cord-002164-oqc9h2lu	2016	We, for this important reason, searched for the RARE consensus sequence (5 ′ -AGGTCA-3 ′ ) 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. In view of (1) 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 3) and (2) 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.
cord-002247-e0z5ypii	2016	On 1 February 2016, 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. The WHO Regional Office for the Western Pacific therefore initiated a rapid survey among national-level public health laboratories in 19 countries and areas to determine regional capacity for ZIKV detection. A total of 28 surveys to national-level laboratories likely to be tasked with ZIKV testing were distributed to 19 countries in the Region (omitting resource-limited countries with Zika virus testing preparedness in the Western Pacific Region Squires & Konings reporting having this capacity. 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.
cord-002341-v4r5d26a	2016	To establish a novel mouse model for ZIKV infection, we compared the clinical, histological, and virological findings of male (group 1) and female (group 2) mice with dexamethasone immunosuppression and ZIKV inoculation with those of the appropriate controls (groups 3 to 8) (Table 1 ). 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-NS1 protein expression on immunohistochemical staining and/or detectable viral load in these tissues. 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.
cord-002581-r7mskri0	2017	title: A human inferred germline antibody binds to an immunodominant epitope and neutralizes Zika virus 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 isolation of 18 plasmablast-derived human mAbs, sorted 12 days post onset of symptoms from a ZIKV-patient in São Paulo, Brazil. Interestingly, one of these mAbs (P1F12) exhibited no nucleotide mutations when compared to its corresponding germline sequences, but still recognized a ZIKV immunodominant epitope and neutralized the virus. Virus capture assay and recombinant E protein ELISA P1F12 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 4G2 (clone D1-4G2-4-15, EMD Millipore) followed by incubation with viral stocks (ZIKV or DENV). Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus
cord-002720-lrkscs71	2017	title: Development and evaluation of a rapid molecular diagnostic test for Zika virus infection by reverse transcription loop-mediated isothermal amplification The assay detected viral RNA at 14.5 TCID(50)/mL in virus-spiked serum or urine samples within 15 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 90 plasma or serum samples and 99 urine samples collected from 120 suspected cases of arbovirus infection in the states of Paraíba and Pernambuco, Brazil in 2016. 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) 14, 18, 19 . 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 15 min) can be used as an indicator of the number of RNA copies in each reaction.
cord-002754-xlk4xpv2	2017	title: Status, quality and specific needs of Zika virus (ZIKV) diagnostic capacity and capability in National Reference Laboratories for arboviruses in 30 EU/EEA countries, May 2016 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 30 countries in the European Union/European Economic Area (EU/EEA) in May 2016. To map ZIKV expertise and identify diagnostic capacity and capability gaps in Europe during the initial phase of the PHEIC in February 2016, 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 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 30 EU/EEA countries.
cord-003041-v9uevz3l	2018	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 (AG129 strain) and cause progressive hindlimb paralysis, as well as severe seizure-like activity during the acute phase of disease. 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 (AG129 strain) and cause progressive hindlimb paralysis, as well as severe seizurelike activity during the acute phase of disease. This study demonstrates that a contemporary strain of ZIKV (PRVABC59) can widely infect astrocytes and neurons in the brain and spinal cord of adult AG129 mice and cause rapidly progressing hindlimb paralysis, as well as severe seizure activity, during the acute phase of disease.
cord-003187-qdbcdn2j	2018	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 5-fluorouracil. We observe that ribavirin, favipiravir, and 5-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. To investigate whether ZIKV replication could be affected by increased mutagenesis, we tested four different compounds known to be mutagenic for diverse viruses, 5-fluorouracil, ribavirin, favipiravir, and decitabine (25, 32, 35, (37) (38) (39) . To investigate whether the antiviral activities observed during treatment with favipiravir, ribavirin, and 5-fluorouracil are connected to their predicted mutagenic activity, we analyzed the mutation frequencies of both ZIKV and USUV rescued after 5 passages in the presence of these compounds.
cord-003284-hjx2d5rq	2018	Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A 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. To analyze the genetic stability of the recombinant ZIKV harboring the point mutation A175V in the coding region of the NS2A protein (rZIKV-RGN-mNS2A), total RNA was purified from Vero cells infected with viruses from passage 1 (P1) to passage 5 (P5) using the RNeasy minikit (Qiagen), according to the manufacturer''s specifications. To investigate whether the reduced RNA synthesis of rZIKV-RGN-mNS2A in Vero cells could result in viral attenuation in vivo, the ability of the mutant virus to induce pathogenesis was analyzed in A129 mice and compared with that of the parental rZIKV-RGN ( Figure 6 ).
cord-003403-ypefqm71	2018	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. 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. 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 [71] . Rapid response to an emerging infectious disease-lessons learned from development of a synthetic DNA vaccine targeting Zika virus
cord-003453-p2buyrcj	2019	In this study, we tested the activity of the natural compounds berberine and emodin for their ability to inhibit ZIKV infection. The supernatant containing infectious virus particles was incubated for 1 h at different concentrations of each compound (berberine: 20-160 µM and emodin: 0.04-40 µM) and subsequently used to infect Vero E6 cells. The supernatant containing infectious virus particles was incubated for 1 h at different concentrations of each compound (berberine: 20-160 µM and emodin: 0.04-40 µM) and subsequently used to infect Vero E6 cells. Vero E6 cells were incubated with berberine or emodin for 1 h at the highest non-toxic concentrations prior to ZIKV infection. Vero E6 cells were incubated with berberine or emodin for 1 h at the highest non-toxic concentrations prior to ZIKV infection. Vero E6 cells were incubated with berberine or emodin for 1 h at the highest non-toxic concentrations prior to ZIKV infection.
cord-003482-f1uvohf0	2019	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. Brain and testicular tissues stained with both goat polyclonal goat anti-Iba1 (green) and monoclonal 4G-2 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 10 dpi in the time course study and 28 day dpi in the pilot study (Fig 9) . Two bat infection experiments were conducted in this investigation; 1) a pilot study to determine susceptibility of Jamaican fruit bats to ZIKV infection, and 2) 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.
cord-004020-qtwcbn7m	2019	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. Gossypol-treated ZIKV was incubated with Vero E6 cells at 37 • C for 1 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). Based on Table 1 , four "hit" natural products, including gossypol, curcumin, digitonin, and conessine ( Figure 2A -D), were selected, since they demonstrated inhibitory activity against ZIKV infection with no obvious cytotoxicity in Vero E6 cells when observed under a microscope. 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).
cord-004022-cr0zskcw	2019	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 [15] [16] [17] . To examine the production of ZIKV Natal RGN SRIPs, the supernatant of replicon-transfected packaging cells was harvested 72 h post-transfection and analyzed using dot-blotting, real-time RT-PCR, and TCID50 assays (Figure 4) . To examine the production of ZIKV Natal RGN SRIPs, the supernatant of replicon-transfected packaging cells was harvested 72 h post-transfection and analyzed using dot-blotting, real-time RT-PCR, and TCID50 assays (Figure 4) .
cord-004053-nmt221tu	2019	Percentages of intrinsic disorder (PIDs) of M and C shell proteins from Zika virus (ZIKV) strains with UniProt accession. 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 [2, 14, 51] . 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 [2, 14, 25, 39, 49, 50] .
cord-004418-08dljap3	2020	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 PRVABC59. 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 a dose dependent neutralizing antibody immune response and an anti-Zika IgG response which correlated with a reduction in ZIKV vRNA post-challenge (Table 2 ). 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. 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 1, 29, 57, and 71), and 30 days post-ZIKV challenge (day 101).
cord-009399-6zpkpdzu	2020	Further, KRRK basic residues of IFITM1 locating at 62–67 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). Finally, IFITM1 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 IFITM1 on the acidification of organelles. Some studies suggest that IFITM proteins may suppress the entry of viruses by inhibiting the hemifusion of viral membrane and host cell membranes [15] or restricting the formation of fusion pores following virus-endosome hemifusion [16] . Alanine scanning and site-directed mutations found that KRRK basic residues were key for the restriction of IFITM proteins on ZIKV and DENV. Significantly, we found that IFITM1 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.
cord-010119-t1x9gknd	2017	Conclusion: The wide distribution in the concentration of bioactive lipids among 405 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. 1 The Department of Blood Transfusion, The PLA General Hospital, 2 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. Weak D types 1, 2 and 3 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).
cord-015352-2d02eq3y	2017	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. 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.
cord-016796-g4kqqpy1	2019	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. Nonetheless, recent studies on respiratory syncytial virus (RSV) developed Luciferase Immunoprecipitation Systems (LIPS) assay to detect IgG Antibodies against Human RSV G-Glycoprotein. 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. 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 novel antibody demonstrates noteworthy specificity to identify H7N9 virus compared to homemade target-captured ELISA, qRT-PCR, and rapid influenza diagnostic test (RIDT) with high sensitivity (Chang et al.
cord-102279-ena1usqv	2020	projections of 3D STED image stacks show high intensity ERmoxGFP and Sec61β-GFP labeling in a 89 CER region and low intensity labeling in PER tubules in mock-infected cells ( Figure 1A ), as reported 90 previously by diffraction limited confocal microscopy (3). Density-based segmentation of the ERmoxGFP-96 and Sec61β-GFP-labelled ER of ZIKV-infected cells showed that the higher density crescent-shaped 97 CER region exhibited significant overlap with dsRNA-positive ER structures relative to the rest of 98 the ER ( Figure 1B ). Morphological comparison of the dsRNA-positive and -negative CER of 133 ZIKV-infected cells with the CER of mock-infected cells showed that the CER was composed of a 134 convoluted network of tubules for both the ERmoxGFP-and Sec61β-labeled ER ( Figure 3B ). 3D STED analysis showed a predominant distribution of both NS2B and 149 NS4B to the CER and more particularly to the dense ZIKV-induced crescent-shaped tubular matrix 150 in ERmoxGFP transfected U87 cells ( Figure 5A ).
cord-102612-xx5l8r9e	2020	ZIKV infection or loss of CEP63 decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection or loss of CEP63 also increased the centrosomal accumulation of the CEP63 interactors, Mindbomb1 (MIB1) and DTX4, ubiquitin ligases that respectively activate and degrade TBK1. In addition to identifying a mechanism by which CEP63 controls the innate immune responses in ZIKV infection, we propose that the altered centrosomal organization caused by altered CEP63 function may contribute to ZIKV-associated microcephaly. ZIKV infection in U87 cells did not affect either the localization or levels of the MCPH-associated proteins PLK4, STIL, SAS6, CDK5RAP2, CEP152, or WDR62 (Supplemental Figure 2A-D) . In summary, we have found that ZIKV NS3 localizes to the centrosome, recruits CEP63 and its binding partners MIB1 and DTX4 to ubiquitylate and degrade TBK1, a key regulator of the innate immune response.
cord-102796-rr8qet8c	2020	We are continuously facing new disease outbreaks, including the new coronavirus (SARS-nCoV-2) in December 2019.The objective of this study was to describe the accumulation of evidence during the 2013-2016 Zika virus (ZIKV) outbreak in the Pacific and the Americas related to aetiological causal questions about congenital abnormalities and Guillain-Barre syndrome. In the 2013-2016 ZIKV outbreak, case reports, case series and basic research studies were published first. A strength of this study is the pre-specified hypothesis about the time to publication of aetiological 139 research and the use of data from systematic reviews that had screened and selected studies that 140 addressed the causal relationship between ZIKV infection and its adverse outcomes. The accumulation of evidence over time in new causal problems seems to follow a hierarchy where 225 case reports and case series were rapidly followed by basic research. Syndrome outbreak associated with Zika virus infection in French Polynesia: A case-control study
cord-253616-7jyui5ca	2020	To investigate the anti-ZIKV activity of harringtonine, we assessed the inhibition of virus infection in Vero cells with MOI = 0.01 under different concentrations of harringtonine for 48 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 1B-D) , indicating that virus propagation was suppressed. Harringtonine (625 nM) was administered at different stages of ZIKV infection with MOI = 0.1, and then the levels of ZIKV RNA in cells, as well as virus titers in supernatants, were determined after 24 h treatment. Harringtonine (625 nM) was administered at different stages of ZIKV infection with MOI = 0.1, and then the levels of ZIKV RNA in cells, as well as virus titers in supernatants, were determined after 24 h treatment.
cord-257539-01s21vh0	2016	Immunofluorescence staining corroborated these results ( Figure 1B ) and additionally, chloroquine decreased the production of infectious ( Figure 1C ) and total ( Figure 1D ) virus particles, including defective viral particles, by ZIKV-infected cells. Incubation of Vero cells with chloroquine at 0 h postinfection had a greater impact on the production of ZIKV particles, decreasing viral RNA 64-fold over the controls ( Figure 3A ). 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 MR766. 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 MR766. Incubation of Vero cells with chloroquine at 0 h post-infection had a greater impact on the production of ZIKV particles, decreasing viral RNA 64-fold over the controls ( Figure 3A ).
cord-260336-kwzo8puo	2019	Here we showed that intraperitoneally administered Z2 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 A129 mice. Student''s unpaired two-tailed t-test was used to monitor the distribution of Z2 in male A129 mouse body and testicular tissue and to analyze the difference of viral RNA level in sera or tissues between Z2-and vehicle-treated A129 mice. ZIKV RNA copies in (A) testes, (B) epididymides, and (C) sperm of Z2-or vehicle-treated ZIKV-infected male A129 mice at day 16 were detected by qRT-PCR. 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.
cord-262944-9k64f0tw	2020	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. 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 explore further the role that NK cells play in specific viral infections in pregnancy TORCH PATHOGENS HCMV Human cytomegalovirus (HCMV) was first described in 1954 by Margaret Smith, who replicated a virus from two newborn babies who had died from cytomegalic inclusion disease (CID) (41) . 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 (131) .
cord-263868-ewnf96cz	2020	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 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. 1c) . 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. 6 ).
cord-272405-jmwn8pdn	2017	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. 1 ). To understand this further, a recently isolated HEV genotype 3 from a chronic hepatitis E patient containing a recombinant virus-host RNA genome was shown to infect cultured human, pig, and deer hepatocytes [39] . 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 [40, 41] . 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.
cord-273065-peqz7okh	2020	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. The recent outbreak of neurological disorders and neonatal malformations associated with Zika virus (ZIKV) infection in Latin America {5}, the yellow fever (YFV) epidemics in Angola and Brazil with importation to China [6] , the ever-expanding West Nile virus (WNV) epidemic in the Americas [7] , the recent emergence in East Africa and global spread of chikungunya virus (CHIKV) [8] , as well as the ongoing and expanding dengue virus (DENV) pandemic in the tropics and subtropics [9] have reinforced the call for action in the fight against emerging and re-emerging arboviral diseases. The vaccine showed high efficacy and good safety in seropositive persons in the 9-45 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.
cord-273326-gmw8gl2r	2018	In this line, and contrary to above mentioned report [73] , CQ, an FDA-approved anti-inflammatory 4-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 [74, 75] . Similarly, by using a drug repurposing screening of over 6000 molecules, it was found that emricasan, a pan-caspase inhibitor that restrains ZIKV-induced increases in caspase-3 activity and is currently in phase 2 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 [82] .
cord-278286-1xk31726	2020	This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) 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). To characterize infection by different ZIKV strains (MR766, PRVABC59 and BeH819015), brain endothelial cells (hCMEC/D3) and neuroglial olfactory ensheathing cells (hOEC and mOEC) were infected at an m.o.i. of 0.1. To examine the virus persistence in neuroglia and human brain endothelial cell cultures, the viral RNA copy numbers in hCMEC/D3, hOEC and mOEC cells infected with MR766, PRVABC59 and BeH819015 for 2 months were quantified using RT-qPCR (Fig. 6) .
cord-283405-aozxvxxs	2018	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. 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. Studies in pregnant nonhuman primates have been instrumental for the identification of CD4 + T cell responses as critical for early control of CMV infection and transmission during pregnancy, 100 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. 125 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.
cord-284646-fhruiw23	2020	Vector competence experiments showed that Ae. aegypti could transmit SPONV when 70 exposed to bloodmeal titers that approximate physiological titers, while Cx. quinquefasciatus nonpregnant, mixed sex 6-to 11-week-old mice lacking type I interferon signaling (Ifnar1 -/-) Ar94 (this is the only strain used in these studies, so it will be referred to hereafter as 86 SPONV); or 10 2 PFU of the highly pathogenic African-lineage ZIKV strain DAK AR 41524 87 (ZIKV-DAK) (Jaeger et al., 2019) . Despite significantly higher maternal 141 viremia observed at 4 dpi with ZIKV-DAK-infected dams, the fact that resorption rates did 142 not significantly differ between the two groups indicates that both ZIKV-DAK and SPONV 143 have a propensity to harm the developing fetus that is independent of the amount of 144 replication in maternal blood. In contrast, ZIKV-DAK-and SPONV-inoculated dams displayed 221 varying degrees of placental pathology with severe effects predominantly observed in the 222 the labyrinth zone, including vascular injury involving maternal and/or fetal vascular spaces, 223 infarction (obstructed blood flow), necrosis, apoptosis, and hemorrhage (Fig. 4) .
cord-290385-0smnl70i	2016	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 dengueor rubella-like in infected individuals. ZIKV RNA could be detected in breast milk and saliva of infected women, although replicative virus particles have not been demonstrated 78, 79 Perinatal transmission of other arboviruses, including DENV, CHIKV, WNV, and YFV, has also been reported. 115,120 74/ 8750 (0.8%) patients with suspected ZIKV infection in the French Polynesia outbreak developed neurological syndromes after presenting with a Zika fever-like illness. 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.
cord-292830-gcfx1095	2018	Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. 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. Fig. 1 shows BSAs and other approved antiviral drugs linked to viral and host targets through viruses they inhibit. Thus, we tested several known BSA agents against (−)ssRNA, (+) ssRNA, ssRNA-RT and dsDNA viruses and identified novel activities for dalbavancin against EV1, ezetimibe against ZIKV and HIV-1, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against RVFV. We identified novel antiviral activities for dalbavancin (against EV1), ezetimibe (against HIV-1 and ZIKV), azacitidine, cyclosporine, minocycline, oritavancin and ritonavir (against RVFV) (Fig. 4) .
cord-294798-ji3p0l4j	2018	We have previously reported isolation of ZIKV [8] , DENV [9] , Human coronavirus NL63 [10] , and Enterovirus D68 [11] from children in this school cohort; however, the cases/outbreaks in these prior publications did not include CHIKV, or cases within the May-August, 2014, time frame of the current study. Viral genomic RNA that was extracted from CHIKV, DENV1-4, 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. The six ZIKV sequences obtained in June 2014 from the CHIKV co-infected patients were highly similar (99.9%) to each other and also cluster within a well-supported monophyletic clade, which, interestingly, includes a divergent strain isolated in 2016 from Guadaloupe (Figs 3B and S3B) .
cord-295351-0zr2e8lh	2020	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. 2) [47] . Following the dissociation of GRP78 that unmasks the GLS, ATF6 translocate to the Golgi apparatus and undergoes sequential proteolytic processing by Fig. 2 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. 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.
cord-298166-045evk7g	2018	As no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer CLR01 may inhibit EBOV and ZIKV infection. 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. Methods describing the effect of CLR01 on pseudotyped lentiviral particles (2.3.), Ebola virus infection (2.4.), the detection of ZIKV infection by a colorimetric MTT assay (2.5.) or by cell-based ZIKV immunodetection assay (2.6.), flow cytometry (2.7.) and confocal microscopy (2.8.) as well as the RNA release assay (2.9.) and the antiviral activity of CLR01 in body fluids (2.10) can be found in the supplement.
cord-300379-db79kb5c	2019	To quantify the ability of ATA to prevent ZIKV-induced apoptosis, tissue culture supernatants from ZIKV-infected Vero and A549 cells were harvested at 24, 48, and 72 h p.i. to measure the level of apoptotic signal as determined by caspase 3 and 7 activities ( Figure 5B) . ZIKV-infected cells showed FIGURE 4 | Aurintricarboxylic acid inhibition of ZIKV replication: Vero (A) and A549 (B) cells (24-well plate format, 2.5 × 10 5 cells/well, triplicates) were infected (MOI 0.1) with Paraiba/2015. In this study, we demonstrated that ATA (Figure 1 ) has limited toxicity (Figure 2) and an effective and dose-dependent antiviral activity against ZIKV infection (Figures 3, 4) in both monkey kidney epithelial Vero and human alveolar A549 cells. Notably, ATA can prevent ZIKV-induced CPE and apoptosis in both cell lines ( Figure 5 ) and has broad anti-viral activity against representative ZIKV strains from the African (Uganda/1947 and Nigeria/1968) and the Asian/American (Puerto Rico/2015 and French Polynesia/2013) lineages (Figure 6) .
cord-300459-tu2xrt9x	2018	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 . 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). 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., 2017; Magnani et al., 2017; Robbiani et al., 2017; Stettler et al., 2016; Wang et al., 2017b; Zhao et al., 2016) .
cord-309275-soffxxqu	2020	title: DEFA1B inhibits ZIKV replication and retards cell cycle progression through interaction with ORC1 KEY FINDINGS: Through human transcriptome array (HTA) we found the defensin alpha 1B (DEFA1B) expression was significantly increased within exosomes isolated from ZIKV infected A549 cells. Further studies demonstrated that DEFA1B interacted with the origin recognition complex 1 (ORC1) which is required to initiate DNA replication during the cell cycle and increased DEFA1B expression decreased the ORC1 level in the cell nuclei. SIGNIFICANCE: Together, our results demonstrated that the anti-ZIKV activity of DEFA1B can be mediated by exosomes, and DEFA1B interacts with ORC1 to retard cell cycles. Both qPCR ( Figure 3K ) and western blot ( Figure 3L ) data showed no significant changes of ZIKV replication between untreated HEK293T cells and cells with up-regulated DEFA1B group. Our data showed the ZIKV induced exosomes could be internalized into recipient cells and inhibit the cells'' DNA replication to retard cell cycles.
cord-310004-h9ixhhzz	2020	A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-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-2. 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 H1N1, ZIKV, HIV-1, SARS-CoV-2, EV-A71, human adenovirus 5 (AdV5), and severe fever with thrombocytopenia syndrome virus (SFTSV) (Fig. 1C and fig. Using our previously established proximal differentiated threedimensional (3D) human airway organoids (AOs) for predicting the infectivity of influenza viruses in humans (14) , we confirmed that ACA reduced virus replication by >4 logs (Fig. 2D) , with markedly decreased expression of viral nucleoprotein (NP) antigen (Fig. 2E) .
cord-311007-0i1abjfa	2016	High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. 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. 3B) . In contrast to parental virus RT-PCR products (Fig. 3B) , these sequences carried a single silent G3127A mutation that was inserted during intron cloning, which indicated that these RNAs were generated from the MR766 plasmid. The addition of supernatants from wild-type plasmid-transfected or parental virus-infected 293T cells resulted in readily detectable levels of viral proteins.
cord-319781-6thdg2up	2020	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. We review evidence for the following viruses: Ebola, Zika, West Nile, pandemic influenza, severe acute respiratory syndrome (SARS), and SARS-corona virus-2 (SARS-CoV-2). 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-2 (SARS-CoV-2) ( Table 1) . 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-2.
cord-321312-hgp2jwbl	2020	title: Fluorescent Quantum Dots-Zika Virus Hybrid Nanoconjugates for Biolabeling, Bioimaging, and Tracking Host-Cell Interactions 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. For biolabeling and bioimaging virus-host cell interactions in vitro, ZIKV_ZAIS conjugates were incubated with VERO cells. 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. ZAIS-CMC fluorescent conjugates showed insights of ZIKA virus-host cell interactions
cord-326512-iex98lr1	2019	title: Convalescent patient-derived monoclonal antibodies targeting different epitopes of E protein confer protection against Zika virus in a neonatal mouse model To examine antibody response in a patient infected with ZIKV, we used single-cell PCR to clone 31 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. The SHM rates of these heavy chains compared with their predicted germline sequences were relatively low, at 4.51% for 7B3H, 3.47% for 1C11H, and 4.17% for 6A6H, which is lower than that of antibodies isolated from annual trivalent inactivated influenza vaccine (TIV) donors [34] and chronic human immunodeficiency virus (HIV)-1 patients (>30%) [27, 35] . In a separate experiment, an unrelated mAb, 2G11, which is specific for H7N9 influenza virus, showed no protective effects on ZIKV-infected neonatal SCID mice (data not shown). Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus
cord-330743-o11d0aa1	2020	Herein, we identified 2 secreted bacterial lipases from a Chromobacterium bacterium, named Chromobacterium antiviral effector-1 (CbAE-1) and CbAE-2, with a broad-spectrum virucidal activity against dengue virus (DENV), Zika virus (ZIKV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV) and herpes simplex virus (HSV). Incubation of the culture supernatant but not the bacterial lysates resulted in significant suppression of DENV ( Figure 1B ) and ZIKV ( Figure 1C ) infectivity in Vero cells, indicating that an extracellular effector(s) secreted by Csp_BJ was responsible for viral inhibition. DENV, ZIKV, HSV-1 and SARS-CoV-2 virus stocks were diluted to 50 plaque-forming units (pfu) per ml and incubated untreated or with a serial dilution of the CbAEs in five-fold steps at 37°C for 1 hr before being added onto Vero cell monolayers for 2 hr of infection.
cord-332473-ec8lu2a3	2017	In cells treated with salubrinal prior to Ars-induced stress, ZIKV-infected cells present higher levels of phospho-eIF2α as compared to mock-infected control (Fig 6C, compare lanes 6 and 8) . Zika virus inhibits stress granule assembly treated mock-infected cells (Fig 6E) . C. Vero cells were infected with ZIKV or mock-infected and treated at 24 hpi with 75 μM salubrinal for 3 h to block the dephosphorylation of eIF2α and then treated with 500 μM Ars for 1 h to induce cellular stress. D. Vero cells were infected with ZIKV or mock-infected and at 24 hpi were treated with 75 μM salubrinal for 3 h and then oxidative stress was induced by treatment with 500 μM Ars for 1 h. Vero cells were infected with ZIKV or mock-infected and treated at 24 hpi with 10 μM sal003 for 3 h to block the dephosphorylation of eIF2α and then treated with 500 μM Ars for 1 h to induce cellular stress.
cord-338136-nbtkl5cx	2020	Reflecting on the findings of Petridou and colleagues, 1 describing imported ZIKV cases to the UK between 2016-2018, confirmed at the Rare and Imported Pathogens Laboratory, we look back to the 2015-2017 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. 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. Travel Medicine and Infectious Disease requires that all authors sign a declaration of conflicting interests. Travel Medicine and Infectious Disease requires that all authors sign a declaration of conflicting interests.
cord-340907-j9i1wlak	2020	Here, based on a novel statistical framework and a large-scale genomic analysis of 2,625 viruses from all classes infecting 439 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. Figure 3A and B depicts the average number of under-represented sequences of size m ¼ 3, 4, and 5 nucleotides, identified in few subsets of viruses in both the original and random variants of the virus. A sampling analysis that we performed (see Supplementary document, Section 2.8) suggests that the number of under-represented sequences identified in dsDNA viruses matches their genomic size, when compared with RNA viruses. 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.
cord-341907-vql8e2j3	2019	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. 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 2C ), suggesting that fusion of hFc to the EDIII subunit vaccine did not affect the generation of ZIKV-specific IgG antibodies. 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 2C ), suggesting that fusion of hFc to the EDIII subunit vaccine did not affect the generation of ZIKV-specific IgG antibodies.
cord-345238-p841weif	2020	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-2/COVID-19 pandemic. 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 [7, 8] . 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-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Lastly, concurrent epidemics like the SARS-CoV-2/COVID-19 or other respiratory pathogens/illnesses can overwhelm health care systems and further complicate clinical-epidemiological diagnoses.
cord-354546-lgkqwm6u	2016	7 By far, Aedes aegypti is considered the principal transmission vector of ZIKV, 8 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. 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. 28 A previous study Figure 4 Phylogenetic tree based on E gene sequences of Zika virus isolates. 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 29 days after symptom onset. In previous research, E gene sequences of ZIKV isolates were usually utilized to construct phylogenetic trees 19 based on experience from molecular study of dengue virus.