected c d sensitization at least dilution steps further. the median normal level of rbc-bound c d was abc (range - abc, n ). the assay enabled demonstration of specific c d-sensitization in the patient; the level of rbc-bound c d in the sample was significantly elevated ( , abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt gene. in humans, the abo and gbgt genetic loci are located on chromosome q , and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors ) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt genes have been constructed of vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion vol. supplement s the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c , e-, e and k -. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d , c-, e-, partial c , partial e . the probable rhce genotype, rhce*ce-jal/rhce*ce g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant woman and her newborn carolina bonet bub* , maria giselda aravechia , thiago costa , marilia sirianni , eduardo bastos , leandro santos , lilian castilho and jos e kutner . hospital israelita albert einstein, hemocentro unicamp background/case studies: rhd*weak d type is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion ) the c. g>c change (p.g a), which characterizes the rhd*weak d type allele is a splicing variant that induces skipping of the whole rhd exon . we report an altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- /ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d w c-c e e . the samples showed weak hemagglutination reactions ( / ) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon in both dna samples. sequencing showed the c. g>c change and the intronic c. - t>a and c. - t>c substitutions, which are associated to the rhd*weak d type allele. conclusion: our results showed that c. g>c associated with c. - t>a and c. - t>c variations had probably a functional impact on splicing inducing exclusion of exon in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type . background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than % of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with , patients and studies from other regions (brazil, egypt and france) with patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was to years and for the other countries to years. available data from us studies included a total of alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens ( . %, . %, . %, . %, . % and . % respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of . % ( . to . , % ci) vs. . % for non-us studies ( . - . , % ci) (p . ) and a transfusion vol. supplement s more alloantibodies per transfused patient ( . vs. . , p . ). similarly, the number of alloantibodies per transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort ( Á vs. . , p Á ). average number of rbc units transfused per patient in the us was also higher compared to data from france ( vs. , p . ). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* , melissa grohotolsky , lisa deblass , bala carver and kip kuttner . health network laboratories, miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: year old white mennonite female g ,p presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at . although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g (r g r) cells. both rhce*c and rhd genes encode ser which determines g expression. rare rhd variant antigens lacking ser are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e c e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r r , r'r, r g r and rr cells. this patient is predicted to be r r (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is a transfusion vol. supplement s excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* , christa voliva , kathy fletcher , heather vaught and tracie ingle . indiana university health, indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology ; : - ) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were > dat positive, were weakly dat positive and were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman . - . ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: bb, bg and bp. the ph of all eluates ranged from . - . with the highest percentage of eluates at a ph of . ( %). sixteen of the eluates tested yielded the same results in both automation platforms (concordance of %). four eluates with different results are summarized in table . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results ( ) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in led to the discovery of human blood groups. in the abo system > alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor ( units over years) who is actually a w . study design/methods: donations were tested with the pk instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns , and and exons and . specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table . tests with anti-a, -a , -b anti-a,b were negative as were the a cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table . the significant changes were found in exons and . in exon there was a nucleotide (nt) deletion of g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly ala. mutations in exon included a nt substitution causing a pro -leu change and a nt deletion c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o. . /abo*aw. ] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a and b cells. this abo discrepancy was caused by the presence of anti-a in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer > : . (schmidt, nacarrow et al. ) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. a transfusion vol. supplement s extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: edta blood tubes collected from random blood donors were used to extract dna from microliters of whole blood on day , and days post collection. blood samples were stored at - c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a /a using a nanodrop (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result ) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the samples, samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from . to . ng/ul. all readings with the exception of ( . ng/ ul) had concentrations > ng/ul. interestingly, the one that was < ng/ ul on day , yielded > ng/ul on day and post collection. over the next months, sets of samples were extracted and tested by hea. eighty-three ( . %) failed extraction and ( . %) failed hea. none of the samples that failed extraction were or days post collection; none of those that failed hea were days post collection; . % were > < days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas , carlos villa , rachel davis-rauser* , helen carpenter and vrunda patel . university of pennsylvania, hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in . communications suggest all patients receiving therapy would have a positive antibody screen because cd is a common antigen expressed on red blood cells. currently, patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at : dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for negative patients was observed up to a : dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a y/o g p at approximately weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [ , ] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m n-s s phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency > . ), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [ , ] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [ ] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays a transfusion vol. supplement s that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in dna samples from chronically transfused patients with scd, in patients with thalassemia and in dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in levels: ( ) rh and k matching; ( ) extended matching and ( ) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for % of our thalassemic and scd patients at level , % for scd patients and % for patients with thalassemia at level and % for patients with scd and % for patients with thalassemia at level . the patients were transfused with a median of . rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to - % with c e k matching and < % with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube ( drops plasma, minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to minutes and specimen volume to drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of clinically significant antibodies were detected using sprca technique, as well as warm autoantibodies and nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being % specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions ( versus ), it identified more clinically significant antibodies ( ) than liss ( ). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd on rbcs patricia a arndt* , anthony salazar and regina m. leger . american red cross blood services, long beach memorial medical center background/case studies: monoclonal anti-cd , e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or aminoethylisothiouronium bromide (aet). chapuy et al described ( ) and validated ( ) ), and % aet (ph . ) as per the aabb technical manual, th ed. these treated and untreated rbcs were stored in alsevers at c and tested on days , , , and by two methods: ) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from total dara patients were tested with reactivity - ), and ) flow cytometry using phycoerythrin (pe)labeled anti-cd . rbcs were also tested on days and or with a serum containing anti-k by peg iat. results/findings: the . m dtt in ph . pbs had a final ph of . and the ph of the commercial . m dtt was . . results are in table ; flow cytometry results from days , and (data not shown) were similar to those from days and . rbcs treated with . m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd by flow cytometry for up to days after treatment. rbcs treated with . m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening ( - %) of reactivity with pe anti-cd . background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon and ) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr ) in gestational week . the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of pregnant women were included. nip rhd was positive in / ( %), negative in / ( %) and inconclusive in / ( . %). compared to the postnatal rhd type, / ( . %) of nip rhd results were false positive (fp) and / ( . %) were false negative. in / ( %) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n ) at gestational week was . ( -and -percentiles: . - . ). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p , ). the fraction of ccfdna was calculated for randomly selected nip rhd true positive cases. median ccfdna ratio was . (the distribution had a highly positive skew, -and -percentiles: . - . ). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r , ; p . ). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of , suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only in , donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, lan-rbc units were transfused over days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day , the patient had symptomatic anemia with hemoglobin (hb) . g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of . g/dl was maintained for days. the antibody screen was negative on day post-transfusion, but strongly panreactive on day , with a positive dat (igg , c ) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from . g/dl on day to . g/dl by day with no bleeding identified, and increase in total bilirubin and ldh (peak . mg/dl and u/l on day ) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day with good response (hb . g/dl). the patient remained stable and was discharged to a skilled nursing facility days later. conclusion: transfusion of lan rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* , andrea gerner , lynne stewart , carol sostok , mollie bell and gregory r halverson . st. elizabeth healthcare, hoxworth blood center background/case studies: anti-f was first described in by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the th antigen assigned to the rh blood group system (isbt rh ). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused unit o-rbcs. two weeks later the patient received an additional o-rbc. within days the hgb had decreased from . to . g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused r r (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c e f phenotype. the rh phenotype and as was repeated on a sample collected days later. the c typing was micro positive, mixed field only after minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post hour hgb increment from the receipt of a standard unit of blood should be near g/dl (or % hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a . g/dl increase, and the second unit was only . g/dl. the last transfusion of units increased by only . g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of r r units was nearly complete. in a case from , ohto and kariyone (transf. ; vol , no. ) reported a cr Ásurvival study of f rbcs in a patient with anti-f. they showed that the initital survival of f cells was fairly normal, however, after days, there was a sudden increase of red cell destruction, and by day all f cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with . m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to days following the dtt treatment of rrbc. reactions were graded using standard serological grading of (negative) to (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to days. this suggests that dtttreated reagent red cells can be stored for at least days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with . m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract - c, and observed for hemolysis (none was seen) for up to days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted - with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* , monique scott , garcia curtis , ellice wong , alexa j siddon and christopher a tormey . yale-new haven hospital, va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately % to % of persons of african descent is characterized by neutrophil count of < . x /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p< . . results/finding: subjects who were clinically identified as having probable ben included patients (mean age . ; all self-identified as african-american; / were male) and controls included patients (mean age . ; self-identified as african american; ( / male). all of the cases ( %) diagnosed with ben had fy(a-b-) phenotype. mean anc ( . x /ul) and wbc counts ( . x /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p . and . , respectively) compared with controls (mean anc . x /ul ; mean wbc count . x /ul). there was no significant difference in mean platelet counts ( x /ul vs x /ul; p . ) or mean hemoglobin levels ( . g/dl vs . g/dl; p . ) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih- had % concordance for all blood grouping assays. for ahg assays, the ih- detected an anti-jka e, anti-fya warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih- identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih- with anti-igg,-c d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih- analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih- is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of tests were performed on , adult patient samples and donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in . % of samples tested. there were discrepancies, all antibody screening ( false positives, failure to detect a very weak prophylactic anti d and positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing - group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* , kathleen bensing , michael schanen , cindy piefer , randall w. velliquette , christine lomas-francis and connie m. westhoff . immunohematology reference laboratory, versiti/bloodcenter of wisconsin, immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new / / / cecf / / / rhce*ce or rhce*ce compound heterozygotes ce g ce g or ce c, g or ces or ceti / / / ce g ce c, g or c, g / / / ce g ces or cemo or ceek or ceek(var) or cern / / / ce c, g ce c, g or cemo or ceti / / / ce c, g/ce g/ g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi / / / total / / / a transfusion vol. supplement s reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd / and rd / , and a licensed comparator anti-e (p gd ms ), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n ) or edta blood from donors (n ) and were tested using a manual tube method or on a pk automated platform. a score ( ) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table ) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r r , r r , r r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: were rhce*ce that were in trans to rhce*ce; were various rhce*ce plus rhce*ce c compound heterozygotes; were rhce*ce or rhce*ce homozygotes; were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with rhce*cear/rhce*ce c compound heterozygote, and with rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and of rhce*cecf homozygotes were detected using the comparator reagent. rd / and rd / failed with and e variants, respectively (table ) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd / clone. none of the reagents detected e antigen variant expressed on example of rhce*cehar/rhce*ce. conclusion: rd / and rd / anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of blood group genes associated with the expression of blood group antigens from blood group systems. we used the illumina's hiseq / system to perform next generation sequencing first on sureselect-enriched genes from dna reference samples with average target design coverage of . %, and then on haloplex-enriched genes from dna reference samples with average target design coverage above . %. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for blood group genetic variants in these genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads ( . %) were mapped to the target regions relative to the sureselect reads ( . %). the mean sequence coverage depth of the targeted bases was around x for sureselect method and x for haloplex method. some exons, such as rhd exons and , , rhce exon , ermap exons and , cd exons and , cr gene (most exons) and gypb exon , are consistently covered with less than x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than % concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than % blood group genetic variants in selected genes. evidence rhce*cehar does not encode for rh (hr b ) antigen debra j bailey* , trina horn , paul mansfield , najmi qazi , pamela nickle , jessica keller , margaret a keller and jan r hamilton . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in and has a phenotype of c e c e w f w , g , hr w , hr , hr s , rh: , rh: with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh: (hr b ) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c. c>g and rhd c. c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d c e c e . her plasma contained an alloanti-s and an antibody that reacted strongly with all random e k s reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d and dc red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e s k red cells homozygous for the rhd*diiia-ce( - )-d, rhce*-ce c, g, t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce c, g, t/ rhd*diiia-ce( - )-d, rhce*ce c, g, t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr ( of sources) and hr b ( of sources) phenotype. conclusion: the rhd*diiia-ce( - )-d, rhce*ce c, g, t haplotype is one of the rh haplotypes expressed by the original hr b individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between / / and / / . all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately years, patients had htla established at least once by titration studies. serological investigations on a total of samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on samples was successful in rule out in % of cases. in an additional patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only % of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for % of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c. c>t (p.thr met). the dau allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion , : ) recently summarized serologic characteristics and associated anti-d alloimmunization for dau family alleles. we investigated two samples with the c. c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample was from a yo multiracial female. her rbcs reacted s at immediate spin (is) and in iat with immucor gammaclone and series and , and mi at is and in iat with ortho bioclone anti-d. rbcs did not react with of (lhm / & / ) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c. c>t characteristic of dau. rhd sequencing confirmed c. c>t and identified two adjacent changes, c. g>t and c. g>t (c. _ delinstt), in exon encoding p.gly leu. sample rbcs reacted w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series and , and quotient albaclone blend and alpha anti-d. papain treated rbcs were s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c. c>t. sequencing confirmed c. c>t and found a new c. c>t change (p.ser -leu) in exon . the c. t has not been reported, but c. g encodes a stop codon (p.ser stop) in japanese (vox sang , : ). conclusion: we report two new alleles: rhd with c. _ delinstt (p.gly leu) and rhd with c. c>t (p.ser leu), both also carrying the c. c>t (p.thr met) characteristic of the african dau cluster. d antigen associated with p. leu is a partial d antigen with a novel epitope pattern. the p. leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by / commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to . the number and diversity of alleles in the dau cluster supports that the c. c>t change is a major ancestral african background allele (wagner et al, blood , : ). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september to present day we got samples of repeated blood donors who are known to be d negative, c positive and/or e negative from blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon , exon and exon in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon to confirm the existence of c. g>a and c. t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the sample, we identified cases ( . %) of total rhd deletion, cases ( . %) of rhd-ce-d hybrid, and cases ( . %) of rhd variant. of rhd variant were determined to be asian type del with c. g>a variation. cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n ) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon and exon of the abo gene were amplified and sequenced. the abo*b . allele was confirmed by pcr-rflp analysis. results/finding: among subjects with b or ab phenotypes, were genotyped as abo*b . . the abo*b . group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage ( . . ) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other subjects with b or ab subjects, genotyped as abo*b . (n ), abo*bw. (n ), abo*bw. (n ), abo*bw. (n ) and abo*bw. (n ), displayed flow patterns differed from the abo*b . group. the abo*bw. , abo*bw. and abo*b . subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (< % in abo*bw. and abo*bw. subjects and < % in abo*b . subject). both abo*bw. and abo*bw. displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b . genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout , the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in % (n ) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in % (n ), % (n ), and % (n ) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n ), anti-jka (n ), anti-k (n ), anti-jkb (n ), both anti-e and anti-c (n ) (see table ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly in cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study volunteers in the gambela region (nct ). the whole ackr gene was amplified in one reaction covering , base pairs (bp). this primary amplicon was re-amplified using nested primers covering nucleotides. nucleotide sequence was obtained by sequencing reactions and manually annotated using ncbi refseq ng_ . . the sequencing covered bp of both exons, bp of intron, bp of '-flanking region, bp of '-utr, bp of '-utr and bp of '-flanking region and encompassed all the variations present in dbsnp and nhlbi esp databases. results/finding: among the samples, a total of snps, including one novel snp in '-utr were observed. snps occurred in the exons, in 'and 'flanking region, in '-utr and in the intron. all individuals carried the snp indicative of the common fy: phenotype; while individuals were homozygous and was heterozygous for the gata box mutation. no splice site mutation was detected. as individuals were observed as being homozygous or heterozygous for snp, we could unambiguously assign distinct alleles. in the remaining individuals with or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than . kb of the ackr gene and identified at least different alleles. the present study found that the vast majority of alleles ( / ) in the gambela population as defined by snps, were similar to the clinically relevant fy* n. allele, which in turn is defined by only snps at positions c. - t>c and c. g>a. out of the remaining alleles, were similar to fy* with the fy(b ) phenotype and was similar to fy* w. with the fy x phenotype. the high frequency of fy* n. ( %) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique ( %- %). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce( - )-d is the most common hybrid and is found in african blacks. it arose by conversion of exons - of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample (male) and sample (multiracial female), both c c e e (presumed r r ), presented with weaker than expected d typing; is and / at iat. rhd beadchip identified the common african rhd*diiia-ce( - )-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c. c>g and c. g>t (heterozygous) was also detected. as rhce*ce with g and t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons and replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce( - )-d were found in both samples. sample (scd male), d c e c e , by rh beadchip had one conventional rhd and rhd*diii type , and rhce*ce g/ces. as rhd*diiia type has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce( c) exons and had replaced those exons in the common hybrid rhd*diiia-ce( - )-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky and ky . we report two different and novel complex rh rearrangements: two samples thought to be r r had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia( - )-ce. in kind, a sample genotyped as diii type rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce c( - )-diiia( )-ces( - )-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples and have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a -year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at g/l. her pregnancy history was not provided. she had received units of packed red blood cells (rbcs) in the past including unit within the last months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r r , r r , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse polymorphisms which determine antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a ) and lu(b ). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of antigens encoded by the kel gene, is organized into exons. there are approximately kel alleles associated with a kell null phenotype (k ) in which no kell antigens are expressed, and alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons , , , and and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k kp(a-b ) js(a-b ). kel-cdna sequence analysis was performed and detected a single transcript species with c. c, c. c, t, and missing the sequences corresponding to exons , and . amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel* allele. this donor was presumed to have a k phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel* m. . here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a and a cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c. deleted g, characteristic of o alleles, c. t, characteristic of a and some uncommon o alleles, and c. a and c. a, characteristic of b alleles. genomic sequencing of exons and confirmed the presence of an o allele, abo*o del g, t, t), and the presence of a b allele ( g, g, t, a, a, c, and a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* , tatiane vendrame , janaína muniz , diana gazito , afonso cortez , lilian castilho and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the -bp deletion in smim in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting and in samples with reactivity of . dna was isolated from peripheral blood and smim was sequenced. results/finding: from donor samples studied, were serologically vel negative by gel-iat but positive by adsorption-elution, presented a reaction and the remained samples showed a reactivity of . genotyping results showed that the samples with negative results and of samples that presented reaction were heterozygous for the bp deletion and presented the a allele rs in homozygous status. from the of remaining samples with reactivity of , ( %) had the a allele of rs and ( . %) had the a allele of rs . in contrast, in the samples with stronger reactions we found the a allele of rs in ( . %) samples and the a allele of rs in ( . %) samples. conclusion: the molecular changes rs and rs are located in intron distancing nucleotides. this study reinforces the association of the a allele of rs with reduction of vel expression and suggests the involvement of a new rs change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as (rh negative), or ! on the neo or ! on the echo (rh positive) for both series and series anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series and series anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in of ( . %) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in of ( %) of samples. of ( . %) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. of ( %) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining of ( . %) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but < on the galileoneo or positive but < on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* , tatiane vendrame , janaína muniz , rosangela person , lilian castilho , afonso cortez and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of samples were included in the study, being previously genotyped as rhd*dar . , rhd*dar . and rhd*dau . the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from - corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon . rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that of samples ( dar . , dar . and dau ) had rhd genes, were phenotyped as c e-c e and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in dar . samples showed the rhce*-cear/ce s genotype, in dar . samples the rhce*cevs. /ce s genotype and in the dau sample the rhce*ce s /ce genotype. table describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized f antibody (hu f -g ) that binds human cd has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu f -g (anti-cd ) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a year old female with progressive follicular lymphoma who was enrolled in phase b/ trial of hu f g in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a year old male with refractory diffuse large b cell lymphoma enrolled in hu f -g clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing ( with anti-a, non-reactive with anti-b) and the reverse typing ( with both a cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase ( to ), at liss- c ( to ), at liss-polyspecific ahg (m ), and at peg-anti-igg (m to ). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at o c ahg phase. the abo typing of the second case performed after anti-cd administration showed a discrepancy between the forward ( with anti-a) and the reverse ( with both a and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is ( ), at c in liss ( ), and liss-polyspecific ahg (m ). the dat and autocontrol were negative. his genotype was determined to be a /o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o. . / o. . , consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n ) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt and a galt. papain-treated patient rbcs were used to screen donor plasmas (n ) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with polyclonal anti-a,b and a monoclonal anti-b (clone g / ) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors or nor antigens. the patient was le(a-b ) and thus a secretor. a positive crossmatch was seen with % of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type or ) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $ years earlier. the reactions are likely due to uptake of recipient-derived ble b (type ) antigen (isbt no. ), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b ). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of . shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive ( ) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from / / to / / . reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of , columns run as part of antibody screens, , ( . %) columns generated "?" results. assuming seconds of technologist time per "?", we estimate that . hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table ) . in cases, all three columns were visually negative but the analyzer reported reactivity with of cells. all cases had mts-gel tm antibody identification panels performed, of also had a mts-gel tm ficin panel. the yield for the panels performed was two routine panels with weak reactivity against hla cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; were negative. one patient newly demonstrated anti-jka. fifty percent ( / ) of visually negative but analyzer positive samples were tested with gel card lot number , % ( / ) with lot , and % ( / ) with lot . conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from patients ( cross-match samples, units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in / patients. further testing was performed in / . eight were tested for the presence of antibodies at c and confirmed in / . rouleaux formation was observed in / patients, / had reactivity detectable at c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* , sandra nance , david moolten and p. dayand borge . ( ): - ). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this . year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from random allogeneic and autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing . % bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* , anna burgos , virginia lew , sunitha vege , susan veneman , christopher j gresens , jonathan hughes and connie m. westhoff . immunohematology and genomics laboratory, new york blood center, blood centers of the pacific, bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons - , and long range sequencing of exon - ( . kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- / ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m n-individuals (meyer et al. br j haematol. ; : - ) . the st a allele, also described as gyp* , is a hybrid gypb-gypatranscript with the crossover in intron . we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a years old pregnant, african american female g p was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a like phenotype. genetic testing did not support the serological findings of a subgroup and a new abo allele, abo* c that has never been reported in correlation with an a like subgroup was detected study design/method: the patient rbcs were typed with anti-a (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a antibody work up was performed using three different lots of a cells and three lots of a cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and c for min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo* c found in the mother. the previously reported abo* a allele encoded an aspartic acid to asparagine change at position p. consistent with an a weak phenotype. also, at least five other alleles encoding an a phenoytpe consisted of polymorphisms at positions c. through c. , giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a / aweak phenotype is encoded by the variant allele abo* c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since ; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n ) were processed with the automated biorobot m robot using the magattract dna mini m extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of . ng/ml and . respectively. in the second phase of the study (n ), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was - ng/ll and the recommended purity was an absorbance ratio of . - . (a / ) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was % concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also % concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano , izaskun apraiz , maría azcarate , miguel angel vesga , montserrat rubia , mercedes piedrabuena , fernando puente , barbera veldhuisen , ellen van der schoot and m onica l opez* . progenika biopharma, a grifols company, centro vasco de transfusi on y tejidos humanos, banco de sangre y tejidos de arag on, sanquin blood supply research background/case studies: it is well established that weak d , and phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type , rhd*weak d type , rhd*weak d type , rhd deletion, rhd*pseudogene and rhd*diiia-ce( - )-d and itgb gene: hpa a and hpa b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa- blood group typing. study design/method: a cohort of previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications / /ce for a ivd product of list a (! % clinical samples, > % neonatal specimens and ! % weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n , %). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa- predicted phenotype were used for comparison. transfusion results/finding: no system failure, % call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a % concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was %. the following id rhd xt predicted phenotype results were obtained: d negative (n ), no amplification variant (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d types , or not detected (n ). regarding hpa- blood group, the predicted phenotype results obtained by id rhd xt were % concordant with bds results: hpa- a positive (n ) and hpa- a negative (n ), hpa- b positive (n ) and hpa- b negative (n ). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types ( % specificity and % sensitivity for d antigen, hpa- a and hpa- b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value < . (clinical analysis) or < x - (gwa) was considered statistically significant. results/finding: of the cohort patients, ( . %) transfused subjects were included with alloimmunized children < years ( . % of ) and alloimmunized adults ( . % of ). in multivariable logistic regression models, age (or . , p . , for age compared to - ), gender (or . , p . , for female compared to male), transfusion history (or . , p< . , for transfusions compared to - ), site, hemolysis (or . , p . , for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or . , p< . ) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from newborns less than , gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on sample pairs. dat test was negative on sample pairs and two were positive. there was % concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on placental blood samples and heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < , g birth weight newborns. o-( . %), ab ( . %), b-( . %), and a-( . %). among the tested donors, . % were d positive with r r being the most common rh phenotype. in the kell blood group system, . % of the donors were k positive, while the k antigen was found to be . %. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a b ) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a b ) and m n-s s at % and . % respectively. the le a and le b alleles were seen in . % and . % of donors respectively, while lu b -phenotype was found in . % of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a b ) and lu(a-b-) were . % , . % and . % respectively, while the m n-s-s-and m-n s-s-phenotypes were not found. the frequency of the p antigen was found to be at . % similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph . and the supernatant was discharged. a dilution buffer containing % human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with ll of washed red cells. the cell suspensions were incubated for min at c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional min at c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for - nm. events were recorded at a frequency of cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* , mark yazer , nancy m. dunbar and biomedical excellence for safer transfusion (best) collaborative . university of vermont medical center, university of pittsburgh, dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of . study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a : dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a -year and -year period, respectively. one center provided plasma and wb testing data for a -year period. results/findings: in total there were group a plasma units tested and ( . %) had a high titer anti-b. the range of high titer group a plasma units between these three centers was . %- . %. of the wb units tested, ( . %) units had a high titer; / ( . %) of the units had a high titer anti-a, / ( . %) had a high titer anti-b, and / ( %) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with . m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd on reagent rbcs and render them free from plasma anti-cd drug interference. procedures for the preparation of . m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on . m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of . m dtt, was adjusted to ph . , ph . , ph . using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n ) were treated with the . m dtt solutions in parallel by mixing : ratio of packed rbcs to . m dtt solution followed by incubation at c. for up to minutes during treatment, the expression of k antigens was measured every minutes by tube method using different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every minutes for each ph level. the reduction in average scores between different phs were also calculated at every minutes to measure the impact of . m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by ! ) after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . complete loss of k expression was seen after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . the reactivity patterns of k antigen tested with sources of anti-k correlate with each other. the reductions in average scores were seen between to minutes range of dtt treatment time when ph . was raised to ph . ; to minutes range when ph . was raised to ph . ; and to minutes range when ph . was raised to ph . . conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to minutes and/or beyond minutes of incubation. the ph of the . m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of . m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n , ) drawn between july , and june , . these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, , patients were found to possess clinically significant red cell antibodies for an overall incidence of . percent. the three most commonly encountered antibodies were anti-d (n ) . %, anti-m (n ) . %, and anti-e (n ) with a frequency of . %. a total of ( . %) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with instances ( . %) followed by anti-e and anti-c with ( . %), and anti-c, anti-e with ( . %). of the multiple antibodies identified, ( . %) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with or . % of the total. the west had ( . %), the midwest ( . %) and the northeast with ( . %). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of . and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the . thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p . ). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* , lindsy rich , sherry stern , sharon wangen and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was to days. results/finding: of the ntd specimens from the immucor neo, resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all results, it was determined that there was no reactivity and a valid result was present. the other samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the absc specimens that were resulted out as positive on the immucor neo, specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: - with ms (n ), - s with ms (n ), no reaction with ms , dgc , p x (n ). samples tested with a polyclonal anti-c showed a - reactivity. d c e c e cases tested with a polyclonal and monoclonal anti-e (ms , ms , ms , ms ) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c. g>a mutation in exon , predicted to encode the p.gly ser substitution. for apparent r r donors, a f-negative type allowed the prediction of a rhce*ce a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce a allele (c and e in cis) in all samples. d c e c e individuals were reactive s with the original source of anti-rh , slightly weaker when compared to rhce*ce a/rhce*ce rbc samples available from our cryobank ( ). conclusion: our results confirm that the c. g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh ). the locr reactivity appears to be rather similar when coded by rhce*ce a or rhce*ce a alleles. this was quite an unexpected finding, since the p.gly ser substitution is close to the critical amino-acid for c/c expression (p.pro ser). none of our cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce a was reported to code for a partial c (rh:- ), we consider that rhce*ce a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes , or . the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d , , or genotypes. study design/methods: between / and / samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types , , or genotypes, but a transfusion vol. supplement s had evidence of rhd genetic sequences in exon and/or intron in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons - to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients ( %) followed by transfusion patients ( %); % had no clinical indication provided. samples ( %) were found to be weak d type , , or ( , , and samples, respectively). samples ( %) appear to be genetically rhd negative. genetic sequencing was performed on samples; had rhd genetic variants that were not weak d types , , or (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples ( %) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types - . of the samples that had evidence of an rhd gene and did not carry the known weak d types - polymorphisms, ( %) of were found to have other rhd variants, and ( %) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types - variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel , curr opin hematol : ) , that individuals with weak d types , , and are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report months experience with rhd genotyping on samples referred with discrepant or weak d typing investigated from january to april . study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for samples ( . % caucasian, . % african american/african, . % multiracial, . % hispanic, % asian, and . % other). results/finding: rhd genotyping identified weak d types , , and in / ( %) and alleles known to encode partial d phenotypes in / ( . %) (table) . uncommon or rare weak d alleles including types , , , , , , (n ), , , , , and were found in ( . %). the partial d alleles found were diverse, but the largest number included partial rhd*d . (n ) and *dar ( conclusion: in a multiracial cohort of individuals with weaker than expected d typing % were due to weak d types , , or and would not be considered at risk of clinical significant anti-d, but for % there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp rhd* . allele causes discrepant genotyping results for rhce small c sabine scholz* , sandra schneider , sabrina k€ onig , susanne helmig and vicky van sandt . inno-train diagnostik gmbh, rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c ( c) and c ( t) is caused by the snp on position on the rhce gene. the rhd* . allele (also known as rhd cat vii type ) carries the snp t>c on the rhd gene and additionally the snp t>c. this rhd* . allele has been described to partially express rhc on the d polypeptide (faas, transfusion, ) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, ) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles ( t>c, t>c) confirming a rhd* . and one rhd* allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series and anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series and series , and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c. a>g change in exon encoding an amino acid change p.met val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c. a>g (p.met val) change in exon . several snps, deletions, and insertions have been reported with changes in exon . phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c. a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* , juan merayo-rodriguez , christopher lough and nancy eckert . lifesouth community blood centers, life south community blood centers, lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is % in most populations and greater than % in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is . / . and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal (k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to . / . the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient was a yo female, c e c e , whose rbcs reacted by echo and by neo with anti-d , and '?' with anti-d . testing with d and d by tube gave and w on initial spin (is) respectively and by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and w with immucor gammaclone anti-d, and all were at iat. rbcs did not react with two (lhm / & / ) of anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c. c>a change encoding p.his gln. patient was a yo pregnant female, c e c e , whose rbc were w at is and at iat with immucor series and and gammaclone, and moderately reactive, is and iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm / & / ) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon from c. to c. encoding amino acid changes p.ile leu and ser asn. conclusion: we found two previously reported rare alleles: rhd with a c. c>a (p.his gln), previously found in france (lefloch et al. genbank ku ), and rhd*dar with part of exon replaced by rhce, reported in sub-sahara africa (granier et al. transfusion : ) designated rhd*dar(ce :v v-s n) with an allele frequency of . to . . blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu f -g is a human monoclonal igg antibody recognizing cd that is in clinical trials to treat hematologic or solid malignancies. cd is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd is thought to enhance phagocytosis and promote anti-tumor responses. cd is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n ) from patients were tested over the course of month treatment. plasma was tested at immediate spin (is) and by iat with r r , rr, d--, rh mod and rh null rbcs, as cd expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg ) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd was observed in plasma as soon as hour post infusion. plasma reacted to at is and with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker ( and ) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was . in contrast, iat reactivity using gamma-clone anti-igg was only w to , and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd titer was at is and peg iat with gamma-clone anti-igg, but was ! with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n ) were reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after x allo-adsorption with papain treated rr cells, but in some samples low level (micro- ) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu f -g anti-cd therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu f -g is igg . reactivity was observed in all phases and with all test methods. cd is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg , can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in . in august , she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was with untreated and with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:- etc. had been excluded, k-phenotyping revealed a k -phenotype. a total of silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel* n. with c. t encoding p. ter (reported in an individual from austria in ). there are two known k -patients in our country, both homozygous for c. t. the daughter was a c. t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k -donors are available in our country. with the help of the isbt rare donor working party, a k o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* , kshitij srivastava , houda romdhane , saloua jemni yacoub and willy albert flegel . nih, dtm/cc/nih, regional blood transfusion center sousse, regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, a transfusion vol. supplement s since . the tunisian population has the largest known prevalence of weak d type . alleles, occurring in of rh haplotypes, compared to in , or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type . in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of , random blood donors were serologically screened for the d antigen using routine techniques. samples with weak reactivity were tested with a panel of monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of discrepant samples ( . %) were observed and expressed the serologic weak d phenotype. among them, carried an allele of the weak d type cluster ( . %), of which samples ( . %) showed the weak d type . allele. only sample each was found for the weak d types , and and the dvii, while samples showed the consensus rhd sequence. no mutation in any of the rhd exons was detected in another samples. the molecular analysis of the rhce gene showed that out of samples with serologic weak d phenotype ( . %) had a variant rhce allele and the most common associations were: weak d type . linked to rhce*cevs. . ; weak d type . . with cear; and weak d type . to rhce*cevs. , while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost % of the weak d phenotypes in tunisia were caused by alleles of the weak d type cluster, of which % represented the weak d type . allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type . in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type . phenotype. there is a possibility that the rhce*cevs. . allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* , heather simmons , christine lomas-francis , gayane shakarian , sunitha vege , lauren hutelmyer , sandra nance , jessica poisson , nicholas bandarenko and connie m. westhoff . duke university hospital, immunohematology and genomics laboratory, new york blood center, arc pennjersey, american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a , year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with . m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c , k , fy(a ),s ]. reactivity was detected to a titer of ; it was not removed by prewarm technique or by x peg alloadsorption. the adsorbed plasma reacted with . m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k , k , js(b ), kp(a b ) and sc: , . her plasma reacted with k o , mcleod, sc: , rbc samples and dtt-treated sc: rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused aliquots of crossmatch incompatible kp(b ), s rbcs. her post-transfusion dat was with anti-igg, with anti-c d. the eluate reacted with all rbc samples except kp(b ) sample. she tolerated additional aliquots from phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k , k , kp(a b ), js(a b ) and sc: , , discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c. a>t (p.glu val) (kel* . ) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a ). sc sequencing found heterozygosity for a '- g>a change (rs , to % prevalence) and conventional sc* , predicting sc: , , . kel and sc results on the mother were kel* /kel* . , heterozygous for the sc change '- g>a, and her rbcs typed k k kp(a b ), sc , ula , consistent with dna predictions. plasma collected months later was nonreactive at rt and in peg iat. her rbcs were dat and now typed k , kp(a b ), ul(a ) sc and sc ,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel* . homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc: , rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b ). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a -year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs - a)genotype, associated with a jkb null phenotype. anti jk was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a ) and jk(b ) . genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk . however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms a transfusion vol. supplement s (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a b-); kp(a b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon revealed a g>a mutation, fy *n. , known to silence fya. sequencing of kel exons - exposed a silent polymorphism in exon , g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy *n. mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly % of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have genetic variants not previously reported in those of african descent. only was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient . study design/methods: patient was a year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at years of age. anti-b changed from undetectable/weak to strong at the age of years. patient was a month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was / . both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient with - reactions up to years of age. thereafter, abo typing showed mainly strong anti-b. patient had / anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for children on long-term tpn. patient had absent/weak anti-b since birth up to years of age, then developed strong anti-b with no change in feeding regiment and medications. patient had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* and elizabeth hart . brigham and women's faulkner hospital, university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title , cfr part . (a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in . a total of samples were evaluated. each abid was subcategorized; ( ) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and ( ) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of abids were performed on new patient samples. of the new abid samples, ( %) had microscopic absc results. for the previously known antibody patients, there were which accounted for % of the total abids performed. when reviewing the total abid workups, a total of ( %) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the new antibody samples were: conclusion: a total of % of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: ( ) discontinue the use of the microscope, ( ) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or ( ) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than % in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a -yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a -year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only ( - ), and negative with anti-c b, c d reagent. the antibody showed a peak gel-igg iat titer of . results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w ), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from to %, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody anti-p anti-m anti-sd a anti-le b anti-jk a anti-k anti-e anti-c results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of % (type ), % (type ), % (type ), % (type ) and % other than , , or . further investigation was conducted to determine the molecular identity of the «others». out of samples, ( %) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon or both exons and . a surprising amount of samples were discovered to be normal rhd. conclusion: along with sandler et al. ( ) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types , , and obtained in serological weak d, years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was , %. no trali happened in the period. prophylaxis were used in % of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p < . ). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) ( . % vs . %, p < . ) or aes with a non-allergic type inflammation etiology ( . % vs . %, p < . ) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes ( . % vs . %, p . ) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for , , , and weeks were . %, . %, . % and . %, respectively (p . , logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week ( . %) and longer than a week ( . %) (p < . ). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found patients from - , who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these cases, only of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as out of transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was . g/dl but declined to . g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days and with poor responses. on day , routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from . mg/dl to . mg/dl (reference . - . mg/dl), and lactate dehydrogenase was above reportable linearity, > u/l (reference - u/l). testing revealed additional anti-e, anti-jkb, dat c , plasma free hb . mg/dl (reference - . mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b ), one of which was also e . one volume tpe was performed to remove free hb on days , , and using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at . mg/dl on day , decreased to . mg/dl before discharge on day results/findings: twenty three cases were identified, of which had medical records available for analysis. ten ( %) patients were male, the mean age was . years (range - years), ( %) had an underlying hematologic malignancy or bone marrow disorder, and ( %) had a history of coronary artery disease (cad). the implicated units included ( %) red blood cells and ( %) platelets; ( %) patients received a single unit, and ( %) received two or more within the previous hours; the mean volume transfused was . ml (range - ml). the mean time to onset of chest pain was . minutes (sd minutes), with % of patients presenting within . hours and % within hours of starting the transfusion. chest pain was present as the only symptom in % of the cases, and for the other cases the accompanying symptoms included dyspnea ( %), fever ( %), back pain ( %), and hypo-and hypertension ( %). a post-transfusion chest x-ray was performed in % of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in % of cases and showed no findings to suggest acute ischemia. three ( %) patients had a minimal increase in their troponin levels, although had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen ( %) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than minutes in the majority of patients ( %). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts . based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, degree, or anti-human globulin phase. check cells were found to be . these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o to a ) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a -year-old o man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha- antitrypsin deficiency who presented for olt (donor o ). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving units of o rbcs and units of o plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a plasma were transfused to wash out anti-a antibody prior to transfusion of a rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for hours post-transplant. the patient's total estimated blood loss was > l. he received a total of units of rbcs (including a ), units of plasma (including a ), units of cryoprecipitate, and units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o . on postoperative day (pod) , a ts showed predominantly a rbcs with trace o rbcs, as well as very low anti-a igm and igg titers (table ) . he received two additional o rbc units ( each on pod and pod ) with increasing o rbcs on ts and rising anti-a titers. his blood type was unequivocally o by pod . the patient showed recovery of liver synthetic function on pod (factor activity %) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod , the patient had reverted to o with recovery of anti-a titers. at months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* , alfred mingo , maria isabel gonzalez , antoni mena and juan pedro benitez . bst, at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h ) and an oncology center (h ). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of transfusion activity in both h and h shows pse and bca, out of and respectively, since the tss deployment in . retrospective analysis and classification of security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h , nm related to pse accounted for . % of all, being the mistake in concordance between patient identification and prescription order the most frequent ( . %). the nm detected in bcas were . % of all and mostly ( . %) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are . % of all and mostly ( %) the systems detects a not assigned bracelet. for h , nm related to pse accounted for the . % of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent ( . %). the nm detected in bcas accounts for . % of all and in . % occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are . % of all and in . % of them the blood components were assigned to another patient. ( , , , , , , , , , , , , , ) were analyzed via a commercially available elisa. comparison of adequate response to ppv , defined as ! mcg/ml for > serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in patients (alloimmunized, ); pre-and post-vaccine titers were available for patients (alloimmunized, ). of the patients, were on chronic transfusions, were on hydroxyurea, were surgical splenectomized, patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv in the previous years; / also reported previous history -valent sp conjugate vaccine within the last years. baseline pre-vaccination titers (n ) showed no difference between alloimmunized and non-alloimmunized patients (all p-values > . ). in the group with pre-and post-vaccination (n ) titers available, out of ( %) non alloimmunized patients had an adequate response versus out of in the alloimmunized group ( %, p ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from to ). patient harm events recorded within the veritas system from january to july were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of tres per month and per year were found over five years. % of tres are associated with pre-bb activities, % occur within bb, and % are post-bb events. sample collection and handling represent % of total tres. most tres ( %) were reported by bb staff, % were reported by non-bb staff. patient harm analysis revealed an average of four level (near miss), three level (no known harm), and . level (patient harm) per month. no deaths related to tre were detected over the seven month january to july period. patient harm was associated with tres occurring in the bb ( %) and post-bb ( %). these events were reported externally ( %) and by bb staff ( %). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january , and march , was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by and added to the number of individual whole blood plts; apheresis plasma units were multiplied by and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was . the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the -hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions ( [ /l vs. [ /l, p . ). there were no significant differences in the frequency of effective therapeutic ( % vs. %, p . ) and prophylactic ( % vs. %, p . ) transfusions between the prcs and gypcs. we did not find significant differences between prcs and gypcs in cci after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . . ) transfusions, in cc after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . .) transfusions. there were no significant differences between prcs and gypcs also in ma after prophylactic ( . . vs. . , p . ) and therapeutic ( . . vs. . . , p . ) pc transfusions. reduction of the severity of bleeds was obtained in ( %) of the cases after prpc transfusions and in ( %) of cases after gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, and cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in . it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were units administered using the emergency transfusion process in the months before the change was implemented. it was found that / ( %) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month ( components administered), / ( %) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: ) a stable anemic inpatient, ) a stable anemic inpatient to be discharged, and ) an asymptomatic post-operative inpatient. results/finding: we identified canadian tm experts: ( . %) provided a response and most had a primary place of practice in a laboratory setting ( / ; . %). for a stable, non-bleeding, anemic inpatient, . % of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with . % generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period ( - hours), a repeat hemoglobin > hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse ( . %) compared to an inpatient potentially symptomatic due to anemia ( . %). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium- ( cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n ) were divided into two ml aliquots, which were labeled with selected concentrations of s-nhsbiotin ( and lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of . . %). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that nm excitation of phycoerythrin (pe)-sa and high laser power ( mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with lg/ml of biotin resulted in $ , mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, % ci) of in , ( . %). the lld for rbc labeled with biotin at lg/ ml was $ in million ( . %). biotinylation was not associated with increased levels of hemolysis ( . . % before labeling versus . . % after labeling; p . ) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes ( ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer- (hboc- ) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: ( ) wb, ( ) wb % hboc volume (model of two units in an adult), ( ) wb % fdp, and ( ) wb % hboc % fdp. samples ( )-( ) simulated autoresuscitation by adding % plasmalyte to - . susceptibility to lysis was tested with ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of %, %, %, and % volume replacement with hboc and/or fdp, with or without prior % plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb hboc, and wb hboc fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb hboc in autodilution simulation (mean lysis . % vs. . %; p<. ). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc ( %) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even % hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc- , there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in / and / . we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of patients, were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery ( %). pcc was given for warfarin reversal in % of cases. a subset of patients received plasma within hours prior to pcc ( %) or hours after ( %). pcc was most frequently ordered in the or/perioperative service ( %). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in , several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; ) simplify and expedite the process; ) improve communication and expectations to decrease tat; ) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood ( units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of er episodes were received. the average tat from order to delivery at the bedside was reduced by % ( . minutes compared to minutes previously), while the compliance rate for er orders and physician documentation was % ( / ), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february -february . mt was defined as the transfusion of ! rbc units in a -hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at days. results/findings: thirty mts occurred during a one year period. a total of , blood products were transfused during that time period. gender distribution was / ( %) males, and the average age of all patients was with a range of to years of age. surgical patients accounted for / ( . %) mts, and / ( . %) were critical care patients. tumor categories included carcinomas ( / ), sarcomas ( / ), leukemias ( / ) and lymphoma ( / ). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer ( / ) was the most common disease seen followed by sacral chordoma ( / ). mtps were activated in only / ( . %) cases. thirty-day survival was seen in / ( . %) patients. only of mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage ( / ) or perisplenic hematoma ( / ). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a -year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a -year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a -year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* , , rebecca ross , christopher a tormey , , and amit gokhale , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: daratumumab (dara) is a igg monoclonal antibody therapy that specifically targets cd , a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, subjects were identified for analysis. their mean age was . years, with male and female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of % ( / ) prior to administration of dara. of these patients, were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these ( %; / ) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september , through april , were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in , , unique women receiving a total of , units of red cells (rc) in , transfusion episodes were identified. their median age was (range - ). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in ( . %) and ( . %). ( . %) transfusion episodes were associated with the use of units or more rc. as a result, ( . %) episodes resulted in a post transfusion hb ! g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given (< . %) and ( . %) women respectively. upon discharge, ( . %), ( . %) and , ( . %) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to ( . %) women. conclusion: in the present study, it is observed that . % transfusion episodes were given at hb ! g/dl. a substantial number of episodes ( . %) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! g/dl). for iron replenishment and bleeding control, up to . % transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes ( . - . x per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with gy and transfused over - hours within hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* , lee grabner , brenda herdman , robert fallis , amin kabani and charles musuka . canadian blood services, kenora rainy-river regional laboratory program, diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between and before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and % were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels ( . g/dl; p . ); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units ( . vs . g/dl; . vs. . g/dl; both p . ). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients ( . g/dl vs. . g/dl; p< . ). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p . ). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p . and p . , respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than % of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered times in total, affecting patients and users. stratified by location, the majority of triggers occurred in the perioperative areas ( times) and the liver icu ( times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin % iv solution, human albumin % iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* , , mahmut akgul , , hollie m reeves , , robert w maitta , , marcie pokorny , anne capetillo and katharine a downes , . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a -month retrospective study (january-july ) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was - mg/dl with critical value of mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were adult (> years) orders reviewed by tms out of which were approved. of the approved orders, ( . %) were in agreement with tms's estimated dose. of ( . %) orders that were not in agreement with the tms's estimate, ( %) were underestimated and ( %) were overestimated. seventeen of orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of . mg/dl (range . to mg/dl) and a median excess of . mg/dl (range . to mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was mg/dl above target, which is significantly higher with intervention than without (which could have been mg/dl below the target; p< . ). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention ( vs. . mg/dl, p . ). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention ( vs. - . mg/dl, p< . ). seven of ( ) ( ) orders were for critically low fibrinogen (< mg/dl) and of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were and units ( . % and %) i.e. and pools and the most frequent orders in the disagreement group were , , and units ( %, %, % and %). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was . and posttransfusion was . . only % of patients had their inr corrected to . , while % had no change, or had increased inr. (table) . the majority ( %) of patients received units of plasma. the mean plasma dose was ml/kg. there were transfusion reactions reported, non-hemolytic and transfusion associated circulatory overload reactions in which required admission to the icu. two patients experienced bleeding during ir procedures (tips) and developed a hematoma (tunneled central line). the median of inr correction in this study was . with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is . . randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. of the patients experienced bleeding complications indicating that inr of . may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately % of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population ( %) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately % of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july , as follows: units of group a plasma and units of group ab plasma is provided for the massive transfusion protocol (mtp) along with units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed mtps at our institution between july and march . twenty patients ( . %) were transfused with incompatible group a plasma ( group ab and group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april , our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in , bonfils immunohematology reference lab (irl) sent out approximately special platelets for patients with hla antibodies. by , hla platelet orders increased dramatically and the irl sent out over special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over , donors in the database with historical hla typing. however, only approximately of those donors actively donate. in the denver area, one of the most common hla types is a a b b . only of the , donors have this type ( . %). therefore, to fill an hla platelet order request for a common hla type, only donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a a b b , there is only out of , donors ( . %) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a for example, all of the a positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in , approximately % of these special order platelets were pra matched and the remaining % were hla matched by donor recruitment. by , approximately % of special platelets sent are pra matched. this change resulted in a . fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains packed red blood cells (prbcs), fresh frozen plasma (ffp) units, and plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august to march mtps were activated at ucm, of which orders could be traced to the origin: on inpatient floor (including icus), in the operating rooms, in the emergency department, in labor and delivery, and in other procedure rooms. of the prbcs that were issued, were transfused ( % utilized); of the units of ffp that were issued, were transfused ( % utilized); of the platelet packs that were issued, were transfused ( % utilized); of the units of cryoprecipitate that were issued, were transfused ( % utilized). since march , the time of first product issue after the initiation of an mtp has also been tracked. of the events that fall within this time period, ( %), had the first product issued in minutes or less. another ( %) were issued between - minutes, resulting in over % of patients being issued their first blood product within the first minutes. only of ( %) events had an initial time greater than minutes and none were greater than minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($ - %). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* , shailesh macwan , arline stein , jane fischman , nancy nikolis , matthew bank , lennart logdberg , alexander indrikovs , sherry shariatmadar and vishesh chhibber . north shore university hospital, northwell health background/case studies: massive bleeding is generally defined as any patient who requires blood volume replacement within hours and/or receives transfusion of greater than or equal to units in one hour with a transfusion vol. supplement s ongoing bleeding. our mtp was officially implemented in in preparation for an initial verification as a level trauma center by acs. our mtp has the following packages: st pack has a ratio of : : (rbcs, plasma & platelets) and subsequent packs a ratio of : : . our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march to add cryo and pcc at a defined point in the mtp (cryo is included in the rd pack and pcc in the th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received > rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had patients who received > rbc in and . mtp had been activated for all patients and all patients received between . to unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt > seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of mg/dl (table ) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of > rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's -day and -day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective -year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of published cases ( reports) of ta-pls, ( reports) were stem cell and ( reports) were organ transplants. all ( %) stem cell transplants and ( %) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the reports of stem cell ta-pls, actively screened for antibodies in the immediate post-transplant period, and of the reports of organ ta-pls, actively screened for antibodies. these screens detected cases of stem cell ta-pls before hemolysis became apparent and cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* , rosario mallari , marc de asis , elaine shu , jonathan hughes and tho pham , . stanford university, stanford health care, stanford blood center, bloodsource background/case studies: mur antigen is present in - % of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received - rbc units every - weeks to maintain a hemoglobin (hb) level of g/dl. the patient remained stable for months when his hb level acutely dropped to . g/dl. the antibody screen remained negative for an additional months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for of the rbc units he received. units were from an asian donor, and a unit transfused days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of , donors at a hospital-associated blood center located in a region where asians comprise approximately % of the population. results/finding: . % of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for of ( . %) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over % of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* , princess maynie , carol chandler , shelia garret and pampee young . vanderbilt, vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [ ] [ ] [ ] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of samples have been analyzed (table) , rh and non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being . (range - ) times higher. the average fold change for rhd/c/e antibody titers were . , whereas the average fold change for non rh titers was . (range - ). the range for anti d titers was particularly variable, - , whereas for c/e, it was - . the overall reproducibility/precision of the automated analyzer was $ %. to correlate the a transfusion vol. supplement s increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $ times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of . units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to % of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately % of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix . % suspension of a and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, group a, and group b. results/finding: of the whole blood edta samples tested, ( group o and group b) exceeded a high titer threshold of . when the pas samples of these donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of , a % decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the ( %) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these hospitals had a lower overall rate of wastage including their own donations than the other hospitals that did not collect in-house plt. the other hospitals had wastage rates ranging between - %. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* , , christopher j gresens , anne capetillo , hollie m reeves , and katharine a downes , . case western reserve university school of medicine, university hospitals cleveland medical center, bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of < . %. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a -year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure ( / mm hg to / mm hg) followed by hematuria ( ml). chills and rigors resolved; blood pressure stabilized after min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of (igm) and , (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* , leila patricia de sousa fontenele , isabel nagle reis , carolina bonet bub , araci sakashita , raffael zamper , cristiane nakazawa , tatiane almeida omura paula , patricia silva batista , marcio dias almeida , fernanda loureiro de andrade orsi and jose mauro kutner . hospital israelita albert einstein, hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of cases of olt performed between and in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of consecutive patients submitted to liver transplantation between and were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or , - % ci: , - , , p: , ), absence of hcc (or , - % ci: , - , , p: , ), cirrhosis of any cause (or , - % ci , - , -p: , ), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or , %ic , - , ) and retransplantation due to primary non function of the graft (or , %ci , - , , , p: , ) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a /b in group o platelet products. charles k. childers* , mark destree , ashley rose and theresa nester , . madigan army medical center, bloodworks northwest, bloodworks nw, dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a /b in group o platelet products is presented from a large regional blood center collected over - months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in ml edta sample tubes. a single : dilution of plasma was prepared using a hamilton microlab series dilutor using . ml saline diluent and . ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at rpm for seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of is used, approximately % of group o apheresis platelets will have a high titer, most commonly with anti-a . less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their day outdate. after months of testing pspp units and verifying that the products rarely had a high titer ( . %), the blood center stopped performing this testing for pspp units. rh ) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to % (from % on the previous day). his hematocrit did not increase ( %), and over the ensuing hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of % while reducing the number of a rbcs in his circulation by approximately %. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes - days for antibodies to develop and they are short-lived ( - weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the calendar year from hospitals. an additional hospital* provided data for august-december . rbc transfusions in patients < year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/ years was also determined. results/findings: see table . the fraction of all transfused rbcs that were oneg ranged from - % (row f). the percentage of oneg rbcs transfused to oneg patients ranged from - % (row g); thus, non-oneg patients received - % of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients ( %- %; row i). overall use of oneg rbcs could have been reduced by %- % if opos units had been given to all oneg patients >/ years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* , nehad mohammed , marwa aly and nashwa fahmy . national blood transfusion services, nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on multitransfused patients who received blood transfusion chronically at our central blood center. they were thalassemia patients ( bthalassemia patients, one patients with a thalassemia), sickle cell anemia patients and immune hemolytic anemia patients ( auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). oncology patients, chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: out of ( %) alloimmunized patients and out of ( %) non alloimunized patients(p< . ) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june . screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified articles for data abstraction, where articles were transfusion guidelines. there were guidelines ( %) that made a recommendation, for a single unit and for multiple unit transfusion strategy (table ) . review b identified retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or . , % ci . - . ), although heterogeneity was high (i %). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december and february was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the months, units of platelets were transfused to recipients. over the months, a median of units was given to each patient with a range of to . the overall distribution of products used was % plasma, % pr, % pas f and % pas c. thirty percent of patients (n ) received all of their products on a single day. single units were given to patients while , and received , , and units respectively. the distribution by product type was % plasma, % pr, % pas c and % pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the month period (p . ). the distribution by service was different for the groups receiving multiple units. for single units the distribution was % hematologic malignancy, % infusion clinic (nos), % solid tumor medicine, % surgery, and % pediatrics. for those receiving multiple units the distribution was % surgery and % each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of . . conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* , , yvemarie b.o. somsen , maike e. van hezel , marleen straat , robin van bruggen and nicole p juffermans . sanquin research and landsteiner laboratory, academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains mg of iron and % of the rbcs are cleared by macrophages within hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in icu patients who received one rbc transfusion, different iron parameters were measured before and hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il- levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion ( . vs. . mmol/l, p . ). also, the transfusion had no effect on transferrin saturation ( vs. %, p . ), ferritin ( . vs. . mg/l, p . ) and il- levels ( . vs. . pg/ml, p . ). hepcidin levels increased in these icu patients after rbc transfusion ( vs ng/ml, p . ). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (- . vs. . % change, p . ). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il- or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since and pathogen reduced platelets have been available since . in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between and . during this time pas c, pas f and pr went from % to % of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in patients received an average of . units/recipient/month and in the average was . units/recipient/month. the intervening data points for , , and were . , . , and . respectively. the year average was . . the slope of the graph for all points was y - . . . the two sample t-test showed that the plt/recipient/month from to was not statistically different with a p value of . . conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that % to % develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least k/ul. the analysis revealed that the mean of . k/ul (n ) had a percent lower bound confidence interval platelet increment of k/ul (p< . ) results/findings: (median range [ - ]) hla matched leucoreduced irradiated sda platelets were transfused to ( m/ f) patients, median age years (range - ). / ( %) patients showing broad alloimmunization to hla class i/class ii antigens. / ( %) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority / ( %) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); / ( %) female patients had prior exposure via pregnancy and / ( %) had a history of hsct. ( %) platelets were abo identical-platelet increment median k/ul (range - to ), ( %) were abo compatible -platelet increment median of k/ul (range - to ) and ( %) were abo incompatible with platelet increments median k/ul ( range - to ). platelet counts were performed within hours in ( %) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* , renee leblanc , dongfu xie , alice cabe and yanyun wu . overlake hospital, bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about for years (from to ) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than . % of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a -year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of . g/dl (baseline - g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was . g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin ( . to . mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to . g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october to march . patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were rbc products transfused to waiha patients. twenty-three ( . %) patients received at least incompatible unit. the mean age was . years (range - yrs) with % women. ethnic composition was % african-american, % caucasian, and % patients of mixed/other ethnicity. one hundred fourteen ( %) of these products were released as li products and ( %) were compatible. ninetythree ( . %) of the li product transfusions had a post-tfn hct change of < % whereas only ( %) of the compatible product transfusions resulted in a post-tfn hct change of < % (p . , v ( ), exact methods). the mean hct increase in the compatible group was . % per unit vs. a slightly lesser per-unit increase of . % in the li group (p . , t-test, -tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for units. units that were incompatible had a lower mean post-tfn hct rise compared to all other li units ( . % vs. . %); however, this difference was not statistically significant (p . ). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected % per unit more frequently than it was for waiha patients who received compatible products ( . % vs. %). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first -hours from mt onset) was calculated with % and . % control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were mt cases from hospitals ( tertiarylevel, smaller/medium sized acute-care and specialist women's). number of mt cases per hospital ranged from to . patient median age was years (iqr , ), % were male and % required admission to intensive care. the most common clinical groups were cardiac surgery ( % cases), trauma ( %) and gastrointestinal hemorrhage ( %); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the tertiary-level hospitals was % (range % to %) and / ( %) were within the % control limit. cb that required ! rbcs within -hours of mt onset occurred in % of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals ( % versus %; p . ). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of bcs and ml of platelet additive solution iii (pasiii) were used to produce pcs (n ). pcs were stored on a flatbed agitator ( cycles/min) in a temperature-controlled cabinet at c for - days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of mm hg was applied. using clamps, a flow velocity of - ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted - %, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration ( . - . x /l) and number (> x /unit). simulated transfusion had no effect on the percentages of cd p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* , , joan sevcik and joseph e. kiss , . university of pittsburgh, blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution : is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient and and (control). results/finding: all patients were ab blood type. patient , a year old female with recurrent adamts deficient ttp, received courses of tpe (total tpe procedures) for relapse and exacerbation. ten out of procedures were performed with ab and a plasma (average ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of tpe procedures. four out of procedures were performed with ab and a plasma (average . ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with adamts deficient ttp who served as a control, received a total of procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between patients. the trends of hemolysis laboratory data for patient and were comparable with patient . all patients had negative dat. only patient received rbc transfusions. all patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to % of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd negative platelet unit is not available for a patient with anti-cd antibodies sameer khatri* , charles harmon , brian r curtis and chisa yamada . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd ) is one of the identified plt surface ags and deficiency is rare, but found in asians ( - %), sub-saharan africans ( - %) and also in some people from mediterranean descent. two types of cd deficiency have been described. type deficiency is the complete lack of cd on both plts and monocyte-macrophages whereas type deficiency lacks cd on plts with variable expression ( - %) on monocytemacrophages. transfusing plts in a patient with cd deficiency is challenging given the rarity of cd negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than units of apheresis plt units over a week period without any significant increase in plt count. cross-match compatible plt unit found in of units and hla matched units were tried without success. at that point, a cd ab was identified in the serum and the patient's type cd deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was % due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd- negative (but blood type different and hla a transfusion vol. supplement s unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd antibody positive reactivity in serial dilutions has reduced from : to : dilutions and his hla class i pra has decreased to %. he is currently receiving apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to . k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec , , blood donations were screened for b microti by immunetics elisa. of those, ( . %) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between . % and . %. no patient babesia transmission has been reported since implementing this test, but we only had documented babesia ttd cases from - . donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of positive test results, lookback investigations were initiated representing % of positive donations. lookbacks were only performed when there was a donation within months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to % were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented . % loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only . % of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in > % of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for blood donors, were positive with an average load of . x copies/ml of plasma, a median value of . copies/ml of plasma, ranging from to . x copies/ml. pre-transplant viral loads were similar. for transplant candidates, were positive with an average of . x copies/ml of plasma, a median value of copies/ml of plasma, ranging from to . x copies/ml. post-transplant viral loads were remarkably different. for transplant recipients, all were positive with an average of . x copies/ml of plasma, a median value of . x copies/ml of plasma, ranging from to . x copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around - copies were present in > % of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt ( ) . post-ivig: all plt ( ) . post-ivig: cd -negative plt from relative ( ) . post-ivig: single donor apheresis ( ) . post-ivig: cross-match compatible ( ) . post-ivig: flow cross-match compatible plt ( ) . a transfusion vol. supplement s detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv ( / / / ), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows % of specificity, with no false positive results on the control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of tcid /ml for denv- , denv- and chikv and of tcid /ml for denv- , denv- and zikv. finally, the first results obtained on denv( ), zikv( ) and chikv( ) clinical samples show %, % and % correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of , , and were prepared from plasma or serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from : to : ( -fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of or higher and was not eliminated by the addition of a blocking step. pools of or samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of or samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $ cfu/ml of organisms associated with platelet contamination and incubated at room temperature for - hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher ( log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc , were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta d and virtuo the organism was recovered % . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc . however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of ml lrap demonstrated % recovery when loaded onto the virtuo and d ( table ) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus - , and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in , a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as- rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and hrs after amustaline addition, respectively, for titration by plaque assay on vero cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, > . log or log /ml of rrv was achieved, with > . log or > . log /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; - ml), large volume (lv; - ml) and dual storage containers (ds; - ml) designed to treat platelet doses between . and . x . the new triple storage (ts) set was designed to expand the dose range to . x and the maximum volume to ml, generating either or doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or % plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in % plasma/ % pas or % plasma with a final volume of $ ml and a dose of - platelets. these conditions represent inactivation using the lowest amotosalen concentration ( mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of - logs in respiratory secretions of mers patients, and with lower genomic titers of - logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log reduction/ml (log /ml) %plasma/ % pas e .coli . <- . > . e. cloacae . <- . > . k. pneumoniae . < . > . s. aureus . <- . > . blue tongue virus . <- . > . bovine viral diarrhea virus . <- . > . adenovirus- . <- . > . %plasma k. pneumoniae . - . > . s. aureus . <- . > . adenovirus- . <- . > . n a transfusion vol. supplement s abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged times up to days, assessing the infectious titer and genomic titer every rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of ! . log infectious titer. no viral replication was observed after days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* , jen-wei chen , chi-ling chen , sheng-nan lu and pei-jer chen . department of research, head office, taiwan blood services foundation, graduate institute of clinical medicine, college of medicine, national taiwan university, division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by , and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since ) and -sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since ) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during - and - , respectively. age-standardized prevalence and its % confidence interval ( % ci) were calculated with adjustment of who world standard population - . for the incidence study, donors who have donated blood two or more times during - and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its % confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ) during - , and the agestandardized prevalence was also decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ). the agestandardized prevalence of anti-hcv was generally higher in female donors before , but it was significantly higher in male donors at (p-value . ). a total of , hcv rna positive cases, . % of them were anti-hcv negative, identified from , first-time donors during - , and the crude and age-standardized prevalence of hcv rna was . per , ( % ci: . - . ) and . per , ( % ci: . - . ), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value < . ), but no significant difference was found after age standardization (p value . ). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend< . ). in the incidence study, a total of new hcv rna positive cases, . % of them were anti-hcv negative, found from , , donors followed for , , person-years. the incidence of hcv rna was . per , person-years ( % ci: . - . ), and no significant difference was observed between both genders (p-value . ) and between age groups (p for trend . ). conclusion: the prevalence of hcv infection has been dramatically decreased by . % during - . it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* , sahar muhmmad and dalia el dewi . national blood transfusion services, azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is - days, hiv from to days, and hbv from - days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from to , the total number of donor samples to be screened is , the age of the donors ranged from to years, and they were of both sexes (m: f : ).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v ) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of nat yield donations among ( . %) seronegative donors. among these nat yields cases, ( . %) were reactive for hbv, ( . %) were reactive for hcv and ( . %) were reactive for hiv- . we stratified the age of the donors into groups; group a ( - years), group b ( - years) and group c ( - years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p . ; with % confidence interval (ci) . - . & p . ; with % ci . - . respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p . ; with % ci . - . ). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p . ; with % ci . - . ). nat-hcv; did not differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups a and c & p . ; with % ci - . to . between groups b and c). nat-hiv; did not also differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p . ; with % ci . to . ). nat hbv was significantly higher in males (p . ; with % ci . - . ), but the prevalence of either hcv or hiv did not differ significantly between males and females (p . ; with % ci - . - . & p . ; with % ci - . - . ; respectively). conclusion: in this study the nat yield of in assumes more significance when one considers the fact that single donation is used for generating components that can be used by recipients. hence, in effect the nat yield becomes times that is, in . saving recipients from tti out of ( . %) is indeed very significant. results/finding: of the , donors who were tested by our donor center, , ( . %) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at : titer. the screening elia s/co of this donor was . . both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were - transfusion transmitted babesia cases per year from - (table ). in the months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed ( in , donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of years of additional nat testing at blood bank, dmch, ludhiana from september to december . results/finding: results . % ( of ) units were initially nat reactive. these units were further tested, of which . % were discriminated ( hiv, hcv, hbv and co-infections). the remaining . % ( ) were repeat non-reactive and . % ( ) could not be discriminated. overall, nat yield rate was one in , whereas virus-specific nat yield rates were one in , for hiv, one in for hcv, one in for hbv and one in , for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past years has increased the screening sensitivities to check viral load and prevented transmission of probable transfusion transmitted viral infections. assuming % component preparation it saved transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu , , wei mao , tao he , yashan yang , , zhan gao , , chunhong zhang , hongmei liao , jingxing wang , and miao he* , . institute of blood transfusion, chinese academy of medical sciences & peking union medical college, sichuan blood safety and blood substitute international science and technology cooperation base, chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from , voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as e - . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: . gb raw data with , , reads were generated in the dna library. meanwhile, . gb raw data with , , reads were generated in the rna library. after cleaning the human background, reads from bacteria, reads from viruses, and reads from parasites were identified (table ) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table ) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of february to february . thrombocyte concentrates are prepared out of single donation units by the buffycoat method. results/finding: over the period platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* , germ an leparc , phillip c williamson , lani palmer , ben reynolds , maria noedel and lindsey houghton . creative testing solutions, oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february , recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march , . with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february , and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february , , the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in , who reported $ million new cases worldwide, resulting in > , deaths. malaria prevalence is highest in sub-saharan africa, home to % of all infections accounting for % of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of . mm amustaline and mm glutathione (gsh) and a h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with % fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching > % parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at > . log or > . log /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from to days using an fda cleared rapid test (rt). in february , our hospital based transfusion service implemented the use of rt on day , and to routinely extend ap shelf life to days. prior to this, we tested aps by rt on day and transfused day or day units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of day platelets. study design/methods: data were obtained for two -month study periods: october -september (pre-implementation) and february -january (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table . the number of ap transfusions increased by % post-implementation, comparable to a % increase in inpatient admissions and an % increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different ( . pre; . post, p . ). the number of rts performed increased by %. the percentage of transfused units tested at least once by rt prior to transfusion increased by % (p< . ). the outdate rate decreased from % to % (p< . ). ad-hoc ordering decreased from % to % (p< . ). conclusion: use of an approved rt for routine ap outdate extension to day was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* , vito scalia , carla osiowy , michael carpenter , anton andonov and margaret fearon . canadian blood services, public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low ( . and . per , donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of units. hcv nat was in place since (using minipools of ) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at - o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the ' ntr-e and ns b regions. sanger sequencing of these regions represents approximately % of the genome. results/findings: all confirmed positive donations were whole blood donations. there were hbv positive donations. of these, had tested hbv nat positive. genotypes were type a, b, c, d and e. there were samples hbv nat negative but hbsag positive ( were anti-hbc reactive). of these, could not be sequenced and one was genotype a (also anti-hbc reactive). there were samples considered hcv positive. of these, samples were hcv nat positive. genotypes were type a, b, c, b and a. there were also samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert d microbial detection system (bta d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert d (bta d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels ( - cfu/ml) of bacterial species commonly associated with platelet contamination, and replicates ( per instrument) of ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta d and the other into virtuo and incubated until signaled positive by the instruments or for up to days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august . this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a % voluntary donor program since and is the only center in the country that has achieved this goal. results/findings: a total of , blood donations were studied from august to december . in the rbdc, where only voluntary blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of : , for hbv; : , for hiv and : , for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s system. hbv s region was also sequenced. results/finding: obi were found in the , donations. in the viral loads assay, samples were negative and samples' viral loads were lower iu/ml. the mean viral loads was . . (log ) iu/ml in other samples,while the mean viral loads with hbsag /hbv dna samples was . . (log ) iu/ml. samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype ( . %) and the other was hbv c genotype( . %). compared the samples with hbsag /hbv dna ,we found two obi samples carrying with t>c mutation, which could cause an amino acid s f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag /hbv dna , and some unique variation was identified in the obi individuals. a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat th days, month, months& months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may , we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may to dec was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s platform using pools of serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, seronegative donations were screened by nat and a total of hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group - years old showed a large proportion, who accounted for % of reported infections. most of the hbv dna cases (about . %) reached senior high school education. the average hbsag dna positive rate was . % ( / ). incidence among apheresis donors in this period for hbsag dna were . / . these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf with day stability at c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control ( ml) and test components ( ml ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within hr and wbd pf within hr. cryo was manufactured according to site sops and frozen at - c (test ml, control ml ). test and control cryos were thawed at c, and characterized immediately post thaw (t ), and after d storage at c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over d at c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through d storage at c. conclusion: pr cryo can be processed from plasma sources, including pf , and stored at rt for days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf with stability over days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , lynne fleischmann , mirjana sarac , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics, abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects - million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over days using a panel of positive and negative samples. sensitivity was evaluated on presumed antibody positive specimens and specificity was evaluated on random blood donor samples. results/finding: precision was % cv or less for positive samples over days. the overall specificity in a blood donor population was . % ( / ). sensitivity was . % for presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , lynn martin , daniela kaleve , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over days. sensitivity was evaluated using known positive samples, commercially available seroconversion panels, the who standard, hbsag mutants, and hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed positive samples. sensitivity was % for all genotypes. % of the mutants were detected vs % for the comparator assay. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. analytical sensitivity ranged from . to . iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , kevin callear , susan sullivan , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types and (anti-hiv- / ). blood centers require very high throughput anti-hiv- / assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv- / antibodies and hiv- p antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv- / antibodies and hiv- p antigen was evaluated on the abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv- , hiv- and hiv group o antibodies and hiv- p antigen. seroconversion sensitivity was evaluated with commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed antibody positive samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. also, sensitivity was % for antigen positive viral isolate samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , joyce siregar , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who st international standard. seroconversion sensitivity was evaluated using commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from . to . iu/ml. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* , anton vanweert , ed bakker , mark paradowski , jane bryant , tuan bui , joyce siregar , george chen , george schlauder and gregg williams . abbott laboratories, sanguin diagnostics, abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over days using htlv i and htlv ii positive samples. specificity was evaluated using , blood donor specimens from europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot . . results/finding: imprecision was less than . % for positive samples over days. clinical sensitivity was . % ( / ) on preselected htlv i and htlv ii positive samples. the specificity was . % ( , / , ) on a blood donor population and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez , michel garcia* , fernando palomino and guillermo orjuela-falla . national blood bank colombian red cross, universidad del rosario, fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than . ; abbott architect i sr) underwent supplemental testing by immunoblot (either chiron riba hcv . sia or hcv blot . test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: - . , - . , > . band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in . % ( / ) of samples, indeterminate in . % ( / ) and were positive in . % ( / ). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group - . ( . %) compared with the - . ( . %). in samples with indeterminate results, ns _ was the most frequent band detected ( , %). in contrast, the most frequent band in the group of positive results was core ( , %). only one sample from the indeterminate group ( . %) had a strong band intensity ( ), compared with samples from the positive group ( . %). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group ( - . ) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (> ). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns _ and ns _ cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* , ivanka mihaljevic , manuela miletic , miljana stojic vidovic , irena jukic , jane bryant , mark paradowski , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, croatian institute of transfusion medicine, abbott gmbh & co. kg, abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over days using positive samples. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than . % cv for positive samples over days. clinical sensitivity was . % ( / ) on preselected syphilis positive samples. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* , ed bakker , anton vanweert , jane bryant , mark paradowski , tuan bui , lynn martin , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over days evaluating positive samples. sensitivity was evaluated using preselected positive samples and seroconversion panels. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than . % cv for positive samples over days. overall clinical sensitivity was % on preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying more bleeds than the comparator assay. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of , was declared as the public health emergency of international concern by who in feb . in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where . % of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, . % of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately : to : . thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of lm and assayed after illumination with visible light from both sides for , , and min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was . log % tissue culture infectious dose (tcid )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for min, min or min and the losses of the infectivity were further demonstrated by repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial min of treatment whereby ct-value jumped from . (control) to . (mbpt for min) (table ) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* , margaret fearon , susan l stramer , megan l nguyen , france bernier , sheila o'brien , vito scalia , sakina smith , yves gr egoire and boris hogema . h ema-qu ebec, canadian blood services, american red cross, sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in , canadian blood donors in . in a subset of , donor samples the seroprevalence was . %. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately , canadian whole blood donors including , from canadian blood services (cbs) and , from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all a transfusion vol. supplement s donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test ( % lod . iu/ml, % ci . - . ) for use on the cobas v r / system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous months are destroyed. recipients will be traced in the event of any products transfused in the previous months. results/finding: as of april , , of , ( , cbs, , hq) tested samples with valid results have been found hev-nat reactive: donors have been confirmed by further testing to date. confirmation is pending in donor. of the donors, were from quebec, and one each from nova scotia and alberta ( male, female). ages ranged from to years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: ate pork (including who ate pork liver), ate shellfish, ate venison, and drank well water. one donor had no identifiable risk factor. viral loads ranged from to iu/ml, of which were < , were - , and were > iu/ml; were anti-hev igm positive and anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around / . the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about to million cases and to million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august . both thin and thick glass stained blood smears of blood samples with giemsa was examined using microscope. results/finding: of the donated blood samples, ( . %) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p> . ), majority of the blood donors that tested positive belonged to blood group o ( . %). the prevalence of malaria parasite in the study was . %. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert d (bta d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life ( , and days after collection). study design/method: pooled lrap were seeded with low levels of organisms commonly associated with platelet contamination at , and days post collection. the seeded lrap were inoculated into bpa and bpn bottles on different days (not consecutive) alternating between teams of people each. seeded bottles were loaded into a virtuo and a bta d and incubated until declared positive or negative (up to days). additionally, bpa and bpn bottles inoculated with ml of unseeded lrap were tested on the virtuo and the bta d ( and bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of . % for the virtuo and . % for the bta d. the virtuo demonstrated an average improved ttd of . hours, when compared to the bta d in the presence of ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for days barring continued zikv testing and nonreactive results. a total of , donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low ( . %). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn , felicia santa maria , yvette girard , peter bringmann , marion lanteri* and adonis stassinopoulos . microbiology department, cerus corporation, scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in . the rapidly increasing number of infections in brazil, with hundreds of fatalities since december , is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate d-yfv using amotosalen (s- ) and uva light prt of platelet components (pc). study design/method: pc in %pas (n ) or % plasma (n ) were spiked with high titers of d-yfv and treated with s- /uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were . . log pfu/ml for pc in % plasma and . log pfu/ml for pc in % plasma while titers in post-prt samples were <- . . log pfu/ml for pc in % plasma and <- . log pfu/ml for pc in % plasma. inactivation to the limit of detection of > . . log or inactivation of > . . log pfu/ml was achieved for pc in % plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples ( hiv- antibody and hiv- p antigen, and htlv-i antibody and htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were . for hcv, hbc, syphilis, cmv igg, and chagas, . for hiv ag/ab and htlv i/ii, and . for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were . % (antibody sample) and . % (antigen sample) for hiv ag/ab combo; . % (htlv i antibody sample) and . % (htlv ii antibody sample); - . % for anti-hcv, - . % for chagas, - . % for hbsag, - . % for anti-hbc, - . % for syphilis, and - . % for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of ng/ml. robustness of the abbott prism methods to biotin interference c fischer , r schneider , w leonard , m cobb , g schlauder , g williams , m zuske m janulis* . transfusion medicine, abbott diagnostics, wiesbaden, germany, add diagnostics, transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from to colony forming units (cfus)/bag (i.e. . to . cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of - cfu/ml over the days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, hours (day ) after collection a sampling volume of spiked platelets ( . - cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate ( . %). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann , frank tolksdorf , wiebke handke , thomas h. m€ uller and axel seltsam* . german red cross blood service nstob, maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from buffy coats using the additive solution ssp (macopharma) with a residual plasma content of %. for inactivation kinetics, pcs (n ) were spiked with bacteria to a final concentration of approx. colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately . cfu/ml. bacteria were allowed to grow for h in the pcs at c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log reduction factors ranged from to for enterobacter cloacae ( . . , pei-b-p- ), pseudomonas fluorescens ( . . , pei-b-p- ), staphylococcus aureus ( . . , pei-b-p- ), and streptococcus bovis ( . . , pei-b-p- ). pcs (n for each species) spiked with these different bacteria species were efficiently sterilized ( out of ). treated pcs remained sterile during storage for days, while bacteria in non-treated pcs grew to high titers of - cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july , to july , was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than . were considered significant. results/finding: a total of , consecutive blood donors were screened between and . the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was . %, . %, . % and . % respectively. the hiv-hbv co-infection was higher among blood donors ( . %) followed by hbv-hcv co-infection whish accounts about ( . %). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of - . in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was , . ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a month old infant patricia davenport* , geeta paranjape and laurie sutor , . carter bloodcare, ut southwestern medical center background/case studies: in at a large pediatric hospital, a month old infant was supported for days by extracorporeal membrane oxygenation (ecmo). over this time blood products were transfused. about days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of units of red blood cells. approximately weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of : . the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* , andrea j linscott and donny dumani . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from - , % of transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a -year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day of hospitalization showed no growth after five days. on day , the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the ml unit had been aliquoted via sterile connecting device days prior for a pediatric patient. all ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was . c. within minutes of starting the transfusion, the patient's temperature increased to . c and subsequently reached a maximum of . c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at - c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of determinations of a low positive control in run. inter-assay variability was determined by testing at least representative negative production pool samples, at least low positive sample (about s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of . and were . and . respectively. the hbsag assay detection limit was . iu/ ml for source plasma and . iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at - c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between : , to : , , depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than % for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than %. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on donors ( - days after the index donation) - donors in the follow up study and tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all donors. denv antibodies were negative in donations and equivocal in . our initial reactive rate is higher than that reported to date for the procleix zikv tma of per , [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since . over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late , for which the program consisted of types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june , bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with ml of plasma, vs. ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, ; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation # : updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation # : implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over , donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on - year olds and premenopausal women (ages - ) donors. on average, - year olds donate . times a year and premenopausal women donate . times a year. if both of these groups were limited to donating once a year, a total of , donations from - year olds and , donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin # - , the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for - year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a . hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: % ( / ) of the simulation group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of hybrid group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of online only students improved their post-test scores and had an average likert scale rating of . (good). the average changes in scores were statistically significant within all training groups (p value < . ). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p< . ) and the hybrid group (p< . ). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in . all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july through january were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a -month period, cases of complex antibody identification workups ( %), transfusion reactions ( %), consultations for blood component utilization ( %), and deviations from standard operating procedures and massive transfusion protocols ( %) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $ , of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* , david lancaster , dianne geary , robert scott , anh thu nguyen , adam garcia , raina shankar , leslie buchanan and tho pham . stanford health care, stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to ( ) streamline the ordering process to accurately reflect inventory status and transfusion practices and ( ) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a -month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a -month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: ( ) the ratio of units transfused per week to the number stocked (t:s), ( ) the number of products ordered as stat, and ( ) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. lines of code were written for both programs, including class modules and distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were . , . , and . before the pilot period compared with . , . , and . during the pilot period. these differences did not reach statistical significance (p . ). we also monitored the number of stat ordered products before and during the pilot period, which were and stat units per week, respectively (p . ). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were and units, respectively (p . ). an estimated hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to . fte and $ , per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* , yulia lin , troy thompson , allison collins and sheena scheuermann . ontario regional blood coordinating network, sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that of the province's hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in indicated that % plan to implement or already have implemented the ptqip and % of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in . publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* , rana hajjeh and cees th. smit sibinga . world health organization regional office for the eastern mediterranean, international quality management (iqm) consulting background/case studies: more than million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between and ; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may in tunisia. results/finding: we found publications on disaster from five countries in the region and publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries ( . %) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated - % of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. ( , , and for - ) . the number of collections per registered trt donor varied significantly, ranging from to therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from . to . between and . conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from through . it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of . after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a % response rate (n ). of these, . % have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. . % of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although . % of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only % of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate % ( units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in , apheresis red cells (arc) represented . % of total red cell collections at our center. hae mcs ln was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs operators first and then operators new to apheresis with a training goal of % of mobile staff. validation of the alyx began / / and took approximately days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs machines were removed from service / / . alyx go-live occurred / / . additional operator training continued through september . results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $ . each providing an estimated annual savings of $ , . conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately . % and decrease our kit costs by %. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october -march . background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! g/l) and for females (minimum interdonation interval increased from to days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october , changes in rebooking of donation appointments in december and culminating with eprogesa criteria changes on march , . both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by . percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from . % in the weeks pre-implementation to . % in the weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were . % in september, . % in october/november, and . % from december to march, . conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past years, , blood products, derived from , procedures, were distributed to different investigators in over laboratories. whole blood was the most common product ( . %), followed by unmanipulated mononuclear cell collections ( . %), and elutriated monocytes or lymphocytes ( . %). less common requests included platelets ( . %), plasma ( . %) and granulocytes ( . %). adverse donor reactions were infrequent ( . % of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable % collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in % plasma. the corresponding pathogen reduction system used for the study has kits with different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than . x , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than x /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of x / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of . x and platelet concentration of x /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of . x and . x at the volume of mls and platelet yield of . x , . x , and . x at the volume of mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* and steve cihura . bonfils blood center, bbc / bsi background/case studies: in , the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated devices with the following criteria in mind: ) device disposable costs, ) reagents/controls/quality control, ) objective hgb/hct measurement, ) portability and durability for a mobile environment, ) ease of use, ) donor experience, ) battery life, ) validation requirements plans, ) blood center suitability, and ) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february with a targeted implementation date of july . after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately %. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to . % in and . % in . during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in , the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may . conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* , ayumi araki , hiromi sanyoshi , hiromi kanai , hiroya kikuchi , katsushi tsukada and kazuhide mure . hokkaido red cross blood center, japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from individuals who donated platelets during the -month period between february and august , and data from the donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the men ( . %) and of the women ( . %) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the donors in the vvr group was . . %, which was significantly higher than the . . % in the non-vvr group. at a maximum dbf threshold of %, sensitivity for discriminating between vvr and non-vvr donors was . % and accuracy was . %. when % dbf was used as the alert level, alerts were issued for donors, including in the vvr group. therefore sensitivity for predicting vvr was . % and specificity was . %. mean time from alert to diagnosis in the vvr group was . . minutes, and accuracy of the alert was . %. some of the vvr could not be predicted even the value of maximum dbf exceeded %. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* , , salam abdus and shabrina shah . inova blood donor services, inova fairfax medical campus, inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every to weeks from december to march . blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of patients, females and males, who underwent a total of rbcx from october to march , using an average number of rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of healthy volunteers were collected before blood donation and after blood donation immediately, day, week, weeks, and weeks among men and weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component ( c ) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at weeks among men and weeks among women, while c significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover week after blood donated and reached their levels before blood donated within weeks among men and weeks among women. conclusion: the biomarkers mutually changed over the course of weeks among men and weeks among women. donating ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low % split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate percent and increased concurrent plasma collections by percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october . after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in and , a total number , rns and rns completed the questionnaires, giving a response rate of . % and . % respectively. the overall mean score in was . points (range to ). the mean score in was . points (range to ). the percentage of rns having perfect scores of increased from . % in to . % in . table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; purposively selected blood prescribing clinicians and nurses from hospitals in countries of the human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at . level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r . ; p . ; df ) and accessibility (r . ; p . ; df ) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f( , ) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, junior faculty co-investigators from west coast institutions each had months to create a minute powerpoint presentation on a fundamental tm topic, after which other members had months to review and edit. therefore, each member created and reviewed presentations (three total steps). during each step, members wrote multiple-choice questions for those particular topics. in the end, each topic would have quiz questions to assess learning. at completion, evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: ) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; ) pre and post-lecture question validated examination (best collaborative) to assess learning; ) resident in-service examination trends specific to tm. results/finding: six presentations were developed as of the abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables and . abo leaders pre-test data could not be obtained for institution b, and trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsÕ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the minute vignette performance and the minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean %) when compared to the pre-test scores (mean %) ttest p< . . during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and % reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between . to . (in the range of - ). nps varies between and . according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased - from . to . . conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* , anne eder , beth a. dy and mary o'neill . american red cross, georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about in , apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. , a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to , hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the months before and after launching the educational outreach. results/finding: the web based course was completed by more than participants; were physicians. based on a review of the evaluations, the course was highly valued with % of participants rating it excellent or very good. the blood center physicians gave over presentations to hospital customers. reporting of suspected strs in increased by % compared to the prior year. the increased reporting came from specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required - minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, .) subscription-based e-learning utilizes - minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june , with a new equipment module assigned each month for the following five months. the series concluded in december with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of . on a -point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with positive and negative comment. level : learning the average score of users increased % from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of . and a t-stat value of . . level : results while equipment-related errors decreased by % after training, there is not enough data to demonstrate a statistical significance. conclusion: our level and evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october , a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n ) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was %. of those, % endorse that fbc creates a climate of respect within our transfusion practice, % believe it has improved communication between work units, and % feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only % responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: students of undergraduate semester and students of semester participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with % of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, / / from - am. there were attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, / / from - am. there were attendees, including regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) % as patients with fcr % may benefit from delaying the procedure for performance in the future. validation process included ( ) a deming regression to globally assess the predicted vs. actual results and ( ) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| %. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during days ( academic hours) the trainees can attend lectures, discuss the methodical materials, participate in seminars, interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, with the group capacity a transfusion vol. supplement s of up to people the number of medical specialists who have attended the training is nearly . results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of - ; being least satisfied/comfortable and being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of total technologists, technologists took the pre esd survey and technologists took the one year post esd implementation survey. table shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, ( . %) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were unplanned sop deviations; in the year after esd implementation there were only deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from to . the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in % plasma is broken up in to two days. on day platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for - hours. on day products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day and day . in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: of employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was . for the two month training period the daily average rose to . conclusion: our "flip the switch" training plan for implementing prt platelets in % plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly % of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as % of an activity. in we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of we performed a supply inventory on all departments to plan future purchases and control residual stocks. in , we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter (q / ) with last quarter (q / ). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q / blood bags were used compared to in q / , demonstrating an activity " . %. price negotiation resulted in . % readjustment. both indicated an estimated cost " . % with a possible impact of over us$ , . we have identified a real cost # . % in q / , representing an overall # . % and us$ , . (r$ , . ) savings. conclusion: economy had deteriorated in our country in with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih- tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v , statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in . in the - nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on % individual and % for the other four levels ( % for each). the bonus (%) is calculated based on the iis as follows; category a: % (iis > %), category b: % (iis: -< %), category c: % (iis: -< %) and category d: % (iis < %). on the pilot implementation, the individual scores for staff ranged from % to %. the iis were % to %. the number of staff in each bonus categories were , % (category a) and , % (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* , deborah r fludd and sandra j nance . background/case studies: rare donors are defined as a blood type occurring in less than in people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in , there were , active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the ( . % of total active donors) returned contact cards alerting ardp of changes in calendar year , ( . %) were donors moving from one ardp facility to another, ( . %) were donors no longer eligible to donate, and an additional ( . %) were address changes. other changes were ( . %) reactivated donors and ( . %) donors who we were notified were deceased, or did not want to be listed in the ardp. in , new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in was ( ), which would be . % of the new donors submitted. conclusion: with nearly a % response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in , donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* , barbara j bachman , mike leamy , susan olson and candace williams . university of florida college of medicine, bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih- (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two ( ) runs of one to six ( ) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing , types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. regardless of quality or speed metrics evaluated, the ih- demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < . ). ih- process steps and time studies addressed in the table below did not account for the ih- reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih- ( % reduction, a difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* , kimberly monnin , barbara j bachman , kyla warren , susan olson and candace williams . clinical pathology labs, bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih- tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of separate test runs of or samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately , type & screens (t&s). the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih- demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < . ). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih- (difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih- tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih- and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih- with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v . statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/findings: using automated result verification, only . % out of , samples evaluated for abo/rh testing would require visual verification, resulting in a % reduction in operator touchpoints (p < . ) and a labor saving of minutes ( : hh:mm) for abo/rh testing. for , antibody screens, automatic validation of results would result in . % reduction in operator touchpoints (p < . ) and a labor savings of minutes ( : hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by % annually and peaked at % in mid . to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an week period in late . hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than day shelf life remaining. units with tie tags attached were in hospital inventories for up to months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was % of the hospitals surveyed. mean percent o neg rbc gross sales for a month period before, during, and after the survey was . %, . % and . %, respectively. mean percent o neg net sales during the -month survey fell to . % compared to an average of . % in the months prior. during the -month survey period o neg rbc monthly return rate increased to an average of . % compared to an average of . % in the months prior. for the months after the survey the average o neg rbc return rate further increased to . % while mean percent o neg rbc net sales trended slightly upward to . %. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* , jeremie gachelin , veronique ollivier , thibaut mutin , xavier telot , benoit ho tin noe and sandra sanfilippo . aenitis technologies, hôpital bichat, inserm u background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac ) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than % (p< . ) and a purity of platelets close to . %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over . blood donations for an area with more than million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in , orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in , when we moved from this equipment to atreus c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in we changed to atreus c (terumo bct) and finally in , we moved to reveos system (terumo bct). since the changes in , our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in , bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during and . conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of - patients and an average, round-trip travel time of approximately -minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about % of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november , the hb has significantly improved the turnaround time of rbc issue -from -minutes to less than -seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately rbc per month out of the window for non-surgical patients. this has been reduced to approximately rbc per month, a % average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in , they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the st rescue squadron ( rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the rqs master sergeant in january . we asked what rqs's order and delivery expectations were. he said sporadic use and the blood order would be rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the rqs needs. staff was trained based on data from january meeting. we contacted the rqs in september to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the rqs association with a civilian blood center. based on his field experiences, he changed the blood order from to rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and rqs performed a mock run on october , and we felt prepared for any future events. results/finding: on november , , the rqs was deployed to a civilian aeromedical evacuation. we anticipated a rbc order. the actual order was rbcs and ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at : am by the rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november th , we established a maximum blood order of rbc and liquid plasma, noting future orders may request fewer products, yet meet the preferred rbc; plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table ). the highest mean fib concentration was mg/donor unit; lowest mean fib concentration was mg/donor unit. all sites had a mean fib concentration at least mg/donor unit above the fda minimum requirement of mg/donor unit. fifteen of blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; manufactured pooled donor cryoprecipitate. most froze plasma in a - c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every minutes for a hour period or until the temperature exceeded c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached > c in minutes as shown in table . the control thermometer recorded temperatures maintained at - c for the entire hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at - c for more than minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: % conventional (c-pc), % pr-pc, and mix of % c-pc/ % pr-pc. the model predicts a modest ($ %) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january . a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august , inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january . a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from to (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in , zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within weeks (sept. phase ) and nationwide within weeks (nov. phase ). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools ( -donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab had % and lab had % of clients requiring universal zkv testing. we evaluated a -month test result upload performance period to determine the impact of zkv test implementation. results/finding: during , lab upload time performance ranged from % to . % from january to july; upload time performance fell between august through november, returning to . % performance in december. lab upload time performance ranged from . % to . % january to august. performance fell september through december . % - . %. lab experienced a low of % upload time performance during phase when there was a rapid implementation; % clients required zkv nat. improved performance was observed during phase , with a % increase in zkv clients. for lab : phase experienced a modest decline of upload performance ranging from . % to . % with . % of clients implementing zkv nat. performance was . % in phase , when an additional . % of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* and colleen a. aronson . advocate lutheran general hospital, acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in of the hospital transfusion services. the sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the sites which implemented the verax pgd test perform testing on all day and day platelets to be issued for transfusion. this abstract summarizes the data collected for the first weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day , day , and those that were tested twice. inventory reports were reviewed for platelets issued on day or day that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february ( week of performing the test), . % of all platelets issued by the participating transfusion services were day or day platelets. in march that number dropped to . %. it is expected that this number will level off at some percentage at or below . % with further data collection. in february . % of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day or day ) will likely level off at some number at or below . % due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, of the sites performing testing are level trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed %. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n ). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days , , and of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was ( . . )%, relative change rate of hsr was ( . . )%, the residual leukocytes were ( . . ) . the ph, hsr, and the cd p expression of pooled platelet concentrates before and after filtering were ( . . ) vs ( . . ), ( . . )% vs ( . . )% and ( . . ) % vs ( . . )%. there is significant change for wbc after filtering (p< . ). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table . conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb - ) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of ml and ml. two users then evaluated the ss tubing segment types with ml or ml samples for a total of data points. samples were collected into the attached ml or ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january , a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by %. the platelet outdate rates dropped after implementing the platelet inventory tool from % ( units) to % ( units); a % decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of fte to . fte, lowering fte by %. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* , jaclyn mckay , jennifer curnes and rowena punzalan , . bloodcenter of wisconsin, children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: ( ) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, ( ) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within -hours after collection. this tight hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight -hour completion time constraint for cryo production (capacity expansion). in particular, during the th -quarter of , a blood processing region was able to process about more cryo units/month (an increase of %) at a slightly lower collection cost (cost avoidance), resulting in an approximately % reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the -the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after -day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from to days in the usa using an fda cleared rapid test (rt). in august , our hospital based transfusion service began using a rt on day and to routinely extend ap shelf life to days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of -day ap. study design/methods: data were obtained for two study periods: september -february (pre-implementation) and september -february (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from -day to -day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by % post-implementation while inpatient admissions and surgical volume increased by % and %, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by % post-implementation and the outdate rate decreased from % to % (p< . ). ad-hoc ordering was not statistically different between study periods (p . ). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different ( . and . , respectively, p . ). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years ( ) ( ) ( ) ( ) ( ) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was , , and units respectively. the mean number of discarded rbc units of the five years of the study exceeds % of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below mg/dl, and so the rejection threshold for lipaemia is level equal to or more than mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including rbcs, plasma products and apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from mg/dl to mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* , , rebecca ross , debra mraz , anne baker , zenna neal , melanie champion and edward l. snyder , . johns hopkins university school of medicine, yale university, yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, pr-sdp were transfused at our hospital (out of a total of platelets transfused). after months of scale-up, pr-sdp were approximately % of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($ mg per g) to the content in pr-sdp (< ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september , the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from donors was collected in k edta tubes. plt concentrations were determined at the qc department using the coulter act diff hematology analyzer (beckman coulter). sample tubes were stored at - c and measured at , and hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd ( ml; n ) or dpd units ( ml; n ). plt pools were stored at - c under mild agitation for seven days except for dpd, which were split in two -ml bags after h. samples were taken on days and . ph, po and pco , hypotonic shock response (hsr), extent of shape change (esc), cd p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at h ( pltx /l), h ( pltx /l) and h ( pltx /l) postdonation. dpd can be stored in the same collection bag for h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits ( - pltx / l) before splitting. on day , lactate and pco concentrations increased, and po decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march . there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted % increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* , colleen vincent and patricia kopko . university of california -san diego, american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only - % of double platelet collections meet requirements for pathogen reduction treatment. blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: , apheresis platelets annually), which includes two hospitals ( inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week ) and the bone marrow transplant (bmt) ward (week ). an e-mail communication explained the change to all physicians and nurses. in phase , we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase , we expanded usage to include the inpatient bmt ward. in phase , we lifted all restrictions so prp could be used throughout the health system, with the goal to reach % prp within months. results/finding: in phase (weeks - ), we requested prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the cancer center. in week , excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase . in phase (weeks - ), we formally expanded issuing of prp to include the inpatient bmt ward and requested prp products weekly. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the phased-in areas. in phase (weeks - ), we began issuing prp throughout the health system. our supplier provided an average of prp weekly (range - ), and prp constituted % of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table ) resulting in a cost savings of $ k. an additional cost savings of approximately $ k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as a transfusion vol. supplement s we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* , james r stubbs , scott a hammel and manish gandhi . mayo clinic, mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing % pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of . , . , and . x may end up below a . in the final storage bag and would need a post-processing sample to ensure the product met criteria at ! . x platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a . yield but ended with a yield below . . these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe- d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that of results were . or . x / mcl with the exception being the wbc failure with a count of . . further monitoring of the wbc counts discovered a result of . which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of . will be tested on the adam to confirm a leukoreduced product. we also discovered of ( %) of the . preprocessing yields products ended with a post processing yield < . . we decided to increase the yields requiring post processing samples to include the . . conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of ! . x platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from / - / were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need ) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in ; the revised prediction method (estimated clinical need ) was then evaluated retrospectively using the same data sources covering / - / and then compared to the prior method. results/finding: the average number of platelets transfused from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted amount and the number of units used was - . u/d. % days ( d/month) were under-predicted (average: u/d). % of days ( ) were under-predicted by ! u (average: u; max: u ( x)). the average number of platelets used from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted and units used was a . u/d. % days ( d/ month) were under-predicted (average: . u/d). one day ( %) over this period was under-predicted ! u ( u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages ( % a %), reduced the number of days under-predicted ( % a %), and decreased the discrepancy on those under-predicted days ( . u a . u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* , maria noedel , nancy haubert , kenneth hudson , larry morgan , robert shaw , tracy fickett , jamie jue , valerie winkelman , sally caglioti , german leparc , and phillip c williamson . background/case studies: on / / , fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within weeks; nationwide in weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on of manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for clients within weeks, and an additional clients within weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the -week period but overcome during the -week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* , hala samuel boules , fatemah saleh al matroud , rabab hussien ali dashti , hanan alawadhi and reem al radwan . kuwait central blood bank, kuwait central blood bank, kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of rbcs baby units were randomly chosen to be traced to their final deposition from the year till . half of them ( units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb ( units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is % efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (< days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march . results/findings: a total of products were tested. fifteen units ( %) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all by the vendor and none were true positives by re-culture. of the units that were successfully tested, were tested again on day for use on day ( %). there were platelets transfused ( %) and expired after day ( %). the cost to test the products including controls was $ , and our calculated cost to produce products would be $ , . if we had needed to import products to meet needs, the cost would be roughly $ , without shipping costs which are estimated at $ , . . we averaged expired platelet products per month (range - ) before verax testing and (range - ) after implementation. conclusion: using verax point-of-care testing saved platelet products from discard. the cost savings were $ , . from importing and $ , from producing a replacement for those products. the average discard rate per month went from to after verax implementation. extending platelet shelf life to days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for data, units were investigated without the use of secure texting. of these, units were identified as preventable wastage, and wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: ) product not released after procedure/ or when patient stabilized ( ) ) product returned outside of appropriate temperature range ( ) ) clinician unaware product was assigned ( ). thus far in , wastage records have identified units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, responses thanked the transfusion service for the information, and in instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: . % dmso, plas-a, % hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) . %dmso: plas-a : %hsa : : . plas-a and hsa are kept at room temperature ( - c, rt) and refrigerated at c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point . c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total tests. at least minutes cooling after dmso, before adding the next reagent. see table: ( ) after directly adding . % dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. ( ) in tube, autologous plasma first, dmso next, powder-like precipitates. ( ) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. ( ) & ( ) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at c. ( )&( ) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the c group is slightly milder/slower than rt group. so hsa should not be added first. ( )&( ) trace of hsa(< ml) was mixed into the plas-a bag ( ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive -month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from . % to . %. with an annual platelet usage of approximately , units, this reduction equates to approximately units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $ , to $ , , per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* , russell thorsen , rosaline ma , antonio g insigne , amy decourten , florence panganiban , patricia mckean , cyril jacquot , sara bakhtary and ashok nambiar . ucsf health, children's national medical center background/case studies: plasma (ffp, pf , pf rt ) stored at - c outdates hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (< months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited ( hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk and richard gammon* . oneblood, oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average - minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average - minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from % to %). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp- using the low glycerol ( %) freezing method and frozen at - c within six days of collection. thawing occurred in a c water bath in the following order: o positive and o negative on january ; o positive and o negative on february ; and o negative on february . deglycerolization occurred on site using the acp- with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $ - and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $ per unit to produce and distribute. drbcs have a shorter shelf life, days versus the days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp- 's and deglycerolize four units at a time. in january and february , it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c izekial butler* , karen leighton , scott jones and rachel beddard . qualtex laboratories, biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c instrument. precision of the new assay parameters was determined by testing replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from iu/ml to iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of . to . iu/ml. stability of samples was determined by testing samples stored at - c and - c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from . to . . so, precision was acceptable since the %cv for all samples tested was %. the mean values for the samples tested in the accuracy study were all % of the expected value which was much lower than the acceptance criteria which was % of the expected value. the linearity of the assay was acceptable with a r ! . %. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to days at - c and up to month at - c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for days at - c and stored up to one month at - c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, ) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april , to march , . local research ethics board approval was obtained. results/finding: patients received ivig for itp at smh over the study period for a total of unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding ( , %), pre-operative or antepartum care ( , %), a platelet count of less than and contraindication to corticosteroids ( , %). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between - x /l. patients received ivig for a likely diagnosis itp while a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. / ( %) of infusions consisted of g/kg over days; the remainder of infusions consisted of g/kg. of those who received g/kg, of patients ( %) had evidence of partial remission after a first g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to staff members in early february of . twenty-two technologists responded for a % response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, % indicated that workspace size was insufficient and % that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* , neil bangs and kimberly sanford . vcu health system, virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from / / to / / to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (< hours, > hours < hours), and if a second sample was required. results/finding: a total of t&s orders were initiated from the ed in this time period. ( . %) patients were not subsequently transfused any type of blood product within hours of tsd and ( . %) patients were not subsequently transfused any type of blood product within hours of tsd. a total of ( . %) patients required a second sample. of these patients requiring a second sample, ( . %) were not subsequently transfused any type of blood product within hours of tsd and ( %) were not subsequently transfused any type of blood product within hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within hours of tsd amounted to an estimated $ , in unnecessary patient charges and approximately . nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* , debra berry , yunchuan delores mo and gay wehrli . university of virginia health system, children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* , elizabeth halperin , sharon breining and mona papari . acl laboratories/ advocate hospitals, advocate health care, itxm background/case studies: a large midwest hospital system with level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and months of data were evaluated that contained events. results/finding: there was an equal number of events that were initiated in the ed and the or ( ). male patients were involved % of the time and % of time the patients expired. trauma of some type was the majority of the cause but . % of the cases involved gi bleed and only . % were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average . with the post hgb average of . . ratios of : for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of : . it was found that the rbc: plasma was . : , rbc: plt was . : and rbc to cryo was . : . use of txa was only . % and cacl was utilized in . % of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* , jody a barna , donald e ulinski and nancy m. dunbar . dartmouth hitchcock medical center, dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july , we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august and november (pre-implementation) , newly identified clinically significant antibodies were resulted for patients compared to patients between the months of august and november (post-implementation). the tat for allergy alert entry for both periods is shown in table . we observed that % of allergy comments were performed within hours in the post-implementation period versus only % pre-intervention (p . ). using the new process, nearly all of the alerts were entered into the emr within hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* , shailesh macwan , nancy nikolis , arline stein , janelle richardson , manju bagu , lennart logdberg , alexander indrikovs , vishesh chhibber and sherry shariatmadar . north shore university hospital, northwell health background/case studies: our institution is a tertiary care facility performing over cardiovascular surgeries (cvs) in , an increase of % after the healthcare system cvs integration in . transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december , the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after reported events in q that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: . open discussions and collaboration between blood bank and cvs nursing teams . mapping the process using flowcharts for additional blood orders from cvs. . identify bottlenecks and brainstorm solutions. . a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. . the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. . follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period / / - / / the blood bank has received verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* , tracie ingle and heather vaught . indiana university health, indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter - and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with - o, d negative red blood cells over days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series , anti-d series , a cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion ; : - who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for % and % of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february -june as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june -october . results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table ) . this led to a reduction in observed overall sli ( . . days vs . . days, p< . ) and odr ( . % vs . %). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. ( )) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january and december thirty-five ( ) notifications were sent to physicians using epic letters and of those, fourteen ( ) responded to the epic notification and five ( ) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* , denden benabdessadek , annu george and alexandra jimenez . westchester medical center, new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of orders were reviewed. approximately, % of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases ( %), but of the issued products, all were returned to the blood bank in % of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> type and screens (t&s) per day] blood banks , respectively. our laboratory which serves a large -bed multispecialty academic hospital and receives - t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of . . and . . minutes , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was % longer and had a larger standard deviation (s.d.) than the published trial result of . . . transfusion vol. supplement s abstract during phase i visionv r performed panels. during phase ii visionv r performed of the visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* , john roback , ronald arkin , michael bartlett , robert geiger and jaxk reeves . university of georgia, emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over , emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a % target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of participants responded to the survey ( . % response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n- - df) of the null hypothesis that the mean vs. the alternative that the true mean is> . overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on run cycles. ct was comprised of metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc ( : ), xm ( : ), cb ( : ) and dat ( : ). by implementing the future state, an average $ . min. lt and vt is saved on each sample loaded for ts equating to a % labor reduction over the current state. a % improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a % lt reduction. on average, a min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $ type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing -column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on patient samples for abo/d typing and antibody screening; of which at least had a positive antibody screen. out of the , had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which were d(-) and were d( ). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the patient and donor samples tested ( % concordance). all samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave % sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for samples at different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* , craig fletcher and peter millward . beaumont hospital, beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short -day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october and quality data was reviewed from august to december . the collected data was then analyzed using descriptive statistical methods. results/finding: data from indicates platelet wastage comprised % of total received platelets and % of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units months preimplementation of the report was units, compared to units months post-implementation and units months post-implementation. wastage rates improved from % (wasted yearly platelets/total received yearly platelet units) in , the year of report implementation, to post-implementation rates of % in and % in (see table) . importantly, this occurred despite a greater than % increase in platelet inventory between and and resulted in cost savings of over $ , in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* , lisa marie button , lori scanlan-hanson , karen koch , janet finley , deepi goyal and camille van buskirk . mayo clinic-rochester, mayo clinic, mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in and were / (events/ed transfusions - ). study design/method: electronic ordering for the ed was implemented march st . any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients < kg and pediatric patients > kg. a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for years (april -march ), and during that time there was instance of blood being ordered for an unintended patient . % ( / ). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in , / ( . %) units were transfused in the ed but not charted in the patient's medical record. in , / ( . %) transfusions were not charted. however, in , the first full year of electronic transfusion order capability, only / ( . %) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level trauma center, with over inpatient beds and over operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option (verbal) for verbal orders and coolers set up; and option (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by % with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* , steven zibrat , geoffrey wool and angela treml . university of chicago medicine, university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in , blood bank accounted for % of all rejected clinical laboratory samples, yet comprised only % of total laboratory volume; % of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was . %. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september . results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of . %, three times the national average of . %. under the new system, rejected blood bank samples decreased from an average of % to % of all rejected laboratory samples, a % decrease. implementation of the new process produced a net savings of $ . k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ). in the postimplementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ; p . ). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* , marcus holme , johnathan bakst , gunta musa and angela treml . university of chicago medicine, university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an minute turnaround time (tat). in april of , the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased %. tat analysis of a representative one week sampling per month showed an increase in outliers from per month to per month. average monthly tys samples performed is . these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was / / . results/finding: the average number of outliers decreased % from per month to . further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $ , for fiscal year . conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from - days with a mean of . days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of - days with a mean of days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* and randy levine . northwell health, lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and only of those units ( %) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, additional irradiated units were issued ( / ; %). post-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and all of those units ( %) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus of the ( %) total units were ordered as irradiated. again, additional irradiated units were issued ( / ; %) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* , charlotte van dyke , dina garza van hoose and rachel beddard . biobridge global, south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january , to april , , , total collections were flagged for additional qc by our trima accels and haemonetics instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in , the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: ) risk assessment, ) quality control plan and ) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* , rachel jug , kimberly ingersoll , nicholas bandarenko , nicole guinn and jessica poisson . duke health pathology, duke university hospital, duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced : : transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july -december . data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the : : ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* , nancy nikolis , linda benison , ruthmire thelusca , renee liberty , sherry shariatmadar , alexander indrikovs and vishesh chhibber . north shore university hospital, northwell health background/case studies: our blood bank (bb) processes approximately , specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june , a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed / . the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of . the implementation of this new process has led to a decrease in the number of unacceptable specimens up to % quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from % to %, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* , , morgan rockwell , joseph hagan , jun teruya , and shiu-ki hui . texas children's hospital, baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within hours pre and post transfusion were evaluated. patients ( - years) receiving prophylactic ptx from july to december admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < ml/kg for patients < kg and one apheresis unit (au) for patients > kg; therefore, patients were separated into groups: < kg and > kg. a significant proportion of orders for both < kg and > kg did not meet patient platelet threshold criteria (p< . ). conclusion: ptx threshold above ir for both groups were ( kg) and % (> kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than % of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p> . ) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only . % of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated ( x ) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately patients ( x ) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts ( . %) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by % with the communication category average rpn having the greatest reduction of %. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* , karen king and joseph sweeney . rhode island hospital, lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different ( minutes for the provuev r and for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over minutes are shown in the table. the results show a reduction in tat by minutes with a % reduction of tat greater than minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* , , lorna orengo , monique scott and christopher a tormey , , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as in , blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as % prior to interventions, but may potentially be reduced to as little as . %. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of < %, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged . %. reasons for specimen rejection were divided into groups: ) hemolysis, ) blood bank witness collection form errors, ) quantity not sufficient, abstract ) duplicate sample, and ) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table ) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a . % rejection rate with only rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s system christopher shahan* , christina dejesus , mosi mccall , fallon hampton , tangi herring , judy davis , anjali patel , sonya gomillion and bonnie maltby . qualtex laboratories, qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual - hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently % of whole blood donor testing turnaround time delays are due to issues and failed runs on the s system and we have technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s system. study design/method: the number of technician related failed runs on the s system were tracked from / / thru / / . a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s system. after counter measures were implemented, the number of technician related failed runs decreased from to . failures per week, which was a % decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by %. patients who were transfused for pre-transfusion hgb > g/dl with resulting post-transfusion hgb > g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: patient charts were reviewed. were excluded for bleeding and cardiovascular instability. / ( . %) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors ( . % vs . %, p . ) and anemia of chronic disease ( . % vs . %, p . ) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb . g/dl vs . g/dl, p< . ). inappropriately transfused patients also had higher median post-transfusion hemoglobin ( . g/dl vs . g/dl, p< . ). moreover, lab evalutions revealed association with lower folate levels (median . nmol/l vs . nmol/l, p . ). / ( . %) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. / providers were responsible for . % of all inappropriate transfusions. / appropriately-transfused patients experienced an fnhtr. deaths unrelated to transfusion occurred ( in appropriate, in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* , willem martin smid and ashley john duits . red cross blood bank foundation, sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* , dee dee cassidy , jed b gorlin , and nancy l van buren , . hennepin county medical center, innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant or more minutes may be required for transit of units often released in less than minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july . data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january -june , and post change included august -december . july data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of rbc/month in coolers. post change this dropped to rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of rbc/month were returned ( %). post change, the average rbc/ month returned was ( %), this represents an absolute % reduction in number of returned products. each rbc dispensed and returned takes approximately minutes to complete paperwork and transport, therefore this change saved an average of minutes per month. it was also noted that the average rbc/month transfused was for baseline and post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of - minutes (estimated) was reduced to - minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over patent-applications have been filed related to "transfusion medicine" and over related to "transfusion alarm", during the last years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an / inch color cmos sensor, providing effectively . mp, a . mm lens, ir-cut, day/night minimum illumination . lux/f and viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood a transfusion vol. supplement s abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h. , video frame rate (fps) - /s, refresh rate hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/ . /b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p p is provided. typical v power-supply, sized x x mm and weighing g. client software is required. the ir range is - m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a gb sd-card. pan/tilt-horizontal o and pan/tilt-vertical o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot ( cfu), multishot cfu or highdose k organism preparations at a low level (< cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < days and the fungal organisms in < days. the overall agreement was . % in bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* , diane schafer , debra brown , jesse cox , scott koepsell and sara shunkwiler . nebraska medicine, the nebraska medical center, university of nebraska medical center background/case studies: anticipating the implementation of the new ( th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september , ), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling in months post implementation compared to in the months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late ' s and in early , intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january , to june , . data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: % were female and % were less than weeks of gestational age. none had co-existing g pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn ( %) and rhesus-hdn ( %). antibodies most often implicated in rh-hdn were anti-d ( / ), anti-d and anti-c ( / ) and anti-c ( / ). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was g/kg (range from , g/kg to , g/kg). neonates received one to four ivig administrations. table shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, required an et for rh-hdn and for abo-hdn. forty-five ( %) patients needed top-up transfusions during hospitalisation and until three months of age: with abo-hdn and with rh-hdn. the mean number of transfusions was three (range: to ). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over % case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a -part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate ( each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, georgia institute of technology ap reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to a) all adult males (am), b) women of non-childbearing age (wncba), and c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to a) women of childbearing age (wcba) and b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed %) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap saving apheresis platelets through use of verax point of care testing jennifer rhamy* and rebecca wride . st. mary's regional blood donor center, st. mary's regional medical center background/case studies: our rural hospital-based blood center serves hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between and per day in ), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson and jun teruya , . texas children's hospital, baylor college of medicine ap vision titers --easier or problematic? (table ) . results/findings: post intercept, t had volumes of - ml, with % hemoglobin (hb) recovery. t had -fold less extracellular protein than c. after days of storage t had higher atp and na than c while lactate and hemolysis were lower. hct, ph, k and glucose were equivalent between t and c on d . d hemolysis for t was . - . %, while for c it was . - . %. t and c atp was > mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table ) . hematocrit (hct, %) . . * . . . . . . hemoglobin (g/unit) not measured hemolysis (%) . . * . . . . * . . ph ( c) . . * . . . . . . total atp (mmol/g hb) . . * . . . . * . . k (mm) . . * . . . total tested total plts issued feb mar totals table: . resident reports to the intranet "drop box" increased from . % to . % to %, each over month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from . per day or , dpmo to . per day or , dpmo. this is a statistically significant difference since the p-value calculated was . . conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately , specimens per month. since , the requirement of having a second blood type on record was met by: . utilizing the historical blood type and the current specimen, or . having second type performed on same specimen by different technologist, and . each type and screen specimen signed by staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards th edition, # . . . a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april , the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding: . there was a minor adjustment period with more phone calls made to blood bank to explain the process. . there was minimal impact on turn around times for release of components. . aborh retype workload decreased from to ( % to % of t&s volume) per month. . unnecessary blood draws minimized, improving patient experience. . no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as to %. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our bed hospital, a retrospective chart review was performed ( / / - / / ) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< grade" or "> grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples ( %) were > titer results higher, while the majority was titer results different ( %). the cost analysis is summarized in table . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated % decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc. samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another samples were measured with fc in ucc maribor. results/finding: samples ( rbc, plt, ffp-all leucocyte depleted and non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from , - , ; plt from , - , ; ffp from , - , ; and for non-leucocyte depleted ffp from , - , (table ) . samples ( rbc, plt, ffp -all leucocyte depleted and nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from , ; plt from , , ffp from , ; and for non-leucocyte depleted ffp from , - , (table ) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than x /unit for leucocyte depleted or x / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control.