key: cord- - nj f authors: ambrose, rebecca k.; gravel, jennifer l.; commins, margaret a.; fowler, elizabeth v.; mahony, timothy j. title: in vivo characterisation of five strains of bovine viral diarrhoea virus (subgenotype c) date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: nj f bovine viral diarrhoea virus (bvdv- ) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. to date many subgenotypes have been reported for bvdv- , currently ranging from subgenotype a to subgenotype u. while bvdv- has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. as an example, bvdv- subgenotypes a and b are frequently detected in north america and europe, while the subgenotype c is rarely detected. in contrast, bvdv- subgenotype c is by far the most commonly reported in australia. despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. the aim of this study was to characterise the in vivo properties of five strains of bvdv- subgenotype c in cattle infection studies. no overt respiratory signs were reported in any of the infected cattle regardless of strain. consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. the quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. further studies are required to fully understand the variability and importance of the bvdv- subgenotype c. bovine respiratory disease (brd) is the most important disease of intensively finished cattle. while multiple factors contribute to the likelihood of cattle developing brd, a generally accepted model for brd development is a primary viral infection predisposing cattle to more severe secondary bacterial infections. several viruses have been associated with an increased risk of brd, including bovine herpesvirus (bohv- ), bovine viral diarrhoea virus (bvdv- ), bovine respiratory syncytial virus and bovine parainfluenza virus. of the key brd associated viruses, bvdv- is the most genetically diverse with subgenotypes, bvdv- a to bvdv- u, having been reported [ ] . yesilbag et al. [ ] recently reviewed the geographical distribution of the bvdv- subgenotypes. this analysis highlighted several unusual trends in the distribution of the bvdv- subgenotypes. as an example, the vast majority of genotyped strains identified in australia have been classified within the subgenotype c [ , ] . this is in contrast to the usa where the subgenotype b is dominant, but the subgenotype a is also frequently reported [ ] . while in europe the most common subgenotypes are a and b, also common are subgenotypes d, e, f and h [ ] . however, the distribution of subgenotypes can vary between countries within europe, for example subgenotype b and d were recently reported to be the most frequently identified subgenotypes in germany [ ] . strains of the subgenotype c have been rarely reported in the usa or europe. the drivers of these distributions are unclear, but do not appear to be influenced by vaccine use [ ] . several studies have suggested that the subgenotypes, at least in part, also reflect the antigen diversity between strains using cross-neutralisation assays [ , , ] . clearly the degree of cross-protection afforded by the subgenotype(s) included in a bvdv- in a vaccine against the subgenotypes circulating in a cattle population is an important issue. the overall biological importance of the bvdv- subgenotypes remains to be fully elucidated since the majority of published studies have focused on the bvdv- a and bvdv- b genotypes. it is reasonable to accept that the subgenotypes of bvdv- share similar properties with respect to their capacity to infect susceptible ruminants, and their contribution to the development of more severe clinical disease outcomes such as brd and the birth of persistently infected calves following transplacental infection. all of these outcomes are reported in cattle populations regardless of what subgenotype is present. the evaluation of any variation of the in vivo properties of bvdv- subgenotypes has received little attention. to assist in developing a better understanding of the importance of the bvdv- subgenotypes, the aim of this study was to assess the in vivo properties of five bvdv- strains from the poorly studied subgenotype c. no overt clinical scores were recorded for any of the trial animals, challenged or unchallenged with the bvdv- c strains used in this study (data not shown). for all groups, except the cattle infected with bvdv- c strain ns , there was a general trend for an increase in the average rectal temperature on day of the experiment, prior to any viral exposure, compared to the temperatures from day to day (figure a -f). the reason(s) for this increase are unclear. the cattle had been inducted into the containment facility seven days prior to the commencement of the trial and were therefore considered to have been well adapted/acclimatised to the environment. however, on day there were extra staff and equipment present while the cattle were being inoculated which included extra handling of the animals/cattle potentially contributing to this apparent increase. due to this anomaly, the day temperatures were used as the reference point in the subsequent statistical comparisons within each treatment group. the daily rectal temperatures were monitored for the trial cattle for days following experimental infection. overall the mean rectal temperature results were variable ( figure ). animals (n = ) infected with bvdv- c strain pi exhibited a significant temperature elevation on day post infection (p < . , figure a ). for animals infected with bvdv- c strain trangie (n = ), the temperature increase was gradual from day to day post infection, although the increase was only statistically significant on day (p < . , figure b ). animals infected with bvdv- c strain ao (n = ) exhibited elevated mean temperatures on day and day post infection, with only the day mean temperature being statistically significant (p < . , figure d) . a similar temperature profile was observed for bvdv- c strain ns (n = ), with elevated temperatures on day and day , with only day being statistically significant (p < . , figure d ). although there was a suggestion of an elevated mean temperature on day post-infection for the cattle (n = ) infected with vr this was not statistically significant ( figure e ). the rectal temperatures of the contact animals (n = ) did not exceed . • c during the experiment (figure f ). no statistical comparisons were undertaken for this group due the low animal numbers. animals infected with bvdv- c strain trangie (n = ); (c) animals infected with bvdv- c strain ao (n = ); (d) animals infected with bvdv- c strain ns (n = ); (e) animals infected with bvdv- c strain vr (n = ); (f) uninfected animals (n = ). the asterisks above selected days indicate significant differences compared to day post-infection for that group of cattle. level of significance; * p < . ; ** p < . ; **** p < . . the course of the bvdv- c nasal shedding by the trial cattle was assessed by testing extracts from nasal swabs collected from day to day and serum samples collected on day and day post-infection with a bvdv- specific quantitative real-time (qpcr). a summary of these results expressed as the threshold cycle (ct) is shown in table . overall, the distribution of positive nasal swabs was variable between and within the groups of cattle infected with the different strains of bvdv- c. the earliest that virus was detected in nasal swabs was day post-infection for animals infected with pi ( of animals), trangie ( of animals) and ao ( of animals). one nasal swab from an animal infected with strain pi tested positive at day post infection. the bvdv- c strain ao was the virus most consistently detected in the nasal swabs with all infected animals reacting with the qpcr at day and day post infection, and three of the four animals also reacted in the qpcr on day ( table ). the serum samples collected from animals infected with ao at day post-infection also reacted and were deemed to be positive for bvdv- c (table ). (b) animals infected with bvdv- c strain trangie (n = ); (c) animals infected with bvdv- c strain ao (n = ); (d) animals infected with bvdv- c strain ns (n = ); (e) animals infected with bvdv- c strain vr (n = ); (f) uninfected animals (n = ). the asterisks above selected days indicate significant differences compared to day post-infection for that group of cattle. level of significance; * p < . ; **** p < . . the course of the bvdv- c nasal shedding by the trial cattle was assessed by testing extracts from nasal swabs collected from day to day and serum samples collected on day and day post-infection with a bvdv- specific quantitative real-time (qpcr). a summary of these results expressed as the threshold cycle (ct) is shown in table . overall, the distribution of positive nasal swabs was variable between and within the groups of cattle infected with the different strains of bvdv- c. the earliest that virus was detected in nasal swabs was day post-infection for animals infected with pi ( of animals), trangie ( of animals) and ao ( of animals). one nasal swab from an animal infected with strain pi tested positive at day post infection. the bvdv- c strain ao was the virus most consistently detected in the nasal swabs with all infected animals reacting with the qpcr at day and day post infection, and three of the four animals also reacted in the qpcr on day ( table ). the serum samples collected from animals infected with ao at day post-infection also reacted and were deemed to be positive for bvdv- c (table ) . table . detection of bovine viral diarrhoea virus subgenotype c in extracts from cattle samples using quantitative real time pcr (qpcr). nasal swabs (n) and serum (s) samples were analysed using qpcr. reactive samples were deemed positive for bvdv- c with the cycle threshold value shown (shaded cells). samples which did not react (> ) with the qpcr were deemed to be negative (−) for the virus. with respect to the other groups, bvdv- c was consistently detected in nasal swabs collected on day , day and day post-infection from two of the six animals infected with bvdv- c strain pi ( table ) . one of the animals in this group tested positive (nasal swabs) from day to day post-infection and the swabs from day and day were also positive. for cattle infected with bvdv- c strain trangie, one of the four animals tested positive on day post-infection, while all the other samples were negative throughout the sampling period (table ) . of the animals infected with bvdv- c strain ns , two animals had positive nasal swabs. one of these animals was positive on day . the strain ns was detected sporadically between day and in samples from animal . the serum samples from day and day from this animal were both reactive with the qpcr assay (table ) . two animals infected with bvdv- c strain vr tested positive for the virus in a sporadic manner. the remaining animals did not return any positive results ( table ) . none of the sample extracts from the two contact animals reacted with the qpcr during the sampling period (table ) . bvdv- c was not detected via qpcr in the nasal swab or serum samples collected from all animals on day , day , day and day post-infection and were deemed to be negative (data not shown). attempts were made to isolate the bvdv- c strains from the nasal swabs collected at day post-infection from selected animals. no bvdv- c was detected in any of the culture supernatants by qpcr after three passages of animal (pi infected and tested positive from day to day ), animal (trangie infected), animal (ao infected) or animal (ao infected). with respect to the other groups, bvdv- c was consistently detected in nasal swabs collected on day , day and day post-infection from two of the six animals infected with bvdv- c strain pi ( table ) . one of the animals in this group tested positive (nasal swabs) from day to day post-infection and the swabs from day and day were also positive. for cattle infected with bvdv- c strain trangie, one of the four animals tested positive on day post-infection, while all the other samples were negative throughout the sampling period (table ) . of the animals infected with bvdv- c strain ns , two animals had positive nasal swabs. one of these animals was positive on day . the strain ns was detected sporadically between day and in samples from animal . the serum samples from day and day from this animal were both reactive with the qpcr assay (table ) . two animals infected with bvdv- c strain vr tested positive for the virus in a sporadic manner. the remaining animals did not return any positive results ( table ) . none of the sample extracts from the two contact animals reacted with the qpcr during the sampling period (table ) . bvdv- c was not detected via qpcr in the nasal swab or serum samples collected from all animals on day , day , day and day post-infection and were deemed to be negative (data not shown). attempts were made to isolate the bvdv- c strains from the nasal swabs collected at day postinfection from selected animals. no bvdv- c was detected in any of the culture supernatants by qpcr after three passages of animal (pi infected and tested positive from day to day ), animal (trangie infected), animal (ao infected) or animal (ao infected). monocytes: seven days post-infection, animals infected with bvdv- c strain pi had significantly reduced concentration of monocytes compared to the pre-infection sample (p < . , figure a ). no significant changes in the numbers of monocytes were detected for any of the animals infected with the remaining bvdv- c strains or the contact animals ( figure ). (e) (f) (e) (f) (e) (f) platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day postinfection (p < . , figure c ). platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day post-infection (p < . , figure c ). platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day postinfection (p < . , figure c ). (a) (b) (c) (d) all trial animals were monitored for the development of bvdv- specific antibodies throughout the course of the experiment. virus specific antibody was first detected on day post-infection with of the infected animals testing positive. however, none of the animals infected with strains trangie or ao had detectable antibodies by this time point. by day post-infection, of the animals had detectable bvdv- specific antibody ( table ). all the bvdv- c challenged animals had detectable bvdv- antibodies by day post-infection ( table ). one of the animals infected with strain ao was positive on day but subsequently tested negative on day (table ) . no bvdv- antibody was detected in the serum samples from either of the two contact animals at any of the sampling time points (table ) . table . serological responses (igg) of cattle challenged with one of five strains of bovine viral diarrhoea virus genotype c. the level of virus specific igg were determined using a commercial elisa and assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++). all trial animals were monitored for the development of bvdv- specific antibodies throughout the course of the experiment. virus specific antibody was first detected on day post-infection with of the infected animals testing positive. however, none of the animals infected with strains trangie or ao had detectable antibodies by this time point. by day post-infection, of the animals had detectable bvdv- specific antibody ( table ). all the bvdv- c challenged animals had detectable bvdv- antibodies by day post-infection ( table ). one of the animals infected with strain ao was positive on day but subsequently tested negative on day (table ) . no bvdv- antibody was detected in the serum samples from either of the two contact animals at any of the sampling time points (table ) . table . serological responses (igg) of cattle challenged with one of five strains of bovine viral diarrhoea virus genotype c. the level of virus specific igg were determined using a commercial elisa and assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++). animal id infection pi − − − + ++ +++ ++ − − + +++ ++++ +++++ ++++ − − + +++ ++++ ++++ +++ − − − ++ +++ ++++ ++++ − − − ++ ++ ++++ ++++ − − − +++ +++ ++++ ++++ trangie − − − +++ ++++ ++++ +++ − − − ++ +++ +++ +++ − − − +++ +++ ++++ +++++ − − ++ +++ +++++ +++++ +++++ ao − − ++ +++ ++++ +++++ ++++ − − +++ − +++++ +++++ +++++ − − +++ ++++ +++++ +++++ +++++ − − − ++ ++++ +++++ ++++− − + + +++++ +++++ ++++ − − − − ++++ ++++ ++++ − − + ++++ +++++ +++++ +++++ vr − − − − ++ ++ ++ − − +++ +++ +++++ +++++ +++++ − − − + + ++ ++ contact − − − − − − − − − − − − − − several studies have explored the potential links between the subgenotypes and antigenic variation through cross neutralisation studies [ , , ] . clearly, knowledge of any such relationship is important as it would facilitate the selection of vaccine components to match the circulating bvdv- subgenotypes, while also enabling the ongoing monitoring of field strains to detect any change in the dominant subgenotype. the importance of the bvdv- subgenotypes with respect to the in vivo biology has received minimal attention and more research is required to define commonalities and divergences between each group such as virulence. several of the parameters measured in this study showed similar effects of the bvdv- c strains on their bovine hosts. these commonalities were not unexpected as the strains used in this study were all bvdv- subgenotype c. currently, there are no specific criteria proposed to evaluate bvdv- virulence, however clinical signs (respiratory and/or digestive), biphasic pyrexia, biphasic leukopenia and thrombocytopenia have been reported for bvdv- subgenotypes a, b, d, e and k from various countries [ ] [ ] [ ] [ ] [ ] . in the current study, no respiratory or digestive clinical signs were observed in the bvdv- c inoculated cattle. while a significant pyrexia was identified in cattle infected with four of the five bvdv- strains, however, there was no evidence of a biphasic pyrexia (figure ). four of the five bvdv- infected groups exhibited significant leukopenia at day post-infection, three of which were biphasic (figure ). only the group infected with vr did not have detectable leukopenia. as vr was the only cytopathic bvdv- strain included in the current study, further research is required to determine why this was the case. with respect to thrombocytopenia, only the cattle infected with strain ns had a significant loss of platelets that was detected days after infection (figure d ). collectively these data suggest the bvdv- subgenotypes c evaluated in this study have low virulence in transiently infected animals under the experimental conditions utilised. one additional parameter which is commonly considered in the assessment of viral virulence is transmission capacity [ ] . there is general agreement that there is either no or limited horizontal transmission of bvdv- between transiently infected cattle [ ] [ ] [ ] . sarrazin et al. [ ] concluded that the field strains of bvdv- subgenotype a and b evaluated in their study were unlikely to play an important role in transmission. the detection of bvdv- c in the nasal swabs of infected cattle the current study was sporadic and where detected the results suggested low quantities of virus ( table ). the range of ct values from nasal swabs and serum samples in the current study were . to . and . to . respectively (table ) . previous studies have evaluated the use of qpcr to differentiate persistently and transiently infected animals. hanon et al. [ ] estimated that a ct value below . from a blood sample would identify all persistently infected animals, although this value was likely to misclassify some transiently infected animals as being persistently infected. while hay et al. [ ] estimated that a ct value below from serum was indicative of an animal being persistently infected with bvdv- . the results of the current study, suggest a ct value of is too high for the differentiation of transiently and persistently infected animals based on a single sample. noting that both prior studies utilised field samples for these estimates, thus direct comparison to the current study requires caution. the use of these estimates, would also require sample preparation and analyses to be comparable, particularly volume of sample extracted and subsequently used in the qpcr assay. associated with these results, the two uninfected control animals included in the study did not test positive for bvdv- or seroconvert to bvdv- over the course of the experiment. unchallenged animals could only be included in one of the containment rooms of the study for logistical reasons. consequently, there were no uninfected animals penned with the animals infected with bvdv- c strain ao or strain pi , the viruses most consistently detected in nasal swabs and in the highest quantities (table ) . collectively, these results suggest that minimal amounts of the challenge viruses were present in the nasal secretions of the infected animals and as a result the risk of virus transmission to other animals was very low for these bvdv- c strains. evans et al. [ ] recently reported the absence of horizontal transmission from sheep experimentally infected with an australian strain of bvdv- subgenotype c to sentinel sheep. the possibility of animals transiently infected with the bvdv- c strains used in these studies producing and shedding sufficient quantities of virus to facilitate transmission if subjected to stressful conditions cannot be excluded. it has recently been demonstrated that the bvdv- strain h (subgenotype a) was only transmitted to sentinel animals when the infected animals were immunosuppressed with dexamethasone [ ] . if the low risk if transmission from transiently infected animals extends to all bvdv- subgenotype c it could have important implications in the implementation of effective bvdv- control plans with persistently infected animals as the sole source of virus [ , [ ] [ ] [ ] . further research is required to determine if any bvdv- subgenotype c strains replicate sufficiently in the nasal epithelia at levels to facilitate transmission to susceptible sentinel animals. the bvdv- c strains pi and ao would be excellent candidate viruses for these studies. while the detection of virus in nasal swab and serum samples was sporadic, the cattle were clearly infected as demonstrated by the serological analyses. infected animals started to seroconvert by day post-infection with of the infected animals testing positive for bvdv- specific antibody. the number of positive animals increased by day , with all infected animals being antibody positive by day . these data are consistent with other bvdv- infection studies [ , ] . there did not appear to be anything specific to the bvdv- c strains used in this study in relation to the serological data with animals from all groups becoming seropositive in a fourteen day period and all animals being seropositive by day . the impacts of some bvdv- c strains on cattle in the current study may have been under-estimated due to the smaller number of cattle in each group, particularly for strain vr where one animal was withdrawn from the experiment immediately prior to commencement of the trial for ethical reasons (lameness). the number of cattle in this study was constrained by the capacity of the facility and need to evaluate the properties of several bvdv- subgenotype c strains. another potential limitation of the current study was the viral inoculums used were quantified using rt-qpcr of the final cell culture supernatants. while previous studies have demonstrated correlations between rt-qpcr results and measures of in vitro infectivity such as plaque forming units and/or % cell culture infectious dose (tcid ) for other viruses, such relationships have not been reported for bvdv- as yet [ ] [ ] [ ] [ ] . future studies which aim to directly compare the in vivo properties of the bvdv- c strains used in the study would need to establish the relationship between rt-qpcr results and measures of in vitro infectivity to enable the standardisation of the challenge doses. future studies will be required to better understand the relationships between the bvdv- subgenotypes and virulence. strong et al. [ ] also identified that the challenge dose can influence the clinical outcomes of cattle challenged with bvdv- a. data was also reported which suggested an influence of calf age on clinical outcome. as a consequence, future studies aiming to characterise the interactions of bvdv- and its bovine host should aim to do so under a standardised challenge system, including route of infection, challenge dose (where possible multiple doses) and age of animals. it is imperative that the subgenotype of the bvdv- isolate(s) used also be included. while the current study has focused on the respiratory component of the bvdv- c infection, it is well accepted the virus can have profound impacts on the reproductive capacity of individual animals and cattle herds overall. the bvdv- c isolate trangie was used in several earlier studies that reported the capacity of this virus to significantly impair bovine reproductive function [ ] [ ] [ ] [ ] . the capacity of specific bvdv- strains and/or subgenotype groups to cause both respiratory and reproductive disease are yet to be investigated, and may be required to fully understand this important cattle pathogen. this study is the first to characterise the in vivo properties of bvdv- strains confirmed as belonging to the subgenotype c. interestingly, the overall impacts of the infection of the different strains on the infected cattle were in general similar to those reported for other bvdv- subgenotypes with transient pyrexia, leukopenia, and quantities of virus in nasal swabs which are unlikely to facilitate horizontal transmission [ ] [ ] [ ] [ ] [ ] ] . of the bvdv- c strains examined in this study pi had the most consistent impact on the experimentally infected cattle and is a strong candidate for use in cattle studies to further define the in vivo properties of the subgenotype c, including direct comparisons to strains of other subgenotypes. all experimental procedures involving animals were reviewed and approved by the university of queensland animal ethics committee, approval number qaafi/ / /mla. the bvdv- c isolates used in the cattle trial are described in table . viral inoculums were prepared by adding µl of primary stock of each bvdv- c strain to culture medium of subconfluent monolayers of mdbk cells in tissue culture flasks ( cm ) and incubated at • c in a % co atmosphere for seven days. the supernatants were clarified at g, aliquoted and stored at − • c until required. as the aims of this study did not include intergroup statistical comparisons, the viral supernatants were used as harvested to inoculate cattle at the maximum possible titre. cattle were sourced by veterinary health research ltd. pty (armidale, nsw, australia). the animals were black angus and to months of age. prior to enrolment into the study, cattle were tested multiple times and confirmed negative for serological evidence of prior bvdv- exposure/infection. elisas were performed using the bio k elisa, as described by the manufacturer (bio-x diagnostics, jemelle, belgium). seven days prior to commencement of the trial, cattle (n = ) were moved into the large animal pc facility at the queensland animal science precinct (gatton, qld, australia). the cattle were randomly assigned to one of four pens ( × ) with two pens per room ( table ). the rooms are operated independently, including separate air handling systems. to minimise the risk of virus transmission between rooms, separate teams of staff were used to maintain/care for and collect samples from the animals in each room daily. on day , the rectal temperature for each animal was recorded and two blood samples collected via the jugular vein. the cattle groups were inoculated with one of the bvdv- viral strains as shown in table . briefly, the animal was restrained with the nose elevated and the viral inoculum ( ml) was slowly dripped into each nostril. the nose was held in this position for to s and the animal then released. two animals in room were not inoculated with virus. clinical assessments: cattle were monitored from day to day post-infection for clinical signs in respect to nasal discharge, coughing, behavior/demeanour and loss of appetite (feed residue). temperature: the rectal temperatures for each animal was recorded from day to day . the expected rectal temperature of healthy cattle was . • c [ ] . nasal swabs were collected from day to day , day , day , day , day and day . nasal swabs were immediately placed on ice for transport back to the laboratory for storage at • c until required. nasal swabs were immersed in µl of pbs containing × antibiotic-antimycotic (thermofisher scientific, waltham, ma, usa) and gently agitated. the swab was subsequently removed and discarded. a µl aliquot of this resuspension was used for total nucleic acid extraction using the dneasy blood & tissue kit (qiagen, hilden, germany) as described by the manufacturer, except for the exclusion of rnase a. total nucleic acid extracts were also prepared from aliquots ( µl) of cattle serum samples, collected as described below, using the same methodology. sample extracts prepared from nasal swabs and sera were analysed by qpcr for the presence of bvdv- rna as previously described [ ] . samples yielding a ct value ≥ were deemed to be negative for bvdv- . an aliquot of the resuspended nasal swab from day post-infection from selected animals were utilised for virus isolation. aliquots, µl and µl of the nasal swab resuspension diluted : with pbs were added directly to culture medium of subconfluent monolayers of mdbk cells in well plates and incubated at • c in a % co atmosphere for seven days. the monolayers were freeze/thawed once and a µl aliquot of the culture supernatant added to new subconfluent monolayers of mdbk cells in well plates and incubated at • c in a % co for seven days. this process was repeated three times. total nucleic acids were extracted from a µl aliquot of each culture supernatant and tested using qpcr for the presence of bvdv- as described previously. blood sampling: blood for serum harvesting ( ml bd vacutainers™, bd biosciences, franklin lakes, nj, usa) and blood cell count analyses ( ml edta bd vacutainers™, bd scientific, franklin lakes, nj, usa) were collected on day , day , day , day , day , day , day and day post infection. serum samples were tested for the presence of bvdv- specific antibodies using the bio k elisa, as described by the manufacturer (bio-x diagnostics, jemelle, belgium). the level of virus specific igg in each serum sample was assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++) according to the manufacturer's instructions. whole blood samples were submitted to the veterinary science diagnostic services (school of veterinary science, university of queensland, gatton, qld, australia) for analyses of the cell populations using standard blood smearing and cell counting methodologies. data generated from the animal trial were analysed 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bovine viral diarrhea virus infection around the time of insemination on the reproductive performance of cattle a single amino acid is critical for the expression of b-cell epitopes on the helicase domain of the pestivirus ns protein a manual for the primary animal health care worker; food and agriculture organization (fao multiplex real-time rt-pcr detection of three viruses associated with the bovine respiratory disease complex the authors declare no conflict of interest. the funding sponsors 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- - xbu hnq authors: slingenbergh, jan title: animal virus ecology and evolution are shaped by the virus host-body infiltration and colonization pattern date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: xbu hnq the current classification of animal viruses is largely based on the virus molecular world. less attention is given to why and how virus fitness results from the success of virus transmission. virus transmission reflects the infection-shedding-transmission dynamics, and with it, the organ system involvement and other, macroscopic dimensions of the host environment. this study describes the transmission ecology of the world main livestock viruses, in total, a mix of rna, dna and retroviruses. following an iterative process, the viruses are virtually ranked in an outer- to inner-body fashion, by organ system, on ecological grounds. also portrayed are the shifts in virus host tropism and virus genome. the synthesis of the findings reveals a predictive virus evolution framework, based on the outer- to inner-body changes in the interplay of host environment-transmission modes-organ system involvement-host cell infection cycle-virus genome. outer-body viruses opportunistically respond to the variation in the external environment. for example, respiratory and enteric viruses tend to be associated with poultry and pig mass rearing. ruminant and equine viruses tend to be more deep-rooted and host-specific, and also establish themselves in the vital inner-body systems. it is concluded that the framework may assist the study of new emerging viruses and pandemic risks. animal viruses may be split into transmissible and persistent viruses. it has been proposed that transmissible viruses correlate with replication and virulence and that virus persistence instead permits a lower transmission rate [ ] . this insight builds on earlier work suggesting that virus persistence may pave the way for virus-host symbiosis [ ] . viruses are considered essential agents within the roots and stems of the tree of life [ ] . for example, rna viruses in vertebrates tend to broadly follow the evolutionary history of their hosts that began in the ocean and extended for hundreds of millions of years [ ] . the symbiotic virus-host relationships can take many forms, from antagonistic to mutualistic, and viruses, like other symbionts, lie on a continuum that can shift with environmental changes [ ] . the present study seeks to take these insights to the next level. starting point in the analysis is the link between virus propagation and transmission success. virus transmission may be considered to present the backbone of virus ecology, determining the viruses selected for. unfortunately, the current classification of animal viruses emphasizes the importance of virus genomic architecture and the host cell infection cycle [ ] . less attention is given to why and how virus fitness results from the virus transmission success. for example, an animal virus may become established in the upper respiratory tract and transmit via aerosols to the next host. an enteric virus features a fecal-oral cycle. a skin virus may transmit on the basis of touch. a virus colonizing the distal urogenital tract may transmit during sexual contact. hence, an analysis of the virus transmission success requires a consideration of the overt clinical signs of infection, the gross pathology, the matching virus shedding profile, and of the ensuing modes of transmission. the current analysis explores how the virus molecular world and the macroscopic dimensions of the host environment are intertwined, integral to one and the same virus transmission ecology. in vertebrate hosts the more vital organ systems are shielded off from external aggressors, small or large. it may be assumed that also the host immune defenses are structured to ensure that harmful viral pathogens remain confined to the outer-body environment, the epithelia. epithelial viruses interface with the external environment and respond to the variation encountered here. opposingly, infiltrative viruses establish in the inner-body environment and are expected to evolve towards a more intimate virus-host relationship. given that the virus-host relationship changes with the position of the virus in the outer-to inner-body continuum the analysis focuses on the extent of virus host-body infiltration. the virus organ system tropism is assumed to evolve in harmony with the virus cell tropism. this may be inferred from the dichotomy in the release of viruses from epithelial cells. apart from direct cell-to-cell transmission [ ] , viruses may be released from the apical cell surface and so end-up in the outer-body environment. these viruses colonize mucosae and skin. in contrast, viruses released from the basolateral cell surface infiltrate underlying tissues. these viruses end-up in the lymph drainage, enter the blood circulation and so may infect any of the internal organs. infiltration of and establishment in the inner-body environment may translate in additional, non-epithelial transmission modes. for example, virus establishment in the reproductive organs may translate in intrauterine or lactogenic transmission [ ] . in birds, virus may be shed into yolk or albumen and so transmit vertically [ ] . virus circulation in the bloodstream may enable virus transmission via needles or arthropod vectors [ ] . taking an invasion ecology perspective, the host-body is viewed as a mosaic of organ systems. viruses and organ systems are virtually portrayed in an outer-to inner-body fashion, based on the outer-to inner-body shifts in the virus infection-shedding-transmission characteristics which, in turn, result from the shift in virus organ system tropism. it is assumed that the nature of the virus-host interaction changes with the position of the virus in the outer-to inner-body continuum. the study describes the transmission ecology of the world main livestock viruses. the rationale for selecting the world main livestock viruses relates to the host damaging effects of these pathogens, the overt clinical signs and gross pathology, translating in prominent virus shedding and obvious virus transmission modes. moreover, because of the major economic impact of these diseases, the causative viruses and the corresponding infection-transmission dynamics have been well studied. placed in a wider perspective the analysis builds on the growing perception that viruses deserve to be viewed as evolving living entities [ ] [ ] [ ] . as biological replicators viruses require a propagation strategy in order to become transmitted to the next host [ ] . for this, a virus may turn host damaging or instead evolve a friendly, persisting virus-host relationship [ ] . the analysis entailed an iterative process. as a first step, the one-to-three scores allocated to the livestock viruses for the four ecological variables were examined in more detail. the scores are shown in figure s b . the variables comprise the extent of virus host-body infiltration, the length of the infection period, the infection severity level, and the virus environmental survival rate. the one-to-three infiltration score reflects the organ systems involvement in infection-transmission and concerns, respectively, virus transmission based on the involvement of the epithelia, transmission involving epithelia and internal organs, and transmission involving just internal organs. the score for the length of the infection period reflects, respectively, acute, acute plus persistent, and persistent infections. likewise, the score for the infection severity level concerns a case fatality of less than one, one to ten, and above ten percent. the score for the virus environmental survival rate refers to the number of days that the virus remains infective outside the host-body, ranging from up to three, three to ten, to over ten days. with one exception, the variables did not increase or decrease in value together. just the association between the extent of virus host-body infiltration and the length of the infection period was found to be monotonic. spearman correlation yielded an r = . and p < . . it was thus found that viruses infiltrating internal organs either cause persistent infections or a combination of acute and persistent infections. conversely, persistent viruses either colonize internal organs or a combination of internal organs and epithelia. hence, the length of the infection period appears to present a measure for the extent of virus host-body infiltration. as a next step, the eleven virus families in the study were grouped and ranked a-d on the basis of the infiltration scores allocated to the individual family viruses, see figure . the transmission of the viruses belonging to the orthomyxoviridae and the paramyxoviridae was found to strictly result from the involvement of the epithelia. the transmission of the viruses belonging to the coronaviridae, the picornaviridae and the poxviridae was in part modulated also by the internal organ systems. the transmission of the viruses belonging to the arteriviridae, the flaviviridae, the herpesviridae, plus also the single infectious bursal disease virus (ibdv), resulted from epithelial modes as well as internal organ systems involvement. finally, the transmission of the single bluetongue virus (btv) plus the viruses belonging to the retroviridae family either reflected the involvement of epithelia plus internal organ systems or of just internal organ systems. a spearman correlation of the a-d virus family specific infiltration ranking and the length of the infection period scores yielded an r = . and p = . the result indicates that the interrelationships among virus families may be defined in ecological terms and that the virus families may be neatly lined up in an outer-to inner-body fashion, virtually. one to ten, and above ten percent. the score for the virus environmental survival rate refers to the number of days that the virus remains infective outside the host-body, ranging from up to three, three to ten, to over ten days. with one exception, the variables did not increase or decrease in value together. just the association between the extent of virus host-body infiltration and the length of the infection period was found to be monotonic. spearman correlation yielded an r = . and p < . . it was thus found that viruses infiltrating internal organs either cause persistent infections or a combination of acute and persistent infections. conversely, persistent viruses either colonize internal organs or a combination of internal organs and epithelia. hence, the length of the infection period appears to present a measure for the extent of virus host-body infiltration. as a next step, the eleven virus families in the study were grouped and ranked a-d on the basis of the infiltration scores allocated to the individual family viruses, see figure . the transmission of the viruses belonging to the orthomyxoviridae and the paramyxoviridae was found to strictly result from the involvement of the epithelia. the transmission of the viruses belonging to the coronaviridae, the picornaviridae and the poxviridae was in part modulated also by the internal organ systems. the transmission of the viruses belonging to the arteriviridae, the flaviviridae, the herpesviridae, plus also the single infectious bursal disease virus (ibdv), resulted from epithelial modes as well as internal organ systems involvement. finally, the transmission of the single bluetongue virus (btv) plus the viruses belonging to the retroviridae family either reflected the involvement of epithelia plus internal organ systems or of just internal organ systems. a spearman correlation of the a-d virus family specific infiltration ranking and the length of the infection period scores yielded an r = . and p = . the result indicates that the interrelationships among virus families may be defined in ecological terms and that the virus families may be neatly lined up in an outer-to inner-body fashion, virtually. next, the organ system tropisms of the viruses belonging to each family were collectively fitted and with the naked eye aligned with the figure line-up of families. for this, the within-group, alphabetical family order was adjusted to secure an optimal visual match. as indicated in figure , from outer-to inner-body the virus organ system appears to shift from the respiratory plus the alimentary tract to the skin, the distal urogenital tract or cloaca, the peripheral nerves and ganglia, the reproductive organs system, the lungs, to the immune plus the circulatory systems. hence, there are indications that both viruses and organ systems may be lined up in an outer-to inner-body fashion, virtually. next, the organ system tropisms of the viruses belonging to each family were collectively fitted and with the naked eye aligned with the figure line-up of families. for this, the within-group, alphabetical family order was adjusted to secure an optimal visual match. as indicated in figure , from outer-to inner-body the virus organ system appears to shift from the respiratory plus the alimentary tract to the skin, the distal urogenital tract or cloaca, the peripheral nerves and ganglia, the reproductive organs system, the lungs, to the immune plus the circulatory systems. hence, there are indications that both viruses and organ systems may be lined up in an outer-to inner-body fashion, virtually. next, the a-d family ranking was converted into a one-to-four virus infiltration score applicable to individual viruses. further, these scores are shown in supplementary figure s b. to make the scoring compatible with the a-d family ranking, the one-to-four scores reflects, respectively, virus transmission strictly based on epithelial modes, primarily based on epithelial modes, involving epithelia and internal organ systems, and primarily involving internal organ systems. there are several differences with the a-d family ranking shown in figure . among the viruses of family group b, tgev, aev, fmdv, and lsdv received a score of three for transmitting on the basis of the involvement of both epithelia and internal organs. pev and svdv of group b were considered primarily epithelial and so received a two score. both these viruses are persistently shed in feces, including in the absence of clinical signs, indicating a systemic infection component. the herpesviruses of group c were split into two. bhv- , dev, ehv- , and gahv were considered primarily epithelial while ehv- , gahv- , and shv- were considered to involve epithelia and internal organs. among the d group viruses alv was considered to involve epithelia and internal organs, unlike caev, jsrv, and mvv, for which the involvement of the epithelia did not appear to contribute to the overall virus transmission success. the latter viruses were allocated a score of four. when the new, one-to-four virus infiltration scores were matched to the scores for the length of the infection period spearman r became . , and p = . when the somewhat atypical, vector borne next, the a-d family ranking was converted into a one-to-four virus infiltration score applicable to individual viruses. further, these scores are shown in supplementary figure s b. to make the scoring compatible with the a-d family ranking, the one-to-four scores reflects, respectively, virus transmission strictly based on epithelial modes, primarily based on epithelial modes, involving epithelia and internal organ systems, and primarily involving internal organ systems. there are several differences with the a-d family ranking shown in figure . among the viruses of family group b, tgev, aev, fmdv, and lsdv received a score of three for transmitting on the basis of the involvement of both epithelia and internal organs. pev and svdv of group b were considered primarily epithelial and so received a two score. both these viruses are persistently shed in feces, including in the absence of clinical signs, indicating a systemic infection component. the herpesviruses of group c were split into two. bhv- , dev, ehv- , and gahv were considered primarily epithelial while ehv- , gahv- , and shv- were considered to involve epithelia and internal organs. among the d group viruses alv was considered to involve epithelia and internal organs, unlike caev, jsrv, and mvv, for which the involvement of the epithelia did not appear to contribute to the overall virus transmission success. the latter viruses were allocated a score of four. when the new, one-to-four virus infiltration scores were matched to the scores for the length of the infection period spearman r became . , and p = . when the somewhat atypical, vector borne bluetongue virus was removed from the correlation, r remained . for the one-to-four scoring, became . for the a-d virus family specific ranking, and . , with p = × − , for the one-to-three infiltration ranking. next, all of the above findings were considered in conjunction with the literature data on the transmission ecology collated for each of the viruses in figure s a . pieced together on this basis was an outer-to inner-body line-up of viruses by organ system or combination of organ systems, guided by the one-to-four virus infiltration score, the corresponding virus organ system tropism, the matching virus transmission modes, length of the infection and shedding periods, infection severity level, and virus environmental survival rate, see figure and, also, figure s d . bluetongue virus was removed from the correlation, r remained . for the one-to-four scoring, became . for the a-d virus family specific ranking, and . , with p = × − , for the one-to-three infiltration ranking. next, all of the above findings were considered in conjunction with the literature data on the transmission ecology collated for each of the viruses in figure s a . pieced together on this basis was an outer-to inner-body line-up of viruses by organ system or combination of organ systems, guided by the one-to-four virus infiltration score, the corresponding virus organ system tropism, the matching virus transmission modes, length of the infection and shedding periods, infection severity level, and virus environmental survival rate, see figure and, also, figure s d . for the epithelial, outer-body viruses it turned out that the length of the infection and shedding periods, as well as the virus environmental survival rate generally increased from respiratory tract to alimentary tract to skin. the respiratory viruses transmitted on the basis of aerosols, direct contact or fomites. alimentary tract viruses were found to transmit on the basis of a fecal-oral cycle, through direct contact, contamination of feed and water, or involving fomites, persons and vehicles. viruses infecting both respiratory and alimentary tract featured a mix of these transmission modes. mostly, these viruses caused rather severe infections. among the skin viruses, the more infiltrative viruses affecting all layers of the skin caused slowly healing lesions. the transmission of these deep-rooted skin viruses was found to rely on abrasion or biting flies rather than on direct touch or on indirect contact, more typical for superficial skin lesions. some of the epithelial viruses are shed in feces over a prolonged time period, also in the absence of clinical signs, and these infections were considered to feature a systemic component. next, the epithelial herpesviruses establishing latently in peripheral nerves and ganglia were found to cause a recurrence or persistence of the mucosal and/or skin infection, including of the distal urogenital tract and external genitalia. virus infiltration of the inner-body environment frequently implicated the genital tract or reproductive system in general. this was found to be the case for the rna, the dna and for the retroviruses in the study. virus establishment in the reproductive system translated in seminal transmission, haphazard abortion, late term abortion, stillbirth, birth of infected, yet apparently healthy offspring or, also, lactogenic transmission. the vertical transmission modes were common among the utmost deep-rooted viruses, the viruses infiltrating also the immune and circulatory systems. some of the utmost infiltrative viruses featured an absence of epithelial transmission modes and were environmentally labile. virus infiltration of the immune system associated with immune-suppression, severe infections, neoplasia, or instead with in-apparent, persistent infection. virus infiltration of immune and circulatory systems associated with iatrogenic transmission modes. virus circulation in the bloodstream facilitated arthropod borne transmission. as indicated in figure , the transmission of the bluetongue virus, the sole arbovirus in the study, was considered somewhat atypical because the virus usually causes a transient infection in the ruminant host while in midges remains infective for life. hence, the involvement of the biological vector complicates a direct comparison with the transmission ecology of the remaining viruses. the finding that virus environmental survival in the outer-body environment increased from respiratory tract to alimentary tract to skin and decreased with the shift from the outer-to the inner-body environment prompted a re-examination of the relationship between the extent of virus host-body infiltration and virus environmental survival. virus infiltration scores two-to-four, running from primarily epithelial transmission, to transmission involving also internal organ systems, to transmission primarily involving internal organ systems, were matched to the one-to-three virus environmental survival rate scores, yielding an r = − . and p = < . . the indication that at least in broad terms the extent of virus inner-body infiltration correlated with a loss of virus robustness was applied in the virus ranking, along with the other factors. furthermore, the outer-to inner-body shifts in virus host tropism and virus genome were examined. underlined in figure are ruminant and equine viruses, contrasted to the remaining, poultry and pig viruses. excluded from the host tropism correlations was the multiple-host fmdv. the remaining viruses all formed part of either of the two virus host groupings. it was found that the rna, dna and retroviruses broadly line up in an outer-to inner-body fashion. virus host tropism and the virus genome line-up were matched. additional correlations concerned the virus host tropism and the four virus ecological variable scores, as well as the virus genome type and the four virus ecological variable scores. the extent of the host-body infiltration was found to increase from rna to dna to retrovirus, with r = . and p < . . from rna to dna to retrovirus the ruminant and equine viruses gained in prominence, with r = . and p < . , and the infection severity level decreased, with r = − . and p < . . moreover, the ruminant and equine viruses were found to cause less severe infections than the poultry and pig viruses, with spearman r = − . and p < . . hence, from outerto inner-body, the virus genome type and host tropism appear to shift in concert, along with the infection-transmission dynamics. the synthesis of the findings is presented in figure . the host environment frames the virus transmission modes and, with it, explains the organ system involvement, the specifics of the host-cell infection cycle, and the virus genome. vice versa, the virus life history explains the virus genomics, the host-cell infection cycle and, with it, the macroscopic level virus-host interactions and the host population ecology. the interplay of the host environment-transmission modes-organ system involvement-host cell infection cycle-virus genome changes from outer-to inner-body, resulting in two opposite virus evolution pathways, respectively for generalist and specialist type viruses. the extent of the host-body infiltration was found to increase from rna to dna to retrovirus, with r = . and p < . . from rna to dna to retrovirus the ruminant and equine viruses gained in prominence, with r = . and p < . , and the infection severity level decreased, with r = − . and p < . . moreover, the ruminant and equine viruses were found to cause less severe infections than the poultry and pig viruses, with spearman r = − . and p < . . hence, from outer-to inner-body, the virus genome type and host tropism appear to shift in concert, along with the infection-transmission dynamics. the synthesis of the findings is presented in figure . the host environment frames the virus transmission modes and, with it, explains the organ system involvement, the specifics of the host-cell infection cycle, and the virus genome. vice versa, the virus life history explains the virus genomics, the host-cell infection cycle and, with it, the macroscopic level virus-host interactions and the host population ecology. the interplay of the host environment-transmission modes-organ system involvement-host cell infection cycle-virus genome changes from outer-to inner-body, resulting in two opposite virus evolution pathways, respectively for generalist and specialist type viruses. implied by figure is that the crowding conditions observed in poultry and in pig husbandry tend to attract horizontally transmitting respiratory and enteric viruses. the pathogenicity level of the viruses evolves to match the dynamics in host abundance and contact rate. at the molecular level, these rna viruses become released from the apical surface of epithelial cells directly into the implied by figure is that the crowding conditions observed in poultry and in pig husbandry tend to attract horizontally transmitting respiratory and enteric viruses. the pathogenicity level of the viruses evolves to match the dynamics in host abundance and contact rate. at the molecular level, these rna viruses become released from the apical surface of epithelial cells directly into the outer-body environment. thus, proliferative virus replication, generalized infection of respiratory plus enteric mucosae, profuse virus shedding, and swift onward transmission all go hand-in-hand. a diametrically opposite scenario is given by relatively stable host environments observed in ruminant and equine husbandry, with parent stock and their young grazing together in the open, not unlike wild herbivore ecologies. the viruses attracted and selected for establish in the vital inner-body systems and transmit vertically, via needles or via bloodsucking arthropods. at the molecular level, virus establishment in the vital body systems is matched by low replication rates and minor or slowly evolving host damage. the utmost infiltrative viruses in the study are the retroviruses. in addition, some of other rna viruses are deep-rooted. the dna viruses in the study take an intermediary position. it has been established that epithelial viruses are highly evolvable, more so than inner-body viruses [ ] . epithelial viruses are responsive to the dynamics in the environment external to the host-body. this may be illustrated on the basis of the genetically related virus pairs in the study. for example, the influenza virus circulating in horses (eiv) generates a transient, dry cough supporting swift virus transmission via aerosols [ ] . in pigs, the virus (siv) causes coughing and sneezing, resulting from significant mucus production [ ] . the virus transmits on the basis of close direct contact, in line with the social behavior and body size of pigs. the rinderpest virus (rpv) in cattle and buffaloes primarily colonizes the alimentary tract and transmits on the basis of direct muzzle-to-muzzle contact [ ] . in small ruminants, the identical peste des petits ruminants virus affects also the respiratory tract and transmits also via aerosols. likewise, the lumpy skin disease virus (lsdv) in cattle causes persistent, deep, necrotic skin plugs and transmits via biting insects, mechanically. in sheep and goats, the virus (sgpv) causes transient lesions [ ] . the caprine arthritis-encephalitis virus (caev) and the maedi-visna virus (mvv) present an example of closely related lentiviruses establishing in the inner-body organ systems of sheep and goats. the viruses display overlap in host tropism and both transmit mainly vertically via colostrum and milk. the difference between the two viruses mainly concerns the differential inner-body virus organ system tropism. projected on a long evolutionary timescale, inner-body viruses tend to become locked in within the host body. this internalization may turn progressive when the epithelial transmission modes are being replaced by internal organ system-based modes. virus establishment in the reproductive system translates in vertical transmission, in turn enhancing virus-host co-evolution [ ] . virus infiltration of also immune and circulatory systems may yield in-apparent, persistent infections, indicating low levels of pathogenicity and/or enhanced host tolerance. the division between virus and host may become blurred and given enough time the two may become one [ ] . the nature of species jumps differs between generalist and specialist type viruses. for example, an opportunistic, epithelial virus of wildlife origin is likely to be found circulating in livestock before becoming first detected in humans as host. this has been the case for influenza [ ] , henipah [ ] and mers corona viruses [ ] . further, the sars corona virus infected civet cats raised as food animals before appearing in humans as host [ ] . in contrast, more infiltrative viruses establish in the vital inner-body systems. specialist viruses circulating in the bloodstream of non-human primates may directly jump to humans as host, as a result of complex ecological, socio-economic, demographic and other drivers. examples comprise hiv-aids [ ] , chikungunya [ ] , and zika viruses [ ] . hence, knowing how species jumps differ for the different host ecologies may assist the study of pandemic risks. a subtotal of livestock viruses of global animal health significance was extracted from the oie-listed diseases, infections and infestations in force in [ ] . livestock infections and diseases resulting from virus spill-over from wildlife were excluded from the analysis. the common livestock hosts, described in the colloquial oie terminology, comprise horses, donkeys, cattle, buffaloes, sheep, goats, swine, chicken, turkeys, ducks, and geese. the total of livestock viruses belong to eleven different families and form a mix of rna (n = ), dna (n = ), and retroviruses (n = ). shown in figure s a for each of the viruses are the virus family, virus genomic architecture, virus name in full, abbreviated, and the common names given to the infection or disease. also presented is a brief summary on the transmission ecology for each virus, with references to the primary livestock host, the virus organ system tropism, the length of the infection and shedding period, the infection severity level, the transmission modes, and the virus environmental survival rate. presented in figure s b are one-to-three scores allocated to the viruses for four ecological variables. the variables comprise the extent of virus host-body infiltration, the length of the infection period, the infection severity level, and the virus environmental survival rate. the one-to-three infiltration score reflects the organ system involvement in infection-transmission and concerns, respectively, virus transmission based on the involvement of the epithelia, transmission involving epithelia and internal organs, and transmission involving just internal organs. also shown is a one-to-four virus infiltration score, an outcome of the iterative analysis process and reflecting, respectively, virus transmission strictly based on epithelial modes, primarily based on epithelial modes, involvement of epithelia and internal organ systems, and of primarily internal organ systems. the score for the length of the infection period reflects, respectively, acute, acute plus persistent, and persistent infections. likewise, the score for the infection severity level concerns a case fatality of less than one, one to ten, and above ten percent. the score for the virus environmental survival rate refers to the number of days that the virus remains infective outside the host body, ranging from up to three, three to ten, to over ten days. also indicated in figure s b is the virus host range as observed in both livestock and wildlife. figure s c lists the literature sources on which figure s a ,b is based. the analysis concerned an iterative process. as a first step, the one-to-three scores allocated to the viruses for the four ecological variables were examined in more detail and the monotonic associations subjected to spearman correlation. just the scores for the virus host-body infiltration and for the length of the infection period were found to increase in value together. next, given the coarse match between virus infiltration and persistence, it was examined how this relationship played out at the virus family level. for this, the eleven virus families in the study were grouped and ranked a-d on the basis of the one-to-three infiltration scores allocated to the individual family viruses. also, this a-d infiltration ranking was held against the length of the infection period scores. next, given the indication, from the above, that the virus families may be neatly lined up in an outerto inner-body fashion, it was examined how the organ system tropisms of the family viruses aligned with it. for this, the organ system tropisms of the viruses belonging to each family were collectively fitted and with naked eye aligned with the a-d family groups. for this, the alphabetical family order within the family groups was abandoned in order to obtain an optimal visual match. the result confirms that also the organ systems may be lined up in an outer-to inner-body fashion, virtually. next, the a-d family ranking was converted into a one-to-four virus infiltration score applicable to individual viruses, as described in section . , and also these scores were matched to the length of the infection period scores. next, since the infiltration-persistence match for the individual viruses was found to be about as strong as for the virus families, the viruses were individually lined up in an outer-to inner-body fashion, irrespective the family origin, strictly on ecological grounds. for this, all of the above obtained results were considered in conjunction with the literature data on the transmission ecology collated for each of the viruses in figure s a . pieced together on this basis was an outer-to inner-body line-up of viruses by organ system or combination of organ systems, guided by the one-to-four virus infiltration score, the corresponding virus organ system tropism, the matching virus transmission modes, length of the infection and shedding periods, infection severity level, and virus environmental survival rate. the finding that virus environmental survival in the outer-body environment increased from respiratory tract to alimentary tract to skin and decreased with the shift from the outer-to the inner-body environment prompted a re-examination of the relationship between the extent of virus host-body infiltration and virus environmental survival. virus infiltration scores two-to-four, running from primarily epithelial transmission, to transmission involving also internal organ systems, to transmission primarily involving internal organ systems, were found to match with the one-to-three virus environmental survival rate scores. the indication that, at least in broad terms, the extent of virus inner-body infiltration correlated with a loss of virus robustness was applied in the virus ranking, along with the other factors. next, furthermore examined were the outer-to inner-body shifts in virus host tropism and virus genome. for this, the ruminant plus equine viruses were contrasted to the poultry plus pig viruses. excluded from the host tropism correlations was the multiple host fmdv. the remaining viruses all formed part of either of the two host groupings. it was found that rna, dna and retroviruses broadly line up in an outer-to inner-body fashion. virus host tropism and the virus genome line-up were matched. additional correlations concerned virus host tropism and the four virus ecological variable scores, as well as virus genome type, and the four virus ecological variable scores. it was found that from outer-to inner-body, the virus genome type and host tropism appear to shift in concert, along with the infection-transmission dynamics. the collective results above served the compilation of the predictive framework for animal virus evolution shown in figure , discussion section. the online rho calculator https://www.socscistatistics.com/tests/spearman/default.aspx was used for the spearman correlations. this software has been audited by established statistics packages. virus ecology: a gap between detection and prediction. emerg. 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good viruses: viral mutualistic symbioses cell tropism predicts long-term nucleotide substitution rates of mammalian rna viruses equine influenza (infection with equine influenza virus) in world organisation for animal health (oie) manual of diagnostic tests and vaccines for terrestrial animals, oie technical disease cards, swine influenza. oie technical disease cards, rinderpest. oie technical disease cards, sheep pox and goat pox (oie, ) transmission modes and evolution of the parasitism-mutualism continuum on the concept and elucidation of endogenous retroviruses spatiotemporal distribution and evolution of the a/h n pandemic influenza virus in pigs in france from to : identification of a potential swine-specific lineage transmission of henipaviruses mers coronavirus: diagnostics, epidemiology and transmission beyond the cut hunter: a historical epidemiology of hiv beginnings in central africa a scoping review of published literature on chikungunya virus zika virus: history, emergence, biology, and prospects for control oie-listed diseases, infections and infestations in force in world health organization in animal health yearbook fao-oie-who this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: i am grateful to epke le rütte, lenny hogerwerf, anneke engering, marjan leneman, jelle bruinsma, dorothea van ooyen and marleen slingenbergh for discussions. the author declares no conflict of interest. sponsors had no role in the design, execution, interpretation, or writing of the study. key: cord- -p rz fy authors: nabil, nehal m.; erfan, ahmed m.; tawakol, maram m.; haggag, naglaa m.; naguib, mahmoud m.; samy, ahmed title: wild birds in live birds markets: potential reservoirs of enzootic avian influenza viruses and antimicrobial resistant enterobacteriaceae in northern egypt date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: p rz fy wild migratory birds are often implicated in the introduction, maintenance, and global dissemination of different pathogens, such as influenza a viruses (iav) and antimicrobial-resistant (amr) bacteria. trapping of migratory birds during their resting periods at the northern coast of egypt is a common and ancient practice performed mainly for selling in live bird markets (lbm). in the present study, samples were collected from wild birds, representing species, which were being offered for sale in lbm. all birds were tested for the presence of aiv and enterobacteriaceae. ten samples collected from northern shoveler birds (spatula clypeata) were positive for iav and pcr sub-typing and pan ha/na sequencing assays detected h n , h n , and h n viruses in four, four, and one birds, respectively. sequencing of the full haemagglutinin (ha) gene revealed a high similarity with currently circulating iav in egypt. from all the birds, e. coli was recovered from . % and salmonella from . %, with – % and – % isolates being resistant to at least one of seven selected critically important antimicrobials (cia), respectively. the presence of enzootic iav and the wide prevalence of amr enterobacteriaceae in wild birds highlight the potential role of lbm in the spread of different pathogens from and to wild birds. continued surveillance of both aiv and antimicrobial-resistant enterobacteriaceae in wild birds’ habitats is urgently needed. wild birds represent a sentinel reservoir for a wide range of viral and bacterial pathogens, with the potential to infect domestic birds and disseminate over long distances in a short time [ ] . therefore, wild birds play a crucial role in the introduction, maintenance, and dissemination of pathogens globally, including zoonotics [ ] [ ] [ ] . several studies have indicated the transmission of antimicrobial resistant (amr) bacteria from sewage, manure, contaminated water, and feeds from domestic birds as well as other animal and human sources to wild birds. these populations can then become reservoirs, recombination melting pots, and potential long-distance spreaders of antibiotic-resistant bacteria and resistance genes [ ] [ ] [ ] [ ] . furthermore, wild birds, especially waterfowl, represent the natural reservoir for influenza a viruses (iav), which mostly present in the low pathogenic (lpai) forms, including the h and h subtypes that were transmitted and maintained in domestic birds, to convert to high pathogenic avian influenza (hpai) [ , ] . however, wild birds could also be a direct source of hpai as in the case of the introduction of h n and h n to egypt [ ] . the other way around has been reported in outbreaks of hpai-h n in asia, where hpai subtypes have spilled over into wild bird populations from domestic birds in live bird markets (lbm) [ ] . multiple iav subtypes (hpai-h n , hpai-h n , and lpai-h n ) are co-circulating in egypt, resulting in huge economic losses. notably, both hpai-h n and h n were first detected in wild birds from a wetland in the damietta governorate in northern egypt shortly before spreading to domestic birds [ ] [ ] [ ] . in contrast, lpai-h n was first detected in domestic birds in giza [ ] . however, recent evidence of h n reassortment with eurasian aivs circulating in wild birds was detected in pigeons, presumably due to co-infection with viruses from poultry and migratory waterfowl [ ] . wild migratory birds land in wetland areas in egypt that are close to human activities including backyard and commercial farming, especially on the northern coast. these areas are where most of the cases have been confirmed [ ] . the presence of antimicrobial resistance (amr) in wild birds screened in egypt has been confirmed with high homology, with samples collected from adjacent water, and both lack the homology with the human isolates [ ] . despite the relatively low number of samples, a recent study [ ] suggests that water contaminated with domestic animal sewage could be a possible source of antimicrobial genes in bacteria found in wild birds. the incidence rate of avian influenza in egypt is particularly high in lbm as compared to backyard and commercial farms sampled birds. lbms are very common and widely distributed all over egypt due to the cultural preference for the consumption of freshly slaughtered poultry [ , , ] . some lbms in egypt sell migratory birds since trapping of such birds is common amongst local communities for commercial sale in lbm in the north coast cities. this is where they come into close contact with domestic birds [ ] . taken together, wild birds and lbm represent an important hub for avian influenza epidemiology and a potential reservoir for amr in egypt. in the present study, samples were collected from wild birds sold at lbm in the north coastal governorates and tested for the presence of iav and antimicrobial resistant enterobacteriaceaeis. ten birds out of were positive ( . %) for the iav m gene by real-time pcr. when subtyped, one remained untyped, four were h n , four were h n , and one was h n (supplementary figure s ). no co-infection was detected in any of the collected samples. all positive samples were detected in northern shoveler birds (spatula clypeata) sampled in gamasa on december showing mild diarrhoea but with absence of respiratory or neurological clinical signs (table ) (supplementary table s ). h n : phylogenetic analysis of the full ha gene of the four h n viruses indicated that they clustered together within clade . . . group b (figure ) with recently isolated egyptian h n strains from domestic birds. sample clustered with strains with the characteristic amino acid substitution l previously reported in recent egyptian samples [ ] . all four samples possessed multibasic cleavage sites in the motif plrekrrkr/glf and six potential glycosylation sites at positions , , , , , and (h numbering). all samples had q and g that indicated preferential binding to α - sialic acidlinkages (avian receptors). the mutations r s and l s were detected in the receptor binding sites of samples and and samples , , and , respectively. mutation t a was detected in antigenic site a of sample . further mutation at position y n was found in sample . total pathogens , , x for peer review of h n : phylogenetic analysis of the full ha gene of the four h n viruses revealed clustering within group b of g -like eurasian sub-lineages (a/quail/hong kong/g / -like). sample , , and clustered with the most recent egyptian strains available on genbank, while sample clustered with earlier strains circulated in ( figure ). all four samples possessed monobasic cleavage sites in the motif parssr/glf. mutations h and l have been detected in samples , , and , which indicated a preference of - α sialic acid receptor (human-like). alternatively, sample possessed q , which is typical of an avian h n signature. no other significant mutations were detected in comparison to other egyptian h n strains except mutation t in sample , which was located at the receptor-binding site. h n : phylogenetic analysis revealed grouping of the h n positive sample with the eurasian gene pool ( figure ) represented by (a/duck/hunan/ / (h n ), as previously described [ ] . blast analysis showed a close relationship (> % nucleotide homology) to a recent wild bird virus from china (a/wild bird/wuhan/cdhn / (h n ) and water wildfowl virus from korea (a/water wildfowl/korea/m / (h n ) and egyptian h n (a/eurasian-teal/egypt/p - / ). this is suggestive of a eurasian wild bird origin. genome analysis revealed that the h n viruses are low pathogenic to chickens with a monobasic cleavage site (pqietr/glf).the presence of q and g (h numbering) on ha indicated a preference of avian (α , -linked sialic acid receptors). using netnglyc . server six potential, n-linked glycosylation sites in ha were identified. out of live wild bird samples, e. coli was recovered from samples, while salmonella was recovered from samples. nine of all samples showed salmonella and e. coli co-infection (supplementary table s ). unlike influenza, e. coli and salmonella were isolated from almost all the wild bird species sampled (table ) . e. coli: serological identification revealed presence of o , o , o , and o in , , , and of e. coli positive samples, respectively (table ). o and o serotypes are usually associated with avian pathogenic e. coli (apec), while o and o are considered as potentially producing lethal toxins to humans. enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec), and enterohemorrhagic e. coli (ehec) are widely prevalent in e. coli positive birds, while enteroinvasive e. coli (eiec) was only recovered from one bird (supplementary table s ). table s ). other enterobacteriaceae: klebsiella pneumoniae has been recovered from samples collected from apparently healthy birds. enterobacter from birds. proteus from birds and citrobacter from birds (table ) and (supplementary table s ). antibiotic resistance: seven antibiotics that were considered as highest priority critically important antimicrobials for human medicine (who-cia) and commonly used in egypt were selected and tested by all positive e. coli and salmonella positive samples. data showed that %, %, %, . %, . %, . %, and . % of e. coli isolates and %, %, . %, . % . %, . %, and . % of salmonella isolates were resistant to ampicillin, ciprofloxacin, doxycycline, erythromycin, streptomycin, tetracycline, and amoxicillin, respectively (table ) . wild birds represent the natural host of every known subtype of type a influenza, with the exception of h n and h n , which were reported solely in bats [ ] . wild birds are responsible for the transcontinental spread of influenza viruses over thousands of kilometers and spill over to domestic birds and threaten human health. few aiv subtypes become as well adapted to poultry as the panzootic goose/guangdong lineage h nx viruses, the recent chinese h n viruses, and multiple eurasian h n lineages and present a major threat to veterinary and human health, while others quickly disappeared [ ] . after long journeys from europe and asia, millions of exhausted wild birds rest in the wetlands of northern egypt, especially around where the river nile drains into the mediterranean sea. in this area, the trapping of wild birds is a very ancient and common practice for commercial sale in live bird markets [ ] . both residences of wild birds and offering them for sale in live bird markets represent a considerable risk for the spillover of the infection to domestic birds and possibly vice versa. in the present study, samples were collected from trapped wild birds offered in lbm, representing wild bird species. only northern shoveler birds were positive for type a influenza virus as evidenced by real-time pcr. the northern shoveler (spatula clypeata) is considered an important aiv reservoir and is known to have high rates of aiv prevalence [ , ] . to our knowledge, different aiv subtypes have been recovered from northern shoveler in egypt and these include h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , and h n [ , [ ] [ ] [ ] [ ] . all of them present in low pathogenic forms. alternatively, in the present study, hpai-h n , lpai-h n , and lpai-h n subtypes have been recovered from , , and birds, respectively. hemagglutinin gene sequencing and phylogentic analysis showed higher similarity and clustering with currently circulating h n , h n , and h n genotypes in domestic fowl. the same findings were previously reported in egypt [ ] , where h n and h n viruses were detected in migratory mallards and were genetically closely related to aiv circulating in domestic poultry. in the same context, hpai has been occasionally reported in wild birds near to affected poultry flocks, but these bird have played a minor or no role in the dissemination of the virus [ ] . we believe that northern shoveler acquire enzootic influenza infection via direct contact with domestic birds in lbm with the absence of clinical signs, which was previously reported [ ] . furthermore, we reported that all positive birds sampled on the same day from the same market had influenza with very high similarity to enzootic viruses, and this scenario suggests a single source of infection. in contrast, the pandemic h n and h n introduced to egypt in late and , respectively, were first detected in a eurasian green-winged teal (anas crecca) which was found dead in the same area [ , ] . in this case, viruses spread to domestic birds where they evolved and became markedly diverged from the original virus introduced via wild birds. altogether, it is unlikely that captured infected wild birds can play a role in the dissemination of the virus to other destinations. however, it is worth mentioning that it is still a considerable risk factor of exporting enzootic h n and h n via migration. further, wild birds in lbm could act as mixing vessels for different influenza subtypes that may lead to reassortment. different surveillances strategies should be developed to find out whether the enzootic viruses are able to disseminate to wild birds from lbm or in their free habitat as well. environmental sampling should include fresh fecal sampling and onsite birds sampling is required for insight analysis of the ability of domestic birds to disseminate enzootic viruses to wild birds in their habitat. wild birds play a crucial role as potential reservoirs of enteric human and animal pathogens and vectors of antimicrobial resistance transfer. information regarding the prevalence of bacterial pathogens and their antimicrobial pattern is limited for the wild birds in egypt. in the present study, profiles of enterobacteriaceae members were investigated in all sampled wild birds using standard methods. fourteen members of the enterobacteriaceae family were recovered. e. coli and salmonella were recovered from . % and . % of all tested birds, respectively. serotyping of the isolated e. coli revealed the wide prevalence of certain e. coli serotypes, which are more frequently associated with avian collibacillosis such as o and o [ ] , and shiga toxin-producing serotypes such as o , o , and o [ , ] were also recovered. potentially, a higher prevalence of salmonella spp. has been recovered in this study with a prevalence of . % (n= / ), when comparing to previous studies [ ] [ ] [ ] . furthermore, a high prevalence of both e.coli and salmonella were detected in waterfowl, especially in the northern shoveler, where . % and . % of tested samples were positive for e.coli and salmonella, respectively, with highly diversified e. coli and salmonella serovars, demonstrating the possible particular importance of the northern shoveler in the dissemination of bacterial pathogens of human and veterinary importance as well as aiv in egypt. although the wild birds were not directly exposed to antimicrobial agents, they get infected [ , ] and act as a reservoir and disseminator of resistant bacteria [ , ] . the who has categorized the antimicrobial reagents into critically important, highly important, and important antimicrobials [ ] . in the present study, seven of the most commonly used critically important antimicrobials (cia) in egypt were selected to test all e. coli and salmonella isolates. in the present study, resistance to cia occurred in %- % of e. coli strains and %- % of salmonella strains. the high rate of resistance of e. coli isolates to streptomycin, tetracycline, and amoxicillin ( %, %, and %, respectively) compared to %, %, and . %, respectively, which was recently detected in wild birds in switzerland, reflects the role of wild birds as a mirror for the unwise use of antimicrobial agents [ ] . samples were collected from migratory birds offered for sale in lbm ( from damietta and from gamasa) during the winter season (between december and february ) ( table and supplementary table s ). both cities are located atthe north-eastern corner of the nile delta. damietta is located between the damietta branch of the river nile and lake manzala on the northern coast of egypt. gamasais located on the northeast coast toward the west of damietta branch. this area hosts significant numbers of overwintering waterfowl each year andis frequented by poultry from surrounding households and commercial poultry farming. furthermore, this area had positive influenza cases in domestic birds just before the sampling period, as confirmed by the reference laboratory for veterinary quality control on poultry production (rlqp). the sample size was limited to the agreement of fishermen or bird sellers. however, to guide field researchers and ensure effective sampling, only live migratory birds that were caged or tied close to a live bird and offered for sale in lbm were sampled. representative images of sampled birds and their clinical signs were reported. two cloacal swabs were collected from each bird, the first of which was placed individually in cryovials containing viral transport medium for iav detection. the second swab was kept in non-selective media for bacterial isolation and identification. samples were stored on ice once collected and were transported rapidly to rlqp for laboratory processing. the sampling plan and experimental procedures were reviewed and approved by rlqp scientific and ethics committee (agreement no. ), in accordance with guidelines of the ministry of agriculture and land reclamation and the ministry of the environment, egypt. swabs from each bird were individually subjected to rna extraction using a qiaamp viral rna mini kit (qiagen, gmbh, hilden, germany) in accordance with the manufacturer's instructions and extracted rna quantity and purity were measured by nano droptm spectrophotometer. reverse transcription and amplification were performed using the quantitect probe rt-pcr kit (qiagen, gmbh, hilden, germany) and analyzed using the mx p real-time pcr system (stratagene, la jolla, ca, usa). the primers and probes that were used to amplify the aiv matrix gene (m) and the thermal profile were previously described [ ] . positive samples for the m gene were tested for ha-h , h , h , and h in accordance with the procedures previously described by [ ] , [ ] , [ ] , and [ ] , respectively. the neuraminidase n , n , and n were tested as previously described by [ ] and [ ] . for molecular pathotyping and confirmation of pcr subtyping, a pan-ha rt-pcr targeting the ha cleavage site of influenza a viruses followed by direct sequencing were used, as previously described by [ ] . neuraminidase subtyping was confirmed by pan-na rt-pcr assay followed by direct sequencing, as previously described by [ ] . positive samples were subjected for newcastle disease virus and coronavirus detection by quantitative rt-pcr, as previously described in [ ] and [ ] , respectively. one hundred µl of the original cloacal swabs were inoculated once into embryonated chicken eggs and allantoic fluids were harvested in accordance with oie recommendations [ ] .collected allantoic fluids were tested for aiv by the haemagglutination (ha) assay and pcr targeting of the m gene before full ha sequencing. in brief, rna was extracted using a qiaamp viral rna mini kit (qiagen, gmbh, hilden, germany) in accordance with the manufacturer's guidelines. the first-strand cdna was synthesized using superscript iii reverse transcriptase (invitrogen, waltham, ma, usa) usinguni- universal primer [ ] . synthesised cdna was amplified with specific primers that cover all ha genes [ ] . pcr products of expected sizes were purified from agarose using a qiaquick gel extraction kit (qiagen, gmbh, hilden, germany) in accordance with the manufacturer's instructions. sequence reactions were performed using the big dye terminator version . cycle sequencing kit (applied biosystems, foster city, ca, usa). sequence reactions were purified using centrisep spin columns (thermo fisher, waltham, ma, usa) and were then analyzed using the genetic analyzer (applied biosystems, usa). the retrieved sense and antisense sequences were assembled and trimmed and a consensus was generated using the geneious®software, version . . [ ] . full ha sequences of all samples were compared with the pan ha sequence results, which were verified using an online basic local alignment search tool (blast) (https://blast.ncbi.nlm.nih.gov/blast.cgi). to perform phylogenetic analysis, full ha sequences of h , h , and h sequences epresenting different genotypes were retrieved from genbank and gsaid, with special reference to genotypes recently found circulating in egypt and neighboring countries, and aligned with sequences from the present study. the alignment was performed with clustal w in mega software version [ ] . phylogenetic trees were constructed with the same software using maximum likelihood, with the substitution model general time-reversible with gamma-distribution (gtr+g) and bootstrap replicates. the trees were viewed using fig tree v . . (http://tree.bio.ed.ac.uk/software/figtree/). cloacal swab samples were placed in tubes containing buffered peptone water (oxoid, basingstoke, uk) and transported in an icebox to the laboratory in the rlqp-gamasa branch. bacteriological examinations for the detection of enterobacteriaceae were performed in accordance with the reference procedure [ ] . the biochemically confirmed e. coli and salmonella isolates were serologically identified in accordance with [ ] and [ ] , respectively. unidentified isolates were subjected to further biochemical tests in accordance with [ ] . for antimicrobial susceptibility testing, seven critically important antimicrobials [ ] that are commonly used in egypt were tested using the agar disc diffusion method on muller hinton agar plates in accordance with the reference procedure [ ] . altogether, wild birds in lbm represent a potential reservoir for influenza viruses and amr, which represents a big risk, especially if we bear in mind that a lot of domestic birds that are offered for sale at lbm are mainly used for restocking, which will lead to further dissemination in the backyard system. however, it is still not fully confirmed whether wild birds acquired enzootic aiv and cia resistance from other domestic birds in the lbm or through the intake of polluted water or food contaminated with human or animal waste in their habitat or from backyard birds near to their habitat. on that basis, further epidemiological studies include environmental samples from wild birds' habitat, onsite wild birds sampling, plus samples from surrounding domestic birds in backyards, lbm, and from sellers using appropriate sanitation measures. this is necessary to understand the mode of transmission of resistant bacteria and enzootic aiv to wild birds and their ability to maintain and disseminate infection into the environment. further, the high prevalence of antibiotic-resistant bacteria and presence of diversified e. coli serotypes, salmonella 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access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would also like to thank the fishermen and bird sellers who were involved in the present studies for their cooperation in field sampling and data collection. also, we thank the biotechnology bacteriology and gene analysis units' members in rlqp (dokki and gammasa branches) for their technical support. we also thank john hammond and andreas alber from the pirbright institute for their constructive criticism of the manuscript. ahmed samy is currently supported by the bill and melinda gates foundation [ - ] and bbs/e/i/ . mahmoud m. naguib is currently supported by the swedish research council vr (grants number - ). the authors declare no conflict of interest. key: cord- -r t uowz authors: ly, hinh title: lassa fever: viral replication, disease pathogenesis, and host immune modulations date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: r t uowz despite major discoveries made in the last few decades about lassa fever, there are still many unresolved key issues that hamper the development of effective vaccines and therapies against this deadly disease that is endemic in several west african countries. some of these issues include the lack of a detailed understanding of the viral and participating host factors in completing the virus life cycle, in mediating disease pathogenesis or protection from disease, and in activating or suppressing host innate and cellular immunity against virus infection, as well as of the animal models required for testing vaccines and therapeutics. this special issue is devoted to understanding some of these important issues and to exploring the current status of the research and development in combating lassa fever. since the first case of the new coronavirus disease in (covid- ) was reported on february in algeria (north africa), the overall numbers of reported cases and deaths have been increasing exponentially in recent weeks in africa, with all member states of the who african region being affected, and more than half of the countries in the region are experiencing community transmission (https://www.afro.who.int/health-topics/coronavirus-covid- ). as of may , the highest case load has been observed in west african regions at % of the total number of cases ( , cases with a case fatality rate (cfr) of . %), followed by the southern african region at % ( , , cfr . %), the north african region at . % ( , cfr . %), the central african region at % ( , cfr . %), and the east african region at % ( , cfr . %). a major concern is that the current covid- pandemic might become endemic in some countries in africa and around the globe. it is important to note that other infectious diseases, such as hiv, malaria, and lassa fever, have already been endemic for many decades in africa, in particular in sub-saharan africa. it is, therefore, important not to lose sight of these ongoing pandemic and endemic infections and the potential of the co-circulation of these deadly human pathogens in these regions where health care and social and political conditions (in some countries) are highly unstable. in this special issue, that is devoted to understanding some of the important issues about lassa fever that is endemic in several west african nations, there are seven timely contributions in the form of original research and review articles on lassa fever's viral replication, the disease pathogenesis and protection, host immune modulations, and other related hot topics. in particular, three review articles contributed by leading researchers in the field that cover important concepts, including how different animal models can be developed for understanding lassa fever's disease pathogenesis and pathologies and for the evaluation of candidate vaccines and antiviral therapies [ ] , how to improve the breadth of host immune responses to lassa vaccination [ ] , as well as how different virus and host factors orchestrate the intricate processes of lassa virus (lasv) entry and genome replication [ ] . in addition to these insightful review articles, two research articles deal with novel strategies of developing vaccines for lassa fever, including the use of the lasv-like particles composed of the viral matrix (z) and envelope glycoprotein complex (gpc) expressed by the modified vaccinia ankara virus to protect mice against lethal virus challenges [ ] and the use of a candidate lasv vaccine known as ml that is composed of the reassorted genome between the pathogenic lasv and the non-pathogenic mopeia virus (mopv), which shows its highly attenuated phenotype in rodents [ ] . an intriguing observation from this study is that the persistent infection of cells with ml can result in the generation of interfering viral particles that can induce a potent level of cell-mediated immunity and can strongly interfere with the replication of arenaviruses, such as lasv, mopv, and lymphocytic choriomeningitis virus (lcmv). there are two other research papers that cover some hot topics in the field. one such paper uses state-of-the-art mass spectrometry to identify novel phosphorylation sites in the lasv z protein [ ] . in particular, residues of two serines (s , s ) and a tyrosine (y ) located in the flexible n-and c-terminal regions of the protein are found be phosphorylated. two of these residues, y and s , happen to be located in or directly adjacent to the so-called late domain with the ppxy motif, which is known to be required for mediating optimal progeny virion release from the infected cells. the authors showed that phosphorylation of these amino acids served as an important regulatory mechanism of virus release and that host-driven, reversible phosphorylation processes might play an important role in the regulation of lasv z protein function in virus assembly and release. another important step in the life cycle of lasv is viral entry, which is a two-step process that involves the viral envelope glycoprotein complex (gpc). the gp subunit (gp ) of the gpc first binds to the cell surface receptor before the viral particle is engulfed into the cellular endosome. a drop in ph in the endosome triggers gpc structural rearrangements which lead the gp subunit (gp ) of the gpc to form a six-helix-bundle that helps fuse the lysosomal membrane with the lasv envelope membrane, allowing the lasv genome to enter the cellular cytoplasm for replication and transcription. in order to identify amino acid residues in gp that are crucial for the process of lasv entry, willard and colleagues performed a semi-saturated alanine scanning mutagenesis of amino acid residues of gp [ ] . they tested these mutant gpcs for efficient gp -gp cleavage, cell-to-cell membrane fusion, and transduction into cells expressing the known cellular receptor (α-dystroglycan) and other secondary lasv receptors. using this systematic experimental approach, the authors successfully identified seven gp mutants that could mediate efficient gp -gp cleavage, yet they were unable to effectively transduce cells, which suggests that these key residues are critically important for gp function in the process of lasv entry into cells. improving the breadth of the host's immune response to lassa virus virus-host interactions involved in lassa virus entry and genome replication. pathogens a single dose of modified vaccinia ankara expressing lassa virus-like particles protects mice from lethal intra-cerebral virus challenge attenuated replication of lassa virus vaccine candidate ml in stat- -/-mice. pathogens host-driven phosphorylation appears to regulate the budding activity of the lassa identification of residues in lassa virus glycoprotein subunit that are critical for protein function the author declares no conflict of interest. key: cord- -kczj se authors: yang, bo; zhang, xiaohui; zhang, dajun; hou, jing; xu, guowei; sheng, chaochao; choudhury, sk mohiuddin; zhu, zixiang; li, dan; zhang, keshan; zheng, haixue; liu, xiangtao title: molecular mechanisms of immune escape for foot-and-mouth disease virus date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: kczj se foot-and-mouth disease virus (fmdv) causes a highly contagious vesicular disease in cloven-hoofed livestock that results in severe consequences for international trade, posing a great economic threat to agriculture. the fmdv infection antagonizes the host immune responses via different signaling pathways to achieve immune escape. strategies to escape the cell immune system are key to effective infection and pathogenesis. this review is focused on summarizing the recent advances to understand how the proteins encoded by fmdv antagonize the host innate and adaptive immune responses. foot-and-mouth disease (fmd) is an acute and highly contagious disease affecting the cloven-hoofed animals, such as pigs and cattle. the pathogen that causes fmd is known as fmd virus (fmdv), a single-stranded positive-sense rna virus that is classified into the genus aphthovirus in the family picornaviridae [ ] [ ] [ ] . the pathogen causes vesicular disease of mouth and feet in susceptible animals [ ] . the high mutation rate of the genome of fmdv and the rapid proliferation has led to the rapid evolution of the virus and the formation of seven main serotypes [ ] [ ] [ ] . the antigenic diversity among the serotypes poses challenges to the research of efficient and cross-protective vaccines [ ] . the genome of fmdv contains an open reading frame (orf) that encodes a polyprotein precursor, and it is cleaved into four structural proteins and non-structural proteins by viral autoproteases and host protease [ , ] (figure ). upon infection of the host, a virus will face the attack from the host's immune response. in the long-term battle with the host immune response, the virus has evolved and developed a series of immune escape mechanisms to overcome the killing and inhibition from the host immune system. the mechanism of virus immune escape can be divided into three categories: ( ) enable the virus to avoid the recognition of humoral immune response; ( ) interfere with the function of cellular immune response; ( ) interfere with the host's immune response to the virus [ ] . all these strategies would be exploited by the virus for replication and spreading to other hosts. as a highly contagious and fast-spreading virus, fmdv has multiple ways to evade the killing by the immune system [ ] , which makes it difficult for controlling the virus. viral capsid protein vp and leading protein l pro can inhibit the production of interferon (ifn) and innate immune response by interacting with soluble resistance-related calcium-binding protein (sorcin) or host transcription factor adnp [ , ] . recently, new mechanisms and functions of fmdv proteins inhibiting innate immunity have been discovered. ddx (a kind of rna helicase), participate in rna metabolism and ribosome synthesis is reported to involve in this new mechanism. the interaction between fmdv a and ddx suppresses the host innate immunity by reducing the phosphorylation of irf [ ] . in addition, nucleotide-binding oligomerization domain (nod ), a member of the nucleotide-binding oligomerization domain-like receptor (nlr) family [ ] , activates the nf-κb and ifn-β signaling pathways during fmdv infection and inhibits the replication of fmdv in infected cells [ ] . fmdv b, c, and c pro inhibit the expression of nod protein, which antagonizes the antiviral response [ ] . reportedly, multiple structural and non-structural proteins of fmdv escape the killing of the host immune system. this review summrized the molecular mechanisms of immune evasion caused by fmdv proteins. the present study aimed to fill the gaps of knowledge on fmdv immune evasion mechanism, providing the basis for the prevention and control strategies for fmdv. pathogens , , x for peer review of figure . schematic of the genome and polypeptide processing of fmdv [ , ] . the fmdv genome contains an orf of about kbp, indicated by the shaded rectangle. each region within the orf rectangle represents a single protein. the flank of orf is a long ' untranslated region ( '-utr) and a short '-utr. b covalently binds to the '-end. upon infection of the host, a virus will face the attack from the host's immune response. in the long-term battle with the host immune response, the virus has evolved and developed a series of immune escape mechanisms to overcome the killing and inhibition from the host immune system. the mechanism of virus immune escape can be divided into three categories: ( ) enable the virus to avoid the recognition of humoral immune response; ( ) interfere with the function of cellular immune response; ( ) interfere with the host's immune response to the virus [ ] . all these strategies would be exploited by the virus for replication and spreading to other hosts. as a highly contagious and fast-spreading virus, fmdv has multiple ways to evade the killing by the immune system [ ] , which makes it difficult for controlling the virus. viral capsid protein vp and leading protein l pro can inhibit the production of interferon (ifn) and innate immune response by interacting with soluble resistance-related calcium-binding protein (sorcin) or host transcription factor adnp [ , ] . recently, new mechanisms and functions of fmdv proteins inhibiting innate immunity have been discovered. ddx (a kind of rna helicase), participate in rna metabolism and ribosome synthesis is reported to involve in this new mechanism. the interaction between fmdv a and ddx suppresses the host innate immunity by reducing the phosphorylation of irf [ ] . in addition, nucleotide-binding oligomerization domain (nod ), a figure . schematic of the genome and polypeptide processing of fmdv [ , ] . the fmdv genome contains an orf of about kbp, indicated by the shaded rectangle. each region within the orf rectangle represents a single protein. the flank of orf is a long untranslated region ( -utr) and a short -utr. b covalently binds to the -end. the p structural protein of fmdv was cleaved into three main viral structural proteins vp , vp and vp by c pro protease during the later translation and modification. vp protein was further cleaved into vp and vp proteins by protease c. interestingly, the vp protein is a cleavage precursor of vp and vp [ ] . the last step in the production of mature virions is the cleavage of ab (vp ), which converts residues of the n-terminal into vp and the remaining into vp , although some copies of vp may be retained in the intact virions [ ] . previous studies reported that vp protein of fmdv inhibits the activation of type i ifn signaling pathway by interacting with irf [ ] (figure , table ). however, further studies are essential to assess the combination of vp to irf to restrain the production of ifns. since then, it is reported that vp proteins of fmdv interact with poly (rc) binding protein (pcbp ) to promote the replication of fmdv [ ] (figure , table ). the pcbp can recruit e ligase aip that contains the hect domain into the polyubiquitin and degrades mav [ ] . the vp protein of fmdv suppresses the host's innate immunity by cooperating with pcbp to suppress the activation of ifn-β promoter. vp protein promotes the formation of pcbp -virus-induced signaling adapter (visa) complex, enhances the degradation of visa mediated by pcbp , and promotes the replication of fmdv [ ] . in addition, the vp structural protein of fmdv is necessary for the correct assembly of the virus [ ] . pathogens , , the fmdv capsid protein, vp , was further cleaved into vp and vp proteins by protease c. according to the structure of the virus, the vp protein of fmdv is localized on the inner surface of the capsid [ ] . although vp does not stimulate the production of neutralizing antibodies independently, it contains t and b cell epitopes, which could be recognized by a variety of haplotype mhc molecules and exhibit high immunogenicity [ ] [ ] [ ] . therefore, the combination of fmdv vp and vp could be used as a backup antigen for the development of a universal vaccine [ , ] . in addition, the vp protein plays a crucial role in immunosuppression. the recombinant fmdv vp -vp protein has been reported to have an inhibitory effect on the innate immune function of mouse peritoneal mast cells, putatively mediated by mannose receptor [ ] . furthermore, nucleoside diphosphate kinase (nme ) regulates the function of p to prevent tumor metastasis and progression and inhibit the metastasis of several malignant tumors [ ] [ ] [ ] . the role of nme in viral infection is not yet clarified. recent studies have demonstrated that nme has antiviral activity and enhances p -mediated transcription, while p regulates the expression of many antiviral genes to perform antiviral functions. however, fmdv vp does not directly interact but degrades nme through macroautophagy [ ] [ ] [ ] (figure , table ). this phenomenon promotes the interaction between p and mdm (mdm is a negative regulatory factor of p ), while on the other hand, it enhances the mif-mediated inhibition of p activity, thereby impeding the antiviral response [ ] . autophagy is an ancient and conservative biological process, which exists in almost all eukaryotes. through continuous research, it has been found that autophagy can selectively degrade intracellular redundant or harmful substances [ ] [ ] [ ] , thus affecting the pathogenesis of some diseases. autophagy can also degrade invading microorganisms (such as bacteria, virus, and parasites) [ ] , and is one of the immune mechanisms against pathogenic infection. it has been proved that a variety of viruses can activate autophagy and be swallowed and degraded [ ] . not only that, after virus infection, host cells resist virus infection by releasing inflammatory factors and activating innate and adaptive immune responses, and autophagy also plays an important role in these defense responses [ ] . in the long process of coexistence of virus and host, autophagy pathway has become one of the targets of virus-versus-host immunity. the inhibitory effect of virus on autophagy is also in many ways. for example, some studies have shown that after prrsv infection, the type i microtubule-associated protein light chain (lc -i) is transformed into lc -ii, which activates the autophagy mechanism and leads to the accumulation of autophagosomes by preventing the fusion of autophagosomes and lysosomes. autophagosomes can act as replication sites to enhance prrsv replication [ , ] . vp is one of the structural proteins of fmdv, localized on the surface of the virus. types o, a, and c fmdv vp contain several antigenic sites that present immunological significance [ , [ ] [ ] [ ] . in addition, the amino acid substitution on the vp b-c loop of fmdv type asia not only mediates the significant antigenic diversity but also alters the replication ability and pathogenicity of the virus. for example, the single asp-to-asn substitution at vp position will reduce the virus replication ability and virulence [ ] . however, the exact reasons for this result need to be further studied. a recent study demonstrated that the interaction between fmdv vp and hspb activates the eif s -atf pathway, which in turn, inhibits the akt-mtor pathway, leads to autophagy, and promotes virus replication [ ] (figure , table ). autophagy has been proposed to provide a membrane platform for virus replication complexes or mediate the virus assembly and release [ ] . thus, autophagy plays a crucial role in the replication of fmdv, and the expression of related vp mutants decreases the level of autophagy. therefore, vp -induced autophagy may be one of the mechanisms of fmdv infection. autophagy regulates type i ifn signaling machinery and plays a vital role in antiviral innate immunity [ , ] , and atg -atg conjugate inhibits the production of type i ifn during vsv infection [ ] . thus, it can be speculated that fmdv vp induces autophagy to increase the replication of fmdv, which might be achieved by blocking type i ifn signal. also, the correlation between vp -induced autophagy and host antiviral immunity of fmdv needs to be investigated further. the changes in some sites on the surface of the vp protein of fmdv affects the cellular tropism and adaptability of the virus; for instance, the replacement of (glu to gly) alters the binding characteristics of the virus to cells [ ] . the positively charged lysine residue at the vp site of fmdv a can increase the adaptability of bhk- cells [ ] . furthermore, the change in some sites of vp would also affect the stability and immunogenicity of fmdv. for example, vp h y replacement reduces the acid sensitivity of the capsid of fmdv type asia , makes the h y mutant of fmdv resistant to acid, and enhances the immunogenicity of virions [ ] . the tyrosine at position of vp mutated to phenylalanine (y f) enhanced the thermal stability of the virus. this mutant presented optimal immunogenicity, and neutralizing antibodies could be induced by immunizing guinea pigs [ ] . thus, identifying these specific sites of vp protein in fmdv provides an idea for the preparation of a heat-resistant and immunogenetically superior fmd antigen. the vp protein is the major surface protein on the fmdv and the primary antigen that elicits the neutralizing antibody response. the fmdv vp stimulates the host to produce cd + t cell responses with cross-protection against multiple serotypes of fmdv. therefore, vp protein or its antigenic determinants have become a research hotspot in the development of novel vaccines [ , , ] . fmdv protein aqueous soluble recombinant dna-derived vp (rvp ) binds to integrin induces apoptosis, and fmdv rvp may selectively act as an effective human tumor apoptosis factor by regulating akt signal pathway [ ] . the vp that interacts with host proteins can either enhance or inhibit the production of ifn in cells. first, it was found that vp interacts with soluble resistance-related calcium-binding protein (sorcin), a negative regulator in the innate immune signaling pathway, through yeast two-hybrid and immunoprecipitation experiments. also, vp binds to sorcin and activates the transcription factor stat . stat inhibits the activation of ikk and nf-κb pathway, thus inhibiting the expression of type i ifn and cytokines [ ] (figure , table ). second, the host protein kinases are essential regulators of virus interaction and play a crucial role in virus replication. tpl (tumor progression locus ), a serine/threonine-protein kinase, promotes the activation of ifn-β signaling pathway by increasing the phosphorylation of irf . tpl phosphorylation site thr is vital for promoting irf -induced ifn-β signal activation. vp inhibits the protein expression of tpl phosphorylated at thr , thereby inhibiting the irf -activated ifn-β signaling, while vp reduces the mrna levels of tpl -mediated ifn-β and some isgs ( figure , table ). third, another study proved that vp suppresses the ifn-β signaling pathway at the irf level by inhibiting the irf phosphorylation, dimerization, and nuclear translocation ( figure , table ). another study suggested that the activation of the mitogen-activated protein kinase (mapk) pathway is essential for fmdv replication. fmdv vp interacts with host ribosomal protein sa (rpsa) to continually activate the mapk signal pathway and promote virus replication by inhibiting the rpsa-mediated function [ ] (figure , table ). as a structural protein of fmdv, vp plays a crucial role in virus assembly [ ] . it blocks the ifn signal transduction, promotes the replication of fmdv, and inhibits the host immune response. in addition, vp inhibits the protein and mrna expression of innate immune junction molecule, visa. it interacts with the visa protein to inhibit the formation of visa-regulated complex, thereby inhibiting the dimerization and phosphorylation of irf , inhibiting the expression of antiviral genes induced by ifn-β, and promoting fmdv replication [ ] (figure , table ). previous studies have focussed on the effect of fmdv after treatment of type i ifn. also, it was demonstrated that fmdv vp inhibits the type ii ifn signaling pathway. furthermore, vp interacts with the host protein kinase jak protein and degrades the jak protein through lysosome pathway, inhibits the activation of the jak-stat pathway, and reduces the ifn-induced antiviral gene expression [ ] (figure , table ). during evolution, some host substances can act on viral proteins, inhibit viral replication, and resist infection. reportedly, the fmdv infection stimulates the expression of mir- , which indirectly induces the degradation of fmdv vp protein through the proteasome pathway and strengthens the host immune response to inhibit the replication of fmdv [ ] . moreover, the induction of mir- is earlier than the full activation of nf-κb and irf / [ ] . nonetheless, fmdv infection-stimulated expression of mir- would be a research hotspot in the future. however, the direct goal of mir- has not yet been determined. recently, it has been reported that tank-binding kinase (tbk ) degrades the vp protein of several types of small ribonucleic acid virus, including fmdv, through its kinase and e ubiquitin ligase activity, while p-tbk is highly enriched in the mir- overexpression cells [ ] . thus, mir- may target negative regulatory factors of tbk . it has been reported that single or co-transfection of micrornamir- a- p and mir- a- p in porcine cell lines followed by infection of fmdv resulted in a decrease in viral protein synthesis and virus production. so, mir- a- p and mir- a- p are potential natural biotherapies against foot-and-mouth disease virus [ ] . interaction between vp and hspb activates the eif s -atf pathway, which leads to autophagy and promotes virus replication [ ] . sorcin soluble resistance-related calcium binding protein vp can bind to sorcin to inhibit the activation of ikk and nf-κ b pathway [ ] . tumor progression locus ; a serine/threonine protein kinase vp inhibits the protein expression of tpl phosphorylation site thr , thereby inhibiting the promotion of irf -activated ifn-β signal by tpl . interferon regulatory factor vp suppresses ifn-β signaling pathway at irf level by inhibiting irf phosphorylation, dimerization, and nuclear translocation. ribosomal protein sa vp interacts with rpsa to maintain the activation of mapk signal pathway and promote virus replication [ ] . visa innate immune junction molecule vp inhibits the expression of visa protein mrna, and interacts with visa protein to inhibit the formation of visa-regulated complex, thereby inhibiting the dimerization and phosphorylation of irf [ ] . janus kinase vp can interact with jak protein and degrade jak protein to inhibit the activation of jak-stat pathway [ ] . fmdv l pro , a papain-like protease, is the first translated protein from the fmdv genome and coexists in two forms in vivo and in vitro, lab pro and lb pro [ ] [ ] [ ] . l pro , a key virulence factor of fmdv, suppresses the host immune response and achieves immune escape. l pro can cleave host translation-related proteins or interact with host transcription factors to inhibit the synthesis of antiviral factors. l pro can target cleavage/degradation pattern recognition receptors, the proteins of the interferon pathway, nf-κb pathway, and stress-related pathway. in addition, l pro acts as a deubiquitinase (dub) and deisgylase. first, l pro specifically cleaves the eukaryotic initiation factors (eifs), gi and gii. the loss of integrity of eif gi and eif gii hinders the formation of eukaryotic cellular translation initiation factor f(eif f) complex, while eif f complex significantly affects the cell cap-dependent translation [ ] , thereby preventing the recruitment of capped mrna in host cells and inhibiting the synthesis of antiviral molecules of innate immunity [ ] . fmdv rna starts translation in a cap-independent manner through the internal ribosome entry site (ires) elements [ ] [ ] [ ] . therefore, fmdv can make use of host protein synthesis mechanism to quickly synthesize virus protein and complete virus reproduction. the interaction between l pro and activity-dependent neuroprotective protein (adnp) is crucial in the process of infection and promotes fmdv replication by inhibiting the expression of ifn and ifn stimulated gene (isg) [ ] . however, whether the processing of adnp by l pro is performed directly by l pro or by related enzymes induced or activated by l pro is yet to be deduced. the present study further elucidates the mechanism though which fmdv evades the immune response by interaction with transcription factors. second, fmdv l pro downregulates the expression of nf-κb and irf / , which in turn, interferes with the transcription of ifn-α /β mrna. fmdv l pro induces the degradation of p /rela, the core component of nf-κb, which destroys the integrity of nf-κb and downregulates the transcription of ifn-β in host cells, thus inhibiting the host immune response [ ] . however, the degradation mechanism of p /rela by l pro is still unclear, and the products of p /rela digested by l pro have not been identified. in addition to destroying the integrity of nf-κb, l pro also decreased the expression of irf / , the key factors of virus-triggered ifn-α/β secretion that inhibit the production of dsrna-induced type i ifn [ ] . third, porcine ifn-λ , a type iii ifn, inhibits the replication of fmdv. however, after screening, the lead protease l pro exerts a robust inhibitory effect on the activity of ifn-λ promoter induced by poly(i:c) by inhibiting the rig-i/mda pathway and interfering with the activation of interferon regulatory factors (irfs) and nf-κb. fmdv l pro alone can inhibit the expression of dsrna-induced ifn-λ , suggesting a new mechanism of fmdv antagonizing ifn-λ -mediated innate immune response [ ] . fourth, in addition to its conventional papain-like protease activity, l pro acts as a deubiquitinase (dub) and deisgylase. lb pro has deubiquitination activity, and the deubiquitination functional sites are highly conserved among the seven serotypes of fmdv. these motifs could significantly inhibit the ubiquitination of key molecules of innate immune signaling pathway such as retinoic acid-inducible gene i (rig-i), tank-binding kinase (tbk ), tnf receptor-associated factor (traf ), and traf , thereby inhibiting the innate immune response and achieving immune escape [ ] . lb pro selectively cleaves the c-terminal peptide bond of isg and exposes an easily detected glygly epitope on the substrate of the modifier, which provides a new method for monitoring fmdv [ ] . a new study shows that abolishing/reducing the deisgylase/dub activity of l pro causes viral attenuation independently of its ability to block the expression of ifn and isg mrna [ ] . the latest research shows that l pro 's ability to cleave rlr signaling proteins but not its deubiquitination/deisgylation activity correlates with the reduced ifn-β gene transcription [ ] . fifth, laboratory of genetics and physiology (lgp ), an innate immune sensor promotes the interaction between viral rna and mda , thus producing antiviral signals [ ] . recently, it has been reported that lgp is the biphasic main activator of many innate immune genes that induce the production of ifn by a cascade effect [ ] . however, fmdv l pro cleaves lgp and blocks the effect of lgp -mediated ifn-β induction [ ] . these features represent a new approach of immune escape and provide a basis for in-depth research on the role of lgp in anti-fmdv response and the interaction between mda and lgp -l pro . sixth, recent studies have demonstrated that the stress response was inhibited during fmdv infection. fmdv l pro targets the sg (stress granule) scaffold proteins g bp and g bp to antagonize the formation of sg, a potentially significant antiviral signaling platform [ ] [ ] [ ] [ ] that regulates the integrated stress response [ ] . however, the l pro -mediated sg inhibition mechanism of fmdv might not be the only one in the cells infected with fmdv. although several studies have suggested the antiviral effect of sgs, their exact role as an antiviral signal platform is not yet clarified. therefore, these studies demonstrated that fmdv l pro inhibits the host immune response and promotes the fmdv replication through various mechanisms ( figure , table ). fmdv b protein is a hydrophobic transmembrane viroporin with oligomeric transmembrane pores that can destabilize the integrity of the host cell membrane, disrupt host cell ca + balance, induce host cell autophagy, and promote virion release [ , ] . fmdv b may play an active role in virus immune escape because of its viroporin characteristics. previous studies have shown three pattern recognition receptors, retinoic acid-inducible gene i (rig-i), melanoma differentiation-associated factor (mda ), and lgp that bind to viral rna. of these, rig-i and mda recognize different structures of rna to activate the antiviral signal transduction, and lgp regulates this process [ , , ] . targeted studies have found that rig-i and lgp inhibit the fmdv replication, and lgp significantly inhibits the inflammatory response of fmdv-infected cells. fmdv b protein suppresses the expression of pattern recognition receptors rig-i, mda , and lgp , inhibiting the host antiviral response and promoting fmdv replication. b protein directly interacts, reduces the protein levels, and inhibits the antiviral effect mediated by rig-i, mda , and lgp ( figure , table ). this reduction does not depend on proteasome, lysosome, or autophagy pathway, and the specific molecular mechanism is yet unclear. in addition, the study on whether b reduces the level of other junction molecules in rig-i like receptor (rlr) signaling pathway showed that although fmdv b does not mediate the decline in tbk and irf in the rlr signaling pathway, it inhibits the phosphorylation of tbk and irf , followed by suppression of the expression of type i ifn [ ] [ ] [ ] (figure , table ). the results of yeast two-hybrid screening and immunoprecipitation showed that one of the two host proteins that could interact with fmdv b protein is the eukaryotic translation elongation factor γ (eef g) [ ] . a previous study confirmed that the decrease in eef g affects the synthesis of some membrane proteins necessary for vesicle formation, and its mislocation reduces the synthesis of membrane proteins [ ] . the b protein of fmdv is associated with increased cell membrane integrity and membrane permeability [ ] . therefore, it can be speculated that eef g assists b in producing virus-induced vesicles and inducing cell lysis. however, further studies are required to substantiate these findings. the other host protein is cyclophilin a, which degrades fmdv l pro and a protein that suppresses the innate immune. strikingly, the interaction between b protein and cyclophilin a directly inhibits the degradation of l pro and a protein by cyclophilin a, thereby inhibiting the host immune response [ ] (figure , table ). recent studies have shown that cyclophilin a also promotes the ubiquitination of rig-i, and thus enhances the innate immune response [ ] . however, whether the interaction between b protein and cyclophilin a affects the ubiquitination of rig-i is yet to be elucidated. another study showed that fmdv b protein interacts with nod to reduce the expression of nod protein, for which the b carboxyl-terminal - region was essential, thus inhibiting the activation of nf-κb and ifn-β signaling pathways ( figure , table ). the decrease in nod is not related to the cleavage of eif g, induction of apoptosis or proteasome, nor lysosome or caspase pathways [ ] . fmdv protein c is a highly conserved polypeptide of amino acids (aa) [ , ] . subsequent studies proved that the c protein plays a critical role in virus replication. guanidine hydrochloride, a molecular antagonist of protein c, suppresses the synthesis of viral genetic material in small ribonucleic acid virus-infected cells [ , ] . three host proteins beclin , vimentin, and nod interacting with fmdv c, were screened by yeast two-hybrid assay. beclin is related to the formation of autophagosomes and the fusion of autophagosomes to lysosomes [ , ] . protein c interacts and inactivates beclin , which in turn, inhibits the fusion of autophagosomes containing fmdv and lysosomes, thereby preventing the degradation of the virus [ ] (figure , table ). vimentin plays a role in the lysosomal degradation of proteins and has been shown to be related to autophagosomes [ , ] . in the early stage of fmdv infection, vimentin forms a cage-like structure around c in order to facilitate virus replication. additionally, the expression of the dominant-negative (dn) form of vimentin significantly reduces the replication ability of fmdv. the replication of fmdv requires a complete vimentin network. however, the exact mechanism of governing vimentin cage formation and dissolution remains to be elucidated [ ] . the interaction between fmdv protein c and nod reduces the level of nod protein to help the virus evade the immune response, and the carboxyl-terminal - and - regions of c were essential for the interaction [ ] (figure , table ). strikingly, the interaction between c and beclin or nod evades immune response through different pathways, which contributes to the replication of fmdv. the interaction between c and cellular vimentin is crucial for the replication of fmdv, albeit the specific pathway is not yet clarified. the a protein is a conserved -aa polypeptide of fmdv, larger than other picornaviruses [ ] . fmdv a protein is related to host tropism and virulence, and the deletion of a alters the virus tropism and virulence. reportedly, a single amino acid change in a endowed fmdv with a new adaptive phenotype, and fmdv a protein is associated with membrane correlation and regulation of host protein secretion [ , ] . plaque assay and virus titeration showed that the stable expression of a or ab protein enhances the replication of fmdv. however, the infection level of fmdv decreased after the transient expression of ab protein. these findings suggested that a and ab play a crucial role in the replication of fmdv [ ] . fmdv a has been proved to interacts with cellular protein dctn by the two-hybrid method. dctn is a subunit of the dynactin complex, a cofactor for dynein. dynactin-dynein complexes are related to the transport of intracellular organelles. the overexpression of dctn and the disruption of dynactin-dynein complex significantly reduces the production of fmdv in infected cells. fmdv replication seems to require a complete dynamic protein cell pathway [ ] . in the previous study, fmdv a protein mutantageness study based on reverse genetic technique revealed the effect of amino acid mutation in a protein on the interaction between a protein and dctn was detected by yeast two-hybrid technique. the data show that a could be bound to dctn when amino acid was alanine or leucine, but when amino acid was mutated to proline, which destroy the binding between a and dctn [ ] . interestingly, both the fmdv o/taw/ strain with pldg peptide from - aa on a and the recombinant fmdv o c a virus with deletion of residues - on a lacked the binding site of the host protein dctn . also, the replication rate in primary bovine cells was slower than that of parental virus strains. however, no significant change was detected in the replication rate of either of the two viruses and their parent virus strains in porcine cells [ , ] . thus, it could be speculated that the binding of fmdv a and host dctn might be related to the host tropism of the virus, but the slight difference in dctn among species could be attributed to the range of hosts of fmdv. the recombinant fmdv with pldg residues - on a produced a delayed and mild disease in cattle, suggesting that a-dctn interaction might play a role in the virulence of the bovine virus. in addition, the virus recovered rapidly during infection and regained dctn binding, suggesting that the interaction between fmdv a and dctn is vital for virus replication in cattle [ ] (figure , table ). however, the a-dctn combination needs further exploration. the type i ifn reporting system was utilized to confirm that the a protein inhibits the activation of the ifn-β signaling pathway. further studies demonstrated that a protein interacted with innate immune molecules rig-i, mda , and visa, inhibited the expression of innate immune junction molecules, such as rig-i, mda , and visa, and inhibited the formation of signal transduction complex, thereby escaping the host innate immune system [ ] (figure , table ). the overexpression of the fmdv a inhibited the sendai virus-triggered activation of irf [ ] . a recent study found that the interaction between dead-box helicase (ddx ) and fmdv protein a increases the interaction between ddx and irf and enhances the ability of fmdv a to inhibit irf phosphorylation ( figure , table ). also, fmdv a inhibits the activation of the ifn-β promoter and isre by reducing the phosphorylation of irf and increasing the replication of fmdv. however, the overexpression of ddx cannot significantly reduce the phosphorylation of irf . thus, we speculated that ddx also inhibits the production of ifn through another different pathway, thereby promoting virus replication [ ] . unlike other picornaviruses that encode a single b copy, fmdv encodes three similar but different b proteins ( b , b , and b ), which are ubiquitous in all fmdv isolates [ ] . the effective replication of fmdv in bovine cells requires a full-length a and three vpg ( b). the deletion of the b coding sequence adversely affects the rna replication of fmdv, and the viral activity requires highly conserved b protein [ , ] . although fmdv lacking b and b can also reduce the viral rna synthesis, the growth of the virus on pig-derived cells is only slightly reduced, and the disease of pigs has also been slightly alleviated [ , ] . these studies showed that b is more critical than b and b in maintaining virus rna replication. the efficiency of rna replication is maximal when three b copies coexist, and the absence of b and b might affect the virulence and host range of fmdv. however, the integration of the three proteins in replication needs to be studied further. fmdv b significantly reduces the levels of ifn-β, isg , and il- levels and the activation of ifn-β, nf-κb, and isre promoters induced by poly(i:c), indicating that fmdv b is also a viral escape protein, which inhibits the response of type i ifn in cells. subsequently, b blocked the interaction between rig-i and trim , thus inhibiting the ubiquitination of rig- and the formation of the rig-i-visa complex, which inhibits the ifn signaling pathway [ ] (figure , table ). further studies have shown that fmdv b reduces the expression of type i ifn by inhibiting the visa signaling pathway via interaction with the visa protein ( figure , table ). fmdv c protein has been identified as a protease. it cleaves not only most of the virus precursor proteins [ ] but also the host proteins to block/inhibit the cellular defense mechanism and promote virus replication. fmdv protein c pro cleaves the related host proteins, inhibits the transcription and translation of host cells, or promotes the translation of virus rna, thus promoting virus replication. for example, fmdv protein c pro is related to the cleavage of histone h , as it removes n-terminal amino acid residues from histone h . the amino terminal of h is related to the regulation of chromosomal transcriptional activity in eukaryotic cells. the cleavage of cpro to h inhibits the transcription of host cells and ultimately hinders the translation of host cells [ ] , which is beneficial for the virus to escape antiviral immune response ( figure , table ). in addition, fmdv protein c pro also cleaves the host translation initiation factors, eif g and eif a, which are components of the cap-binding complex eif f, which inhibits the synthesis of host-related antiviral proteins [ ] ( figure , table ). however, it can only partially cleave eif g and eif a but not completely block the translation of host cells [ , ] . unlike the cleavage of eif g by l pro in the early stage of infection, the cleavage of eif g and eif a by fmdv protein cpro requires the accumulation of c protein. hence, it occurs in the later stage of infection cycle, and the cutting site of eif g by c pro is different from that mediated by l pro [ ] . in addition, the cleaving of eif a may be disadvantageous to the virus, and the translation of viral rna requires eif a [ ] . the fmdv protein c pro can also cleave the -kda src-associated substrate during mitosis (sam ), a unique rna-binding protein (figure , table ). truncated sam spreads to the cytoplasm, combines with fmdv rna and attaches to ires to enhance the translation of the virus rna. however, the titer of fmdv was reduced -fold after transfection of sam -targeted sirna molecules, indicating sam might not limit to enhancing virus translation, and sam may play a variety of roles in fmdv infection [ ] . fmdv protein c pro directly or indirectly degrades vital immune molecules, thus inhibiting/blocking the expression of ifn and antiviral genes. fmdv protein c pro can degrade pattern recognition receptors, rig-i and lgp , thus inhibiting the production of antiviral factors and promoting fmdv replication [ , ] ( figure , table ). it also degraded the modulator necessary for innate immune key molecule nemo, the vital regulator of nf-κb, a junction protein necessary for activating nf-κb, and the ifn regulatory factor signaling pathway. this cleavage impaired the activation of irfs and nf-κb and inhibited the expression of downstream antiviral genes ( figure , table ). the cysteine protease activity of c pro is necessary for c pro to cleave nemo [ ] . reportedly, fmdv c suppresses the jak-stat signaling pathway, thus inhibiting the antiviral response induced by ifn. another study found that c protease activity promoted the degradation of kpna , the nuclear localization signal receptor for tyrosine-phosphorylated stat , to block the nuclear translocation of stat /stat to inhibit the jak-stat signaling pathway [ ] (figure , table ). autophagy-associated protein atg -atg is shown to be associated with the replication of fmdv. in the process of fmdv infection, atg -atg upregulates the anti-virus nf-κb and irf signaling pathways, thereby inhibiting the proliferation of fmdv. fmdv protein c pro antagonizes the host antiviral immunity and suppresses autophagy by degrading atg -atg [ ] (figure , table ). furthermore, atg -atg enhances the expression of host protein kinase pkr, a serine-threonine kinase, which can be induced by ifn and activated by double-stranded rna (dsrna). consequently, it blocks the synthesis of cell and virus protein, inhibits the replication of fmdv, and exerts a significant antiviral effect [ ] . moreover, fmdv c pro protein induces pkr degradation through the lysosome pathway and inhibits the pkr-mediated antiviral effect by downregulating the pkr protein. no interaction occurred between fmdv c pro and pkr [ ] (figure , table ). recent studies have shown that c pro decreases the level of nod , thus inhibiting the antiviral effect induced by nod , and the reduction of nod induced by c pro depends on its protease activity ( figure , table ). no interaction occurred between fmdv c pro and nod [ ] . taken together, fmdv c pro exerts its immunosuppressive function and suppresses the innate immunity of the host via a myriad of cascades. can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity. fmdv a and b reduce the expression of junction protein visa at the transcriptional or protein level. l pro , b and a can directly or indirectly target irf to inhibit interferon production. c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production. l pro and c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation. l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis. c can promote virus replication by regulating autophagy. eif g: eukaryotic initiation factor g; nf-κb: nuclear factor kappa b; irf : interferon regulatory factor ; irf : interferon regulatory factor ; rig-i: retinoic acid inducible gene i; tbk : tank binding kinase i; traf : tnf receptor-associated factor ; traf : tnf receptor-associated factor ; adnp: activity-dependent neuroprotective protein; lgp : laboratory of genetics and physiology ; mda : melanoma differentiation associated factor ; cypa: cyclophilin a; nod : nucleotide-binding oligomerization domain ; dctn : dynactin ; ddx : dead-box helicase ; sam : kda src-associated substrate during mitosis; visa: virus-induced signaling adapter. can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity. fmdv a and b reduce the expression of junction protein visa at the transcriptional or protein level. l pro , b and a can directly or indirectly target irf to inhibit interferon production. c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production. l pro and c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation. l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis. c can promote virus replication by regulating autophagy. eif g: eukaryotic initiation factor g; nf-κb: nuclear factor kappa b; irf : interferon regulatory factor ; irf : interferon regulatory factor ; rig-i: retinoic acid inducible gene i; tbk : tank binding kinase i; traf : tnf receptor-associated factor ; traf : tnf receptor-associated factor ; adnp: activity-dependent neuroprotective protein; lgp : laboratory of genetics and physiology ; mda : melanoma differentiation associated factor ; cypa: cyclophilin a; nod : nucleotide-binding oligomerization domain ; dctn : dynactin ; ddx : dead-box helicase ; sam : kda src-associated substrate during mitosis; visa: virus-induced signaling adapter. l pro cut eif gi and eif gii, thus preventing the recruitment of capped mrna and inhibiting the synthesis of antiviral molecules [ , ] . nf-κb nuclear factor kappa b l pro induce the degradation of p /rela, which is the core component of nf-κb [ ] . interferon regulatory factor / l pro decreased the expression of irf / to inhibit the production of type i ifn induced by dsrna [ ] . rig-i tbk traf traf retinoic acid inducible gene i; tank binding kinase i; tnf receptor associated factor tnf receptor associated factor l pro can significantly inhibit the ubiquitination of key molecules of innate immune signaling pathway such as rig-i, tbk , traf , and traf [ ] . adnp activity dependent neuroprotective protein l pro and adnp interact to promote the replication of fmdv by inhibiting the expression of ifn and isg [ ] . lgp laboratory of genetics and physiology l pro can cleave lgp and block the effect of lgp -mediated the production of ifn-β [ ] . g bp and g bp stress granule scaffold proteins l pro targets to cleave the sg scaffold proteins g bp and g bp to antagonize the formation of sg [ ] . retinoic acid inducible gene i; melanoma differentiation associated factor ; laboratory of genetics and physiology ; b protein suppress the expression of rig-i, mda and lgp , inhibiting host antiviral response [ , ] . tbk ; irf tank binding kinase i; interferon regulatory factor b suppress the phosphorylation of tbk and irf , and then inhibit the expression of type i interferon [ ] . cypa cyclophilin a the interaction between b protein and cyclophilin a directly inhibits the degradation of l pro and a protein by cyclophilin a [ ] . nod nucleotide-binding oligomerization domain b protein can interact with nod to reduce the protein level of nod , which inhibit the activation of nf-κb and ifn-β signal pathways [ ] . beclin involve in the fusion of autophagosomes to lysosomes c interacts with beclin to induce beclin inactivation, which inhibits the fusion of autophagosomes of containing fmdv and lysosomes [ ] . nod nucleotide-binding oligomerization domain the interaction between fmdv protein c and nod reduces nod at the protein level to help the virus evade immune response [ ] . a protein increases the interaction between ddx to inhibits the activation of ifn-β promoter and isre by reducing the phosphorylation of irf [ ] . retinoic acid inducible gene i; b blocked the interaction between rig-i and trim , thus inhibiting the interferon signal pathway [ ] . visa virus-induced signaling adapter b reduces the expression of type i interferon by inhibiting visa signal pathway by interacting with visa protein. histone h related to the transcription of host cells the cleavage of c pro to h inhibits the transcription of host cells and ultimately hinders the translation of host cells [ ] . eif g and eif a host translation initiation factors c pro is involved in the cleavage of eif g and eif a, thus inhibiting the synthesis of host-related antiviral proteins [ ] . kda src-associated substrate during mitosis c pro can also cleave sam . truncated sam spreads to the cytoplasm and meets the fmdv rna and attaches to the ires to enhance the translation of the virus rna [ ] . retinoic acid inducible gene i; laboratory of genetics and physiology ; protein c pro can degrade rig-i and lgp [ , ] . nemo nf-κb necessary regulator c can also degrade nemo to impaired activation of irfs and nf-κb [ ] . kpna the nuclear localization signal receptor for tyrosine-phosphorylated stat c promoted the degradation of kpna to block the nuclear translocation of stat /stat to inhibit jak-stat signal pathway [ ] . autophagy associated protein fmdv protein c pro antagonizes host antiviral immunity and suppresses autophagy by degrading atg -atg [ ] . pkr a serine-threonine kinase c pro induces pkr degradation and inhibits pkr-mediated antiviral effect by down-regulating pkr protein [ ] . nod nucleotide-binding oligomerization domain c pro induces the reduction of nod , thus inhibiting the antiviral effect induced by nod [ ] . the untranslated region ( utr) of fmdv is about bp, which is larger than that of several small rna virus of the family picornaviridae. it contains s-fragment, poly (c), pseudoknots (pks), the cis-acting replication element (cre) and internal ribosome entry site (ires). studies have shown that the special structure of the untranslated region is essential for the translation and replication of the virus genome [ ] . the first element of the end is the s-fragment with approximately nt long, the sequence can fold and form the stem ring. it is speculated that this structure can block the function of host exonuclease, thereby maintaining the stability and replication of the virus genome [ ] . s fragment is related to the interaction between virus and host protein. some studies have confirmed the direct correlation between the degree of s fragment deletion mutation and the attenuated phenotype. the fmdv mutant with bp deletion in the upper part of the s fragment loop was highly attenuated in vivo [ ] . fmdv with deletion of s fragment induces higher expression of ifn-β and isg mrna, so it is concluded that s fragment of fmdv is necessary for host cell replication and regulation of innate immune response [ ] . downstream of s-fragment is the variable length poly (c) region. studies on the viral genome have shown that a certain threshold length of poly (c) is correlated to the viability of the virus, but there is no evidence that the length of the poly (c) chain is directly related to virulence [ ] . the end of ploy (c) is pseudoknots. some studies have shown that the deletion of -nt in pks reduces the pathogenicity of o/cha/ / strain of fmdv in bovine cells and bovines, and the artificial deletion of bases will reduce the pathogenicity of o/me-sa/panasia strain fmdv in bovine. this deletion occurs naturally in the region of the porcinophilic cathay topotype fmdv genome. it is suggested that the natural absence of pks area may be the reason for the transformation from bovines to pigs as a vector for the transmission of fmdv. it is concluded that the pseudoknots region of fmdv untranslated region is the decisive factor of virus tendency and virulence [ ] . the cis-acting replication element (cre) is a stem-loop structure containing conserved aaaca motifs [ , ] . it has been confirmed that fmdv cre is necessary for genome replication, and cre has been found to be adjacent to ires, which indicates that cre may play a role in coordinating translation and replication, but this still needs further confirmation [ ] . fmdv -utr consists of a nt structural sequence folded into two independent stem loops and a poly (a) tail of variable length [ , ] . related studies have shown that the -utr directly binds to the s-fragment and ires elements of the -utr at different sites, and fmdv -utr affects virus replication and virulence by enhancing the activity of ires elements [ ] . moreover, genetic studies have shown that the recombinant fmdv with missing structural sequence of -utr cannot be recovered [ ] , and it is concluded that the structural region of -utr is essential for the infectivity and replication of fmdv. in addition to the direct rna-rna interaction, - -end bridging, which is also related to protein-protein and protein-rna interactions, it has been found that cellular proteins pcbps and p can directly bind to s-fragment and -utr [ ] . it is inferred that - -end bridging may play an important role in the replication of fmdv. fmdv is a highly contagious virus that infects almost all cloven-hoofed animals, showing vesicles on the foot and mouth, skin erosion on the mucous membranes, fever, weight loss, pacing, and salivation, severely threatening the development of animal husbandry. however, in addition to causing acute infections and diseases, fmdv can be asymptomatic carriers in some cases, which might lead to another outbreak of fmd, making prevention and control challenging and costly. high infectivity, wide geographical distribution, wide host range, short-term immunity without serotype cross-protection, multiple modes of transmission, and persistent infection render the control and eradication of this disease rather difficult. therefore, study the molecular mechanism underlying fmdv evading immunity is imperative for the control of an epidemic situation. the immune system includes innate immunity and acquired immunity, which is a major protective system against the invasion of pathogenic microorganisms, surveillance, and removal of foreign bodies. fmdv suppresses the function of the immune system at the initial stage of infection, such that the virus can proliferate rapidly in the respiratory system and spread to its natural infection site [ ] . in terms of evading the humoral immune system, each serotype of fmdv is prone to antigenic variation, which makes the virus escape from the neutralizing antibodies [ ] . in the aspect of inhibiting cellular immune response, fmdv infection can cause the decrease of host lymphocytes and is accompanied by severe viremia, which will eventually lead to the destruction of t cells and fmdv infection inhibits the function of dendritic cells and weakens the ability of dendritic cells to process them into antigens [ , ] . previous studies have shown that, mhc class i molecule expression on the surface of cells was suppressed at min after fmdv infection, indicating that the cells infected with fmdv will immediately lose the ability to present mhc-i-related viral peptides to t lymphocytes. this mechanism would facilitate the virus escape from the host's cytotoxic immune response. limiting the killing effect mediated by nk cells is also an important mechanism for fmdv to evade the cellular immune response. some studies have shown that the responsiveness of porcine nk cells decreases significantly - days after fmdv infection, and then returns to normal [ ] . strikingly, nk cells isolated from infected pigs could not secrete ifn-γ [ ] . the research on fmdv interference with immune effect and suppression of innate immunity has been widely studied. some proteins of fmdv (l pro , b, a, b, c) can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity [ , [ ] [ ] [ ] ] . fmdv vp , vp , a, and b reduce the expression of junction protein visa at the transcriptional or protein level [ , , ] . fmdv l pro , vp , vp , b, and a can directly or indirectly target irf to inhibit interferon production [ , , , ] . vp and c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production [ , ] . fmdv proteins l pro and c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation [ , , , , ] . in addition, it is interesting that l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis [ ] . fmdv protein vp and c can promote virus replication by regulating autophagy [ , ] . these mechanisms provide opportunities for rapid transcription and translation of fmdv. in the previous studies on fmdv, hek cells have been widely used in in vitro experiments because of its highly transfected efficiency. however, hek cells are not fmdv susceptible cells and there are species differences between hek cells and fmdv susceptible cells. therefore, the use of hek cells for fmdv-related research has some limitations. in summary, fmdv has evolved a variety of ways to evade the immune response in the long-term combat with the host immune system. although there are many breakthroughs in the research on the immune escape of fmdv, many mechanisms underlying the fmdv-affected host immunity have not yet been elucidated, and the interaction between fmdv protein and host protein need to be explored further. in addition to the interaction between the virus and host protein, exploring the mechanism of synergistic inhibition of immune response by multiple viral proteins is of great significance for the development of specific drugs and new vaccines. previous studies mainly focused on the effect of fmdv with respect to innate immunity. however, there are a few studies on acquired immunity, and these need to be supplemented further. also, persistent infection of fmdv needs to be investigated intensively in the future. it was the goal of the literature study to summarize the current knowledge and to point out future research directions and define the scientific questions that remain to be elucidated to gain better knowledge of immune responses against fmdv and its immune escape mechanisms. foot-and-mouth disease: host range and pathogenesis foot-and-mouth disease foot-and-mouth disease: past, present and future molecular epidemiology of foot-and-mouth disease virus the generation and persistence of genetic variation in foot-and-mouth disease virus foot-and-mouth disease virus evolution: exploring pathways towards virus extinction emergence and selection of rna virus variants: memory and extinction understanding the molecular epidemiology of foot-and-mouth-disease virus molecular basis of 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positively charged lysine residue at vp position allows for the enhanced adaptability of foot-and-mouth disease virus serotype a in bhk- cells single amino acid substitution of vp n d or vp h y confers acid-resistant phenotype of type asia foot-and-mouth disease virus mutation in the vp gene of p - a capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype o enhanced immune response of dna vaccine (vp -pcdna) adsorbed on cationic plg for foot and mouth disease in guinea pigs induction of a cross-reactive cd + t cell response following foot-and-mouth disease virus vaccination vp of foot-and-mouth disease virus induces apoptosis via the akt signaling pathway foot-and-mouth disease virus capsid protein vp interacts with host ribosomal protein sa to maintain activation of the mapk signal pathway and promote virus replication the vp structural protein of foot-and-mouth disease virus inhibits the ifn-β signaling pathway foot-and-mouth disease virus structural protein vp degrades janus kinase to inhibit ifn-gamma signal transduction pathways host microrna mir- suppresses foot-and-mouth disease virus replication by promoting vp degradation and enhancing innate immune response the e ubiquitin ligase tbk mediates the degradation of multiple picornavirus vp proteins by phosphorylation and ubiquitination host microrna- a is antagonistic to the progression of foot-and-mouth disease virus infection two initiation sites for foot-and-mouth disease virus polyprotein in vivo the two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities identification of critical amino acids within the foot-and-mouth disease virus leader protein, a cysteine protease sonenberg, n. eif initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation foot-and-mouth disease virus leader proteinase involvement of c-terminal residues in self-processing and cleavage of eif gi divergent picornavirus ires elements extremely efficient cleavage of eif g by picornaviral proteinases l and a in vitro l protease from foot and mouth disease virus confers eif -independent translation for mrnas bearing picornavirus ires degradation of nuclear factor kappa b during foot-and-mouth disease virus infection foot-and-mouth disease virus leader proteinase inhibits dsrna-induced type i interferon transcription by decreasing interferon regulatory factor / in protein levels foot-and-mouth disease virus (fmdv) leader proteinase negatively regulates the porcine interferon-λ pathway the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase irreversible inactivation of isg by a viral leader protease enables alternative infection detection strategies impairment of the deisgylation activity of foot-and-mouth disease virus lpro causes attenuation in vitro and in vivo dissecting distinct proteolytic activities of fmdv lpro implicates cleavage and degradation of rlr signaling proteins, not its deisgylase/dub activity, in type i interferon suppression lgp synergy with mda in rlr-mediated rna recognition and antiviral signaling pum is a biphasic negative regulator of innate immunity genes by suppressing lgp innate immune sensor lgp is cleaved by the leader protease of foot-and-mouth disease virus stress granules and cell signaling: more than just a passing phase? translation inhibition and stress granules in the antiviral immune response stress granules regulate double-stranded rna-dependent protein kinase activation through a complex containing g bp and caprin the stress granule protein g bp recruits protein kinase r to promote multiple innate immune antiviral responses foot-and-mouth disease virus leader protease cleaves g bp and g bp and inhibits stress granule formation viroporin activity of the foot-and-mouth disease virus non-structural b protein functional analysis 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fraction before lysosomal degradation foot-and-mouth disease virus modulates cellular vimentin for virus survival role of nonstructural proteins a and b in host rangeand pathogenicity of foot-and-mouth diseasevirus susceptibility to viral infection is enhanced by stable expression of a or ab proteins from foot-and-mouth disease virus interaction of foot-and-mouth disease virus nonstructural protein a with host protein dctn is important for viral virulence in cattle a partial deletion in non-structural protein a can attenuate foot-and-mouth disease virus in cattle evaluation of infectivity and transmission of different asian foot-and-mouth disease viruses in swine foot-and-mouth disease virus non-structural protein a inhibits the interferon-β signaling pathway comparative genomics of foot-and-mouth disease virus vpg gene amplification correlates with infective particle formation in foot-and-mouth disease virus domain disruptions of individual b proteins of foot-and-mouth disease virus do not alter growth in cell culture or virulence in cattle foot-and-mouth disease virus b protein inhibits type i interferon pathway signaling by blocking the interaction of rig-i with trim foot-and-mouth disease virus protease c induces specific proteolytic cleavage of host cell histone h foot-and-mouth disease virus c protease induces cleavage of translation initiation factors eif a and eif g within infected cells cleavage of translation initiation factor ai (eif ai) but not eif aii by foot-and-mouth disease virus c protease: identification of the eif ai cleavage site sequential modification of translation initiation factor eif gi by two different foot-and-mouth disease virus proteases within infected baby hamster kidney cells: identification of the cpro cleavage site dominant negative mutants of mammalian translation initiation factor eif- a define a critical role for eif- f in cap-dependent and cap-independent initiation of translation the nuclear protein sam is cleaved by the fmdv c protease redistributing sam to the cytoplasm during fmdv infection of host cells foot-and-mouth disease virus c protease cleaves nemo to impair innate immune signaling cpro of foot-and-mouth disease virus antagonizes the interferon signaling pathway by blocking stat /stat nuclear translocation foot-and-mouth disease virus infection suppresses autophagy and nf-kb antiviral responses via degradation of atg -atg by cpro regulation of innate immunity through rna structure and the protein kinase pkr foot-and-mouth disease virus induces lysosomal degradation of host protein kinase pkr by c proteinase to facilitate virus replication biological function of foot-and-mouth disease virus non-structural proteins and non-coding elements foot-and-mouth disease virus -terminal s fragment is required for replication and modulation of the innate immune response in host cells infectious foot-and-mouth disease virus derived from a cloned full-length cdna the pseudoknot region of the untranslated region is a determinant of viral tropism and virulence of foot-and-mouth disease virus the rhinovirus type genome contains an internally located rna structure that is required for viral replication identification of an rna hairpin in poliovirus rna that serves as the primary template in the in vitro uridylylation of vpg identification and characterization of a cis-acting replication element (cre) adjacent to the internal ribosome entry site of foot-and-mouth disease virus translation and replication of fmdv rna the end of the foot-and-mouth disease virus genome establishes two distinct long-range rna-rna interactions with the end region enhanced ires activity by the utr element determines the virulence of fmdv isolates deletion or substitution of the aphthovirus ncr abrogates infectivity and virus replication the pathogenesis of foot-and-mouth disease in pigs morphologic and phenotypic characteristics of myocarditis in two pigs infected by foot-and mouth disease virus strains of serotypes o or a. acta veter effect of foot-and-mouth disease virus infection on the frequency, phenotype and function of circulating dendritic cells in cattle loss of plasmacytoid dendritic cell function coincides with lymphopenia and viremia during foot-and-mouth disease virus infection natural killer cell dysfunction during acute infection with foot-and-mouth disease virus cell mediated innate responses of cattle and swine are diverse during foot-and-mouth disease virus (fmdv) infection: a unique landscape of innate immunity discriminating self and non-self by rna: roles for rna structure, misfolding, and modification in regulating the innate immune sensor pkr the authors would like to thank the anonymous editors and reviewers for their valuable comments and suggestions that helped improve the quality of this manuscript. the authors declare no conflict of interest. key: cord- -oq bc u authors: felten, sandra; klein-richers, ute; hofmann-lehmann, regina; bergmann, michèle; unterer, stefan; leutenegger, christian m.; hartmann, katrin title: correlation of feline coronavirus shedding in feces with coronavirus antibody titer date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: oq bc u background: feline coronavirus (fcov) infection is ubiquitous in multi-cat households. responsible for the continuous presence are cats that are chronically shedding a high load of fcov. the aim of the study was to determine a possible correlation between fcov antibody titer and frequency and load of fecal fcov shedding in cats from catteries. methods: four fecal samples from each of cats originating from german catteries were examined for fcov viral loads by quantitative reverse transcriptase polymerase chain reaction (rt-qpcr). additionally, antibody titers were determined by an immunofluorescence assay. results: cats with antibodies were more likely to be fcov shedders than non-shedders, and there was a weak positive correlation between antibody titer and mean fecal virus load (spearman r = . ; p = . ). antibody titers were significantly higher if cats shed fcov more frequently throughout the study period (p = . ). when analyzing only fcov shedders, cats that were rt-qpcr-positive in all four samples had significantly higher antibody titers (p = . ) and significantly higher mean fecal virus loads (p = . ) than cats that were rt-qpcr-positive in only one, two, or three samples. conclusions: the cats’ antibody titers correlate with the likelihood and frequency of fcov shedding and fecal virus load. chronic shedders have higher antibody titers and shed more virus. this knowledge is important for the management of fcov infections in multi-cat environments, but the results indicate that antibody measurement cannot replace fecal rt-qpcr. feline coronavirus (fcov) is a viral pathogen infecting cats worldwide. it is highly contagious [ ] : nearly % of cats become infected when exposed, usually horizontally via the fecal-oral route [ , ] . additionally, the virus can persist in excretions in the environment for up to several weeks [ ] , rendering indirect transmission, e.g., via caregivers, a possible route of infection. the prevalence of fcov in the cat population is very high; this is especially true for multi-cat households, in which prevalence can be as high as % [ ] [ ] [ ] . only about - % of fcov-infected cats go on to develop fcov more frequently throughout the study period and ( ) whether cats shedding fcov in all four fecal samples collected would have higher antibody titers and shed higher concentrations of fcov than cats that shed virus only intermittently. indeed, the results obtained suggest that in cats from german catteries, the antibody titer is correlated with mean fecal virus load. cats with antibodies were more likely to be fcov shedders than non-shedders. cats with higher antibody titers shed fcov more frequently and with a higher mean fecal virus load than cats with lower fcov antibody titers or without antibodies. cats shedding fcov in all four fecal samples had higher fcov antibody titers and shed higher concentrations of fcov than cats shedding fcov intermittently. however, there were nine cats without antibodies that shed fcov. thus, antibody testing cannot replace fecal testing by rt-qpcr. anti-fcov antibodies were detected in of the cats ( %) included in the study (figure ). all catteries homed at least one cat with antibodies. fcov rna could be detected in at least one of the four fecal samples collected at intervals of - days by rt-qpcr in of the included cats ( %), and all catteries homed at least one cat that was shedding fcov in at least one fecal sample. of the cats without detectable antibodies, nine cats ( %) shed fcov at least once. of the cats with antibodies, / ( %) with an antibody titer of : , / ( %) with a titer of : , and / ( %) with a titer of : shed fcov at least once. none of the cats had an antibody titer of : . most ( %) of the cats that were not shedding fcov either did not have antibodies ( / ; %) or only a very low titer of : ( / ; %). nevertheless, / ( %) cats that were rt-qpcr-negative in all four fecal samples had an antibody titer of at least : . fecal virus load ranged from . × to . × per gram (g) of feces (median . × per g feces) in cats shedding fcov (table ) . cats with higher anti-fcov antibody titers also had significantly higher mean fecal virus loads. there was a weak positive correlation between the figure . antibody titers of cats not shedding feline coronavirus (fcov) and of cats with one, two, three, or four fecal samples positive for fcov rna by quantitative reverse transcriptase polymerase chain reaction (rt-qpcr). fecal samples with weak positive rt-qpcr results were considered positive; samples below the limit of quantification were considered negative. of the cats without detectable antibodies, nine cats ( %) shed fcov at least once. of the cats with antibodies, / ( %) with an antibody titer of : , / ( %) with a titer of : , and / ( %) with a titer of : shed fcov at least once. none of the cats had an antibody titer of : . most ( %) of the cats that were not shedding fcov either did not have antibodies ( / ; %) or only a very low titer of : ( / ; %). nevertheless, / ( %) cats that were rt-qpcr-negative in all four fecal samples had an antibody titer of at least : . fecal virus load ranged from . × to . × per gram (g) of feces (median . × per g feces) in cats shedding fcov (table ) . cats with higher anti-fcov antibody titers also had significantly higher mean fecal virus loads. there was a weak positive correlation between the antibody titer and the calculated mean fecal virus load (mean of all four fecal samples) (spearman r = . , % confidence interval . - . ; p = . ). table . fecal feline coronavirus (fcov) load per gram of feces of cats with different antibody titers detected by quantitative reverse transcriptase polymerase chain reaction (rt-qpcr). fecal fcov load was calculated as the mean of all four fecal samples from each cat. the two cats with only one weak rt-qpcr-positive or one sample below the limit of quantification and three rt-qpcr-negative fecal samples each were excluded from analysis. in addition to shedding larger amounts of fcov with their feces, cats with higher antibody titers also shed fcov more frequently. antibody titers were significantly higher in cats with more frequent than in cats with less frequent fcov shedding (p = . ; figure ). however, when comparing the cats in different groups (based on their frequency of fcov shedding), this difference was only significant between cats that were rt-qpcr-negative in all four fecal samples and cats that were rt-qpcr-positive in all four fecal samples (dunn's test; p < . ). two cats that were rt-qpcr-positive in all four fecal samples did not have antibodies and two cats that were rt-qpcr-negative in all four fecal samples had a high antibody titer of : . when considering the mean fecal virus load and frequency of fcov shedding, there was a slight tendency that cats shedding fcov more frequently shed higher concentrations of fcov rna, but that association was not significant (p = . ; table ). table . mean fecal feline coronavirus (fcov) load per gram of feces of cats with different frequencies of fcov shedding detected by quantitative reverse transcriptase polymerase chain reaction (rt-qpcr). fecal fcov load was calculated as the mean of all four fecal samples from each cat. fecal samples with weak positive rt-qpcr results were considered positive; samples below the limit of quantification were considered negative. the two cats with only one weak rt-qpcr-positive or one sample below the limit of quantification and three rt-qpcr-negative fecal samples each were excluded from analysis. one sample rt-qpcr-positive . × - . × (median . × ) two samples rt-qpcr-positive . × - . × (median . × ) three samples rt-qpcr-positive . × - . × (median . × ) four samples rt-qpcr-positive . × - . × (median . × ) fifty-eight of the cats ( %) were positive for fcov rna by rt-qpcr in at least one of the four fecal samples collected. of those cats, ( %) were rt-qpcr-positive in all four fecal samples. these cats shedding in all four fecal samples had significantly higher antibody titers than cats that were rt-qpcr-positive in only one, two, or three fecal samples (p = . ; table ). moreover, when comparing shedders with four rt-qpcr-positive fecal samples and shedders with one to three rt-qpcr-positive fecal samples, shedders with four rt-qpcr-positive fecal samples shed significantly higher mean fecal virus loads ( . × - . × viral copies per g feces, median . × ; p = . ; figure ) than shedders with one to three rt-qpcr-positive fecal samples ( . × - . × viral copies per g feces, median . × ). pathogens , , x of moreover, when comparing shedders with four rt-qpcr-positive fecal samples and shedders with one to three rt-qpcr-positive fecal samples, shedders with four rt-qpcr-positive fecal samples shed significantly higher mean fecal virus loads ( . × - . × viral copies per g feces, median . × ; p = . ; figure ) than shedders with one to three rt-qpcr-positive fecal samples ( . × - . × viral copies per g feces, median . × ). fecal samples with weak positive rt-qpcr results were considered positive; samples below the limit of quantification were considered negative. the two cats with only one weak rt-qpcr-positive or one sample below the limit of quantification and three rt-qpcr-negative fecal samples each were excluded from analysis. the aim of this study was to investigate a possible correlation between fcov antibody titer and fecal virus shedding in healthy cats living in german catteries. the majority of antibody-positive cats mean fecal virus load per gram (g) of feces of cats with four rt-qpcr-positive samples was significantly higher compared to cats with only one, two, or three rt-qpcr-positive samples (p = . ). mean fecal fcov load was calculated as the mean of all four fecal samples from each cat. fecal samples with weak positive rt-qpcr results were considered positive; samples below the limit of quantification were considered negative. the two cats with only one weak rt-qpcr-positive or one sample below the limit of quantification and three rt-qpcr-negative fecal samples each were excluded from analysis. the aim of this study was to investigate a possible correlation between fcov antibody titer and fecal virus shedding in healthy cats living in german catteries. the majority of antibody-positive cats in this study ( / , %) shed fcov in their feces. however, the study also showed that this assumption is not absolute, since / ( %) of cats without antibodies also were fcov shedders and pathogens , , of / ( %) of the cats with a high antibody titer of : did not shed fcov. previously, only % antibody-positive cats were reported to shed fcov [ ] . a possible reason for this disagreement is the way in which shedding was confirmed in the two studies. in the present study, fcov rna was detected in fecal samples by rt-qpcr. this technique is very sensitive. in the previously published study, rt-pcr was not yet available, so the classification of a cat as shedder occurred via its infectivity to other cats. more precisely, kittens of antibody-positive queens were monitored for the development of antibodies and it was shown that % of the litters developed antibodies, indicating viral shedding occurred in around % of the queens [ ] . of course, this approach is not directly comparable to the testing of feces by molecular methods such as rt-qpcr, as performed in the present study. according to previous studies, fcov antibody titers are significantly higher in cats that shed fcov with their feces than in cats that do not shed fcov [ , , ] . additionally, it was proposed that cats without fcov antibodies are not shedding fcov and that, as a consequence, it would be safe to introduce an antibody-negative cat into a fcov-free household [ , , ] . however, a smaller number of studies reported discordant results, refuting a correlation between fcov antibody titer and the likelihood of fcov shedding [ ] [ ] [ ] . pedersen et al. [ ] tried to explain this discrepancy by different ways of data evaluation. correlation between antibody titer and fecal virus shedding was significant when shedding and non-shedding cats were looked at as groups. conversely, there was a substantial overlap when evaluating individual cats [ ] . this, at least in part, is also true for the cats in the present study. most cats with a high antibody titer of : were shedding fcov (and shed fcov in all four fecal samples), but two cats with a titer of : did not shed fcov in any of the four fecal samples. even more discrepancy could be seen among the cats without antibodies: / of the cats without antibodies shed fcov at least once during the study period. this is a result that, despite the initial belief that antibody-negative cats would never shed fcov [ ] [ ] [ ] , has been reported in the literature before [ , ] . it is possible that the contamination of fecal samples by other fcov-positive cats had occurred in the same multi-cat household. in order to avoid contamination, rectal swabs could be used for testing purposes by cat breeders [ ] , but this would reduce the amount of available fecal material. contamination of samples in the laboratory cannot fully be excluded, but seems rather unlikely given that extensive quality controls were included in the rt-qpcr protocol. more likely, it is conceivable that serum/plasma samples were obtained early in an infection, at a time before the cats developed antibodies, but at which they already shed fcov with their feces. shedding of fcov before the time of antibody development has already been reported in kittens [ ] . since serial antibody measurements were not performed in the present study, such scenario cannot be confirmed. alternatively, the documentation of fecal fcov shedding in antibody-negative cats could be explained by localized infection. it is known that fcov replication can stay confined to the gastrointestinal tract in some fcov-infected cats, resulting in fecal fcov shedding without the development of antibodies [ ] . this might also have occurred in the cats in the present study. antibody testing and the separation of antibody-negative cats in the past has been suggested as method to clear fcov infection from a breeding cattery [ ] or prevent the introduction of fcov infection into the fcov-free geographical area of the falkland islands [ ] . however, as clearly shown by the results of the present study, although the antibody titer can give an idea on a cat's shedding status, a negative antibody titer in a cat does not exclude fecal shedding. thus, the introduction of cats into fcov-free environments on the basis of a negative antibody test still bears the risk of introducing a fcov-shedding cat. on the other hand, the separation of all antibody-positive cats might not be necessary, because not all cats with antibodies shed fcov with their feces. the next question to be answered was whether cats with higher antibody titers also shed higher concentrations of fcov rna with their feces. in this study, there was a weak positive correlation between the quantity of antibodies and the mean fecal virus load determined by rt-qpcr, indicating that cats with higher antibody titers were more likely to shed fcov more intensely compared to cats with low antibody titers and cats without antibodies. it is possible that a higher amount of viral replication, as demonstrated by a higher fecal virus load, also leads to increased antibody production, pathogens , , of and thus, higher antibody titers. however, since there is some overlap, this correlation is only weak, demonstrating that antibody measurement alone is not sufficient to differentiate high intensity from low intensity shedders. so far, there is only one report, which was not published in a peer-reviewed journal, that also suggested a correlation between fcov antibody titer and the intensity of fecal virus shedding [ ] . clarification of such information, however, is of practical importance, since especially high intensity shedders are a concern in environments where many cats are housed together in a limited space. these cats shedding large amounts of fcov pose a high risk of transmission to non-infected cats, and in order to reduce infection pressure in multi-cat environments, the contact of non-infected cats to litter trays of high intensity shedders must be avoided [ , ] . however, the routine practice of testing four sequential fecal samples or rectal swabs taken one week to one month apart for fcov by rt-qpcr, while increasing the likelihood of correctly identifying non-shedders, leads to a prolonged period of time, in which the shedding status of cats in the multi-cat environment is unknown [ , ] . this is especially problematic in rescue shelters, where new cats with unknown fcov status are to be introduced. antibody titers (for which only one blood sample is necessary), in contrast, are available much faster and therefore could help to distinguish high intensity from low intensity shedders or cats not shedding fcov. the results of the present study indicate that the risk of shedding large amounts of fcov will increase with the level of a cat's antibody titer, and a cat without antibodies most likely will not shed large amounts of fcov. this understanding might help to at least estimate whether strict quarantine measures are indispensable or not for each individual cat. the practice of separating high intensity shedders, low intensity shedders and non-shedders in a cat population in order to eliminate fcov infection is being proposed, but not without controversy [ , ] . it has been suggested that antibody measurement might be of importance in breeding catteries, in which kittens might be initially protected from infection by maternally-derived antibodies (mda), that are transferred by nursing from antibody-positive queens, but can become infected as soon as mda wane. isolation of pregnant queens before birth and early weaning of kittens at an age of five to six weeks, before the loss of mda, has been proposed [ , ] . however, this protocol has been questioned, since the adequate socialization of isolated kittens is a concern and fcov infection can occur as early as two weeks of age [ ] . after fecal-oral fcov infection, three possible shedding patterns have been observed. cats can shed fcov intermittently [ , , , , , ] . most likely, this is only partially caused by intermittent fecal excretion, but also by re-infection with either the same or a different fcov strain throughout their lives [ ] . some cats will shed fcov for weeks or months and eventually cease fecal shedding. up to % of cats will shed fcov persistently for a prolonged period of time and sometimes even lifelong [ , , , , ] . the exact percentage of cats following each pattern is unknown and likely varies depending on the epidemiological situation and virulence of the infecting fcov strain. in order to correctly characterize a fcov-shedding cat for eradication purposes, sequential fecal samples must be obtained and four fecal samples, collected - days apart, were obtained from each cat in this study. results indicate that cats had significantly higher antibody titers if they shed fcov more frequently. when considering the results of the individual cats, it becomes apparent that most cats ( %) that did not shed fcov in any fecal sample either did not have antibodies ( / ) or only had a low antibody titer of : ( / ), whereas most cats with a high antibody titer of : ( / ; %) shed fcov in all four samples. nevertheless, although there was a tendency for cats shedding fcov more frequently (possibly due to continuous re-infection) to have higher antibody titers, again there was some overlap, and the correlation was only significant for cats shedding in all four fecal samples compared to non-shedders. it was also determined whether cats shedding fcov in all four samples had higher antibody titers than cats shedding fcov in only one, two, or three fecal samples. the latter cats probably eliminated the infection at some point, shed intermittently or became re-infected. in this context, it is of special interest to evaluate whether measurement of the antibody titer can indicate if a cat likely is continuously shedding fcov, and thus, poses a permanent risk of infection to other cats. it could be shown that, in this population, cats that were rt-qpcr-positive in all four samples had significantly pathogens , , of higher antibody titers than cats, that were rt-qpcr-positive only intermittently in one, two, or three fecal samples. additionally, cats shedding fcov in all four samples also had significantly higher mean fecal virus loads than cats shedding only intermittently. this confirms the results of one study performed as part of a doctoral thesis, which followed fcov-infected cats over a period of weeks, and could demonstrate that the amount of fcov rna shed was significantly higher with higher shedding frequency (defined as % of rt-qpcr-positive fecal samples) [ ] . twenty-four cats were rt-qpcr-negative in all four fecal samples, indicating that they persistently did not shed fcov. these cats either did not have contact to fcov, which seems rather unlikely given the fact that all of the included catteries harbored at least one fcov-shedding cat and the virus is highly contagious [ , , ] . alternatively, these cats could have been resistant to fcov. resistant cats never shed fcov and do not develop fcov antibodies or only very low titers [ ] . the mechanism for this is unknown [ ] . interestingly, however, of the non-shedders in this study did have antibodies. mostly, these cats had low antibody titers. a possible explanation for this could be that the cats had been infected previously, but ceased shedding before the beginning of the study and antibodies persisted for a longer period of time. the longest period documented in the literature for which a cat remained antibody-positive after it stopped fcov shedding was months and decreasing antibody titers were demonstrated in studies following cats ceasing to shed fcov over time [ ] . it is possible that antibody titers were in the process of declining in the cats in the present study as well. this study had some limitations. first, blood samples were obtained from each cat at only one time point. therefore, it was impossible to follow the cats and the development of their antibody titers over time. this would have been especially interesting in cats that ceased to shed virus or in persistent viral shedders. a second limitation is the fact that most cats were housed very closely, and each cattery housed at least one cat that shed fcov. thus, cross-contamination between the individual cats cannot fully be excluded, even though cat breeders acted with great caution to separate fecal samples. thirdly, in the past, the term chronic fcov carrier has been used for cats shedding fcov for at least nine months [ ] . cats in the present study were followed for a maximum of four months. therefore, it is not known whether cats shedding fcov in all four fecal samples were true fcov carriers or might have ceased shedding after the end of the study period. the study was performed prospectively and included cats originating from german catteries. catteries were distributed all over germany. a cattery was defined as a cat household with at least five cats and at least one intact queen for breeding. breeders collected samples from a variable non-predefined number of cats in their cattery. breeds included british shorthair (n = ), bengal (n = ), birman (n = ), maine coon (n = ), scottish fold (n = ), norwegian forest (n = ), turkish van (n = ), and turkish angora (n = ). the study protocol was approved by the responsible veterinary authority (regierung von oberbayern; reference number . - - - . - - ) . cat breeders collected four fecal samples from the cats, at varying time intervals from - days between collections. samples were frozen at − °c until examination. all fecal samples were examined for fcov load per g of feces by rt-qpcr. a mean fecal virus load was calculated for each cat from all rt-qpcr-positive samples. for rt-qpcr, total nucleic acid was extracted from fecal samples by qiaamp dna blood biorobot mdx kit on an automated qiagen platform (qiagen gmbh, hilden, germany) according to the manufacturer instructions, with slight modifications. the rt-qpcr and total nucleic acid extraction procedures were adapted from previously published protocols [ , ] . a quantitative real-time pcr based on the b gene [ ] was performed as a singleplex reaction at a commercial reference laboratory (idexx laboratories, ludwigsburg, germany). real-time pcr was run with six quality controls, including: ( ) pcr-positive controls (quantitatively;), using synthetic dna covering the real-time pcr target region (integrated dna technologies idt, coralville, ia, usa), ( ) pcr-negative controls (pcr-grade nuclease free water), ( ) negative extraction controls (extraction positions filled with lysis solution and pcr-grade nuclease free water only), ( ) rna pre-analytical internal sample control targeting feline ssr rrna ( s rrna) gene complex, ( ) a swab-based environmental contamination monitoring control, and ( ) spike-in internal positive control (using lambda phage dna). these controls assessed the functionality of the pcr test protocols ( ) for the absence of contamination in the reagents ( ) and laboratory ( ), the absence of cross-contamination during the extraction process ( ), quality and integrity of the rna as a measure of sample quality ( ), reverse transcription protocol ( and ), and the absence of pcr-inhibitory substances as a carryover from the sample matrix ( ) . the interpretation of rt-qpcr results is demonstrated in table . if rt-qpcr was initially weak positive (threshold cycle (ct) > ), rt-qpcr was repeated in duplicate. depending on the results of this duplicate repetitive analysis, results were then categorized (table ). questionable rt-qpcr results occurred in four cats. the first cat had one fecal sample that was weak positive by rt-qpcr (low concentrations of fcov rna were detected but viral load was below the limit of quantification); the other three fecal samples of this cat were rt-qpcr-negative. the second cat had two rt-qpcr-negative, one rt-qpcr-positive and one weak positive fecal sample. the third cat had three rt-qpcr-positive and one fecal sample below the limit of quantification. the fourth cat had three rt-qpcr-negative and one fecal sample below the limit of quantification. weak positive samples were considered positive for further analysis. samples below the limit of quantification were considered negative for further analysis. the two cats with only one weak rt-qpcr-positive or one below the limit of quantification and three rt-qpcr-negative fecal samples each were excluded from all analyses involving fecal virus load, since mean fecal virus load could not be determined. additionally, one serum and/or plasma sample was obtained from each of the cats, around the time of collection of the last fecal sample of each cat. only a serum sample was available from cats, only a plasma sample was available from cats, and both a serum and a plasma sample were available from four cats. serum and/or plasma antibody titers were determined by an indirect immunofluorescence assay as previously described [ , ] , using pd- cells (swine origin) infected with transmissible gastroenteritis virus (perdue strain). infected cells were mixed with uninfected cells; the latter served as internal negative control. cat samples were tested at dilutions of : , : , : and : . the fluorescein isothiocyanate (fitc) conjugated secondary antibody (rabbit anti cat igg (h + l), nordic-mubio, sustern, netherlands; lubio sience gmbh, luzern, switzerland) was diluted at : . the serum samples and the conjugate were each incubated at • c for h in a humid chamber, followed by three phosphate buffered saline (pbs) wash steps, and drying of the slide surface with absorbing paper, preventing the wells from drying out. a positive control (aliquoted serum sample of a fcov-antibody-positive field cat) and a negative control (aliquoted serum from a specified pathogen free fcov-antibody-negative cat) were run with each slide. the slides were covered with a cover slide, fixed with a few drops of drops of pbs-glycerol ( : ) and read using a fluorescence microscope (leica dmlb, leica microsystems, heerbrugg, switzerland). the antigen preparations used to prepare the slides were tested for the absence of contaminating viruses by rt-pcr and pcr, as previously described [ ] . the correlation between level of antibody titer and mean fecal virus load was determined by spearman's correlation. kruskal-wallis test including dunn's post-test was performed in order to determine a possible correlation between antibody titer and the frequency of virus shedding in an individual cat. kruskal-wallis test was also performed to evaluate a possible correlation between frequency and amount of fecal virus shedding. mann-whitney u test was applied when comparing cats shedding in all four fecal samples collected and cats shedding in only one to three fecal samples, in order to determine a possible correlation between the frequency of fcov shedding and antibody titer and between the frequency of fcov shedding and mean fecal virus load. statistical analysis was performed using graph pad prism version . (graphpad software, san diego, ca, usa). in this study, cats with antibodies and especially cats with higher antibody titers were more likely to shed fcov; additionally, they shed fcov significantly more frequently throughout the study period and had significantly higher mean fecal virus loads than cats with lower antibody titers or without antibodies. cats shedding fcov in all four fecal samples collected had significantly higher antibody titers and significantly higher mean fecal virus loads than cats shedding fcov intermittently. however, it is important to note that even antibody-negative cats shed fcov, and only % of the antibody-positive cats shed fcov with their feces. therefore, the measurement of the antibody titer can help in managing fcov infection in catteries or multi-cat households, but cannot replace the examination of fecal samples by rt-qpcr. rt-qpcr and antibody titer taken together can give a more reliable picture of the status of an individual cat than each test 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(malvinas) cats from feline coronavirus infection the epidemiology of feline infectious peritonitis in catteries control of feline coronavirus infection in kittens recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium persistence and evolution of feline coronavirus in a closed cat-breeding colony one-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses on the serological diagnosis of feline infectious peritonitis seroepidemiology of feline infectious peritonitis virus-infections using transmissible gastroenteritis virus as antigen antibody induction after combined application of an adjuvanted recombinant felv vaccine and a multivalent modified live virus vaccine with a chlamydial component key: cord- -plptulfb authors: tilocca, bruno; soggiu, alessio; greco, viviana; piras, cristian; arrigoni, norma; ricchi, matteo; britti, domenico; urbani, andrea; roncada, paola title: immunoinformatic-based prediction of candidate epitopes for the diagnosis and control of paratuberculosis (johne’s disease) date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: plptulfb paratuberculosis is an infectious disease of ruminants caused by mycobacterium avium subsp. paratuberculosis (map). map is an intracellular pathogen with a possible zoonotic potential since it has been successfully isolated from the intestine and blood of crohn’s disease patients.since no cure is available, after the detection of the disease, animal culling is the sole applicable containment strategy. however, the difficult detection of the disease in its subclinical form, facilitates its spread raising the need for the development of effective diagnosis and vaccination strategies. the prompt identification and isolation of the infected animals in the subclinical stage would prevent the spread of the infection.in the present study, an immunoinformatic approach has been used to investigate the immunogenic properties of map proteins. these proteins were chosen according to a previously published immunoproteomics approach. for each previously-described immunoreactive protein, we predicted the epitopes capable of eliciting an immune response by binding both b-cells and/or class i mhc antigens. the retrieved peptide sequences were analyzed for their specificity and cross-reactivity. the final aim is to employ the discovered peptides sequences as a filtered library useful for early-stage diagnosis and/or to be used in novel multi-subunit or recombinant vaccine formulations. bovine paratuberculosis, also known as johne's disease (jd) is an infectious disease of ruminants caused by mycobacterium avium subsp. paratuberculosis (map). it is characterized by chronic and progressive granulomatous enteritis. the infected animals initially show normal appetite and food consumption, but the intestinal wall thickening and the impaired nutrient absorption cause a reduced feed-conversion rate and a progressive weight loss. milk yield is also negatively affected by the progression of the infection. nevertheless, clinical manifestations do not involve all infected animals [ ] [ ] [ ] ; the subclinical stageof infection can last from to years [ ] and, despite the absence of visible symptoms, animals in this stage can shed map and spread the disease [ , [ ] [ ] [ ] . for these aforementioned reasons, this pathology leads to significantly increased veterinary costs worldwide [ , , ] . the causal agent of jd is map. it is considered a zoonotic pathogen [ ] because of its possible link with crohn's disease. map infection affects animals and there is considerable evidence that might be a co-cause of human crohn's disease [ ] . map isolation from the intestine and blood of crohn's disease patients has extensively documented. more precisely, map presence was found to be seven times higher in crohn's disease patients than what has been found in patients with any other bowel inflammation [ , ] . map also infected animals and crohn's disease patients show similar alterations of the immune system response reinforcing the hypothesis about the analogy between the two [ ] [ ] [ ] [ ] . map is a slow-growing bacterium, commonly acquired via the fecal-oral route by both animals and humans [ ] . despite the pathogenetic mechanism of map, infection has not been fully understood, it has been demonstrated that its acid resistance enables it to survive in the gastric environment, allowing its entrance in the intestinal tract. at the ileal level, map invades the lymphatic system overlying peyer's patches. this stimulates the host's immune response that, besides activating the humoral response, promptly phagocytizes map into macrophages [ ] . as an intracellular pathogen, map can either survive into macrophagic cells or being killed and disassembled to present its antigens to t-lymphocytes [ ] . evidence from multibacillary jd revealed a massive humoral antibody response along with a tendency to suppress the cell-mediated immune response [ , , ] . whereas, a recent comparative study between two groups of cows, one in the sub-clinical and the other in the clinical stage, highlighted an increased t-cell activity in the first group of animals compared to the second one [ ] . studies on cattle at the early stage of map infection revealed an upregulation of class i mhc molecules, suggesting a pivotal role of these molecules in the very beginning of the infective process [ ] ; this is of great interest for both diagnosis and prophylaxis-oriented studies. figure provides an overview of the major immunological mechanisms triggered bymap infections. to date, jd diagnosis relies on both direct (map culture, pcr, microarrays etc.) and indirect (elisa) detection of map from feces, milk and necroscopy-derived tissues. however, all the available diagnostic methods suffer from sensitivity (especially in the sub-clinical phase) that strongly reduce their robustness and efficient applicability on large-scale control programs. the failure to detect the subclinical infection makes it difficult to timely apply the control measures necessary to block the spread of the infection within the same, and to other, herds. a thorough comprehension of the etiopathogenetic mechanisms of map infection and host response would be beneficial for diverse research scenarios, providing guidance for the design of map-specific diagnostic tools capable of jd diagnosis in the subclinical phase. from this perspective, a previous study from our research group [ ] employed an immunoproteomic approach to investigate and select map-specific immunoreactive proteins. here, incubation of map proteome with sera from infected bovines highlighted several possible candidate immunoreactive proteins. these candidates represent a good starting point for an immunoinformatic analysis of their sequences in order to find the best immunoreactive sub-sequences and epitopes. this would provide a library of peptides that might be useful for novel prophylactic strategies and/or for their potential application in the immune-based detection of map. the rapid development of the bioinformatics tools and databases provides the possibility to detect the antigenic/epitopic regions of given protein sequences. this innovative strategy for the in-silicoanalysis is time-and cost-effective compared to the "old-fashioned" laboratory-based approach. recently, carlos et al. [ ] and rana et al. [ ] applied immunoinformatics-based studies to detect class ii mhc epitopes possibly useful for the control of jd in a rapid and cost-effective manner. over the last decade, these computational approaches lead to the achievement of successful epitopes prediction in several research fields as virology [ , ] , bacteriology and cancer research [ ] . in this study, previously-selected immunogenic proteins [ ] were studied via several immunoinformatics approaches aiming at the detection of the most promising peptide sequences useful for diagnostic purposes. the parameters taken into account were affinity for both the humoral antibody binding and the class i mhc molecule binding. we predicted the most suitable peptide sequences and discuss their potential employment in the design of innovative control measures against jd, with a specific focus on the early diagnosis of jd and/or potential use in novel specific vaccine formulations. pathogens , , x for peer review of over the last decade, these computational approaches lead to the achievement of successful epitopes prediction in several research fields as virology [ , ] , bacteriology and cancer research [ ] . in this study, previously-selected immunogenic proteins [ ] were studied via several immunoinformatics approaches aiming at the detection of the most promising peptide sequences useful for diagnostic purposes. the parameters taken into account were affinity for both the humoral antibody binding and the class i mhc molecule binding. we predicted the most suitable peptide sequences and discuss their potential employment in the design of innovative control measures against jd, with a specific focus on the early diagnosis of jd and/or potential use in novel specific vaccine formulations. the peptide sequences of the previously-identified immunoreactive proteins [ ] were used to recall the novel protein identifiers in the ncbinr protein database. because of the continuous evolution of the data repositories and the increasing knowledge on their entries, some protein accession numbers were re-classified into other identifiers. table summarizes the blast-based alignments of the peptides performed to line up the selected proteins to the current identification system. all pblast alignments matched at % with the reference protein kept. the low e-value of each alignment supports the attribution of the immunoreactive proteins to the novel identifiers. the peptide sequences of the previously-identified immunoreactive proteins [ ] were used to recall the novel protein identifiers in the ncbinr protein database. because of the continuous evolution of the data repositories and the increasing knowledge on their entries, some protein accession numbers were re-classified into other identifiers. table summarizes the blast-based alignments of the peptides performed to line up the selected proteins to the current identification system. all pblast alignments matched at % with the reference protein kept. the low e-value of each alignment supports the attribution of the immunoreactive proteins to the novel identifiers. once the updated protein identifiers are inferred, the major immunogenic domains of the selected proteins were predicted through an immunoinformatic approach. prediction of the linear b-epitopes provided a list of epitopes capable of eliciting antibody production (supplementary table s ). all the selected proteins showed relevant epitopes from an immunogenic point of view. a large number of short epitope sequences is predicted for each immunogenic protein; whereas, an average of six candidate epitopes (min -max ) meeting the threshold of a minimal length of aminoacids is predicted for each of the selected immunogenic proteins. figure depicts the ten immunogenic proteins of map along with the relative distribution of the predicted b-epitopes. whole protein calculated immunogenic potential based on the type-b epitopes prediction indicates the "hypothetical protein map_ c" (aas ) as the most immunogenic one. this evidence is supported by its highest number of predicted epitopes and the highest average epitope length ( figure ). on the other hand, the fructose-bisphosphate aldolase (eta ), reported the lowest number of predicted epitopes along with the lowest epitope sequence length. regardless of the number of predictions, candidate epitopes are evenly mapped over the full sequence length of the immunogenic proteins, suggesting a good versatility of the predicted sequences ( figure ). pathogens , , x for peer review of prediction of binding affinity for the diverse class i bolas histocompatibility antigens predicted a high number of peptide sites. the full list of class i mhc epitope prediction is provided in the supplementary table s . epitope prediction from the previously selected immunogenic proteins yielded a total of peptides, each of which scoring a peculiar binding affinity. peptides scoring a binding affinity among the top . % are considered as strong binders (sb); whereas, peptides with a percentile rank comprised between . % and % were labelled as weak binders (wb). sorting all the entries using the "sole" wb and sb resulted in a total of candidate epitopes when considering all the mhc haplotypes for the ten immunogenic proteins (supplementary table s ) . for a better evaluation of the most suitable map epitopes, we focused our attention towards the sequences that are most commonly recognized by the immune system effectors (i.e., bola haplotypes and, in turn, cd + t-cells). figure lists, for each of the tested immunogenic protein, the shared epitopes among the mhc haplotypes. pathogens , , x for peer review of prediction of binding affinity for the diverse class i bolas histocompatibility antigens predicted a high number of peptide sites. the full list of class i mhc epitope prediction is provided in the supplementary table s . epitope prediction from the previously selected immunogenic proteins yielded a total of peptides, each of which scoring a peculiar binding affinity. peptides scoring a binding affinity among the top . % are considered as strong binders (sb); whereas, peptides with a percentile rank comprised between . % and % were labelled as weak binders (wb). sorting all the entries using the "sole" wb and sb resulted in a total of candidate epitopes when considering all the mhc haplotypes for the ten immunogenic proteins (supplementary table s ) . for a better evaluation of the most suitable map epitopes, we focused our attention towards the sequences that are most commonly recognized by the immune system effectors (i.e., bola haplotypes and, in turn, cd + t-cells). figure lists, for each of the tested immunogenic protein, the shared epitopes among the mhc haplotypes. summarizes bolas haplotypes predicted to bind each sequence as means of a color scale that depends on the binding affinity. peptides "amlqdmail" belongs to heat shock protein (caa . ); "aqldlsnal" and "hmlrhqqir" belong to hypothetical protein map_ c (aas . ); "arleppgpl" belong to hypothetical protein map c (aas . ); "gvfnleatl" and "neraveeal" belong to the protein fixa (aas . ); "rlleiepal", "aagqigysl" and "alegvvmel" belong to the malate dehydrogenase protein (p . ); "amqtlpqvl" and "rmaqtgspl" belong to phosphoglucosamine mutase (q s . ); "nqielhpll", "teravsaal", "amdaceasl" and "tqsytplal" belong to uncharacterized oxidoreductase map_ (q vk . ); "emfdlihqm" and "amrkwessm" belong to fructose-bisphosphate aldolase (eta . ); "glyefftpl", "gmigltqal" and "tqmtaaipl" belong to -ketoacyl-acp reductase (ajk . ); "fitwrgipl" and "nqsaiatyl" are peptides of the hypothetical protein ega _ (azp . ). the vast majority of the listed epitopes are classified as sb; while eight of them, belonging to the proteins malate dehydrogenase (p ), uncharacterized oxidoreductase map_ (q vk ) and hypothetical protein ega _ (azp ), are to be considered as wb on the basis of their affinity rank. regardless of the binding affinity, all these sequences are predicted to be commonly bound by a plurality of mhc haplotypes. an average of (min -max ) suitable epitopes are selected for each of the tested protein. such epitopes are predicted to be recognized by five diverse bolas out of the six mhc haplotypes used for the computer-based prediction; except for the immunogenic proteins fixa (aas ) whose epitopes can be bound by four bolas out of six. the bola-hd , bola-jsp. and bola-t c are able to recognize all the selected epitopes sequences . selected class i mhc binding peptides. the heat map displays the selected t-epitopes and summarizes bolas haplotypes predicted to bind each sequence as means of a color scale that depends on the binding affinity. peptides "amlqdmail" belongs to heat shock protein (caa . ); "aqldlsnal" and "hmlrhqqir" belong to hypothetical protein map_ c (aas . ); "arleppgpl" belong to hypothetical protein map c (aas . ); "gvfnleatl" and "neraveeal" belong to the protein fixa (aas . ); "rlleiepal", "aagqigysl" and "alegvvmel" belong to the malate dehydrogenase protein (p . ); "amqtlpqvl" and "rmaqtgspl" belong to phosphoglucosamine mutase (q s . ); "nqielhpll", "teravsaal", "amdaceasl" and "tqsytplal" belong to uncharacterized oxidoreductase map_ (q vk . ); "emfdlihqm" and "amrkwessm" belong to fructose-bisphosphate aldolase (eta . ); "glyefftpl", "gmigltqal" and "tqmtaaipl" belong to -ketoacyl-acp reductase (ajk . ); "fitwrgipl" and "nqsaiatyl" are peptides of the hypothetical protein ega _ (azp . ). the vast majority of the listed epitopes are classified as sb; while eight of them, belonging to the proteins malate dehydrogenase (p ), uncharacterized oxidoreductase map_ (q vk ) and hypothetical protein ega _ (azp ), are to be considered as wb on the basis of their affinity rank. regardless of the binding affinity, all these sequences are predicted to be commonly bound by a plurality of mhc haplotypes. an average of (min -max ) suitable epitopes are selected for each of the tested protein. such epitopes are predicted to be recognized by five diverse bolas out of the six mhc haplotypes used for the computer-based prediction; except for the immunogenic proteins fixa (aas ) whose epitopes can be bound by four bolas out of six. the bola-hd , bola-jsp. and bola-t c are able to recognize all the selected epitopes sequences among the immunogenic proteins. on the other hand, the bola-t a is not showing any binding affinity to the epitope sequences; while bola-d . and bola-t b fail to bind the epitopes of aas protein (figure ). the class i mhc epitopes as of figure are further aligned against both the mycobacteria and cow databases to assess the specificity of the predicted epitope sequences for map. complete list of alignments is available in the supplementary table s . sequence alignment highlighted that the peptide amdaceasl and amrkwessm respectively of the "uncharacterized oxidoreductase map_ " (q vk ) and "fructose-bisphosphate aldolase" (eta ) proteins are the most specific for map. specifically, amdaceasl scores % identity with the map and the mycobacterium aviumcomplex (mac); whereas, hits with other mycobacteria specimens are featured by a lower sequence identity (below %) and a far higher e-value when compared with map and mac hits ( . vs. . , supplementary table s ) . similarly, the peptide amrkwessm scores % sequence identity with map and mac and only less than % of sequence similarity is scored by the alignments with other mycobacteria. the e-value supports the robust alignment against the map and mac (e-value . ) in spite of the other alignments (e-value > ) further supporting the hypothesis on the specificity of this peptide sequence (supplementary table s ). concerning the alignment of the peptides against the cow database, both amdaceasl and amrkwessm did not score relevant matches with any of the cow proteins. several hits were matching with discontinuous sequences of the cow proteins database with high e-values (supplementary table s , topic better commented in the discussion section). the host's immune response to map infection is complex and heterogeneous. debates on the sequelae of immunological events following map infection are currently ongoing. nevertheless, it seems widely accepted that the early stage of the infection elicits an important cell-mediated response. once map is phagocytized, its antigen presentation is accomplished through the loading of the processed antigen onto mhc molecules. the bovine mhc genes complex (i.e., bovine leukocyte antigen, bola) is carried in the chromosome and represent a fundamental component of the bovine immune system that allows the recognition and presentation of a virtually infinite number of antigens [ ] (figure ). such a high versatility relies on diverse factors, including the polygenetic origin of the mhc genes, the codominance of the parental alleles, the polymorphism of the genetic variants and the peptides/proteins splicing [ ] . class i mhc molecules recognize, bind and present peptide antigens from intracellular pathogens to cd + t-lymphocytes [ ] . in this view, class i mhc molecules and cytotoxic t-lymphocytes (ctl) are likely to play a pivotal role in the early stage of the map infection [ ] . thus, of potential interest for early diagnosis-oriented studies and the design of efficient vaccine formulations. class i mhc peptide antigens are to be considered among the main triggers of the cell-mediated responseand their specific immunostimulation would lead to a more efficient prophylactic outcome. nevertheless, a study from rana etal. highlighted an important involvement also of the humoral response to map infection, other than the adaptive immunity mediated by the class ii mhc molecules [ ] . huge efforts have been made to optimize diagnostics for the efficient detection of map by means of both direct and indirect methods [ , , ] . the slow-growing rate of map along with the reduced sensitivity of the culture-based methods raised the need to develop alternative diagnostic strategies. pcr-based diagnosis targeting the multicopy insertion sequence is held the promise of fast detection of map in both environmental and clinical samples. however, the presence of is -like sequences in other bacterial specimens resulted in a reduction of the pcr specificity. this, along with the elevated costs of the reagents, equipment and procedures, precludes the pcr applicability in large-scale programs [ ] . among the indirect methods, elisa-based detection of anti-map antibodies enables faster diagnosis time but still suffer from drawbacks related to sensitivity and specificity. although great improvements have been made in optimizing elisa kits to reduce cross-reactions with environmental mycobacterium strains [ , ] . still, this method suffers from a lack of sensitivity. moreover, the high antigen similarity between map and mycobacterium bovis obstacles the discrimination between bovines infected with tuberculosis and inoculated with live or attenuated paratuberculosis vaccines [ ] . this promotes the seek of molecular target(s) capable of offering a more robust diagnosis. the present work describes a companion study that relies on previously-obtained datasets of our research group [ ] . employing an immunoproteomic approach, we experimentally validated the whole map proteome for its capability of being complexed by the antibodies naturally occurring in sera of infected bovines. ms-based identification of the immunogenic proteins enabled the detection of protein candidates whose protein sequences have been now further investigated for their immunogenic features. we employed an immunoinformatic approach for further focusing on the peptide sequences, potentially involved in the immunostimulation. a key point of the immunoinformatic approach is the prediction of the protein epitope sequences. epitopes prediction can be based on several features such as physical-chemical properties and structural folding of the primary protein sequence [ ] [ ] [ ] . the present investigation is mainly focused on linear epitopes because protein-antibody complexes were selected through two-dimensional electrophoresis ( -de) and western blotting; thus, on linearized proteins [ , ] . however, the application of other mass spectrometry technologies is quickly developing in the field on immunoproteomics [ ] there are still significant limitation to map on a large scale conformational epitopes. as expected from the previous experimental data, all the screened protein sequences showed the capability of being recognized by both b-cells and class i bolas. the comprehension of recognition and binding of map by the host immune cells is still controversial. some studies document a relevant humoral response to map infections. on the other hand, other pieces of evidence describe the importance of the cell-mediated response to control map growth [ ] [ ] [ ] [ ] [ ] . from our perspective and, according to the collected evidence, map-targeted antibodies could play a key role in the specific and sensitive detection of this pathogen in the subclinical stage of the infection. b-cell epitopes prediction highlighted the "hypothetical protein map_ c" (aas ) protein as the most active in stimulating antibody production. this finding is in agreement with our previous study [ ] , where this protein showed a high level of immunoreactivity exclusively against the serum of the map infected animals. to the best of our knowledge, this protein was not described before as an antigen and, according to our dataset on its functional domains, it is possible to hypothesize that it is part of a surface-associated dehydrogenase with oxidoreductase activity involved in pentose phosphate pathway [ , ] . the fructose-bisphosphate aldolase (eta ), instead, is described as less prone to elicit antibody production. this is consistent with its intracytoplasmic localization and with its major role in the central metabolism. despite its cellular localization, several moonlighting properties have been described as part of its multiple functions [ ] [ ] [ ] . interestingly, b-cell epitopes prediction highlighted a homogenous distribution of multiple peptide sequences throughout all the proteins primary sequences (figure ). this suggests the potential usefulness of the selected proteins for a variety of implications where two or more epitopes are needed in a single protein molecule (e.g., sandwich elisa, and other indirect diagnostic tests ensuring higher sensibility) [ , ] . prediction of the class i bolas binding peptides confirmed the immunogenicity of the previously studied proteins. similarly to hlas, bolas are highly polymorphic proteins; thus, including a plurality of bolas while computing the peptide binding affinity would benefit the robustness and reliability of the prediction [ , ] . among the class i mhc epitopes predicted in the present study, the hypothetical protein ega _ (azp ) differs by only one amino-acidic residue from the map membrane protein c (v kre ), whose immunogenic properties have been already demonstrated by both our previous investigation and other studies [ , , ] . it is, indeed, a surface-exposed protein involved in the mechanism of invasion of the epithelial cells [ , ] . its expression is upregulated when the map is exposed to the physicochemical conditions similar to the intestine environment and the specific block of this protein reduces the virulence up to % [ ] . interestingly, this protein is among the entries classified as wb suggesting that more immunogenic properties can also be exploited by the other wb protein besides the others predicted as being sbs. moreover, we specifically focused on the sole peptide sequences whose binding affinity is shared among multiple bolas. in this manner, the most suitable epitope sequences are likely to have a broad recognition in a higher portion of the bovine population [ , ] . epitopes identified with this approach are of potential interest for diverse purposes and studies, including the investigations aimed at elucidating the order of immunological events following the map infection, and shedding light on the controversial aspect of suppression, or not, of the cellular-mediate immune response following map infection [ ] . to prove selected epitopes as suitable candidates for the unbiased diagnosis of map infection, we aligned the peptides sequences against a database comprising the closest taxonomically-related bacteria. such alignment generated a steep reduction of the number of input sequences and returned two peptides suitable for a specific diagnosis of map. these two candidates were not overlapping with other mycobacteria other than mycobacterium avium complex (mac). the described approach resumes the pipeline of an in-silico method, therefore, empirical tests will be required for the definitive assessment of differential diagnosis capability of the selected sequences. the sequence alignment against the host-specific protein (i.e., the publicly available cow proteome) fails to identify significant sequence identities. the only alignment hits observed (supplementary table s ) were not continuously overlapping and showed a low percentage of identity and a high e-value. acknowledged the prediction of linear epitopes, the matching of our candidates with gapped sequences of cow is likely to be of a negligible relevance since regarding amino acid residues that are not laying in a concatenate order. thus, we speculate that the candidate epitopes suggested in this study are of potential value for the design of either multi-subunit or recombinant vaccines to confer protection against the first-time infection of the calves by map. nevertheless, confirmatory experimental trials are warmly encouraged especially to assess the specificity between map infected animals and bovine with tuberculosis [ , ] . although less significant, a certain level of identity hasbeen registered with other mycobacterium strains. however, the discrepancy observed in the sequence alignment might be used as the driving force for the differential diagnosis. at this purpose, application of optimized laboratory protocols expecting high stringency condition might be the key to improve the specificity of the diagnostic methods. finally, empirical evaluation of the synergistic effect of both b-cell epitopes and the class i mhc epitopes are desirable. this will aim at the evaluation of the successful diagnosis of map infection at the subclinical stage and at the potential in elicitation of protective immunity. to conclude, the present study describes an innovative pipeline based on the in-silico survey of selected immunoreactive proteins capable to uncover the immunogenic features of each protein. this pipeline was applied to the detection of a restricted number of peptides potentially useful for the diagnosis of jd at the early subclinical stage. obtained results are as well useful for the implementation of innovative vaccination strategies. the obtained results confirmed the immunogenicity observed experimentally through the immunoproteomic approach applied to the map proteome. this evidence demonstrates, once again, reciprocal support between immunoproteomics and immunoinformatics. nevertheless, empirical confirmations are warmly required to test the provided epitope sequences both in-vitro and ex-vivofor the possible detection of the subclinical phase of the infection and for the efficacy of the eventual vaccinal formulations. such experimental tests might also help with the comprehension of the controversial role of the host immune cell-response underlying behind jd. complementation of the linear epitopes array with other conformational ones is also of importance for befitting efficacy and safety of the deliverables empirical confirmation may serve as further proof of the robustness of the immunoinformatics approaches as key contributors in the study of diverse infectious diseases. this would provide reliable scientific support in a safe, rapid and cost-effective approach. the current study focuses on ten proteins whose immunoreactivity has been experimentally investigated by means of an immunoproteomic approach [ ] . brielfy, the map proteome was incubated with sera from infected animals to screen for proteins with immunoreactive potential. the most promising entries were then subjected to ms-based identification. identifiers of the candidate immunoreactive proteins were queried in the ncbi non-redundant (ncbinr) protein database to retrieve the whole protein sequences and export them as a fasta file. update of the protein accession numbers operated by the reference data repository (i.e., ncbi) required the run of a protein sequence alignment for the attribution of the novel protein identifiers (gi numbers). selected peptide sequences of the immunoreactive proteins were searched against the ncbinr database restricted to mycobacterium avium subsp. paratuberculosis (taxid ) and the best hit was used to transform the former protein accession numbers into the novel ncbi protein gis. the list of proteins employed in this study along with their current gi number is provided in table and supplementary table s . prediction of the protein sequences that are likely to elicit antibody production and/or bind class i mhc proteins was performed through two tools that are commonly employed for the epitope prediction [ ] , namely iedb (http://tools.iedb.org/bcell/) and netmhc (http://www.cbs.dtu.dk/ services/netmhc/), respectively for b-and class i mhc epitopes prediction. bepipred algorithm was chosen for the prediction of linear protein epitopes capable of binding b-cells. this employs a combination of a hidden markov model and a propensity scale method [ ] . each protein residue is scored for its epitope behavior and the sole aminoacid with a score greater than or equal to . was considered as a potential epitope. linear peptide epitopes of the least length of aminoacids were selected for this study. prediction of epitopes binding class i mhc molecules was performed through netmhc prediction tool, using the artificial neural network (ann) algorithm [ ] . the algorithm was set for the prediction of nine-aminoacids long peptides capable of binding the following bola alleles: bola-d . ; bola-hd ; bola-jsp. ; bola-t a; bola-t b; bola-t c. the binding affinity of the peptides wasscored, and a percentile rank wasprovided by computationally comparing the score of each queried peptide sequence against , natural peptides of the same length. peptides scoring a binding affinity up to . % were considered as strong binders (sb); whereas, peptides with a percentile rank comprised between . % and % were labelled as weak binders (wb). all other peptides were discarded [ , , ] . the resulting list of selected peptide epitopes was further quality-checked and filtered. for each of the selected proteins, the epitopes shared among the major number of bola haplotypes were kept (i.e., the most commonly recognized in the bovine population), resulting in a consensus list of epitopes to be further used in the study. a summary of the experimental worklow employed in this study is provided in figure . . schematic representation of the immunoinformatic approach. blue and orange arrows refer to the input and output of the data, respectively. data arising from our previous immunoproteomic study [ ] were used to retrieve the protein sequences of the immunogenic proteins, selected on the basis of their capability of being complexed by the immunoglobulins in the sera of the infected cows. the sequence of the immunogenic proteins is subjected to the epitope prediction through dedicated tools and algorithms. the b-epitope prediction was performed via the iedb prediction tool, that provides a list of candidate b-epitopes. the class i mhc epitopes waspredicted via netmhc. this makes use of the bola haplotypes from the data repository (ncbi) as references for computing the linear peptides capable of being recognized and presented by the diverse bolas. the list of potential t-epitopes is further refined by selecting the most commonly recognized epitopes with a relatively high binding affinity. the refined list of epitopes is further tested for sequence specificity and cross-reactivity by pblast alignment versus the mycobacteria and cow protein database which, in turn, arise from the publicly available data repository (ncbi). the list of epitope sequences was further analyzed through the basic local alignment search tool for protein sequences (pblast) [ ] . this tool implements the pam algorithm to compare protein sequences and calculates the robustness of matches as means of expected values (e-value). . schematic representation of the immunoinformatic approach. blue and orange arrows refer to the input and output of the data, respectively. data arising from our previous immunoproteomic study [ ] were used to retrieve the protein sequences of the immunogenic proteins, selected on the basis of their capability of being complexed by the immunoglobulins in the sera of the infected cows. the sequence of the immunogenic proteins is subjected to the epitope prediction through dedicated tools and algorithms. the b-epitope prediction was performed via the iedb prediction tool, that provides a list of candidate b-epitopes. the class i mhc epitopes waspredicted via netmhc. this makes use of the bola haplotypes from the data repository (ncbi) as references for computing the linear peptides capable of being recognized and presented by the diverse bolas. the list of potential t-epitopes is further refined by selecting the most commonly recognized epitopes with a relatively high binding affinity. the refined list of epitopes is further tested for sequence specificity and cross-reactivity by pblast alignment versus the mycobacteria and cow protein database which, in turn, arise from the publicly available data repository (ncbi). the list of epitope sequences was further analyzed through the basic local alignment search tool for protein sequences (pblast) [ ] . this tool implements the pam algorithm to compare protein sequences and calculates the robustness of matches as means of expected values (e-value). this value describes the statistic of matches occurring "by chance"; thus, it decreases exponentially as the score of the match increases. in the pblast, each epitope sequence has been aligned against both mycobacteria (ncbi taxid ) and cow (ncbi taxid ) protein repertoires to evaluate sequence specificity and cross-reactivity (figure ) , of importance while selecting candidate epitopes to be employed for the effective diagnosis and/or prophylaxis of map. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . table s . b-cell binding protein epitope prediction. the file summarizes the b-epitope prediction results. each sheet of the xls file is relative to one of the ten selected proteins. "position" column is relative to the position of each aminoachid along the protein sequence; "residue" indicates the type of aminoacid; "score" is relative to the epitope propensity attributed by the algorithm and "assignment" rank each aminoacid residue as epitope or not depending on the prefixed settings. table s . class i mhc binding protein epitope prediction. the xls file summarizes the results in a table reporting: the predicted affinity (nm); the percentile rank, and the predicted binding core for all the selected alleles. two additional columns summarize the predictions across alleles: harmonic mean of the %rank calculated over all specified alleles (h_avg_ranks); the number of alleles covered by a given peptide (n_binders). table s . selected peptide epitopes alignment against the mycobacteria and cow databases. the xls file provides a summary of the pblast alignment search. full description of the table columns and the alignment criteria is available at https://blast.ncbi.nlm.nih.gov/blast.cgi?page=proteins. table s . full list of the prototypic peptides alignment. the file summarizes the p-blast alignment of the two selected epitopes against both the mycobacteria and the cow database. full description of the table columns and the alignment criteria is available at https://blast.ncbi.nlm.nih.gov/blast.cgi?page=proteins. the authors declare no conflict of interest. defining resilience to mycobacterial disease: characteristics of survivors of ovine paratuberculosis experimental infection model for johne's disease using a lyophilised, pure culture, seedstock of mycobacterium avium subspecies paratuberculosis gene expression profiles during subclinical mycobacterium avium subspecies paratuberculosis infection in sheep can predict disease outcome control of paratuberculosis: who, why and how. a review of countries paratuberculosis in sheep and goats johne's disease) in cattle and other susceptible species temporal patterns and 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approaches new latex bead agglutination assay for differential diagnosis of cattle infected with mycobacterium bovis and mycobacterium avium subsp. paratuberculosis duplex pcr for differential identification of mycobacterium bovis, m. avium, and m. avium subsp. paratuberculosis in formalin-fixed paraffin-embedded tissues from cattle improved method for predicting linear b-cell epitopes gapped sequence alignment using artificial neural networks: application to the mhc class i system in silico identification of epitopes in mycobacterium avium subsp. paratuberculosis proteins that were upregulated under stress conditions protein database searches using compositionally adjusted substitution matrices key: cord- -tqceazdp authors: n. nuñez, luis fabian; santander-parra, silvana h.; de la torre, david i.; de sá, lilian r. m.; buim, marcos r.; astolfi-ferreira, claudete s.; piantino ferreira, antonio j. title: molecular characterization and pathogenicity of chicken parvovirus (chpv) in specific pathogen-free chicks infected experimentally date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: tqceazdp chicken parvovirus (chpv) is an agent frequently associated with runting stunting syndrome (rss). this syndrome has been reported in association with chpv in many countries, including brazil; however, studies characterizing the virus on a molecular level are scarce, and chpv pathogenicity in day-old chicks remains unclear. the aim of the present work was to establish the molecular characteristics of chpv, determine the pathogenicity of chpv in spf chicks and detect and quantify chpv by qpcr in several tissues and chicks of different ages. the experimental challenge was performed at one day of age, and daily and weekly observations were performed and five birds from each experimental group (mock and infected birds) were euthanized to perform the different analysis. chpv genome copies were detected and quantified by qpcr in gut, spleen, thymus, kidney, pancreas, proventriculus and bursa. clinically, the infected group presented with diarrhea h post-infection, which persisted until days of age. the small intestine was distended, and its contents were aqueous and foamy. enteritis and dilated crypts with cyst shapes were observed in intestinal segments. acute pancreatitis associated with lymphocytic nodules, infiltrating lymphocytes and plasma cells between the pancreatic acinus was observed. koch’s postulate was demonstrated and the genetic characterization of the vp gene showed that the brazilian chpv isolate belongs to the chpv ii group. enteric disorders are considered the most important concern to poultry gut health due to their compromising effect on poultry growth. enteric disorders have a multifactorial etiology and are associated with bacteria, fungi, protozoa and viruses [ ] [ ] [ ] [ ] [ ] [ ] . runting-stunting syndrome (rss) is commonly reported in poultry production and is associated with several viruses, such as astrovirus, rotavirus, reovirus, coronavirus and parvovirus, and bacteria, including coccidia [ ] [ ] [ ] [ ] [ ] [ ] . the clinical manifestation of rss is characterized by ruffled feathers, diarrhea, cloacal pasting, apathy, depression, the sequences obtained in this work from the vp gene of chpv generated a sequence of bp, accession number mk . , which corresponds to the complete gene sequence of vp . the obtained vp nucleotide sequence was aligned and compared with previously published sequences of chpv. the analysis of complete vp nucleotide sequences showed high similarity of nucleotides (nt) and amino acids (aa) between the analyzed sequences ( table ). the sequence of the isolated chpv showed - . % nt and - . % aa similarity to sequences from korea; . - % nt and . - . % aa similarity to sequences from the united states; . % nt and . % aa similarity to sequences from china and . % nt and % aa similarity to reference sequences from hungary. compared to tupv sequences, the obtained sequence showed low nt ( . - . %) and aa ( . - . %) similarity, as shown in table . phylogenetic analyses showed two well-defined groups, a chpv group ( % bootstrap) and a tupv group ( % bootstrap). the sequence of the chpv isolate used in the present work clustered with a usa isolate (km . ) (bootstrap %) belonging to chpv group ii; the chpv reference sequence was grouped in a separate branch ( % of bootstrap) with the usa sequence (km . ; figure ). in the experimental infection, depression, lethargy, somnolence, ruffled feathers and diarrhea were observed h post-inoculation. after h of infection, the animals presented with cloacal pasting, dirty and wet feathers at the cloacal region caused by watery feces and ruffled feathers ( figure ). somnolence and lethargy increased, resulting in birds that moved less and had more apathy, in addition to an increase in diarrhea. the clinical signs of enteric disease, mainly diarrhea, were observed throughout the experiment ( table ). the infected animals also showed differing characteristics of the feathers on their wings, namely, the wings were found folded in the outer direction. ten days after infection, animals in the two groups were observed to have different sizes, with the infected animals appearing smaller and stunted compared with animals from the mock group. the mock group did not show any clinical signs and exhibited normal movement and healthy appearance, as described above. outer direction. ten days after infection, animals in the two groups were observed to have different sizes, with the infected animals appearing smaller and stunted compared with animals from the mock group. the mock group did not show any clinical signs and exhibited normal movement and healthy appearance, as described above. the birds of the mock group did not show any macroscopic lesions in the organs at any age examined. the postmortem examination of the challenged birds showed distended coelomic cavity intestinal loops filled with aqueous and foamy content. the intestinal loops exhibited segmentations along the small intestine; there were dilated stretches as well as narrow ones, and the contents were liquid, containing foamy and undigested feed along the length of the intestine. the birds in all age groups presented with intestinal volvulus, in which there was rotation of the duodenal loop segment in the mesenteric axis that resembled a corkscrew ("j" appearance). the mesentery presented opacification in this segment, and there was atrophy of the pancreas. the birds showed persistence of the yolk sac. the rest of the organs did not show any macroscopic alteration (table ; figure ). pathogens , , x for peer review the birds of the mock group did not show any macroscopic lesions in the organs at any age examined. the postmortem examination of the challenged birds showed distended coelomic cavity intestinal loops filled with aqueous and foamy content. the intestinal loops exhibited segmentations along the small intestine; there were dilated stretches as well as narrow ones, and the contents were liquid, containing foamy and undigested feed along the length of the intestine. the birds in all age groups presented with intestinal volvulus, in which there was rotation of the duodenal loop segment in the mesenteric axis that resembled a corkscrew ("j" appearance). the mesentery presented opacification in this segment, and there was atrophy of the pancreas. the birds showed persistence of the yolk sac. the rest of the organs did not show any macroscopic alteration (table ; figure ). the birds infected with chpv showed preserved villous:crypt ratios and mild to moderate hyperplasia of crypts with the presence of crypt bifurcations, which were dilated, elongated and tortuous in different evaluation periods. from the th to nd day after inoculation, the crypts of lieberkühn were lined by a squamous epithelium containing cellular debris and degenerated inflammatory cells that formed structures with a cyst shape; these structures were observed in the table . clinical signs and macroscopic findings at postmortem examination of chickens from chpv infected group. the birds infected with chpv showed preserved villous:crypt ratios and mild to moderate hyperplasia of crypts with the presence of crypt bifurcations, which were dilated, elongated and tortuous in different evaluation periods. from the th to nd day after inoculation, the crypts of lieberkühn were lined by a squamous epithelium containing cellular debris and degenerated inflammatory cells that formed structures with a cyst shape; these structures were observed in the duodenum, jejunum and ileum (figure ) . table shows the distribution of histopathological parameters of the digestive system in both groups. between the th day and th day post-infection, necrosis of crypt cells was observed in all segments of the small intestine (p < . ). furthermore, there was an increased number of mitotic cells in segments of the duodenum and an increased number of intraepithelial lymphocytes from the th to nd day (p < . ). the density of lymphocytes and plasma cells found in the lamina propria of the duodenum was significant (p = . ) from the th to the st day after inoculation, that of the jejunum was significant from the th to st day (p < . ) after infection, and that of the ileum was significant from the st to th day after infection (p < . ). the pancreas showed a loss of zymogene granules; lymphocytic nodules that contained an infiltrate of lymphocytes and plasma cells between the pancreatic acinus were also present, indicating acute pancreatitis. lymphoplasmacytic mesenteritis was observed in the mesentery of the duodenal loop and the peripancreatic area, which is associated with pancreatitis. the birds of the mock group showed segments of the duodenum, jejunum and ileum within the standard of normal histology (table ; figure ). the qpcr assay for the detection and quantification of chpv showed that all analyzed animals were positive for chpv in all collected organs from the first day post-infection. peak titers of chpv was observed in the intestines, for the ileum, followed by the jejunum and duodenum. however, it was very interesting to note that replication of the virus seemed to commence at - days post-infection for the ileum followed by the jejunum and then the duodenum. interestingly, in this study, replication in the pancreas and an elevated number of genome copies were not observed. the gastrointestinal tract (duodenum, jejunum and ileum) showed the highest level of genome copies of chpv at day fourteen post-infection, and the viral concentration decreased through day . the pancreas, thymus, liver, proventriculus and kidney showed basal virus genome titer from the first day until the end of the experiment at days old. the spleen had the highest viral concentration at day , after which the concentration decreased (table ; figure ). figure . the figure compares the viral distribution in the days after infection with the chpv strain according to each organ/tissue. note that peak titer of chpv is greatest in the intestines. however, it is interesting to note that replication of the virus seems to commence at - days post-infection for the ileum followed by the jejunum and then the duodenum. interestingly, multiplication in the pancreas was not observed. in the present work, the pathogenicity, viral tissue distribution and molecular characterization of chpv in chicks from a strain isolated in brazil were determined with a demonstration of koch's postulates according to our previous description [ ] . there are few complete genomes of chpv available, and the complete genome refers to the chpv reference strain abu-p , which was followed figure . the figure compares the viral distribution in the days after infection with the chpv strain according to each organ/tissue. note that peak titer of chpv is greatest in the intestines. however, it is interesting to note that replication of the virus seems to commence at - days post-infection for the ileum followed by the jejunum and then the duodenum. interestingly, multiplication in the pancreas was not observed. in the present work, the pathogenicity, viral tissue distribution and molecular characterization of chpv in chicks from a strain isolated in brazil were determined with a demonstration of koch's postulates according to our previous description [ ] . there are few complete genomes of chpv available, and the complete genome refers to the chpv reference strain abu-p , which was followed by viral genomes from korea [ ] , the united states [ ] and china [ ] . thus, these genomes possess the classical conformation of parvovirus and contain structural and nonstructural proteins; based on their vp gene sequences, three groups of chpv can be identified [ ] . the lack of chpv vp gene sequences could likely be related to the difficulty in chpv dna isolation for whole vp gene sequencing; however, the majority of published works have included a molecular characterization of the ns gene sequence [ , ] . nevertheless, genetic information based on ns gene sequences is not sufficient for discriminatory genotyping because there are few differences among strains [ ] . hence, we report the first molecular characterization of chpv in brazil based on complete vp gene sequence analyses, confirming that the chpv used in the experimental infection belongs to the chpv ii group. around the world, chpv has been reported and detected in healthy and sick chickens, but it has mainly been identified in chickens showing enteric disorders. this virus has the particularity that it is detected relatively more in chicks and young animals [ ] [ ] [ ] [ ] . chpv causes severe enteric problems that are characterized by the presence of diarrhea, high morbidity and mortality [ , ] . in the field, outbreaks that affected chickens showed many pathological alterations in the intestine upon postmortem examination, showing mainly distended intestines filled with aqueous feces and foamy and undigested feed. moreover, the challenge with outbreaks in the field is the association of chpv with other enteric viruses or bacteria, which could decrease the quality of the gut integrity [ , , , , ] . experimental infections with isolated chpv (abu-p ) have demonstrated that the virus causes enteric diseases, resulting mainly in chickens with diarrhea, cloacal pasting, impaired growth, runting and stunting [ ] . as reported in the present investigation, the principal clinical signs present in the infected animals were also diarrhea, cloacal pasting, somnolence, apathy, ruffled feathers, impaired growth, runting and stunting. however, the chpv strain abu-p does not cause any macroscopic lesions in enteric tissues [ , ] . interestingly, this investigation revealed the presence of intestinal volvulus characterized by the rotation of the duodenal loop segment in the mesenteric axis; thus, the duodenal loop was rolled, resembling a corkscrew and pancreatic atrophy was also observed. lesions were previously described in commercial chicken flocks affected with rss and reported by our own group [ ] ; the duodenal loop presented the same features, demonstrating koch's postulates in relation to chpv and experimentally infected chickens. the enteric diseases caused by bacteria or viruses (rss) have the particularity of presenting alterations at the ciliated columnar epithelium, lieberkühn crypts, and in intestinal villi, which show atrophy and fusion or expansion of the lamina propria as a result of inflammatory infiltrates [ , , ] . nevertheless, experimental infections with chpv (abu-p ) have reproduced rss, but microscopic alterations were not found in the intestines or in any other organ [ ] . in the present investigation, we demonstrated the presence of crypt alterations, which were characterized principally by dilated crypts lined by a squamous epithelium that contained cellular debris and degenerate inflammatory cells, a cyst shape and crypt cell necrosis, all of which are present in the segment of the rolled duodenal loop and in other segments of the intestine; thus, these findings microscopically characterize the chpv strain isolated in brazil. chpv was detected initially in the intestinal content or in the intestinal wall [ , , , ] , because the intestine is considered the primary target for enteric virus infection; however, other studies showed the presence of the virus in the spleen and pancreas [ ] , suggesting another tropism of enteric viruses. these results were supported by qpcr targeting the chpv strain usp- - [ ] . furthermore, the presence of chpv was quantified and detected in the three segments of the small intestine (duodenum, jejunum and ileum) and increased after third day post-infection (p.i.) for ileum; after the sixth day for jejunum and after the seventh day for duodenum with high concentrations of genome copies per mg of tissue, however, in the thymus, liver, kidneys, pancreas, proventriculus and bursa, an increase in virus in the tissue was not detected. nevertheless, the peak of the genomic titer was observed between the first-and fourth-weeks post-infection. the presence of chpv in many organs until day showed that the virus could persist for a long time, explaining the continuous reinfections through the presence of chpv in poultry litters [ ] . the total weight of the infected animals showed a significant decrease compared to the mock group, which agreed with previously reported data from experimental infection with abu-p [ ] and showed that the chpv strain isolated in brazil is an important virus causing enteric diseases. other studies should be performed to understand the interaction of chpv with the lymphopoietic organs (bursa of fabricius, spleen and thymus), the exocrine function of digestive organs and certainly the interaction of chpv with chicken immune system cells. a strain of chpv was isolated in spf chicken embryos as described previously and was designated usp - [ ] . an aliquot of a macerated embryo from which the virus was isolated was transferred to a ml microtube containing ml of . m pbs at ph . . the suspension was subjected to three freeze thaw cycles consisting of freezing at − • c for min and thawing at • c for one min; each cycle was followed by homogenization. the sample was centrifuged at , × g for min at • c. the viral dna was extracted from µl of the supernatant of the viral suspension using trizol (thermo fisher scientific, carlsbad, ca, usa) reagent according to the manufacturer's instructions. for molecular characterization of chpv, pcr was carried out in triplicate as described previously [ ] , with some modifications. the amplified products were purified using gfxpcr dna and gel band purification kit (ge healthcare, piscataway, nj, usa) following the manufacturer's instructions. the purified dna was then ligated into the pcr™ . -topo cloning vector (thermo fisher scientific, carlsbad, ca, usa), and the vector was transformed into e. coli top competent cells according to the manufacturer's instructions. the bacteria were cultured on luria bertani (lb) agar containing ampicillin µg/ml. three colonies were cultured in ml of lb broth and shaken at rpm for h. plasmid dna was extracted from the lb broth bacterial suspensions using the qiaprep spin miniprep kit (qiagen, hilden, germany). the plasmid dna was then sequenced in the forward and reverse directions using a bigdye ® terminator v . cycle sequencing kit (thermo fisher scientific, carlsbad, ca, usa) with m primers. sequencing reactions were performed with an abi dna analyzer (thermo fisher scientific, carlsbad, ca, usa). the obtained electropherograms were analyzed using geneious . . software using de novo assembly. the consensus sequences were aligned and compared with other sequences of chpv available in genbank using the clustal w method in clustalx . . software (european bioinformatics institute, saffron walden, uk). the similarity of nucleotides (nt) and amino acids (aa) was determined using bioedit . . . the phylogenetic tree was built using the neighbor-joining statistical method and p-distance substitution model with bootstraps of replications in the mega software package [ ] . eighty (n = ) day-old spf chicks, supplied by ceva (ceva animal health, campinas, brazil), were divided into two groups of forty (n = ) birds: group i was infected with × genome copies (gc) of chpv in µl of pbs, as previously determined through qpcr [ ] , and group ii was mock infected with µl of sterile . m pbs ph . . all chicks were challenged by gavage. both groups were maintained for days in separate isolators under positive air pressure (alesco, campinas, sp, brazil), with water and food supplied ad libitum. the birds were maintained and used in accordance with the guidelines and the approval of the committee on the care and use of laboratory animal resources of the school of veterinary medicine, university of são paulo, brazil, under protocol number # / . the birds were observed daily and scored for clinical signs and mortality. the experimental challenge was performed at one day of age, and daily and weekly observations were performed (day pre-inoculation; daily , , , , , and days post-inoculation; and weekly , , , and days post-inoculation). five birds ( ) at each experimental phase/step were euthanized and subjected to necropsy examination. hereafter, each organ sample was collected separately, and selected organs included the duodenum, jejunum, ileum, proventriculus, pancreas, liver, spleen, bursa, thymus and kidney. the samples for qpcr were initially stored in liquid nitrogen and then stored at − • c until processing. a fragment of each organ listed above from the th to nd day was fixed in neutral-buffered % formalin and embedded in paraffin. sections of µm thicknesses were prepared and stained with hematoxylin-eosin (h&e). the slides were examined by light microscopy. the intestinal segments were examined by observing the villous:crypt ratio, lieberkühn crypt morphology, intensity of mononuclear and polymorphonuclear infiltrate in the lamina propria, presence and distribution of lymphoid follicles, number of intraepithelial lymphocytes present in one hundred enterocytes, and the number of cells in mitosis observed in the crypts in three fields at ×. the microscopic examination of fragments of intestine was semiquantitative according to the parameters described in table . the organs, including the proventriculus, pancreas, spleen, bursa, liver, thymus and kidney, were examined for the presence of inflammatory infiltrate, cell degeneration and other microscopic lesions. three randomly selected birds were necropsied from both the infected and mock groups, as described above, and dna was extracted from mg of tissue from each collected organ. chpv was detected and quantified using qpcr as described by nuñez et al. 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of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses a novel tool for specific detection and quantification of chicken/turkey parvoviruses to trace poultry fecal contamination in the environment chicken parvovirus viral loads in cloacal swabs from malabsorption syndrome-affected and healthy broilers avian parvovirus: classification, phylogeny, pathogenesis and diagnosis genetic characterization of parvoviruses circulating in turkey and chicken flocks in poland naturally occurring parvoviral infection in hungarian broiler flocks investigation into the aetiology of runting and stunting syndrome in chickens periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on the farms phylogenetic diversity of avian nephritis virus in hungarian chicken flocks detection of enteric viruses in pancreas and spleen of broilers with runting-stunting syndrome (rss) isolation and molecular characterisation of chicken parvovirus from brazilian flocks with enteric disorders mega : molecular evolutionary genetics analysis version . for bigger datasets this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare that there is no conflict of interest in this manuscript. key: cord- -m c bvuj authors: ashour, hossam m.; elkhatib, walid f.; rahman, md. masudur; elshabrawy, hatem a. title: insights into the recent novel coronavirus (sars-cov- ) in light of past human coronavirus outbreaks date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: m c bvuj coronaviruses (covs) are rna viruses that have become a major public health concern since the severe acute respiratory syndrome-cov (sars-cov) outbreak in . the continuous evolution of coronaviruses was further highlighted with the emergence of the middle east respiratory syndrome-cov (mers-cov) outbreak in . currently, the world is concerned about the novel cov (sars-cov- ) that was initially identified in the city of wuhan, china in december . patients presented with severe viral pneumonia and respiratory illness. the number of cases has been mounting since then. as of late february , tens of thousands of cases and several thousand deaths have been reported in china alone, in addition to thousands of cases in other countries. although the fatality rate of sars-cov- is currently lower than sars-cov, the virus seems to be highly contagious based on the number of infected cases to date. in this review, we discuss structure, genome organization, entry of covs into target cells, and provide insights into past and present outbreaks. the future of human cov outbreaks will not only depend on how the viruses will evolve, but will also depend on how we develop efficient prevention and treatment strategies to deal with this continuous threat. coronaviruses (covs) were discovered in the s and they were classified under family coronaviridae, which is the largest family within the order nidovirales ( figure ) [ ] . family coronaviridae encompasses two subfamilies: subfamily orthocoronavirinae and subfamily torovirinae ( figure ) [ ] . subfamily orthocoronavirinae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus ( figure ) [ ] . covs are typically harbored in mammals and birds and are common in camels, cattle, cats, bats, and other animals [ ] . alpha and betacoronaviruses circulate in mammals, including bats ( figure ) [ ] . gammacoronaviruses mostly infect avian species the first discovered covs were ibv that causes respiratory disease in chickens and the human covs, human cov- e (hcov- e) and human cov-oc (hcov-oc ), which cause the common cold in humans [ , ] . since the emergence of hcov- e and hcov-oc , several other hcovs were discovered, such as severe acute respiratory syndrome-cov (sars-cov) in , hcov-nl in , hcov-hku in , middle east respiratory syndrome-cov (mers-cov) in [ ] . starting december , there were reports of patients presenting with severe viral pneumonia in the city of wuhan, china [ ] . sequencing of the virus from these patients has identified a novel cov as the causative agent of this respiratory disease [ ] . the novel cov virus ( -ncov) was recently named sars-cov- by the world health organization (who). the disease caused by sars-cov- has been named covid- . prior to , covs were treated as nuisances but never as serious viruses. things changed after the emergence of sars-cov, which caused serious illnesses and deaths in - [ ] . unlike all human covs that cause mild respiratory symptoms, sars-cov, mers-cov, and sars-cov- are associated with serious respiratory diseases [ , ] . since its emergence, the sars-cov- has drawn well-deserved attention from the world. efforts are underway in an attempt to control this new cov outbreak. covs, including the newly discovered sars-cov- , are spherical positive single-stranded rna viruses that are characterized by spike proteins projecting from the virion surface [ , ] . the spherical morphology of the viral particle together with the spike projections led to the name coronavirus from the latin word corona meaning crown, due to the appearance of the virus as a royal crown under the electron microscope [ , ] . covs are enveloped viruses (envelope is a lipid bilayer derived from the host cell membrane) with the viral structure formed primarily of structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, and hemagglutinin- figure . classification of different types of coronaviruses within the family coronaviridae, subfamily orthocoronavirinae, and the respective genera: alpha-, beta-, gamma-, and deltacoronaviruses. the sars-cov- is classified as a betacoronavirus. the first discovered covs were ibv that causes respiratory disease in chickens and the human covs, human cov- e (hcov- e) and human cov-oc (hcov-oc ), which cause the common cold in humans [ , ] . since the emergence of hcov- e and hcov-oc , several other hcovs were discovered, such as severe acute respiratory syndrome-cov (sars-cov) in , hcov-nl in , hcov-hku in , middle east respiratory syndrome-cov (mers-cov) in [ ] . starting december , there were reports of patients presenting with severe viral pneumonia in the city of wuhan, china [ ] . sequencing of the virus from these patients has identified a novel cov as the causative agent of this respiratory disease [ ] . the novel cov virus ( -ncov) was recently named sars-cov- by the world health organization (who). the disease caused by sars-cov- has been named covid- . prior to , covs were treated as nuisances but never as serious viruses. things changed after the emergence of sars-cov, which caused serious illnesses and deaths in - [ ] . unlike all human covs that cause mild respiratory symptoms, sars-cov, mers-cov, and sars-cov- are associated with serious respiratory diseases [ , ] . since its emergence, the sars-cov- has drawn well-deserved attention from the world. efforts are underway in an attempt to control this new cov outbreak. covs, including the newly discovered sars-cov- , are spherical positive single-stranded rna viruses that are characterized by spike proteins projecting from the virion surface [ , ] . the spherical morphology of the viral particle together with the spike projections led to the name coronavirus from the latin word corona meaning crown, due to the appearance of the virus as a royal crown under the electron microscope [ , ] . covs are enveloped viruses (envelope is a lipid bilayer derived from the host cell membrane) with the viral structure formed primarily of structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, and hemagglutinin-esterase (he) protein in some betacoronaviruses [ ] . the s, m, and e proteins are all embedded in the viral envelope; pathogens , , of however, n protein interacts with the viral rna and is located in the core of the viral particle, forming the nucleocapsid [ ] . the s protein is a heavily glycosylated protein that forms homotrimeric spikes on the surface of the viral particle and mediates viral entry into host cells [ ] . in some covs, each monomer of the homotimeric s protein exists as two subunits (s and s ) on the viral particle due to cleavage of s protein by host furin-like proteases during viral replication [ , ] . however, in other covs including sars-cov, s protein forms s and s domains, remains intact on viral particles, and only gets cleaved inside endocytic vesicles during viral entry [ , ] . the m protein is one of the most important proteins in the virion structure. it exists in higher quantities than any other protein in the viral particle, in contrast to the e protein which is found in small quantities within the virion [ ] . the difference in abundancy may due to the fact that m protein gives the virus its shape and is critical together with e protein in orchestrating the assembly of the virus and in forming mature viral envelopes [ ] . the e protein also functions in release of viral particles from host cells, in addition to other functions [ ] . the n protein binds the viral rna and is required for packaging of viral rna into the viral particle during viral assembly [ , ] . as mentioned previously, he is present on the surface of some betacoronaviruses. it is a hemagglutinin similar to influenza virus hemagglutinin (binds sialic acid on host cell-surface glycoproteins) and possesses acetyl-esterase activity [ ] . he characteristics may enhance entry and pathogenesis of coronaviruses that contain such protein in their viral structure. the rna genome of covs is the second largest of all rna viruses, ranging from to kilobases (kb) in size [ ] . the largest genome of all rna viruses is that of the recently described planarian secretory cell nidovirus, pscnv ( . kb genome size) [ ] . viral rna codes for structural and nonstructural proteins [ ] . the structural proteins together with a few nonstructural proteins, with different functions, are coded within the end of the viral genome [ ] . however, the ' two-thirds of the genome codes for nonstructural proteins that are important in viral replication, including the rna-dependent rna polymerase (rdrp) [ ] . once the viral genome is inside the host cell cytoplasm following viral entry, translation of the end of viral rna produces the rdrp, which uses viral rna as a template to generate virus-specific mrnas (subgenomic mrnas) from subgenomic negative strand intermediates [ , ] . subgenomic mrnas share the same ends and the same leader sequence of - nucleotides at their ends [ , ] . translation of subgenomic mrnas leads to production of structural and nonstructural viral proteins [ ] . once sufficient structural proteins and genomic viral rna are formed, viral rna is then assembled with viral structural proteins into virions. viral assembly and budding occur in smooth-walled vesicles in the endoplasmic reticulum-golgi intermediate compartment (ergic) [ ] . s protein is the viral protein that mediates the entry of covs into host cells [ ] . receptor-binding domain (rbd) within the s domain mediates binding to the cognate host cell receptor; however, the s domain mediates the fusion events, between viral membrane and host cell membrane, that are required for entry of covs into host cells [ , ] . the rbd is located at n-terminus of the s subunit as in mouse hepatitis virus (mhv) or at the c-terminus as in sars-cov and mers-cov [ , ] . the tissue tropism of covs is determined by the s protein interaction with the receptors on host cells. several cellular receptors were described as receptors for covs. for example, aminopeptidase n (apn) was identified as the receptor for several alphacoronaviruses [ ] , angiotensin-converting enzyme (ace ) as the receptor for sars-cov [ ] , hcov-nl [ ] and possibly for the newly discovered sars-cov- [ ] , ceacam as the receptor for mhv [ ] , and dipeptidyl-peptidase (dpp which is also known as cd ) as the receptor for mers-cov [ ] . a study on the rbd of sars-cov- s protein showed its similarity in structure to that of sars-cov with some key amino acid differences [ ] . the previous finding suggests that sars-cov- could employ human ace as its cellular receptor. a recent study published in science showed that sars-cov- s protein has higher affinity to ace than sars-cov s protein [ ] . however, the receptor usage and the cellular tropism of sars-cov- need further investigation. the trimeric cov s protein is cleaved by host cell proteases during infection to expose the fusion peptide of the s domain, which induces the fusion of viral and cellular membranes [ , , , ] . fusion of viral envelope with host cell membrane results in the release of the viral genome into the cytoplasm [ , ] . cleavage of s protein occurs at different sites that were identified, in different coronaviruses, to be between the s and s domains (s /s site) and within the s domain proximal to the fusion peptide (s site) [ , , , ] . it is believed that cleavage at both sites is required for viral entry [ ] . different proteases have been identified to cleave the s /s site depending on its amino acid sequence in different viruses. for example, the s /s site of mers-cov s protein (rsvr↓sv) is cleaved by furin after its biosynthesis during viral replication [ ] . sars-cov- has an s /s site (ayt↓m) that is identical to the one in sars-cov [ ] . this sars-cov s /s site has been shown to be cleaved by cathepsin l following receptor binding and during viral entry in late endosomes [ ] . we believe that the sars-cov- s /s site may be cleaved by cathepsin l similar to sars-cov. other proteases such as trypsin, elastase, and tmprss have been shown to cleave sars-cov s protein at other sites between s and s domains [ , , ] . unlike sars-cov, sars-cov- has an additional furin-like protease cleavage site (rrar↓sv) that is n-terminus to the s /s site (ayt↓m), and is absent in sars-cov [ ] . the presence of this furin-like cleavage site in sars-cov- suggests its cleavage by furin during viral egress. in addition to the previous s /s sites, sars-cov- has a furin-like protease cleavage s site (kr↓sf) that is identical to that in sars-cov [ ] . however, there is no evidence that sars-cov s protein is cleaved by furin-like proteases at the s site during viral egress [ ] . similar to s /s site, we believe that sars-covs protein is cleaved at the s site by cathepsin l or tmprss in different cellular locations during viral entry. the previous notion is supported by several studies which showed that cathepsin l and tmprss promote sars-cov entry while their inhibition suppressed infection of permissive cells [ , , ] . given that sars-cov- has the same s site as sars-cov, we believe that processing of sars-cov- s protein at s site is similar to sars-cov. similarly, mers-cov has an s site (rxxr↓sa) that is less efficiently cleaved by furin and most probably cleaved by tmprss or cathepsin l during viral entry [ ] . despite the identification of putative protease cleavage sites in sars-cov- s protein, their relative importance for sars-cov- s protein activation, viral pathogenesis, and cellular tropism needs further investigation. antiviral small molecules that inhibited cathepsin l were able to inhibit sars-cov infections in vitro and that of other viruses that depend on cathepsin l for entry, such as ebola, hendra, and nipah viruses [ ] . the presence of a cathepsin l cleavage site in sars-cov- s protein (s /s site) suggests that cathepsin l inhibitors may be valuable in inhibiting sars-cov- infections [ ] . antibodies against rbd and s domain of sars-cov and mers-cov s proteins have been found effective in neutralizing infections of permissive cell lines in vitro [ ] [ ] [ ] [ ] . in addition, neutralizing antibodies were capable of treating infections in experimental animals and in infected patients during these major outbreaks [ ] [ ] [ ] [ ] . in one study, several sars-cov rbd-specific monoclonal antibodies did not bind to sars-cov- s protein [ ] . another study showed that the sars-cov-specific monoclonal antibody, cr , bound with high affinity to sars-cov- rbd [ ] . the previous studies suggest that there are differences between the two rbds which impact the cross reactivity of many neutralizing antibodies. however, neutralizing antibodies against sars-cov- , once developed, could be promising in controlling the current sars-cov- outbreak. around cases that were infected over nine months (around % fatality) ( table ) [ ] . sars-cov was found to infect unciliated bronchial epithelial cells and type ii pneumocytes and cause fever, cough, shortness of breath, and severe complications such as pneumonia and kidney failure [ , ] . the incubation period for sars-cov was estimated to range from to days, and up to days (table ) [ ] . studies have shown that bats harbor covs that are ancestral to sars-cov (table ) [ ] . civets and raccoon dogs, of chinese local markets, were shown to harbor sars-like covs (table ) [ ] . the detection of sars-related covs (sarsr-covs) in bats and small animals in retail markets may indicate an interspecies transmission from bats to small animals and finally to humans (figure ). studies in bats from different regions of china have identified several sarsr-covs [ ] . the previous finding indicates that sars-cov has been circulating in bats for a long time before genetically changing and jumping to humans. since ace was identified as the receptor for sars-cov, it is not surprising that sars-cov has adapted itself to bind human ace and efficiently infect human cells [ ] . that sort of adaptation required a set of amino acid changes in the rbd of s protein of sars viruses that were circulating in bats [ ] . therefore, we conclude that the human-to-human transmission that was seen during the sars-cov outbreak is attributed to the ability of sars-cov to adapt its s protein (particularly rbd) to efficiently bind to human ace and infect airway epithelia ( figure ). [ ] . civets and raccoon dogs, of chinese local markets, were shown to harbor sarslike covs (table ) [ ] . the detection of sars-related covs (sarsr-covs) in bats and small animals in retail markets may indicate an interspecies transmission from bats to small animals and finally to humans (figure ). studies in bats from different regions of china have identified several sarsr-covs [ ] . the previous finding indicates that sars-cov has been circulating in bats for a long time before genetically changing and jumping to humans. since ace was identified as the receptor for sars-cov, it is not surprising that sars-cov has adapted itself to bind human ace and efficiently infect human cells [ ] . that sort of adaptation required a set of amino acid changes in the rbd of s protein of sars viruses that were circulating in bats [ ] . therefore, we conclude that the human-tohuman transmission that was seen during the sars-cov outbreak is attributed to the ability of sars-cov to adapt its s protein (particularly rbd) to efficiently bind to human ace and infect airway epithelia ( figure ). mers-cov was first described in as a new cov that causes a severe respiratory disease in saudi arabia [ ] . similar to sars-cov, mers-cov infects unciliated bronchial epithelial cells and mers-cov was first described in as a new cov that causes a severe respiratory disease in saudi arabia [ ] . similar to sars-cov, mers-cov infects unciliated bronchial epithelial cells and type ii pneumocytes and causes severe illness of the respiratory tract, which is characterized by fever, cough, shortness of breath, and severe complications such as pneumonia and kidney failure [ ] . the incubation period of mers-cov is quite similar to sars-cov and ranges from to days (table ) [ ] . as of january and since , of infected cases in countries have died (≈ % fatality), which is more than three times the fatality seen in sars-cov infections (table ) [ ] . however, unlike sars-cov, human-to-human transmission of mers-cov is not easy and has not been confirmed except in cases of very close contact with infected patients in health care settings [ ] . mers-related covs (mersr-covs) were detected in bats, suggesting a potential bat origin (table ) [ , ] . mers-cov was transmitted to humans from dromedary camels ( table ) [ ] . studies have also shown that camel mers-cov strains are almost identical to human mers-cov strains [ ] . it was postulated that mers-cov existed in camels at least years ago since antibodies to mers-cov were detected in samples that were collected from camels in [ ] . sequence analyses have shown that mersr-covs' rbds share only - % sequence identity with that of human and camel mers-covs [ ] . similar to the adaptation of sars-cov to human host, mersr-covs that are circulating in bats had to undergo several amino acid changes in rbd of s protein to become capable of infecting camels and humans ( figure ) [ ] . we believe that the amino acid changes in mersr-covs' rbd led to the emergence of mers-cov strains that are capable of binding to human dpp with high affinity, infecting humans, and causing the outbreak ( figure ) . as mentioned previously, the sars-cov- was isolated and sequenced from patients that showed symptoms of respiratory illness and pneumonia in wuhan, china during december . sars-cov- is the third identified human cov that causes severe respiratory illness with symptoms and incubation period resembling that of sars-cov and mers-cov infections (table ) [ , ] . since december , sars-cov- infection rates have been rising in china and worldwide [ ] . similar to sars-cov and unlike mers-cov, human-to-human transmission has been confirmed [ ] . initial cases of sars-cov- infections were somehow connected to the huanan seafood market in wuhan, in the hubei province of china [ ] . in this market, a number of nonaquatic animals were on sale, such as birds, snakes, marmots, bats, and rabbits [ ] . genetic analyses of viral samples from patients with sars-cov- infections revealed that the sars-cov- is a betacoronavirus that has % sequence identity to two bat sarsr-cov: % identity to sars-cov and only % identity to mers-cov [ ] . the previous findings suggest that sars-cov- is a new virus that is distinct from sars-cov and mers-cov but most probably originated in bats, similar to sars-cov and mers-cov [ ] . another recent study confirmed that sars-cov- significantly clustered with a sequence from the bat sars-like cov that was isolated in [ ] . however, the existence of an intermediate host for sars-cov- is still not verified (figure ). to date, the fatality of sars-cov- appears to be less than that observed in sars-cov and mers-cov infections. however, since new cases are confirmed everyday as we write this review, the fatality of this virus may keep changing and will not be accurately calculated until after the end of this outbreak. the virus appears to be more fatal in elderly patients or patients with comorbidities [ ] . however, it is important to note that there could be cases that went undetected, which makes it hard to accurately calculate the fatality of this new virus. our lessons learned from sars-cov and mers-cov outbreaks include the high mutation rates that characterize all rna viruses [ ] , the evolving nature of covs [ , ] , and the ease of transmission from one species to another [ , ] . as mentioned previously, it appears that sars-and mers-covs arose at sometime from ancestral covs harbored by bats (figure ). whereas animals served as intermediate hosts, humans served as terminal hosts (figure ). sars-cov was transmitted to civets and raccoon dogs, and to camels in the case of mers-cov (figure ). they were then transmitted pathogens , , of from these intermediate animal hosts to humans (figure ). the practice of eating raw meat and the close contact between humans and animals are both risk factors for the initiation of a new human cov outbreak. this is due to the constant exposure of humans in these cultures to the ever-changing mutant covs. the first cases of sars-cov- infections were reported in the chinese city of wuhan during december [ ] . although other options have not been completely ruled out yet, it is believed that the sars-cov- stemmed from a large seafood and animal market in wuhan, the capital of hubei province, china [ ] . as for other wet markets in china, live animals are sold, mostly for food or medicine. the attributed medicinal and/or magical uses of wildlife and rare animal parts (such as pangolin scales and tiger paws) are mainly based on traditional chinese medicine (tcm), which has been widely promoted by the current chinese government. however, it is important to note that most of these folk remedies are never prescribed in reputable tcm hospitals. the wuhan market is known to have a lot of exotic animals and exotic animal parts [ ] . thus, sars-cov- disease can be considered a zoonotic disease (like sars) that has initially spread from animals to humans. however, human-to-human transmission has also been confirmed [ ] . it is not well understood why outbreaks of cov infections are mostly occurring in china. we speculate that those viruses may be predominantly circulating in animals in china rather than other animals in different parts of the world. one of the reasons for these sudden outbreaks could be the close interactions with live and wild animals that are consumed as food in wholesale food markets in china. in a very recent study, the genomes of covs isolated from nine patients having viral pneumonia in wuhan were analyzed [ ] . the study showed that the genomes of these viruses differed by less than . percent (more than . % of sequence identity), which indicates that the virus has only recently emerged in humans and has been detected rapidly after its emergence [ ] . as the virus keeps spreading to more individuals, more mutations may arise which can potentially make the virus more virulent and thus constant surveillance will be necessary. the u.s. center for disease control and prevention (cdc) reported that sars-cov- causes a respiratory illness that is characterized by fever, cough, and shortness of breath. radiographs of some sars-cov- patients demonstrated invasive lesions in both lungs [ ] . the cdc also reports that the elderly, individuals with underlying health problems, and people with compromised immune systems are at a particularly higher risk of developing severe pneumonia from the virus [ ] . we believe that it is too early to assess the impact of the new virus on children. sars-cov infections were significantly less common among children than adults, and kids younger than reported much less severe symptoms than patients who were more than [ ] . this may have to do with children being exposed to more cov in school and the outdoors than adults or because of the better overall health status of children as compared to adults. also, children tend to be more up-to-date with vaccinations, which may protect them from secondary infections that are often triggered by the main infections. during the sars-cov outbreak, most schools and factories in china remained open. following the sars-cov- outbreak and confirmation of many infected cases, china responded by locking down residents of wuhan city, banning wildlife trade until the epidemic is over, and attempting to build two new hospitals in the city of wuhan to specifically handle the new outbreak. it is currently unclear if these two hospitals will be able to handle a major outbreak in a city of million residents. there is no definite information about the exact time of the start of the outbreak. that is why it is hard to assess how contagious the virus is (the rate of sustained spread) at the present time. however, it seems likely and plausible that it is highly contagious, based on the mounting data about human-to-human transmission outside china [ ] . in order to be able to precisely assess the rate of sustained spread, information about numbers of cases and deaths (the overall number of patients) will need to be divided by the overall number of people at risk of acquiring the disease (the number of individuals who have been in contact with the patients). once this is determined, the overall risk can be assessed. underreporting or misdiagnosis of cases can negatively impact the calculations and thus delay the ability of public health officials to truly assess the situation. in an outbreak of this magnitude, the most important number that public health experts will be looking for is the basic reproduction number, also known as the r [ ] . this number measures the disease's potential and represents the average number of people who will catch the disease from one infected person in a population that has never had the disease in the past [ ] . in one study, the mean estimate of r was calculated to be between . and . [ ] . another study estimated the r to have a high average value of . [ ] , which is consistent with other groups that reported values from to [ , [ ] [ ] [ ] [ ] . these r estimates for the sars-cov- are consistent with r estimates for sars-and mers-covs (from to ) (table ) [ , ] . the virus is less deadly than sars, which killed about % of the infected patients. because r represents an average, outliers (carriers who infect so many people or carriers who infect nobody) can have a huge impact on the final value of the r . in other words, a bigger r does not necessarily mean more infections. for example, the r for the seasonal flu typically ranges from . to . [ ] , but it still infected many more people than the number of people who got infected with sars-cov. at any rate, any r above should be taken seriously. the goal will be to reduce the r to a value that is below . finally, r estimates can be higher than the "true" r values because of two main reasons: infected people who did not show symptoms and infected people who did not report their symptoms. since r is not an intrinsic property of the virus itself, r estimates tend to be lower in places where there are sound infection control methods and vice versa. typically, r estimates are highest at the beginning of an outbreak and then subside gradually once countries become aware of the outbreak and manage to put in effective control measures to prevent the spread of the virus. the lockdown strategy that china is implementing works best during the early stages of an infection, which is not the case in wuhan, as there are several million people who already left the city before the restrictions were imposed. if we are beyond the early stages, then there can be disadvantages associated with such lockdown on wuhan. among the disadvantages are people evading care to avoid any restrictions on their life. given that the incubation period can be up to days or more, a two-week federal quarantine was ordered for u.s. citizens who were flown back from china [ ] . the action was described by the cdc as precautionary and preventive. for comparison, there were no quarantines ordered in the u.s. for the recent sars-cov or mers-cov outbreaks. the last federal quarantine in the u.s. was ordered back in the to prevent the spread of smallpox from sweden to the u.s. during a smallpox outbreak in sweden [ ] . as shown in table , the number of people infected by the virus has exceeded the global total infected with sars-cov ( individuals) in a nine-month period that extended from until . it will be difficult to control the disease without a level of disruption of air travel. we believe that limiting travel to and from china will be a necessary measure. respiratory viruses spread through respiratory droplets that are produced when an infected person coughs or sneezes. the exact modes of transmission of sars-cov- are not entirely clear at this early stage, but reports of healthcare professionals in china who contracted the disease suggest a highly contagious virus [ ] . prevention of the sars-cov- infections entail precautions that are common to other respiratory viruses. the most obvious measure is to avoid contact with people who are sick. this is especially important due to the contagious nature of the virus. if someone is sick, they should stay home. once they recover, they may consider using disposable face masks (while frequently changing them) and avoiding close contact with coworkers. however, the value of wearing face masks is controversial, to say the least [ ] . surgical masks do not fully protect against airborne viruses as they do not fully seal the nose and the mouth. thus, small droplets, which can travel farther than large droplets and in more unpredictable patterns, can be inhaled around the sides of the masks. the n masks offer a better protection as long as they fit properly. it is worth noting that n masks are not suitable for people with facial hair [ ] . being an enveloped virus, washing hands with water and soap for at least s would be beneficial in deactivating the sars-cov- [ ] . hand sanitizers can also be used if water and soap are not readily available, while touching eyes, nose, and mouth should be prevented [ ] . disinfection of different environmental surfaces, tools, and objects is crucial in limiting the spread of the virus [ ] . the more you use one of these objects or touch one of these surfaces, the more pressing cleaning and disinfection become. public health officials, local health departments, hospitals, doctors, and cdc personnel should work closely with universities and other workplaces to educate and provide needed supplies to contain the spread of the virus. the world health organization (who) declared sars-cov- as a public health emergency of international concern (pheic) on january [ ] . a pheic is an atypical event that constitutes a public health risk and potential for the disease to spread to other countries, thus requiring a coordinated international response [ ] . this designation could help mobilize more resources to the impacted areas. the current outbreak represents the sixth time the who has declared a global emergency since it gained the power to declare an international emergency in [ ] . the previous five times were the h n swine flu, the ebola outbreak in west africa, the polio outbreak, the zika outbreak, and ebola outbreak in the democratic republic of congo [ ] . none of these previous emergencies led a worldwide pandemic. however, we predict that the current outbreak is more likely to become a pandemic. the current diagnostic test for the virus is pcr-based [ ] . since this test typically takes h, a new quicker diagnostic test needs to be developed. it will not be practical to isolate (quarantine) a large number of individuals until results of the pcr-based diagnostic test become available. this can also overwhelm healthcare facilities, already overwhelmed by the increasing number of cases that are discovered every day in china and elsewhere in the world. containing the outbreak before it can spread is the best way to prevent pandemics. border closures and screening at airports and checkpoints are classical measures that were previously implemented in the h n flu pandemic [ ] . this can reduce the spread of the virus but will not be a fool-proof strategy. the reason is that the incubation period of the virus is believed to be as long as days, as was the case with mers-cov [ , ] . this means that carriers of the virus can show up at the border with no apparent symptoms and readily pass through security without raising any red flags. compared to the sars-cov outbreak in , the current increase in air traffic in china and worldwide has likely contributed to the more rapid spread of sars-cov- in . without a quick diagnostic test, there are not many good options that can completely stop the transmission of the virus. once a test is available, cases can be identified and isolated. based on previous genetic experience with sars-cov, scientists will need to quickly develop a vaccine for sars-cov- . the world is hoping to succeed in containing this virus as soon as possible. even after success, there needs to be follow-up with patients who are cured and declared virus-free. this is a lesson we learned from the ebola outbreak, in which some patients who walked out from the hospital "virus-free" were found later to harbor the ebola virus that was carefully hiding itself in other parts of the body, such as the immune-privileged eye [ ] [ ] [ ] . although this hidden ebola virus was no longer transmissible to other humans, it rendered the label "virus-free" incorrect with these individuals. natural disasters bring people together but epidemics and outbreaks split them apart. the sars-cov- is another cov that may lead to a pandemic, if not timely controlled. our current knowledge of this virus suggests an intermediate host; however, human-to-human transmission is confirmed and is of concern. the number of infected cases to date indicates a very rapid and efficient human-to-human transmission. this necessitates quick development of therapeutics that can inhibit coronavirus genomics and bioinformatics analysis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains fatal swine acute diarrhoea syndrome caused by an hku 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/n respirators in health care workers with facial hair: results of the beards (adequate respiratory defences) study evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation efficacy of ethanol against viruses in hand disinfection china coronavirus: who declares international emergency as death toll exceeds ebola outbreak declared a pheic, world waits for next steps global health concern stirred by emerging viral infections first case of novel coronavirus in the united states transmission dynamics, border entry screening, and school holidays during the influenza a (h n ) pandemic the novel coronavirus originating in wuhan, china: challenges for global health governance comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia persistence of ebola virus in various body fluids during convalescence: evidence and implications for disease transmission and control retinal pigment epithelial cells are a potential reservoir for ebola virus in the human eye immune tolerance elicited via unique ocular and oral routes key: cord- -qd qjo y authors: rothan, hussin a.; acharya, arpan; reid, st patrick; kumar, mukesh; byrareddy, siddappa n. title: molecular aspects of covid- differential pathogenesis date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: qd qjo y in the absence of therapeutic interventions, and a possible vaccine candidate, the spread of covid- disease and associated fatalities are on the rise. the high mutation frequency in the genomic material of these viruses supports their ability to adapt to new environments, resulting in an efficient alteration in tissue tropism and host range. therefore, the coronavirus’ health threats could be relevant for the long-term. the epidemiological data indicate that age, sex, and cardio-metabolic disease have a significant impact on the spread and severity of covid- . in this review, we highlight recent updates on the pathogenesis of sars-cov- among men and women, including children. we also discuss the role of the cellular receptors and coreceptors used by the virus to enter host cells on differential infection among men, women, and cardio-metabolic patients. infection with severe acute respiratory syndrome coronavirus- (sars-cov- ) that appeared in december in china has become a global public health threat [ ] . there are no effective vaccines or specific antiviral drugs available to manage covid- disease. generally, coronaviruses pose severe threats to human health and economic stability worldwide. the high mutation frequency in the genomic material of these viruses supports their ability to adapt to new environments, resulting in an efficient alteration in tissue tropism and host range [ ] [ ] [ ] . recent findings support that the binding affinity of sars-cov- envelop spikes to the cellular ace receptor is - times higher than sars-cov- , facilitating the high transmission and infectivity of sars-cov- in humans [ ] . based on the genetic sequence identity and the phylogenetic reports, sars-cov- is significantly different from sars-cov- , and it can thus be considered a novel beta coronavirus that infects humans. sars-cov- most likely developed from the bat origin coronaviruses and was transmitted to humans by yet unknown methods [ ] . coronaviruses (covs) are enveloped positive-sense and single-stranded rna viruses that belong to the coronaviridae family and nidovirales order [ ] . the covs are classified into four genera: alpha-covs (α-covs), beta-covs (β-covs), gamma-covs (γ-covs), and delta-covs here, we discuss the epidemiology of the covid- illness based on the patient groups and the severity of the infection. we also discuss the role of the cellular receptors and coreceptors in virus infectivity, comorbidity with cardio-metabolic syndromes, and the overlap between cardio-metabolic treatments and sars-cov- infection. the epidemiological reports have shown that the death cases of covid- illness are higher in healthy or older adults than children. a study showed that among , covid- confirmed cases, only ( . %) were between - years old, and ( . %) were between - years [ ] . about % of children were asymptomatic, % had a mild illness, and % had a moderate illness. another study showed that the percentage of severe sars-cov- infection among children was % compared to . % in adults [ ] . generally, infected children show milder to asymptomatic covid- disease. adults with severe covid- disease suffer from deadly pneumonia and insufficient supply of oxygen throughout the body, as reported by several frontline clinicians. on the other hand, clinical reports showed that the children are not susceptible to pneumonia caused by sars-cov- infection [ , [ ] [ ] [ ] [ ] . the considerable variation in sars-cov- infection between children and adults raises a question that could help understand the mechanism of covid- disease pathogenesis. although initial reports indicate that sars-cov- positive children only develop a mild disease, new updates from clinics report cases of "severe shock syndrome with hyper inflammation", and "kawasaki-like-disease" [ ] [ ] [ ] . in late april , the royal college of pediatrics from the uk reported systemic inflammatory response in a fraction of sars-cov- positive children with cardiac injury that have overlapping features with kawasaki disease. most of the children had a higher level of c-reactive proteins, d-dimers, troponin, and significant decreases in lymphocyte count [ ] . in early may , clinicians from new york city reported similar findings among a fraction of sars-cov- positive children [ ] . similarly, paris reports indicate an increase in incidences of "kawasaki-like multisystem inflammatory syndrome" among children, which may be related to sars-cov- [ ] . all these findings indicate that all sars-cov- positive children should be carefully monitored for the development of systemic inflammatory responses and associated cardiac injury for better management of the disease. interestingly, the reports showed that the rate of sars-cov- infection is higher in men than women, and the severity of the illness is much higher in men. the percent of disease fatality in men is . %, while in women, it is . % [ , ] . an early study from china showed that of the patients with covid- pneumonia, the combined average age of the patients was · years, while the average age for men was years, and for women, it was only years [ ] . another report showed that of , covid- confirmed cases reported in february , the men comprised . %, women comprised . %, and the median age was years [ ] . the differential pathogenesis of sars-cov- among different groups requires further investigations that focus on the disease's molecular mechanism. understanding the mechanism of covid- pathogenesis would help in the development of an efficient cure. angiotensin-converting enzyme- (ace ) represents the primary sars-cov- entry receptor, and its physiological role is crucial in the progress of covid- illness. lung epithelial cells that express the ace receptor on the cell membrane are the primary target of coronaviruses. binding of the virus spikes to the receptor-binding domain of the ace initiates sars-cov entry into target cells [ , ] . amino acid sequence data shows similarities between sars-cov- and sars-cov- , and the recent reports strongly suggest that the entry of sars-cov- to the host cells is via the ace receptor [ , ] . it is important to note that the ace gene is mapped on the human x chromosome (xp ) [ , ] , and the female hormone β-estradiol increases the expression levels of ace protein, as investigated in ovariectomized female rats [ ] . furthermore, a significantly higher ace expression was detected in older females than male rats [ ] , and such high expression levels of ace could be crucial in preventing kidney injury in the experimental hypertensive model [ ] . previous studies on sars-cov- reported that the binding of viral spike (s) protein to ace downregulates the expression of ace , resulting in a diminished protective role of ace and, subsequently, acute respiratory failure [ ] . furthermore, ace expression is dramatically reduced with aging in both genders [ ] . the levels of ace expression, which could be sex-and age-dependent, have a protective role against lung and kidney injuries that could impact the severity of covid- illness in male vs. females and old vs. young individuals. the initiation of sars virus infection is ace -dependent, and after that, there are ace -independent pathways for cell-to-cell virus transmission [ ] . cell-to-cell spreading has a significant impact on virus infection and pathogenesis. the ace downregulation due to virus infection, as mentioned above, contributes to lung and kidney injuries. we hypothesize that the reduction in ace levels in men is higher than females, which would explain the observed increases in disease severity. ace receptor is coexpressed with tmprss , a cellular transmembrane protease that cleaves the s protein of sars-cov- and sars-cov- into two fragments: s , which is essential for virus attachment, and s , for virus fusion into the target cells [ , [ ] [ ] [ ] . tmprss protein is expressed in many tissues, including the lungs, primarily in the epithelial cells [ , ] . the expression levels of tmprss protein are regulated by levels of androgen and androgen receptors [ ] [ ] [ ] [ ] , suggesting sex-related expression levels of tmprss protein. both women and children have a lower level of androgen and androgen receptors than men, and therefore, tmprss could play a potential role in the severity of covid- pathogenesis in men. this pattern is supported when we take the total deaths in the united states due to covid- and consider the breakdown of age percentages and sex distribution (figure ) [ ] . the percentage of deaths in male patients is higher compared to female covid- patients. thus, it could be possible that the expression levels of ace and tmprss impact virus infectivity and pathogenesis among different groups of individuals, considering the variation in the expression levels in older men compared to the women and children. the severity and mortality of sars-cov- infection are positively correlated with the comorbidity of lung disease, diabetes, cardiovascular diseases and cerebrovascular diseases. a study reported that patients from confirmed cases of covid- had comorbidities of hypertension ( . %), diabetes mellitus ( . %), heart diseases ( . %), and cerebrovascular disease ( . %), and all of the patients showed severe covid- illness [ ] . another report showed that of a group of sars-cov- -infected patients, patients died with comorbidities of diabetes and the severity and mortality of sars-cov- infection are positively correlated with the comorbidity of lung disease, diabetes, cardiovascular diseases and cerebrovascular diseases. a study reported that patients from confirmed cases of covid- had comorbidities of hypertension ( . %), diabetes mellitus ( . %), heart diseases ( . %), and cerebrovascular disease ( . %), and all of the patients showed severe covid- illness [ ] . another report showed that of a group of sars-cov- -infected patients, patients died with comorbidities of diabetes and cerebrovascular diseases ( %) [ ] . another study reported that of severe cases of covid- , about % of the patients experienced chronic hypertension, and % had diabetes [ ] . the susceptibility of cardio-metabolic patients to develop severe covid- illness and the high mortality rate could be linked to the ace function during sars-cov- infection and the cardio-metabolic treatments that may interfere with ace -virus interaction. ace cleaves angiotensin (ang) i to form ang ii within the renin-angiotensin system (ras). ang ii has been recognized as the primary active peptide cleaved by ace , a homolog of ace, to form ang ( - ). ace has high catalytic efficiency, suggesting an essential role in preventing ang ii accumulation while enhancing ang-( - ) formation [ , ] . ace alterations have been described in experimental models of hypertension and diabetic kidney disease, and ace levels were found to be decreased in the setting of hypertension [ ] [ ] [ ] [ ] . ace expression is dramatically reduced with aging in both genders and young adult vs. old [ ] . thus, ace overexpression improves pancreatic islet-cell function, cardiovascular health, blood pressure, and the renal protective arm of the ras [ ] . ace has a therapeutic effect on diabetes, cardiovascular conditions, kidney disease, and several other conditions in which the overactivity of ang ii is undesirable. previous studies on sars-cov- reported that the binding of viral s protein to ace downregulates the expression of ace , resulting in a diminished protective role of ace and, subsequently, acute respiratory failure [ ] . downregulation or malfunction of ace leads to the accumulation of ang ii, resulting in a significant reduction in insulin secretion from the pancreas and the glomerular filtration rate in the kidney [ ] . a previous study by wysocki et al. tested whether a soluble human recombinant ace (race ) may be used to decrease ang ii and increase ang ( - ) levels in plasma and tissues and whether race may be used to prevent ang ii-induced hypertension in mice. interestingly, this study found that race infusion induced a dose-dependent increase in serum ace activity but had no effect on kidney or cardiac ace activity [ ] . however, the role of indigenous or exogenous circulating soluble ace on the progression of covid- still needs further investigation. aging decreased expression of the ace , which also leads to the accumulation of ang ii levels, affecting other body organs like the heart, pancreas, and kidneys. these factors suggest that treatment with ace -activating compounds could enhance hypertension and diabetic kidney disease during infection. the impact of ace activators/inhibitors on the covid- illness requires urgent investigation. the american heart association, the heart failure society of america, the american college of cardiology, and the council on hypertension of the european society of cardiology have urged that the current therapies should be continued at this point of covid- illness as the withdrawal of the arb therapies may be unwise since there is no clinical evidence for the interaction of arb therapies and covid- illness [ , ] . recent reports have supported these recommendations, indicating that the results of clinical studies showed no evidence that ace inhibitors or arbs affected the risk of covid- , and there is no potentially harmful association of ace inhibitors or arbs with in-hospital deaths caused by covid- illness [ ] [ ] [ ] . we conclude that the variations in the expression levels of sars-cov- receptors and co-receptors, due to physiological and co-morbidity conditions, could 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albuminuria in diabetes expression of ace and ace in individuals with diabetic kidney disease and healthy controls angiotensin-converting enzyme and angiotensin-( - ): an evolving story in cardiovascular regulation ace and diabetes: ace of aces? diabetes american college of cardiology. hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid- position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers drug therapy, and mortality in covid- renin-angiotensin-aldosterone system blockers and the risk of covid- renin-angiotensin-aldosterone system inhibitors and risk of covid- key: cord- - chcsqe authors: wang, lihua; madera, rachel; li, yuzhen; mcvey, david scott; drolet, barbara s.; shi, jishu title: recent advances in the diagnosis of classical swine fever and future perspectives date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: chcsqe classical swine fever (csf) is a highly contagious viral disease of pigs, including wild boar. it is regarded as one of the major problems in the pig industry as it is still endemic in many regions of the world and has the potential to cause devastating epidemics, particularly in countries free of the disease. rapid and reliable diagnosis is of utmost importance in the control of csf. since clinical presentations of csf are highly variable and may be confused with other viral diseases in pigs, laboratory diagnosis is indispensable for an unambiguous diagnosis. on an international level, well-established diagnostic tests of csf such as virus isolation, fluorescent antibody test (fat), antigen capture antibody enzyme-linked immunosorbent assay (elisa), reverse-transcription polymerase chain reaction (rt-pcr), virus neutralization test (vnt), and antibody elisa have been described in detail in the oie terrestrial manual. however, improved csf diagnostic methods or alternatives based on modern technologies have been developed in recent years. this review thus presents recent advances in the diagnosis of csf and future perspectives. classical swine fever (csf), a list-a disease classified by the world organization for animal health (oie), is considered as a transboundary animal disease by the food and agriculture organization of the united nations (fao) [ ] . the disease causes high morbidity and mortality in both feral and domestic pigs and can result in significant economic losses to the swine industry worldwide [ ] . currently, it is present in many countries in asia, the caribbean islands, africa, and south and central america ( figure ). it is most likely to be introduced to csf-free countries through inadvertent or deliberate importation of classical swine fever virus (csfv) infected animals, animal products, and animal feed [ , ] . classical swine fever virus (csfv) is the etiologic agent of csf and belongs to the genus pestivirus in the flaviviridae family [ ] . the genome of csfv is a positive single-strand rna of about . kb. it contains untranslated regions at and ends and a single large open reading frame (orf). the orf codes four structural (c, e rns , e , and e ) and eight nonstructural viral proteins (n pro , p , ns , ns , ns a, ns b, ns a, and ns b) [ , ] . based on the nucleotide sequences of -non-translated region ( -ntr) and glycoprotein e , csfvs are divided into three genotypes and sub-genotypes ( . - . , . - . , and . - . ) [ ] [ ] [ ] . as reported, csfv genotype . and genotype . caused the more figure . global distribution of classical swine fever (csf) epidemics, . map based on data from cabi invasive species compendium. wallingford, uk: cab international. available online: www.cabi.org/isc (accessed on ). in addition, we also incorporated the most current csf epidemic information (disease present in japan and romania) from oie, . https://www.oie.int/animal-health-in-the-world/official-disease-status/classical-swine-fever/map-ofcsf-official-status/ names of countries with csf are given in the map. classical swine fever virus (csfv) is the etiologic agent of csf and belongs to the genus pestivirus in the flaviviridae family [ ] . the genome of csfv is a positive single-strand rna of about . kb. it contains untranslated regions at ′ and ′ ends and a single large open reading frame (orf). the orf codes four structural (c, e rns , e , and e ) and eight nonstructural viral proteins (n pro , p , ns , ns , ns a, ns b, ns a, and ns b) [ , ] . based on the nucleotide sequences of ′-nontranslated region ( ′-ntr) and glycoprotein e , csfvs are divided into three genotypes and subgenotypes ( . - . , . - . , and . - . ) [ ] [ ] [ ] . as reported, csfv genotype . and genotype . caused the more recent outbreaks in europe [ ] . sub-genotypes . , . , . , and . are prevalent in asia [ ] , while sub-genotypes . - . are distributed in other separated geographic regions [ , [ ] [ ] [ ] . traditional diagnostics for csf include clinical signs, pathological findings, and antigen and antibody detection [ ] . although unique clinical and pathological observations such as "button" ulcers in the cecum and large intestine mucosa may be found exclusively in csf, other clinical signs and pathological findings in pigs infected with csfv are highly variable and are often similar to that of other viral diseases of pigs, such as african swine fever, pseudorabies, porcine dermatitis, and nephropathy syndrome (pdns), post-weaning multisystemic wasting syndrome (pmws), thrombocytopenic purpura, and various septicemic conditions [ ] . thus, laboratory diagnosis of csf for detection of the specific csfv antigen and antibody is indispensable [ , ] . the wellestablished diagnostic methods of csf such as virus isolation, fluorescent antibody test (fat), antigen capture antibody enzyme-linked immunosorbent assay (elisa), reverse-transcription polymerase chain reaction (rt-pcr), virus neutralization test (vnt), and antibody elisa (table ) have been widely used and well described in the oie terrestrial manual [ ] . recently developed techniques and alternatives have made significant improvements in several key components of csf diagnosis, including less sample and reagents required, less effort and time needed, increased detection efficiency (multiplexing), ease of performing and disposal, automation, and point of care (poc). this review provides an updated overview on laboratory diagnosis of csf and future perspectives. cabi.org/isc (accessed on ). in addition, we also incorporated the most current csf epidemic information (disease present in japan and romania) from oie, . https://www.oie.int/animalhealth-in-the-world/official-disease-status/classical-swine-fever/map-of-csf-official-status/ names of countries with csf are given in the map. traditional diagnostics for csf include clinical signs, pathological findings, and antigen and antibody detection [ ] . although unique clinical and pathological observations such as "button" ulcers in the cecum and large intestine mucosa may be found exclusively in csf, other clinical signs and pathological findings in pigs infected with csfv are highly variable and are often similar to that of other viral diseases of pigs, such as african swine fever, pseudorabies, porcine dermatitis, and nephropathy syndrome (pdns), post-weaning multisystemic wasting syndrome (pmws), thrombocytopenic purpura, and various septicemic conditions [ ] . thus, laboratory diagnosis of csf for detection of the specific csfv antigen and antibody is indispensable [ , ] . the well-established diagnostic methods of csf such as virus isolation, fluorescent antibody test (fat), antigen capture antibody enzyme-linked immunosorbent assay (elisa), reverse-transcription polymerase chain reaction (rt-pcr), virus neutralization test (vnt), and antibody elisa (table ) have been widely used and well described in the oie terrestrial manual [ ] . recently developed techniques and alternatives have made significant improvements in several key components of csf diagnosis, including less sample and reagents required, less effort and time needed, increased detection efficiency (multiplexing), ease of performing and disposal, automation, and point of care (poc). this review provides an updated overview on laboratory diagnosis of csf and future perspectives. virus isolation in cell culture is the oldest laboratory technique for detecting csfv. porcine kidney cell lines (pk- and sk- ) are often used for isolation of csfv [ ] . however, the use of other porcine cells including swine primary cells (pulmonary alveolar macrophages and peripheral blood mononuclear cells) may enhance the chances of obtaining different csfvs with different growth characteristics. since csfv does not cause a cytopathic effect (cpe), the growth of csfv in the cells is usually visualized by using immunological technologies with fluorescent or horseradish peroxidase (hrf)-conjugated antibodies [ ] [ ] [ ] . the cell culture, virus propagation, and staining are labor intensive and time-consuming (weeks). in addition, skilled and experienced personnel and adequate facilities are needed for cell culture, handling csfvs and accurate interpretation of the cpe. these disadvantages make virus isolation less attractive for mass surveillance or rapid diagnosis. however, virus isolation is still considered the "gold standard" for confirming csf clinical cases and the only method for making virus collections (table ). fat is the commonly used staining method for csfv detection. it utilizes fluorescein isothiocyanate (fitc) labeled antibodies to detect csfv proteins in the slices of cryostat (frozen) tissues or fixed cells. anti-csfv gamma-globulins prepared from specific pathogen-free pigs are recommended to be used. these globulins can ensure that most variant csfvs will be captured. the differentiation of csfv from other pestiviruses in fat positive samples, especially bovine viral diarrhea virus (bvdv) and border disease virus (bdv), can be done using rt-pcr with genetic typing or virus isolation in cell culture with specific monoclonal antibody (mab) typing [ , ] . the main advantages of fat are that it is relatively easy and rapid to perform and allows direct visualization of the csfvs in stained tissues. therefore, it is useful for a first laboratory investigation in suspected clinical cases (table ) . several fitc conjugated anti-csfv antibodies (polyclonal or monoclonal) for fat are commercially available for research purposes, such as those from creative diagnostics, bioss inc., biorbyt llc, and so on. however, fat requires highly specialized equipment (i.e., fluorescent microscope) and immunohistochemical staining expertise. it is only recommended to be used in laboratories that have the expertise of performing this technique. the novel viewrna in situ hybridization method can detect csfv rna directly in infected cells [ ] . using rna in an in situ hybridization method and specific probes of csfv rna, the relative location of csfv rna can be visualized in pk cells. the sensitivity of this method was three to four orders of magnitude higher than that of fat. the specificity experiment showed that it was highly specific for csfv (sub-genotypes . , . , . , and . ) and without cross-reaction with other pestiviruses including bvdv, porcine parvovirus (ppv), porcine pseudorabies virus (prv), and porcine circovirus ii (pcv- ). this assay has the potential to be used for testing for csfv in cells. however, it remains to be determined whether this method can be used to detect csfv in swine tissues and it is still expensive and is not commercially available yet. antigen-capture elisa uses anti-csfv antibodies on an elisa plate to capture the csfv proteins [ ] . it has been developed for the rapid screening of large numbers of pigs with clinical suspicion of csfv infection [ , , [ ] [ ] [ ] . commercial antigen-capture elisa kits are available from several commercial vendors including idexx laboratories, thermo fisher scientific, median diagnostics, and so on. these kits are double-antibody-sandwich (das)-based elisa for detecting csfv e or e rns protein in serum, blood, plasma, or tissue extracts ( table ) . antigen-capture elisa is fast (provides results within h), easy to perform, and does not require specialized equipment. it can be applied at a herd level for confirmation of clinical cases or determining infection-free population status (table ) . however, its sensitivity and specificity are lower than most of the other diagnostics, especially the real-time rt-pcr. it is not recommended for testing individual animals and has been increasingly discouraged in recent years. real-time rt-pcr has now replaced the traditional rt-pcr and has become an essential tool in the routine diagnosis of csfv [ ] [ ] [ ] . it is a suitable approach for confirmation of clinical cases and prevalence of infection surveillance for csf (table ) . several commercial real-time rt-pcr kits are available for rapid and specific detection of csfv rna, including idexx realpcr csfv rna mix, virotype ® csfv rt-pcr kit, csfv dtec-rt-qpcr test, adiavet™ csf real time, csfv genesig ® advanced and standard kits, and so on (table ) . these kits use either sybr green or taqman probe to detect the accumulation of amplicon during the exponential phase of the reaction, which can specifically and sensitively test the csfv in serum, blood, plasma, viral culture, tissue, or swabs. the disadvantages of real-time rt-pcr are its high cost and complexity due to simultaneous thermal cycling and fluorescence detection, false positives caused by laboratory contamination from polluted specimens or equipment, and false negatives caused by pcr inhibitors in the sample or degraded rna [ ] . the improved real-time rt-pcrs and advanced alternatives have been designed to help resolve these issues. one-step and automated rt-pcrs can reduce the risk of contamination [ ] [ ] [ ] . the primer-probe energy transfer rt-pcr assay provides a higher specificity by analyzing the melting curve following pcr amplification [ , ] . the loop-mediated isothermal amplification (lamp) assay can accumulate the csfv amplicon under isothermal conditions [ ] [ ] [ ] . the functionalized gold nanoparticles were developed as nanoflare probes for rapid detection of csfv without nucleic acid amplification [ ] . multiplex real-time rt-pcr as a powerful technique has expanded exponentially in the diagnosis of csf in recent years. it is quite useful and convenient for quick and accurate detection of different pathogens in mixed infections, which is common in swine production systems. multiplex rt-pcr assays for rapid detection and genotyping of csfvs [ , ] , simultaneous detection, and differentiation of common swine viruses [ ] [ ] [ ] [ ] have been developed. additionally, multiplex combined high-throughput molecular diagnostic platform, user-friendly electronic microarray, magnetoelastic sensor, and microfluidic detection systems were developed as potential alternatives for detection and surveillance of csfv infection [ ] [ ] [ ] [ ] [ ] . these assays can save considerable time and effort without compromising robustness and sensitivity and can reduce the sample and reagent requirement as well. next generation sequencing (ngs) is a highly sensitive method for generating sequence data and exploring the genetic characters of infectious agents. it has been extensively applied to metagenomics and whole-genome sequencing of infectious viral diseases of livestock [ , ] . by using ngs, researchers found that there might be a long-term persistence of genotype . csfv strains in wild boar in germany [ ] . by analyzing ngs data of csfv isolates of varying virulence in infected pigs, higher quasispecies diversity and more nucleotide variability were found in viral samples from pigs infected with the highly virulent isolates compared to samples of pigs infected with low and moderately virulent isolates [ ] . evolutionary changes in virus populations following the challenge of naïve and vaccinated pigs with the highly virulent csfv strain were studied using the ngs technology and this study found that vaccination imposes a strong selective pressure on csf viruses that subsequently replicate within the vaccinated animals [ ] . the complete genome sequences obtained from ngs can provide detailed genetic information for construction of reliable phylogenetic relationships of csfvs for monitoring the evolution and transmission patterns during field outbreaks or epidemics of csf. one phylogenetic analysis using csfv complete genome sequences from different asian countries indicated that the circulating indian csfv strains belong to different branches of the . sub-genotype [ ] . these data combined those obtained from other different diagnostic tests can be used for meta-analysis of csf prevalence, which is important for the investigation of csf prevalence in different regions [ ] . currently, most of the ngs platforms are expensive to establish and require highly skilled molecular biologists and bioinformaticians. the implementation of ngs is still a challenge and cannot be used as a routine test for disease diagnosis due to cost and the time required [ , ] . however, with the novel and emerging sequencing technologies, cost-effective, user-friendly, and portable ngs will be developed and will act as an effective tool for csf control and prevention. vnt is the gold standard for sensitivity and specificity of antibody detection methods. it can be used for confirmation of clinical cases, prevalence of infection surveillance, evaluation of the immune status post-vaccination, and the efficacy of csf vaccines (table ) [ ] [ ] [ ] . however, vnt is a work-intensive and time-consuming procedure that requires cell culture and a high-containment laboratory that can handle infectious csf virus. in addition, it cannot be automated, thus it is not suitable for mass analysis of samples [ , , [ ] [ ] [ ] . more recently, alternatives have been developed to overcome the disadvantages of vnt. a neutralizing mab-based competitive elisa (celisa) with emphasis on the replacement of vnt for c-strain post-vaccination monitoring was developed in our group. the test principle of this celisa is that the neutralizing mab can compete with c-strain vaccine induced neutralizing antibodies in pig serum to bind the capture antigen c-strain e protein. the established celisa showed % sensitivity ( % confidence interval: . to %) and % specificity ( % confidence interval: to %) when testing c-strain vnt negative pig sera (n = ) and c-strain vnt positive pig sera (n = ) and showed excellent agreement (kappa = . ) with vnt when testing the pig sera (n = ) in parallel. the inhibition rate of serum samples in the celisa is highly correlated with their titers in vnt (r = . , p < . ). the c-strain antibody can be tested in pigs as early as days post vaccination with the celisa. this celisa is a reliable, rapid, simple, safe, and cost-effective tool for sero-monitoring of c-strain vaccination at a population level [ ] . in addition, another group developed a high-throughput vnt by using the recombinant csfv possessing a small report tag and luciferase system. as reported, the vnt titers of the serum can be determined tentatively at days post-infection (dpi) and are comparable to those obtained by conventional vnts at or dpi. this system allows csf virus growth to be easily and rapidly monitored and enabled the rapid and easy determination of the vnt titer using a luminometer, which could be a powerful tool to replace the conventional vnt as a high-throughput antibody test for csfv infections [ ] . antibody elisa is the quickest, easiest, and most widely used technique for serological diagnosis and epidemiological investigation of csf. it is suitable for herd or individual animal csfv infection screening, prevalence of infection surveillance, and immune status checking in individual animals or populations post-vaccination ( table ). the e protein is crucial for inducing an immune response in the host following csfv infection [ ] . detection of e antibodies in the serum of animals is an easy and reliable method for monitoring csfv infection during and after outbreaks and for testing coverage of immunization after vaccination [ ] [ ] [ ] . several commercial csf antibody elisa kits are available including those from biocheck, boehringer ingelheim, cusabio technology llc, idexx laboratories, id vet, indical bioscience, intron biotechnology, median diagnostics inc., thermo fisher scientific, and so on. most of the commercial kits are indirect, competitive, or blocking elisas based on the detection of envelop glycoprotein e specific antibodies ( table ). limitations of these antibody elisas are lower specificity (i.e., cross-reactions with bvdv, bdv, and other pestiviruses) and inability to discriminate animals vaccinated with conventional attenuated vaccines or e -based subunit vaccines. the rationale of genetic diva (differentiation of infected from vaccinated animals) is the identification of genetic differences between vaccine strains and wild-type csfvs. both traditional rt-pcr and real-time rt-pcr (single-plex or multiplex)-based csf genetic diva systems have been developed and evaluated [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . multiplex nested rt-pcr and real-time rt-pcr assays have been developed for differential detection of wild-type virus from c-strain vaccine [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a one-step rt-pcr using taqman minor-groove-binding (mgb) probes was developed to distinguish between attenuated korean lom and wild-type strains of csfv in korea [ ] . a simple rt-pcr based on the t-rich insertions in csfv genome was developed for rapid differentiation of wild-type and at least three attenuated lapinized vaccine strains [ ] . the modified genotype . (including c-strain) real-time rt-pcr assay with a real-time rt-pcr assay that detects all known csfv strains has been successfully used to distinguish c-strain vaccine from the circulating field strains that do not belong to genotype [ ] . the genetic diva approach facilitates a rapid and reliable differentiation of field virus infected from live attenuated virus vaccinated domestic pigs and wild boars. it is especially useful for detection of the infected animals that are incompletely protected by vaccination and will play a critical role for making decisions prior to and during cessation of a control strategy that employs vaccination with csf live vaccines. the ideal serological diva test has the ability to discriminate antibodies induced by csfv infection from the vaccine-derived antibodies, so it can rule out csfv infected pigs from vaccinated pigs [ ] . this can be obtained by detection of specific antibodies against antigens or epitopes that are modified or lacking in a subunit or marker vaccine. it has been shown that antibodies to e rns can be used as an indicator of csfv infection in pigs and the e rns -based elisa can be used as a companion diagnostic test to identify csfv-infected pigs vaccinated with the e -based subunit or marker vaccines [ ] [ ] [ ] [ ] . currently, two e rns elisas are commercially available and have been evaluated as accompanying diva diagnostic tools for e subunit vaccines, cp _e alf, or similar chimeric vaccines. one is priocheck csfv e rns (thermo fisher scientific, waltham, ma, usa); the other is pigtype csfv e rns ab (indical bioscience, gmbh, leipzig, germany) ( table ) . published data on their evaluations showed that priocheck csfv e rns has a sensitivity of - % with sera from csfv infected domestic pigs and a specificity of - % with sera from vaccinated domestic pigs [ ] . in combination with the marker vaccine "cp _e alf", pigtype csfv e rns ab has a sensitivity of . % and a specificity of . % [ ] . however, cross-reactivity with antibodies against other pestiviruses was observed for these two e rns elisas [ , ] . depending on the represented data, these two e rns elisas are recommended to be used on a herd basis and not for diagnostic analysis on samples of single animals. additional approaches or alternatives are undergoing development or further optimization. these include the multiplex microsphere immunoassay [ ] , which is capable of discrimination within epitope-specific antibody populations [ ] and the indirect e rns antibody elisa with pichia pastoris-expressed e rns [ ] . recently, our research group successfully generated a mab against e rns , which can specifically recognize c-strain, but not react with wild-type csfvs or other viruses in the genus pestivirus. a celisa was developed in our group based on the strategy that the c-strain-specific mab will compete with the c-strain vaccine-induced antibodies in pig serum to bind the capture antigen (c-strain e rns ) [unpublished data]. different from the csfv neutralizing monoclonal anti-e antibody based celisa for sero-monitoring of c-strain vaccination at a population level [ ] , this novel anti-e rns mab-based celisa is a valuable tool for measuring and differentiating immune responses to c-strain vaccination and/or infection in pigs. the data about the establishment and validation of this c-strain specific celisa will be published separately at a later date. in brief, suitable tools for serological diva are available. however, there is room for improvement, especially with respect to cross-reactivity issues. user-friendly, cost-effective, rapid, and reliable poc diagnostics (i.e., diagnosis of diseases directly on-site) are indispensable tools for immediate decisions of effective and evidence-based disease control strategies [ ] . for example, dipstick tests are designed to use thin paper/plastic strips coated with specific antiviral antibodies to detect viral antigens in serum and other body fluids. lateral flow assays (lfa) and microfluidic devices are two different and yet more complex technologies that are also based on the biochemical interaction of antigen-antibody. the principles for these three immunochromatographic assays are the same as sandwich elisa and the major difference between them is that the immunological reaction is carried out on different platforms for different assays. poc studies in animal health management are rare compared to human and companion animal medicine. the immunochromatographic assay-based kits including antigen rapid test kit (ring biotechnology co., ltd., beijing, china), lilif™ csfv antibody rapid test kit (intron biotechnology, inc., gyeonggi, south korea), and csfv antibodies rapid test kit (antibodies-online inc., limerick, pa, usa) are commercially available for rapid testing of csfv antigen or antibodies in the field. laboratory-based assays, including the loop-mediated isothermal amplification, combined with a lateral flow dipstick assay [ ] , the immunochromatographic strip [ ] , and duplex lateral flow assay [ ] have been investigated as potential csf poc tools as well. poc diagnostics showed advantages in rapidity and portability, which are the most important parameters considered by farmers and veterinarians [ ] . it is foreseeable that as interests and needs of stakeholders increase and new portable poc technologies emerge, novel and applicable poc diagnostics will be developed for detection, control, and prevention of csf in the field in the near future. although commercial and in-house diagnostics (antigen detection and antibody detection) of csf are available, there is still room for improvement. the authors suggest that the following aspects should be considered: (i) continuously improving the sensitivity, specificity, costs, speed, automation, and poc is necessary; (ii) reference materials (serum bank, virus bank, and non-infectious molecular standards) should be produced and be accessible for validation of the developed csf diagnostics; (iii) the development of diva diagnostics without cross-reaction with antibodies induced by other pestiviruses is critical. the other emerging infectious diseases, such as the spreading of african swine fever in asia and the ongoing pandemic of coronavirus disease (covid- ), may shift focus away from the csf [ , ] . however, as long as csf exists, it will remain a continuous threat to the pig industry worldwide. therefore, international cooperation on surveillance and control of csf becomes even more crucial, both currently and in the future. researchers should continue to work on developing novel rapid and reliable diagnostics to facilitate the surveillance and control of csf. usda ars non-assistance cooperative agreements, grant numbers [ - - - , - - - , - - - , - - - ]; national pork board grant, grant number . the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors 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technology for detection of swine viral diseases african swine fever the outbreak of coronavirus disease (covid- )-an emerging global health threat this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -j rlgbl authors: powell, joshua d.; waters, katrina m. title: influenza-omics and the host response: recent advances and future prospects date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: j rlgbl influenza a viruses (iav) continually evolve and have the capacity to cause global pandemics. because iav represents an ongoing threat, identifying novel therapies and host innate immune factors that contribute to iav pathogenesis is of considerable interest. this review summarizes the relevant literature as it relates to global host responses to influenza infection at both the proteome and transcriptome level. the various-omics infection systems that include but are not limited to ferrets, mice, pigs, and even the controlled infection of humans are reviewed. discussion focuses on recent advances, remaining challenges, and knowledge gaps as it relates to influenza-omics infection outcomes. "omics" data refers the large-scale acquisition of specific "features" in a biological sample, including genes (genomics), gene expression in the form of mrna (transcriptomics), or protein expression (proteomics) that may include specific post-translational modification events (phosphoproteins, glycosylation, etc.). applied to studies of pathogens, -omics analyses can identify hundreds to thousands of signature features that characterize temporal microbial and host response events during the course of infection. features of interest are dependent on the experimental system and hypothesis, but -omics studies often interrogate the expression level of rna or protein for a target of interest in the infected sample versus an uninfected reference control. after data is acquired, bioinformaticists must make sense of these microbial and/or host changes to identify infection-related pathways. additionally, promising novel host gene antiviral candidates that are impacted by the infection process may also be of interest. finally, it may be of interest to elucidate how genetic variants in either the host or the pathogen itself may impact immune responses. as next-generation sequencing (ngs) technologies such as shotgun transcriptomic rna sequencing (rna-seq) replace more conventional microarray analysis, and proteomic approach platforms become more sensitive, quantitative assessment should also become more reliable. in summary, -omics-driven experiments represent a very powerful tool to study the host response to infection, but data interpretation is critical to ensure that meaningful conclusions are reached. this review focuses on the numerous influenza a virus (iav) omics-related studies of host responses that have been published over the last two decades. while vaccination strategies exist to combat the seasonal spread of iav, these viruses can mutate and undergo genetic reassortment that can evade vaccines and other therapeutics. of particular concern is that not just humans, but wild and domesticated birds can spread iav, including recent highly pathogenic bird flu isolates of h and h designation that cause high mortality rates in infected humans. additionally, iav is also endemic in pigs, with the h n global pandemic caused from a swine-to-human spillover event. monitoring multiple reservoirs of iav for zoonotic spillover events into humans therefore requires the characterization of iav isolates on a continual basis and to define host responses-including -omics approaches-in multiple species. as iav strains evolve and sequence information from the iav cannot currently determine pathogenicity, -omics platforms can be a powerful tool to compare emergent iav strain data with previously circulating strains for the purpose of threat characterization. in the last ten years, mass spectrometry instrumentation and methodology advancements have led to considerable knowledge of the host proteomic response to viral infection (for a recent review, see [ ] ). the increasing popularity of ngs technologies such as rna-seq, in combination with further advances in microarray profiling technology has also increased our transcriptomic-rna understanding of virus-host interactions. as -omics capabilities become cheaper and the software to interpret data becomes more user-friendly, the number of laboratories that can perform these dataintensive studies will likely increase. the purpose of this review is to assess both proteomic and transcriptomic findings in the context of iav infection. in the following sections, recent advances and future prospects are discussed. figure summarizes notable published -omics experiments, many of which are discussed in the proceeding sections. a number of published data sets examining host-iav interactions and subsequent meta-analysis are summarized in figure . numerous -omics influenza studies have been funded by the national institute of allergy and infectious diseases (niaid) systems biology for infectious diseases research program [ ] . niaid-related data sets from these virus studies are publically accessible through the influenza research database (www.fludb.org) [ ] . additionally, the national center for bioinformatics (ncbi) gene expression omnibus (geo) serves as a public repository for various high-throughput -omics experimental data [ ] . these data include microarray-based experiments measuring mrna, genomic dna, and protein abundance. geo also provided a repository for non- a number of published data sets examining host-iav interactions and subsequent meta-analysis are summarized in figure . numerous -omics influenza studies have been funded by the national institute of allergy and infectious diseases (niaid) systems biology for infectious diseases research program [ ] . niaid-related data sets from these virus studies are publically accessible through the influenza research database (www.fludb.org) [ ] . additionally, the national center for bioinformatics (ncbi) gene expression omnibus (geo) serves as a public repository for various high-throughput -omics experimental data [ ] . these data include microarray-based experiments measuring mrna, genomic dna, and protein abundance. geo also provided a repository for non-array techniques such as serial analysis of gene expression (sage) and mass spectrometry proteomic data. collectively, geo allows researchers from all over the world access to data for analysis on various software programming packages [ ] . swine influenza viruses (siv) have the zoonotic potential to cause global pandemics in humans, such as the h n pandemic (ph n ) that spread across the globe [ ] . swine are considered a mixing vessel for novel influenza reassortment, as pigs have the capacity to support influenza strains of human, swine, and avian origin. while swine influenza is a public health concern, compared to humans, we have a limited understanding as to swine host responses to influenza infection at the -omics level [ , ] . in , ma and co-workers [ ] performed a whole-genome analysis using swine-specific microarrays to compare transcriptomic pig lung responses of human origin ph n to that of either swine origin ph n and a classical siv strain (ia ). ph n human and pig viruses caused clinical symptoms and upregulation of genes related to immune and inflammatory responses at day post-infection (pi) that were not evident in the classical siv strain. at day pi, expression of cell death and lipid metabolism genes substantially differed in ph n -infected pigs versus those of ia infection. building on their earlier work, in michael katze's lab also compared their human swine microarray ph n (ca / strain) data to mice and macaque lung responses after infection with the same virus [ ] . the authors noted similar clinical courses of infection progression, but they also found significant differences between host species in lipid metabolism and inflammatory gene responses. in particular, ca / virus significantly altered the expression of cholesterol homeostasis genes (lxr/rxr) in swine and mice, and vitamin d receptor genes in macaques. in total, genes were found to be differentially regulated between species. notable swine -omics studies include lin and co-workers' [ ] assessment of swine h n iav and streptococcus suis serotype (ss ) co-infection through microarray analysis. these authors found that co-infected pigs had altered interferon and interleukin receptor expression, as well as differential expression for several chemokine and chemokine receptor genes, along with the differential expression of five tumor necrosis factor (tnf) receptor superfamily genes versus either iav or ss infection alone. li and co-workers [ ] using a/swine/hubei/ / (h n ) also studied influenza infection in swine lungs at day and day pi through microarray. these studies confirmed that toll-like receptor genes (tlrs) and interferon-induced genes (isgs)-which are involved in anti-viral signaling-were heavily impacted after infection. additionally, wilkinson and colleagues [ ] also used microarray analysis to compare infection outcomes in low vs. high birth weight litters with swine h n (tx ) that resulted in differentially expressed genes including il , il , and ccl . these authors also characterized transcriptomic responses based on disease severity in pigs. lastly, while there are minimal large-scale in vivo proteomic experiments, zhu and co-workers [ ] have experimentally infected porcine macrophages with two different swine h n strains. using d-gel electrophoresis and maldi-tof ms/ms, the authors identified up-regulated and down-regulated protein spots associated with molecular biosynthesis and heat shock proteins. considering the global concern of iav circulation in pigs, additional -omics data sets should prove useful to help elucidate potential host factors that are impacted by infection and to identify those iav circulating strains that could pose a threat to human health. numerous avian iav subtypes circulate in domestic and wild birds, with occasional spillover into humans. of particular concern are h n and h n that have been transmitted from poultry to humans in asia, resulting in infections with high mortality rates [ ] . using a chicken k agilent microarray, wang and colleagues studied h n lung infection in broiler chickens and found differentially expressed mrnas compared to mock-infected chickens [ ] . building on these earlier studies, the same group in used a next-generation sequencing rna-seq platform approach with the same h n virus to look at infection in two inbred chicken lines-one resistant to and one susceptible to iav infection. the authors noted that their bioinformatics data analysis inferred that the hemoglobin gene family and cell adhesion molecule signaling pathways play prominent roles in iav disease resistance in chickens. a recent microarray study comparing highly-pathogenic h n versus low-pathogenic h n in the chicken lung provided valuable insight into inflammatory/cytokine host gene response differences related to infection outcomes [ ] . in particular, the authors found differentially expressed host genes after h n infection and host genes after h n infection at days pi, respectively. of note, a study took a similar approach using a nimblegen chicken genome array to analyze , host genes in response to low and high pathogenic recombinant iav infection in chickens [ ] . the authors wanted to assess genes related to survivability, and deduced that cd , rnf b, oasl, zc hav , pla g , gch , and usp were important factors for regulating iav severity. finally, smith and co-workers have explored the transcriptomic sequencing differences between ducks and chickens in high (h n ) and low (h n ) pathogenic iav [ ] . the authors note that unlike ducks, chickens lack the retinoic acid-inducible gene i (rig-i) virus sensor gene, which partially explains severity differences after iav infection between species. additionally, the authors note that differences in the expression of the interferon-induced transmembrane protein (ifitm) family may restrict infection in ducks to a greater extent than in chickens. similar to swine studies, there is also limited proteomic analysis data available for avian species, but sun and co-workers have identified proteins using d-dige (differential gel electrophoresis) followed by maldi-tof/tof-ms in the trachea of iav-infected chickens [ ] . the authors found that two annexin proteins (anxa , anxa ), and a heat shock protein (hspb ) were differentially expressed in infected chickens. of note, zhou and co-workers [ ] , using a similar approach identified up-regulated proteins and down-regulated proteins in the brains of h n infected chickens, as this highly pathogenic virus is known to cause neurovirulence. the most notable protein changes that authors found occurred within cytoskeleton and ubiquitin-proteasome pathway related proteins. ferrets are considered to be an ideal small animal model for iav infection, as they have similar sialic acid characteristics for iav binding to that of humans [ ] . additionally, ferrets are ideal for iav in vivo studies, as they exhibit clinical symptoms of infection and can transmit iav through sneezing [ ] . while the ferret model is used for pathogenesis, transmission, and virulence studies, the host response at the molecular level has only resulted in a handful of publications. last year, tisoncik-go and co-workers [ ] used an integrated high-throughput -omics approach to study the difference between the ph n ca / strain and the highly virulent -h n virus in infected ferrets. in particular, the authors focused on the lipids, metabolites, and proteins in respiratory compartments for highly virulent versus less virulent outcomes in ferret lungs. these findings identified unique lipids in the lung and lipids in the trachea, along with lc/ms analysis identifying proteins in the lung and proteins in the trachea of infected ferrets. key infection findings included significant abundance changes in phospholipids (diacylglycerophosphocholine and diacylglycerophosphoethanolamine species) known to be major constituents of pulmonary surfactant and phospholipid precursors that can be cleaved to form arachidonic acid that would impact iav pathogenesis. additional studies have also characterized the influenza "infectome" through ngs and microarray analysis of ferret lung and lymphoid tissue with h n (a/mexico/ / ) [ ] . this study identified genes that were significantly upregulated, and genes that were downregulated. interferon-stimulated genes were markedly upregulated, including antiviral responses genes cxcl , oas , irf , and rsad , as well as various cytokine and chemokine genes related to immune cell recruitment. ferret-specific microarrays were also used in a study to assess distinct infection profiles for three differing h n influenza strains from the blood of ferrets [ ] . in particular, authors identified a gene expression profile consisting of probes that could classify samples based on both strain and severity of disease. finally, in one of the earlier ferret -omics studies by camp et al. [ ] , the authors identified more than , partial ferret transcripts, including more than gene orthologs known to be involved in the innate and the adaptive immune response through de-novo transcriptome sequencing. the macaque model (rhesus and cynomolgus) for iav infection has resulted in the generation of insightful -omics data sets. a review summarized the historical significance of using the nonhuman primate model for iav infection [ ] . one of the earliest macaque -omics studies in compared uninfected versus infected monkeys using h n a/texas/ / (tx ) [ ] . in this study, microarray profiling for approximately , genes was assessed, and virus-related interferon and cytokine responses were found to be sustained throughout the course of the infection in both lung and blood samples. in this same study, proteomic lc-ms-ms was also assessed from monkey lungs and blood, resulting in a total of , peptides and proteins that were collectively identified. proteins related to infection included several well-known interferon-induced proteins relevant to neutrophil and monocyte/macrophage function. in , a proteomics study compared highly pathogenic h n and -h n versus the less virulent tx seasonal influenza strain [ ] . over , peptides representing proteins were identified, with increased proteins and decreased proteins associated with viral infection. classical protein responders of viral infection included mx , mda , oas , isg , and rig-i. in , cillóniz and co-workers [ ] used whole-genome microarray analysis to compare highly pathogenic avian h n to the pathogenic -h n virus. higher tissue pathology was evident in -h n infected macaques, which demonstrated upregulation of key components of the inflammasome, including nlrp and il- beta. interestingly, these genes were downregulated in animals infected with the h n virus, which resulted in a less severe infection compared to the -h n virus. a study compared ph n ca / infection in aged versus young macaques [ ] . aged animals had higher viral titers and increased cytokine expression in bronchoalveolar lavage (bal) fluid through use of a multiplex elisa. follow up whole-genome microarray experiments-which found genes differentially expressed between young adult and aged macaques-confirmed increased inflammatory gene expression in aged animals as well as the enrichment of protective genes associated with naïve t or b cells in younger animals. mcdermott and co-workers have comparatively assessed h n transcriptomic infection outcomes in calu- human cells, mouse lung, and the macaque lung (discussed further in section . ) [ ] . additionally (and as mentioned earlier), swine, mouse, and macaque lung infections with ph n have also been comparatively assessed at the transcriptome level [ ] . finally, a proteomic approach was used for ph n -infected cynomolgus macaques that identified over differentially expressed proteins in the bal versus uninfected controls [ ] . these authors found that there was a significant increase at day relative to day pi in abundance of interleukins (ils) including il b, il , il , il , il , il , and il , as well as proteins associated with the interferon response versus uninfected monkeys. extensive -omics analysis has been investigated using the mouse model for influenza infection. additionally, increased and earlier expression of cytokine and chemokine-associated genes in mouse lungs infected with -h n was evident through transcriptomic-microarray analysis. in the same lab performed additional transcriptomic profiling and narrowed down the pathogenicity determinant to the pb segment for - h n [ ] . extensive transcriptomics analysis has also been performed comparing pathogenicity differences between h n and h n - in mouse lungs [ ] . the authors found that unlike h n - , h n disseminated to extrapulmonary organs with upregulation of inflammasome genes more so than h n - . in addition, the authors note that h n also upregulated the expression of tumor necrosis factor alpha (tnf-α)-a potent inflammatory molecule; ifn-γ; eukaryotic initiation factor ak (eif ak ); protein kinase rna activated (pkr); and additional chemokines and inflammation-related genes. other notable mouse transcriptomics studies include those of zou and co-workers [ ] , who used an ngs rna-seq approach to assess ph n versus swine origin iav outcomes in mouse lungs. interestingly, the authors found that the swine-origin virus caused higher mouse morbidity with numerous cytokines and interferon-stimulated genes having higher expression level in swine h n infected groups. an additional rna-seq study assessed highly pathogenic h n versus h n in mouse lungs, and the authors found higher pathogen-related transcripts for h n -infected mice at day and [ ] . the authors noted that inflammation pathways were elevated in h n -infected mice versus h n , which was consistent with higher observed viral titres and virulence. finally, morrison and co-workers [ ] used microarray analysis and studied outcomes in the balb/c mouse strain with ph n and three avian strains (h n , h n , and h n ). avian viruses were more pathogenic than ph n , with host gene signatures reminiscent of -h n . coagulation genes and lxr/rxr pathway genes were highly upregulated by ph n , but not by the avian viruses; the authors state that this finding suggests that the avian viruses were potentially suppressing gene expression in these two pathways. of note, the authors also assessed the host responses that could be targeted using fda-approved drugs that could potentially be repurposed to treat h n infection using six potential therapeutics. while mouse models of iav infection can be insightful, not all mouse strains are equally susceptible to infection. in , alberts and co-workers [ ] infected a resistant strain (c bl/ j) and a highly susceptible mouse strain (dba/ j) with h n -pr and performed genome-wide microarray analysis. the magnitude of immune response genes was significantly higher in dba/ j compared to c bl/ j, along with several immune response genes exclusively regulated in dba/ j. the authors also noted a region of chromosome that was significantly different in the expression for both strains. a similar study comparing two strains with disparate iav susceptibility, but using ngs rna-seq, found the irf gene to be a virulence determinant [ ] . of interest, researchers at the university of north carolina have generated a panel of genetically diverse mouse strains referred to as the collaborative cross (cc) (for review see [ ] ) and compared them to pre-cross strains for h n -pr infection outcomes [ ] . by conducting quantitative trait loci (qtl) mapping in combination with transciptomic-microarray profiling, the authors identified a novel allele within the anti-influenza mx gene that was a major determinant of influenza pathogenicity. proteomic approaches to assessing influenza infection in mice are limited compared to transcriptomics, however there is recent progress that may reflect increased capabilities in this field and indicates the potential for future work. last year, hu and co-workers [ ] compared a high versus low pathogenic h n strain in mouse lungs using isobaric tags for relative and absolute quantitation (itraq) coupled lc-ms/ms method. the highly-pathogenic h n strain up-regulated proteins corresponding to inflammatory response, cell death, reactive oxygen species production, and complement response more so that the low-pathogenic h n strain. another study from last year identified over proteins collectively from days , , and pi with h n a/hong kong/x , and found decreased levels of several cell junction proteins but increased levels of tissue metalloproteinase mmp [ ] . in a separate analysis, zhao and co-workers [ ] performed proteomics on d differential gel electrophoresis (dige) protein spots for h n -infected mouse lung tissues using a lethal versus a non-lethal strain. of the differentially regulated proteins for infected versus uninfected controls, were different between highly pathogenic h n versus low pathogenic h n . of note, five virus-related proteins (tgtp, ifit , ifit -l, lcp - , and lcp - ) were upregulated as soon as day pi in the lethal virus-inoculated group, but did not show appreciable upregulation until day pi in the non-lethal group. their results suggest that proteomic approaches can distinguish the lethality of the influenza strain in a mouse model within h of infection. human influenza trials and the accompanying -omics data sets have been documented, but in a rather limited manner. with human trials, there are obvious ethical restrictions to using high pathogenicity influenza strains to infect volunteers, and therefore only safer seasonal iav strains are permitted. furthermore, significant harm to the trial participant has to be avoided, therefore preventing invasive biopsies with usually only serum blood draws and nasal secretion collection permitted. nevertheless, these studies often enable valuable protracted longitudinal sampling on the same patient. building upon previous microarray data sets [ , ] , wang and co-workers [ ] applied a multivariate estimation technique (mset) for h n and h n human infection. the authors were able to monitor the acute phase of infection and quantify the predicted health outcome of infection from the blood of patients. this technique could prove useful, as it could potentially diagnose influenza severity before the patient shows signs of sickness. besides clinical trials, surveillance of active infection has proved invaluable for acquiring -omics data. for example, in - , researchers enrolled healthy adults in a study where the volunteers self-reported and blood draws were performed within h of fever onset. this was followed up with additional sample acquisition at day , , , and . in total, influenza a, influenza b, rhinovirus, and patients with fever but no viral agent were analyzed. collectively, these data sets provide a wealth of information to understand the systemic response to naturally acquired respiratory infection through the analysis of acute versus recovery gene expression [ ] . an additional study included a novel proteomics technology referred to as somascan to quantify different human proteins from the human mucosa (nasal lavages) of influenza patients for the purpose of using this technology as a biomarker tool [ ] . the authors noted that of the differentially expressed proteins between infected versus uninfected patients, many were associated with the recruitment and activation of memory t cells, most notably cxcl , cxcl , and il- . human tissue culture cell lines provide a convenient in vitro means to study host responses to influenza infection. while cell lines cannot recapitulate some clinical aspects of natural infection, they can still be informative. as discussed earlier, comparisons between human cells, mice, and non-human primates do show conserved innate host responses to highly-pathogenic h n [ ] . due to convenience and cost, numerous studies at the -omics level have therefore implemented a cell line approach. another benefit of cell line use is the potential for consistency between experiments and even labs when obtaining -omics data. for example, certain human lung epithelial cell lines such as calu- and a that are widely available allow researchers to compare their new findings to prior data sets, as an immortalized cell line is derived from a single donor. the benefits of cell lines, as well as some of the drawbacks of their use are saved for the discussion section. in , the katze lab at the university of washington studied the transcriptomic responses of human calu- cells infected with either avian-origin h n , h n , h n , or human seasonal h n [ ] . the h n ahuni/ / strain was found to more closely resemble the seasonal h n in transcriptomic responses compared to the other two avian strains. this finding suggests that the h n has at least been partially adapted to humans. importantly, using genome-based drug repurposing approaches, with the ingenuity knowledge database and the connectivity map (cmap) data driven tool, the authors found that already-fda-approved kinase-inhibitors could potentially be repurposed as novel drug targets based on -omics data. this analysis also suggested that sb- and genistein may revert the host response to all four iavs tested. these studies infer the potential for critical pathways for iav adaptation that could be targeted using therapeutics. of note, microarray findings from this study were in agreement with a separate calu- cell infection study that found similar transciptomic profiles for h n and a human h n virus compared to two other avian strains, suggesting human adaptation for h n [ ] . these authors also noted more efficient replication and viral titers for this partially adapted h n bird flu strain at the human temperature of infection ( • c) versus the avian favorable infecting • c temperature. proteomic approaches have also been used on immortalized lung cell lines. coombs and co-workers [ ] have used a stable isotope labeling amino acid (silac) approach in cell culture and high throughput -d hplc mass spectrometry to look at uninfected and infected a cells with the widely used a/puerto rico/ / h n strain. of the proteins identified, were either significantly up-or down-regulated. gene ontology pathway analysis indicated numerous functional profiles that were impacted, including host cell immunity, cell adhesion, metabolism, and signal transduction. the authors noted that key proteins upregulated after infection included mx , ltf, and vim, while those that were downregulated included erc , l cam, and ctnnb . infiltrating cells in the lung (e.g., macrophages and monocytes) can also have unique signatures after iav infection. in a -omics study by lee and co-workers [ ] , macrophages infected with high pathogenic h n versus less pathogenic h n led to stronger type i interferon and tnf-alpha expression. a recent proteomics study also with infected macrophages revealed that iav can cause robust secretions of proteins, including those associated with antiviral cytokines, copper metabolism murr- domain proteins, and autophagy-related proteins [ ] . lastly, in , a proteome and secretome study using cell fractionation and a -plex itraq approach showed dramatic changes in mitochondrial and nuclear proteomes in response to udorn/ and beijing/ / h n infection [ ] . in particular, the authors found cytoplasmic leakage of lysosomal proteins, including cathepsins, results in inflammasome activation, and apoptosis of the macrophage cell. in summary, macrophage studies are informative, as similar to the lung epithelium, macrophages can provide a unique biomarker platform for measuring influenza infection severity. in the discussion section, additional cell types and ways to provide more relevant cell culture platforms to study influenza -omics are discussed. in order for -omics data to be meaningful, the study authors must relate their data sets to that of the underlying biology. various software packages that run within r or python programming languages are currently used for analysis. the stand-alone ingenuity pathway analysis (ipa) platform available through qiagen is the most popular -omics software approach for elucidating the upstream biological causes and probable downstream effects for gene targets of interest [ ] . ipa software has been cited in thousands of articles for the analysis, integration, and interpretation of data derived from proteomic and transcriptomic experiments. while software packages such as ipa can help a researcher interpret their own data, there is still a level of ambiguity in comparing influenza -omics data from two or more different studies. the particular iav strain, time points chosen, and the starting infection dose-referred to as multiplicity of infection (moi)-are all major considerations when comparing results from one study to those of another. for in vivo studies, additional considerations such as the age of the animal, particular animal strain, volume of virus inoculum, and cell/tissue collection method also impact the -omics findings. unfortunately, while determining what signifies a statistically upregulated or downregulated rna or protein is straightforward, study design differences that reflect an -omics "snapshot" of slightly differing phenotypes and methodologies may lead to differing conclusions. as reviewed in [ ] [ ] [ ] , lethal iav infection leads to cytokine dysregulation and upregulation of inflammatory host genes during virus spread. however, comparing severe versus mild infection does not lead to wide-scale differences in cytokine/chemokine gene induction, but rather the magnitude of the response. for example, when mice were infected with h n iavs of different virulence, the host responses differed primarily in magnitude and velocity, rather than if a host gene was activated or not [ ] . while there is no magic bullet gene at one time point to tell you how virulent an iav strain may be, a defined series of influenza-specific signature genes can provide a comprehensive view into iav pathogenicity. for instance, a meta-analysis from studies comparing iav strains as well as sars coronavirus allowed chang and co-workers [ ] to show a relationship between the expression of chemokine subnetwork genes and influenza severity in mice. this type of meta-analysis is promising so future researchers can hopefully analyze a handful of genes to deduce the pathogenicity of an emerging iav strain. as prior virus systems biology reviews have noted [ ] [ ] [ ] , -omics approaches often seek to define virus-host immune responses to better design therapeutics such as antivirals and better vaccine strategies. the field of virus -omics is an evolving discipline that has increased in complexity and popularity over the last decade. comprehensive proteomics 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antiviral innate immunity through the lens of systems biology systems-biology approaches to discover anti-viral effectors of the human innate immune response acknowledgments: this study was funded by the national institute of allergy and infectious diseases (niaid) through a pilot project supplement to grant u ai . pacific northwest national laboratory (pnnl) is a multi-program national laboratory operated by battelle for the doe under contract de-ac - rlo . the authors declare no conflict of interest. key: cord- -xnu pj authors: ahmed, mohammed a. m.; siewe fodjo, joseph nelson; gele, abdi a.; farah, abdiqani a.; osman, shariff; guled, ibraahim abdullahi; ali, abdiaziz mohamed; colebunders, robert title: covid- in somalia: adherence to preventive measures and evolution of the disease burden date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: xnu pj following the covid- outbreak in somalia, strict preventive measures were implemented by the government. we assessed adherence to the government recommendations via two consecutive online cross-sectional surveys between april and july . a five-point adherence score was constructed based on self-reported observance of five preventive measures (physical distancing, face mask use, hand hygiene, mouth covering when coughing/sneezing, and avoidance of touching the face). and responses were analyzed during the first and second survey, respectively. the mean adherence score decreased from . ± . in the first survey to . ± . during the second survey; p < . . more participants experienced at least one flu-like symptom during the second survey ( . %) compared to the first ( . %); however, the proportion of positive covid- tests in the first ( . %) and second survey ( . %) was similar. the ordinal logistic regression model identified the following predictors for high adherence scores: female gender (odds ratio (or) = . ( . – . ), p < . ); being a healthcare worker/student (or = . ( . – . ), p < . ); obtaining covid- information from official sources (or = . ( . – . ), p < . ); and having postgraduate education (or = . ( . – . ), p < . ). conversely, obtaining covid- information from social media and residing in urban settings were associated with lower adherence. targeted and context-specific adaptations of the covid- response may be required in somalia. the novel coronavirus (covid- ) disease outbreak has spread globally during the past months, posing significant public health challenges in virtually all countries. resource-poor settings with the study was carried out in somalia (east africa). two consecutive cross-sectional surveys investigating the adherence to covid- preventive measures were conducted. the first took place from april to may, whereas the second was held from to july . the duration of the second survey was slightly longer because it took more time to recruit as many respondents as during the first survey. this study was part of a network of online surveys organized in several low and middle-income countries by the international citizen project on covid- (icpcovid) [ ] . the icpcovid general online questionnaire was adapted to the somali context by local investigators and was disseminated via social media platforms (in english and somali) to consecutively recruit adult participants (age ≥ years) via a snowball approach. all responses were submitted anonymously directly to the icpcovid platform, mainly using smartphones but also via computers. submitted responses were stored in the secure icpcovid website until data extraction. in addition to socio-demographic information and self-reported adherence to various preventive measures, the questionnaire also included questions about the presence or absence of flu-like symptoms experienced by the respondents. participants were asked whether they had been tested for covid- , and for the results of the test if available. a copy of the full questionnaire used is available as a supplementary material (file s , appendix ). adherence to the government's restrictive instructions for covid- control were assessed using individual and community parameters, configured either as yes/no questions or as likert scales in the questionnaire. a composite adherence score was made based on the respondent's self-reported observance of the following personal preventive measures: physical distancing, face mask use, hand hygiene, coughing hygiene, and the habit of touching one's face (table ) . descriptive statistics are presented using means with standard deviation (sd) for continuous outcomes, and percentages (%) for categorical variables. pearson's chi-squared test was used to investigate associations between two categorical variables, and to compare proportions across the surveys. continuous variables were assessed for normality using the shapiro-wilk test, and compared across groups using parametric tests (t-test, anova) or non-parametric tests (mann-whitney u, kruskal-wallis) as appropriate. both the who clinical definition (fever with at least one respiratory symptom) [ ] and a recently proposed case definition (fever and/or anosmia, with at least one other respiratory symptom) [ ] were used to estimate the covid- burden in both surveys. for multivariable analysis, we constructed an ordinal logistic regression model to investigate factors associated with adherence to national preventive measures against covid- , using the adherence score as the dependent variable. mandatory covariates in the model included socio-demographic features (age, gender, profession, educational level, urban vs. rural residence), and survey timing (first vs. second survey). non-likert covariates which showed a significant association with the dependent variable in univariate analysis were also included in the model. the final model, selected based on the least akaike information criterion (aic) value, was obtained after a backward stepwise process (supplementary file s , appendix ). the significance level adopted was %, and all tests were two-sided. data analysis was performed using the software r, version . . . the study protocol was approved by the university of antwerp ethics committee (ref: / / ) and the mogadishu university institutional review board. informed e-consent (checkbox) was required from each participant before submitting responses. all responses were anonymous and securely stored in a password-protected server in belgium. after data cleaning, over participants were retained in each survey round. their comparative characteristics are summarized in table . a majority of respondents during the second survey ( / , . %) acknowledged participating in the first survey as well. during both surveys, the study population was predominantly male, and most respondents had attained a university level of education. based on data from the second survey only, respondents were geographically distributed across the regions of somalia as follows: ( . %) in benadir; ( . %) in galmudug; ( . %) in hirshabelle; ( . %) in south west; ( . %) in somaliland; ( . %) in jubaland; and ( . %) in puntland. during the covid- pandemic in somalia, the participants reported low to moderate levels of worry/fear about their own health, with mean likert scores on a five-point scale of . ± . and . ± . during the first and second survey, respectively (p < . ). a similar trend was observed regarding their level of worry/fear vis-à-vis the health of their loved ones (mean likert scores on a five-point scale of . ± . during the first survey, and . ± . during the second survey; p < . ). considering the individual covid- preventive measures, the adherence rates ranged from . % for mask use to . % for coughing hygiene ( table ). the mean adherence score decreased from . ± . in the first survey to . ± . during the second survey; p < . . combining available data from both surveys and comparing work habits in the private and public sectors during the covid- pandemic in somalia, we observed that / ( . %) of private employees and / ( . %) of government employees were working from home (p < . ). comparing covid- preventive behaviours across genders revealed that women observed the safety recommendations more frequently than men (see supplementary file s , appendix ). while some respondents reported using face masks all the time when they went out ( . % in survey and . % in survey ), others used masks only sometimes ( . % in survey and . % in survey ), and a minority wore masks at home ( . % in survey , . % in survey ). about one quarter of the participants reported wearing reusable cloth (fabric) masks: . % in the first survey, and . % in the second survey. among the participants who reported not using face masks during our surveys, the following reasons were given: no money to buy face masks in cases ( . %); not knowing where to get the face masks for respondents ( . %); the fact that face masks make them uncomfortable, as reported by ( . %); whereas another ( . %) thought that face masks are not necessary during the covid- epidemic. the mean likert score assessing the difficulty of observing lockdown measures increased from . ± . to . ± . in the first and second surveys, respectively (p < . ). in both surveys, the level of difficulty of staying at home, as required for lockdown purposes, was significantly different across genders, residential setting, and professional status; p < . (table ). overall, ( . %) participants in survey and ( . %) participants in survey reported experiencing at least one flu-like symptom during the two weeks preceding the survey; p < . . headache was the most frequent symptom in both surveys (table ). applying the who's clinical definition [ ] and a recently proposed case definition [ ] for covid- screening (with no consideration of previous contacts with an infected person) revealed a prevalence of suspected covid- cases ranging from . %- . % in the first survey, and . %- . % in the second survey (table ). during the second survey, mean adherence scores varied significantly across geographical regions (p < . ), ranging from . to . . a multivariable model investigating factors associated with the adherence score to covid- preventive measures found that being a female, healthcare worker/student, and obtaining covid- information from official sources significantly increased the odds for higher adherence (table ). this study shows an overall unsatisfactory level of adherence by somali residents to the preventive measures put in place by the government to control covid- transmission. considering that more than half of the second survey participants also took part in the first survey, our findings may indeed reflect the evolution of covid- preventive attitudes in a given cohort of individuals rather than in two different populations. the lower adherence scores during the second survey, compared to the first, indicates that compliance to government measures is decreasing as the covid- epidemic evolves in somalia. it appears that as the testing capacity increases, resulting in more covid- cases being reported during the second survey, covid- fear and adherence to preventive measures are slowly declining. the fact that the prevalence of suspected covid- cases was fairly similar in the different regions of somalia suggests that important community transmission of covid- has been ongoing throughout the entire country [ ] . this was to be expected, given the low adherence to preventive measures. our findings are particularly relevant for the population in the capital city mogadishu, in the benadir region, where over % of the respondents resided (data from survey only). considering that mogadishu is one of the few places in somalia where the government healthcare system has actually deployed a covid- response [ ] , it was not surprising to find relatively lower adherence scores in the other regions (except for galmudug, which had slightly higher adherence scores than benadir). furthermore, the fact that urban residents and university level respondents were over-represented in both surveys makes it difficult to generalize the findings to the entire somali population. it is estimated that less than % of somalis aged years and above have attained university level education [ ] . this leaves a minority of the population fitting the characteristics of our survey participants. notably, residing in rural areas was associated with more difficulties in complying with the government lockdown instructions, and with lower adherence scores. this may be due to the fact that rural residents are often involved in subsistence farming, and staying at home would imply dropping their main source of livelihood. it is therefore important to design targeted, context-specific covid- prevention strategies that could be differentially implemented in different communities even within the same country. the reported level of worry/fear about the respondents' health or that of their loved ones significantly decreased from the first to the second survey. this was accompanied by reduced adherence scores across the surveys ( table ). the initially high anxiety caused by the news of the high mortality of covid- in developed countries with aging populations could be a possible explanation for this trend. however, upon realising that the disease is mostly benign among younger populations like in somalia (barely % are older than years [ ] ), the respondents may not be considering covid- as a major threat anymore. consequently, a progressive laissez-faire attitude was noted during the second survey, with more persons reporting that they have been frequenting public places such as bars/restaurants, places of worship, markets, or even travelled during the seven days prior to participating in the survey. the hypothesis of declining adherence as the epidemic evolves is further supported by the lower adherence scores observed among participants who have experienced flu-like symptoms, as it appears that they are no longer scared of the disease and hence are becoming less careful. although younger persons may be at a lower risk for severe covid- , they can still become infected and transmit the disease to the high-risk groups (elderly and chronically sick individuals). furthermore, covid- deaths have been reported in persons below years, although less frequently compared to older patients [ ] . the who director general recently highlighted that it is difficult to convince young people that they may indeed be at risk of dying from covid- , and urged them also to observe the preventive measures [ ] . in a like manner, the youthful somali population should be sensitized about the importance of adhering to preventive measures as much as possible for the sake of their own health and of the health of their loved ones. about half of the respondents reported wearing face masks during the covid- epidemic. this is far below the % threshold recommended by modelling studies [ ] , and therefore needs to be improved. of note, reusable cloth masks were reported only in about one quarter of participants. cloth masks offer some advantages, such as lower cost and being far less dangerous for the environment when compared to surgical masks, and have therefore been proposed for covid- control especially in resource-poor settings [ ] . aside from the reported difficulties of acquiring masks (such as lack of finances and not knowing where to obtain masks), other major limitations to wearing masks included the associated discomfort and the widespread thought that masks were unnecessary in times of covid- . indeed, masking is a relatively strange practice among the african population, and they should be educated about its importance during the covid- crisis. this is in contrast with asian countries, where masks are culturally accepted as a common hygienic practice [ ] . the multivariable analysis showed several factors associated with adherence to covid- preventive measures. the male gender was associated with poorer adherence, most likely because in the somali community, men are often the breadwinners and are compelled to leave the house even in the midst of the covid- outbreak, in order to work to support their families. this finding is in line with the results of a study in spain, where it was also observed that women were more adherent to covid- preventive measures than men, suggesting a more responsible attitude of women toward the dangers associated with the pandemic [ ] . respondents with a post-graduate educational level, who were probably more informed and knowledgeable about the disease, were more likely to have higher adherence scores compared to their less educated peers. in addition, government employees were more likely to highly adhere to the preventive measures compared to privately employed workers. we surmise that this is because the government agencies were more rigorous in implementing strategies against covid- in the professional milieu (including working from home), compared to private companies, which are often out to maximize profit even at the detriment of employee welfare. therefore, it is necessary that strict protocols that align with covid- safety regulations be implemented and re-enforced in workplaces during the ongoing health crisis. it is equally worth mentioning that obtaining covid- information from official sources (radio, tv, and government announcements) increased the odds of having a high adherence score, whereas information from social media decreased these odds. this highlights the role of misinformation in determining people's adherence to scientifically proven preventive measures to control the covid- epidemic [ ] . previous research has also demonstrated that social media users tend to be more extraverted [ ] , suggesting that such individuals may find it more difficult to stay at home and observe the covid- preventive measures compared to the more introverted non-social media users. it is necessary that our findings be interpreted in the light of some limitations. first, the participants were not a representative sample of the general population in somalia. indeed, the majority of respondents were educated people belonging to the middle to high social classes and most often residing in urban settings, since poor people in remote places may have very limited internet access. based on data from a recent demographic survey in somalia [ ] , our study population constituted a minority (only . % of somalis are aged years and above); our gender distribution was different ( % male in this study vs. . % male in the general population); education levels were biased (> % attended university in this study vs. . % in the general population); and urban residents were over-represented ( % in this study vs. . % in the whole country). second, it is not possible to verify the veracity of responses obtained via a web-based questionnaire. third, the fact that our study was done only at two time points makes it difficult to fully understand the rapidly changing landscape of the covid- epidemic and related attitudes in somalia. more studies, possibly with a longitudinal design, would be necessary to monitor the situation prospectively. our study revealed low adherence to covid- preventive measures among residents of somalia, with a decreasing trend in adherence over time. mass sensitization and targeted interventions (based on the residential setting, profession, educational level, and gender) may be needed to improve preventive behaviours and limit the transmission of the virus. additionally, social media platforms should be leveraged to disseminate accurate information about covid- . a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster asymptomatic transmission, the achilles' heel of current strategies to control covid- strengthening preparedness for covid- in cities and urban settings who coronavirus disease (covid- ) dashboard. available online covid- situation update for the who african region evidence for significant covid- community transmission in somalia using a clinical case definition what works where in prevention of covid- : the case of somalia covid- response in somalia: a comparative look international citizen project covid- world health organization. global surveillance for covid- caused by human infection with covid- virus: interim guidance the somali health and demographic survey somalia: unfpa age-dependent risks of incidence and mortality of covid- in hubei province and other parts of china world health organisation. who director-general's opening remarks at the media briefing on covid- - mathematical assessment of the impact of non-pharmaceutical interventions on curtailing the novel coronavirus mass masking as a way to contain covid- and exit lockdown in low-and middle-income countries rational use of face masks in the covid- pandemic could attitudes toward covid- in spain render men more vulnerable than women? rising above misinformation or fake news in africa: another strategy to control covid- spread who interacts on the web?: the intersection of users' personality and social media use this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the study was supported by a grant from the european research council (erc ). the study sponsor had no role in the design, execution, interpretation, or writing of the study. the authors have no conflicts of interest to declare. key: cord- -f gvwjyh authors: musso, nicolò; costantino, angelita; la spina, sebastiano; finocchiaro, alessandra; andronico, francesca; stracquadanio, stefano; liotta, luigi; visalli, rosanna; emmanuele, giovanni title: new sars-cov- infection detected in an italian pet cat by rt-qpcr from deep pharyngeal swab date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: f gvwjyh the pandemic respiratory disease covid- , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), emerged in wuhan in december and then spread throughout the world; italy was the most affected european country. despite close pet–human contact, little is known about the predisposition of pets to sars-cov- . among these, felines are the most susceptible. in this study, a domestic cat with clear clinical signs of pneumonia, confirmed by rx imaging, was found to be infected by sars-cov- using quantitative rt–qpcr from a nasal swab. this is the first italian study responding to the request of the scientific community to focus attention on the possible role of pets as a viral reservoir. an important question remains unanswered: did the cat actually die due to sars-cov- infection? the world health organization (who) declared covid- disease, caused by severe acute respiratory syndrome coronavirus (sars-cov- ), as a worldwide pandemic [ ] . the first epidemic cluster occurred in china, namely in wuhan city [ , ] , as early as december . as of mid-july , there have been over , , confirmed covid- cases worldwide, with more than % cases in the eu and uk and more than , deaths globally [ ] , (https://www.worldometers.info/coronavirus, accessed on may ). italy was severely affected [ ] , and it was one of the first and hardest hit countries in europe, with over , cases and , deaths reported [ ] , for which reason on march a lockdown was declared for the entire country and progressively stricter restrictions adopted [ , ] . sars-cov- is structurally and functionally closely related to the already known coronaviruses responsible for the middle east respiratory syndrome (mers-cov) and the severe acute respiratory syndrome (sars-cov). in detail, the mechanism of host cell infection is the same for sars-cov and sars-cov , i.e., it depends on the external protein of the virus, the glycoprotein (s) spike, which is able to bind and recognize the human receptor angiotensin converting enzyme (ace ), primed by the transmembrane protease, serine (tmprss ) and/or extracellular matrix metalloproteinase inducer cd [ , [ ] [ ] [ ] . because of the close contact between people and pets, such as dogs and cats, the scientific community started to investigate the possibility of animal-human virus transmission [ ] . furthermore, the animal ace receptor has high human ace aminoacidic sequence identity [ ] [ ] [ ] . it has been shown that some animal species, particularly felines, can occasionally become infected with sars-cov- [ , [ ] [ ] [ ] [ ] . so far, some cases of domestic animal infection have been reported in belgium [ ] , hong kong [ ] , and france [ ] , but no cases had yet been investigated in italy. in particular, viral rna and infectious viral particles were found in the upper respiratory tract of domestic cats after introduction of sars-cov- virus samples through their nasal cavities; nevertheless, none of the infected cats showed clinical signs of the disease. in addition, viral rna was detected in : healthy cats exposed to infected felines, suggesting that they had contracted the virus from the droplets exhaled by infected cats [ , ] . in this study, we identified the natural infection of a cat with sars-cov- for the first time in italy. the rna obtained from the nasal swab was processed by rt-qpcr using two different chemistries, revealing the presence of two sars-cov- genes. finally, part of one gene was further sequenced to evaluate its nature. a sterilized, three-year-old, male european shorthair cat was presented to a veterinary clinic with the owner reporting serious respiratory distress of about three days. the mandatory vaccinations required by the current italian legislation, including viral rhinotracheitis, calicivirus and panleukopenia, had been completed. on physical examination, the cat had severe dyspnea and sialorrhea, kussmaul breathing, and asynchronous chest and abdomen. chest auscultation revealed increased vesicular murmur. due to the presence of pathological infiltration and ground-glass opacity of the lungs, the differential diagnosis suggested an idiopathic interstitial pneumonia of bacterial, viral or mycotic origin. despite amoxicillin and clavulanic acid administration in combination with aerosol, the cat died shortly after admission and the corpse was cremated according to the current health protocol. according to good veterinary practice, a blood sample was collected from the jugular vein and ml was transferred to a sterile tube containing ethylenediamine tetra-acetic acid (edta) for hematological analysis, whereas another aliquot of about ml was collected in a sterile glass tube to obtain serum sample for biochemical analyses. moreover, clinical imaging evaluation was performed using ultrasound and x-ray techniques. given the similar clinical picture and the current situation caused by sars-cov- , a nasal swab was collected to test for sars-cov- . a nasal swab was collected using the virus test kit diagnostics sterile pack swabs universal viral transport system (cod. ryco-vart b , jiangsu rongye technology co., ltd., touqiao town, yangzhou city, china). to enhance rna uptake, a variation to the standard protocol was made: µl of swab buffer was processed in the same column, instead of the original µl. ave buffer and ethanol were added in the same proportions in order to reach a final extraction volume of . ml. then, the total volume was eluted in the same column ten times. for the rest, the extraction process followed the protocol provided by the manufacturers. viral rna from the original swab was purified using the qiaamp ® viral rna mini-kit (qiagen, hilden, germany; cod. ). the extracted rna was quantified by fluorometric technique using the qubit™ rna hs assay kit (thermo fisher, waltham, ma, usa; cod. q ) according to the standard procedure. nasal swab rna reverse transcription was carried out using the quantitect ® reverse transcription kit (qiagen, hilden, germany; cod. ). rt-qpcrs targeting sars-cov- were performed using taqman and sybr chemistries on a rotor-gene q thermocycler (qiagen) to amplify two different sars-cov- genes: the n gene, as indicated by the centers for disease control and prevention (cdc), and the spike gene, respectively. the primers used, their concentrations and the qpcr thermal profiles are listed in table . rt-qpcr targeting sars-cov- was performed with taqman chemistry: the taqman ® probe (qiagen, hilden, germany) was labeled at the -end with the reporter molecule -carboxyfluorescein (fam) and at the -end with the black hole quencher (bhq- ) (eurofins genomics, luxembourg); the reaction was performed using the quantinova probe pcr (qiagen, hilden, germany; cod. ) according to the manufacturer's recommendations. rt-qpcr targeting the sars-cov- spike was performed using sybr rt-qpcr (quantitech primers, qiagen, hilden, germany; cod. qt ) giving an amplicon of bp. finally, amplicons were verified by running the rt-qpcr products on . % agarose gel stained with (canvax, córdoba, spain) greensafe dna gel stain (cod. e ) and fastruler ladder (thermo fisher, waltham, ma, usa; cod. sm ). to exclude human mrna cross-contamination, human beta-actin primers at a final concentration of µm were used as a control at the sybr rt-qpcr (quantitech primers, qiagen, hilden, germany; cod. qt ) giving an amplicon of bp. the amplicons obtained by pcr from the amplification with the n portion gene primers (without probe) were purified using the qiaquick pcr purification kit (qiagen, hilden, germany; cod. ) and quantified using the fluorimeter qubit dsdna br assay kit (invitrogen, carlsbad, ca, usa; cod. ), then ng of the product were sequenced in a seqstudio genetic analyzer (thermo fisher scientific, waltham, ma, usa) using the applied biosystems bigdye terminator cycle sequencing . v (thermo fisher scientific, waltham, ma, usa; cod. ) as previously described [ ] . amplicons were then sequenced in a seqstudio genetic analyzer (thermo fisher scientific, waltham, ma, usa) using the applied biosystems bigdye terminator cycle sequencing . v (cod. , thermo fisher scientific, waltham, ma, usa; cod. ) as previously described [ ] and compared with the reference sequence "mt severe acute respiratory syndrome coronavirus isolated sars-cov- /human/ita/inmi / (complete genome sequence release date: -apr- )" using the blast tool (https://blast.ncbi.nlm.nih.gov/blast.cgi). ethical approval was not necessary as per institutional and national guidelines and regulations. the main biochemical and hematological parameters revealed lower alkaline phosphatase and higher glycemia values, while the blood count test showed relative and absolute neutrophilia (figure a) . the radiographic analysis revealed an unstructured interstitial pattern caused by the widespread presence of pathological infiltrate throughout the lung interstitium. the increased radiodensity of the lung parenchyma, defined as "ground glass", with little or no evidence of the intrathoracic vessels, clearly confirmed this condition (figure b) . furthermore, x-ray imaging revealed interstitial pneumonia with an area of pulmonary opacity (figure b) leading to the suspicion of effusion, excluded by the ultrasound examination (data not shown). the rna extracted from the nasal swab was quantified at . ng/µl and tested in duplicate by taqman probes for the detection of the sars-cov- n portion gene. sars-cov- synthetic rna and nasal swab rna amplified within the th and th cycle threshold (ct), respectively, while no amplification for the no template control (ntc) was detected. the amplification curves and ct, average and sd data are reported in figure s a (supplementary materials). furthermore, the real-time qpcr products were run on a . % agarose gel to confirm the correct size of the amplicon ( figure s a, right) . further evidence of sars-cov- was given by the detection of the spike gene within the th ct during the real-time qpcr assay, while no amplification for sars-cov- synthetic rna and ntc was found ( figure s b ). to ensure that two different real-time activities were conducted on the same rna during the sample collection and processing phases, and to exclude any possible contamination with human rna, a feline housekeeping gene (available in the techne-fcov kit) was used to verify the presence of correct feline rna. the nasal swab sample amplified within the th ct, while there was no amplification for the ntc (figure s c ). at the same time, the feline sample tested negative for the human beta-actin that was used as control. finally, the amplified fragment obtained by endpoint pcr using the same rt-qpcr primers ( figure s a, right) was sequenced using the sanger method to verify the accuracy of the amplification, resulting in a perfect match with the sars-cov- n gene according to the blast tool (https: //blast.ncbi.nlm.nih.gov/blast.cgi) ( figure s ). in late december , the severe acute respiratory syndrome-coronavirus (sars-cov- ), identified as a novel coronavirus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , first caused uncommon pneumonia in humans in wuhan (china) and then rapidly spread internationally. thus, the world health organization (who) named the disease caused by this virus "coronavirus disease " (covid- ) and officially declared covid- as a pandemic [ ] . there is evidence that sars-cov- shares . % of its nucleotide identity with the ratg coronavirus detected in horseshow bats in china [ ] . indeed, first evidence indicates that the infection took place through the consumption of meat derived from infected bats (bat meat is commonly eaten in china). the fact that pets are in close contact is given, but it became especially relevant once it was shown that pets were also susceptible to infection. knowing their susceptibility to sars-cov- is therefore very important as several cases of infected pets have been reported. unlike several studies carried out on dogs, which have shown little, if any, susceptibility to the virus, felines-especially cats-seem to have greater predisposition to the infection [ , ] . in the same way as in other coronaviruses such as sars-cov, the feline coronaviruses described until now seem to be able to change their tissue tropism [ ] . in this study, we report-for the first time in italy-the case of a male european cat positive for sars-cov- . unfortunately, we were not able to confirm the presence of the virus in the lungs of the cat because it was cremated before any tissues could be collected. as such, no association can be made between detection of sars-cov- in the nasal swab of the cat and its clinical condition. sars-cov- may well have been the cause of the cat's death, but it may have equally been an accidental finding [ ] . the cat presented clinical signs of pulmonary disease and the blood test and subsequent x-ray and ultrasound investigation confirmed the diagnosis of severe pneumonia. as the cat's pathology evolved rapidly and harmfully (the animal died in as little as three days), with clinical signs and rate of disease progression similar to human covid- patients, and because previously published papers reported different cases of feline infection [ , [ ] [ ] [ ] [ ] , a nasal swab was collected in order to verify a possible infection with sars-cov- . the identification of sars-cov- virus was supported not only by the qpcr amplification of the commonly used n gene within the th ct, by means of the taqman probe, but also by the amplification, using a more economic sybr green chemistry, of another portion of the sars-cov- virus spike gene-missing in the synthetic rna commonly used as positive control-which may represent a new molecular identification target. at the same time, the feline sample was free from human cross-contamination, as confirmed by positive amplification for a feline housekeeping gene and by the absence of amplification of the human housekeeping gene beta-actin. finally, to verify the presence of sars-cov- , an endpoint pcr was performed with cdc primers targeting n and the products were subsequently sequenced using the sanger method. although the clinical history and the investigations performed clearly show positivity with clinical manifestations for sars-cov- in the lung, the infection source could not be clarified. as a resident of a ground floor apartment, the cat was used to going outside. moreover, it was the only pet living with the owner. probably, the contamination was due to the habit of cats of licking potentially contaminated surfaces or through contact, not detected by the owners, with other unidentified positive cats or positive asymptomatic visitors. at the same time, the presence of sars-cov- in the nasal swab is not enough to assert that it caused the pathological event [ ] . as per health protocols and due to the time required for the swab transport and analysis, the cat corpse was cremated before knowing the test results. for this reason, unfortunately it was not possible to perform histological analysis to confirm that the pet died due to sars-cov , although the concomitant presence of severe pneumonia and virus rna was ascertained. therefore, the link between sars-cov- infection and the cat death remains an open question. to date, the crisis that has affected several world regions seems to be over, but the natural reservoirs of the virus and its high contagion rate, as well as environmental and social conditions may pave the way for a second epidemic wave. particularly, as cats and dogs are more common and in closer contact with humans than bats, they should be checked for sars-cov- when affected by severe pneumoniae, as several al studies have demonstrated the propensity of coronaviruses for interspecies transmission [ , ] . at this point, there is no evidence that cats can spread covid- and owners should not abandon their pets nor compromise their wellbeing [ ] . supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : amplification profiles for the detection of sars-cov- . figure s : sequencing of amplified end-point pcr derived fragment compared with sars-cov- genome. author contributions: n.m., a.c. and g.e. conceived and designed the study. n.m., a.c. and f.a. performed the experiments. s.s., r.v., l.l., s.l.s. and a.f. contributed to data analysis. n.m. and a.c. wrote the draft manuscript, s.s. reviewed and edited the manuscript. all authors have read and agreed to the published version of the manuscript. the authors received no financial support for the research, authorship, and/or publication of this article. world health organization. who director-general's opening remarks at the media briefing on covid- covid- infection: the china and italy perspectives clinical features of patients infected with novel coronavirus in modelling the covid- epidemic and implementation of population-wide interventions in italy chronology of main steps and legal acts taken by the italian government for the containment of the covid- epidemiological emergency structure, function, and antigenicity of the sars-cov- spike glycoprotein sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor structural basis for the recognition of sars-cov- by full-length human ace evidence for 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probes droplet digital pcr as the best sensitive assay fot the sars-cov- detection somatic loss of an ext gene mutation during malignant progression in a patient with hereditary multiple osteochondromas sars-cov- viral load in upper respiratory specimens of infected patients a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china genome composition and divergence of the novel coronavirus ( -ncov) originating in china a novel coronavirus outbreak of global health concern viral load of sars-cov- in clinical samples asymptomatic cases in a family cluster with sars-cov- infection genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of coronavirus disease in china epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study world health organization (who) a pneumonia outbreak associated with a new coronavirus of probable bat origin spike protein fusion peptide and feline coronavirus virulence causation and causal inference in epidemiology cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: we wish to thank stefania stefani and massimo gulisano for their suggestions. the authors declare no conflict of interest.pathogens , , pathogens , , key: cord- -oudj q authors: al-tayib, omar a. title: an overview of the most significant zoonotic viral pathogens transmitted from animal to human in saudi arabia date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: oudj q currently, there has been an increasing socioeconomic impact of zoonotic pathogens transmitted from animals to humans worldwide. recently, in the arabian peninsula, including in saudi arabia, epidemiological data indicated an actual increase in the number of emerging and/or reemerging cases of several viral zoonotic diseases. data presented in this review are very relevant because saudi arabia is considered the largest country in the peninsula. we believe that zoonotic pathogens in saudi arabia remain an important public health problem; however, more than million muslim pilgrims from around islamic countries arrive yearly at makkah for the hajj season and/or for the umrah. therefore, for health reasons, several countries recommend vaccinations for various zoonotic diseases among preventive protocols that should be complied with before traveling to saudi arabia. however, there is a shortage of epidemiological data focusing on the emerging and reemerging of zoonotic pathogens transmitted from animal to humans in different densely populated cities and/or localities in saudi arabia. therefore, further efforts might be needed to control the increasing impacts of zoonotic viral disease. also, there is a need for a high collaboration to enhance the detection and determination of the prevalence, diagnosis, control, and prevention as well as intervention and reduction in outbreaks of these diseases in saudi arabia, particularly those from other countries. persons in the health field including physicians and veterinarians, pet owners, pet store owners, exporters, border guards, and people involved in businesses related to animal products have adopted various preventive strategies. some of these measures might pave the way to highly successful prevention and control results on the different transmission routes of these viral zoonotic diseases from or to saudi arabia. moreover, the prevention of these viral pathogens depends on socioeconomic impacts, available data, improved diagnosis, and highly effective therapeutics or prophylaxis. rudolf virchow , one of the foremost th century german leaders in medicine and pathology [ ] , noted a relationship between human diseases and animals and then introduced the term "zoonosis" (plural: zoonoses) in [ ] . later, the world health organization (who) in specified that "zoonoses are those diseases and infections which are naturally transmitted between vertebrate animals and man" [ ] . venkatesan and co-authors reported that the term zoonosis is derived from the greek word "zoon" = animal and "noso" = disease [ ] . zoonotic pathogens causing different kinds of diseases are of major public health issues worldwide [ ] . these zoonotic diseases include frequent mixing of different animal species in the markets in densely populated areas, and the human intrusions into the natural habitats of animals, have facilitated the emergence of novel viruses. the most important zoonotic viral diseases of which eight were diagnosed (in dead or diseased animals or through antibody detection) on the arabian peninsula over the last years include rabies, middle east respiratory syndrome (mers-cov), influenza virus (ifv), alkhurma hemorrhagic fever, crimean-congo hemorrhagic fever (cchf), rift valley fever (rvf), west nile fever (wnv), and dengue fever virus. among these eight zoonotic viral diseases, two (alkhurma and mers-cov) were first reported in a patient in and , respectively in saudi arabia [ , ] . these two were transmitted later to several other countries, not only in the middle east but also to africa, asia, and europe. rabies is an almost invariably fatal zoonotic disease, which belongs to the genus lyssavirus of the rna family rhabdoviridae. rabies virus is considered an endemic viral infectious disease in animals in saudi arabia. recent scientific data on rabies cases reported in camels at al-qassim region (one of the thirteen administrative regions of saudi arabia) showed that there is an increasing number of this fatal virus disease [ ] . however, the most significant animal bites which have been recorded in saudi arabia were caused by different species of animals including dogs, cats, rodents, and foxes [ ] . later, al-dubaib reported rabies in dromedaries in saudi arabia in and suggested an incidence of about . % for rabies that was reported among camel herdsmen looking after more than animals [ ] . interestingly, another survey was conducted between and in the al-qassim region of central saudi arabia among camels and showed that about . % of clinical rabies incidence is caused by dogs (may be cause it highly used as a perfect guard for camels), followed by foxes; furthermore, the diagnosis of viral rabies in that region was confirmed among dogs, foxes, camels, and cats [ ] . lately, the relevant government authorities (the moh and ministry of agriculture in saudi arabia) in an updated report between and showed that there were a total of , animal bites to humans in saudi arabia [ ] . furthermore, most cases of animal bites were caused by dogs ( . %) and cats ( . %), followed by mice and rats, camels, foxes, monkeys, and wolves [ ] . moreover, dogs, particularly feral dogs and foxes, are considered the most important host for rabies virus; however, bats are also considered as reservoirs of this disease. humans can become rabid by direct contact with animal mucosal surfaces via bites. according to the moh and ministry of agriculture data in saudi arabia, pets are responsible for most animal bites in humans [ ] , and it is well-known that insufficient vaccination coverage of pets are among the most common hallmarks of the endemic status of rabies worldwide [ ] . more recently, many saudi and expatriate families are keeping pets; however, there are limited number of specialized veterinary clinics (~ ) within the kingdom of saudi arabia that have fully licensed veterinary laboratories with state of the art technologies and veterinary staff. globally, almost % of all human deaths caused by rabies occur in africa and asia [ ] . however, saudi arabia, as one of the asian countries, has scarce publications and epidemic data on rabies status [ , ] . moreover, memish et al., between and in saudi arabia, reported the histologic detection of the virus by identifying negri bodies in the brain samples of animal rabies cases. the study showed that among the suspected rabies cases, (~ . % of all cases) were found to be positive; thus confirming rabies cases among dogs, foxes, sheep, camels, goats, wolves, and cows [ ] . furthermore, more recent data confirmed the transmission of rabies virus in saudi arabia by feral dogs [ ] . in spite of these facts, there are very few studies available, and no case of human rabies has been reported in recent decades from saudi arabia [ ] . however, in march , a scientific work was reported as the first confirmed case of human rabies in saudi arabia from makkah city, which has now been published [ ] . indeed, several previous global epidemiological data confirmed that rabies accounted for , to , human deaths per year [ ] , and more than % of these cases occur in children < years of age [ ] . in september , a -year-old saudi man, presented with different clinical features-such as nausea, vomiting, and epigastric pain, with significant features suggestive of gastritis-at makkah hospital. his past medical history was significant for hypertension and diabetes type . during the clinical diagnostic procedure of this case, he developed respiratory distress and tachycardia, for which he was transferred to the intensive care unit [ ] . because, his case worsened with chest pain and ventricular tachycardia he was referred to the king abdullah medical city in makkah for further management. the written diagnostic report indicated that he had acute anteroseptal myocardial infarction, had coronary angiogram which suggested that two-vessels were diseased with left main involvement, and surgical intervention was planned. after the decision for surgery, he was found to have leukocytosis and severe retching while attempting to drink water (hydrophobic behavior), which necessitated further review by the infectious disease consultants based on the patient's clinical symptoms. the consultant team discovered the history of an unprovoked scratch on the patient's face by a dog in morocco a month prior to the admission at the hospital. also, the patient stated that he only received tetanus vaccine. all diagnostic tests including neurologic examination were unremarkable and his saliva polymerase chain reaction (pcr) test confirmed rabies virus. he was administered verorab rabies vaccine and human hyperimmune rabies immunoglobulin ( iu/kg) intramuscularly (im) [ ] . in addition, he had troponin i ( . ng/ml), creatine kinase isoenzyme mb (ckmb) was found ( . ng/ml), and serum glucose ( mg/dl). on the fifth day of hospital, he had recurrent episodes of ventricular tachycardia, progressively worsening of hemodynamic parameters, and he succumbed to his infection on that day. there is no vaccine against rabies recommended for travelers from/to saudi arabia, and no rabies treatment is offered to pet dogs. however, vaccination is given to dogs before they are infected; otherwise they are euthanized if infected. according to a previous study, most patient injuries from animal bites in saudi arabia showed some variations due to the monthly incidence and/or, according to the animal species [ ] . bites by dogs and cats were reported frequently throughout the year, with a decrease in april and between august and october. however, bites by foxes increase between august and september while camel bites were more frequent between december and march of the subsequent year. the same previous study suggest that these seasonal variations of injuries might be due to the saudi population habits, with people going to the desert for leisure activities during good weather periods. laboratory diagnosis of rabies viral disease occur with the use of the rabies virus direct fluorescent antibody test (dfat) on brain samples and hippocampal tissue [ ] . while rabies is considered nearly % fatal, it is also % preventable, and thus vaccination to pets is the key element to prevent the risk of rabies zoonotic infection [ ] . reports of the epidemiology of rabies virus worldwide, and particularly in saudi arabia, suggest that it is on the increase, thus the implication of this virus' potential to spread across borders from high to low prevalence countries was highlighted [ ] . the mers-cov infection is considered to be a new respiratory disease with a dire global concern [ ] . mers-cov infections are caused by a newly emerging coronavirus (cov), belonging to the designated lineage c of betacoronavirus of the rna family coronaviridae. with respect to viral origin and transmission, bats are thought to be the reservoir host of betacoronaviruses, and the african neoromicia bats in particular are the natural reservoir of mers-cov [ , ] . since its emergence in in saudi arabia, when an elderly patient ( years old) with respiratory illness died after admission to a hospital in jeddah [ ] , the disease was subsequently reported to have been transmitted to several countries worldwide, and has affected more than patients with over % fatality [ , [ ] [ ] [ ] . moreover, a -year-old saudi man was admitted to a private hospital in jeddah, saudi arabia in june with a history of fever, severe acute respiratory syndrome with cough, expectoration, and shortness of breath. he did not smoke; and for the disease, which was suggested to be due to an animal transmission of coronaviruses, he was treated with oseltamivir, levofloxacin, and piperacillin-tazobactam. on day , he died [ ] . after this, a -year-old saudi male with hypertension and diabetes with no history of smoking, reported for surgery. at the time of admission, he was asymptomatic. he was initially screened using nasopharyngeal swab, endotracheal aspirate, and serum sample for mers-cov per protocol with the mers rrt-pcr assay. the results confirmed mers-cov infection. he died three days after admission. it was discovered that the patient owned a dromedary camel barn in saudi arabia, and had a history of close contact with camels, as well as a habit of raw milk consumption of an unknown duration [ ] . two studies have suggested a relationship between the infection and contact with dromedary camels [ , ] . in addition to this, serological diagnostic methods have been used to confirm mers-cov infections in dromedary camels for at least - decades and has thus confirmed camels as an intermediate host for this virus [ , ] . thus, in , a novel coronavirus (mers-cov) was isolated from two fatal human cases in saudi arabia and qatar; and since then, more than clinical cases of mers-cov have been identified, and the great majority of the cases were from saudi arabia [ ] . this previous report author raised a thoughtful comment related to the emerging viral diseases "why we need to worry about bats, camels, and airplanes" [ ] . moreover, another study suggested that mers-cov infection is usually transmitted from human's direct contact with dromedary camels, especially when people drink the milk or use camel's urine for medicinal purposes [ ] . more recently, a metagenomics sequencing analysis of nasopharyngeal swab samples from mers-cov-positive live dromedary camels marketed in abu dhabi, united arab emirates, showed at least two recently identified camel coronaviruses, which were detected in . % of the camels in that study [ ] . however, limited human-to-human infections have been reported. the prevalence of mers-cov infections worldwide still remains unclear. in addition to this, the who reported about cases of these infections since june , with about deaths in different countries, worldwide. recently, a study was conducted from june to july , during which samples were collected from mers-cov infected individuals, from the national guard hospital in riyadh (the saudi arabian capital city), the moh in saudi arabia, and other gulf corporation council countries, to determine the prevalence of mers-cov [ ] . the epidemiologic data that were collected, showed that the highest number of cases (about of patients) were reported from saudi arabia (~ %). among the mers-cov cases from saudi arabia, riyadh was the worst-hit area with infected cases ( . %), followed by the western region of makkah where cases ( . %) were reported [ ] . furthermore, this study also showed that the incidence of mers-cov infections was highest among elderly people aged ≥ years [ , ] ; with speculation that there might be certain conditions or factors involved. it is considered that mers-cov infection might have a peculiar gender predisposition [ ] . recent data examined the mortality in patients with mers-cov and the gender relationships, looking at the survival of cases among females and males. it was suggested that males have a higher risk of death [ , ] ; however, this was contradicted by the findings from two other studies which suggested that males have a low risk of death [ ] ; while another survey which examined the influence of gender on -day and -day survival, found a low risk of death especially in the older age group [ ] . on the other hand, badawi et al., suggested that mers-cov infections could be mild and may only result in death among patients suffering from any kind of immune system disorder and/or any chronic disease [ ] . more recently, data regarding the mortality in patients with mers-cov have been published. according to saudi arabia's moh daily statements, dated from february through march , laboratory-confirmed new cases of mers-cov and deaths occurred [ ] . recently, on february , patients infected while hospitalized at riyadh included two men ( and years old) in stable condition, who were not healthcare workers. according to a february update, a new case involved a -year-old man from the city of buraydah who later died. meanwhile, on march , another mers-cov infection in a riyadh hospital patient, a -year-old man who was listed in critical condition and who likewise had contact with camels, as the other two patients, was reported. thus, the moh stated that the spillover from camels is thought to be the main source of mers-cov in saudi arabia, since all these patients were exposed to the animals before reporting ill [ ] . furthermore, an -year-old patient from riyadh, and other two patients who had camel contacts from hail city in the north central part of saudi arabia were listed in critical condition. the illness in these patients was reported on march . according to a march statement, another patient, a -year-old man from najran located in southern saudi arabia, was reported. the man was listed in a stable condition. of these new cases, only one death, involving the -year-old man from riyadh, according to the march moh statement, was reported [ ] . still, much work is needed to detect the mers-cov infection risk in saudi arabia, because data showed increasing number of cases exist among the eight countries including saudi arabia. thus, the emergence of mers-cov in the region and its continuing transmission from - , currently poses one of the biggest threats to global health security [ ] . most cases (over %) reported to date have been from countries in the region (e.g., egypt) notably from saudi arabia, with cases including deaths [ ] . influenza viruses are considered to be important infectious viral diseases, which is caused by three virus types (a, b, and c) [ ] . due to their zoonotic spread, influenza type a infects both humans and animals, and causes moderate to severe illness, with more likelihood of fatalities in young children and the elderly [ , ] . other types of influenza, including type b and c, infect only humans [ ] . furthermore, influenza a viruses, members of the rna family orthomyxoviridae, are further classified into human, swine, and avian influenza viruses. however, during the influenza pandemic, swine influenza virus infected one-third of the world's population (an estimated million people) and caused approximately million deaths [ ] . since , several infections with this virus have been recorded from various areas worldwide, including saudi arabia [ ] . at the end of april , an outbreak of a new type of influenza, a/h n , started in mexico and the usa [ ] . the who declared the pandemic influenza a (h n ) as a "public health emergency of international concern" following the first few initial cases in mexico, and subsequently in the usa [ , ] . in saudi arabia, the epidemiological data for influenza virus were collected using a predesigned questionnaire with the first confirmed pandemics influenza a (h n ) cases identified by the infectious diseases department from the moh, and the database during the period covered from june to july [ ] . however, according to the saudi moh data, the number of laboratory-confirmed cases of the virus in saudi arabia as at december was , , with deaths [ ] . the virus later spread worldwide, causing a pandemic, and the most recorded cases then, as reported by the who in the middle east, were in saudi arabia with , cases, followed by kuwait [ ] , egypt, and oman; with less number of infected patients [ , ] . nevertheless, between and , a serosurveillance outcome of swine influenza virus from egypt provided evidence of laboratory diagnosis and very early confirmation of the virus in human patients [ ] . in saudi arabia, the influenza surveillance system has been established since . moreover, among people with certain chronic medical diseases or conditions, a trivalent influenza vaccine (tiv), which contains inactivated antigens for two different subtypes of influenza viruses (types a and b), became available in saudi arabia [ ] . indeed, h n is now in the post-pandemic period and has become a seasonal influenza virus that continues to circulate with localized outbreaks of varying magnitude in saudi arabia [ ] . a previous data was collected using a predesigned questionnaire for the first cases of pandemic influenza a (h n ) from different hospitals in saudi arabia. the age of patients enlisted in the data ranged from to years. the age groups with the highest percentage of cases were between: and years ( %), and and years ( %). there were males and females, and % patients had some contacts with infected persons within saudi arabia while about % had history of travels into saudi arabia and/or the philippines [ ] . these facts are similar to the previous relationship noted between the occurrence of zoonotic viral diseases and the gender of patients and/or their ages, as reported for another viral (e.g., mers-cov) infection; provided certain conditions are met [ ] [ ] [ ] [ ] . interestingly, among elderly patients, influenza cases were higher in females than males. this relationship with viral infection occurring particularly with respiratory viral diseases, might pave the way and play a big role of more significant importance in the detection of these diseases, taking into account the influence of climate change and the different environmental factors [ , , ] . nevertheless, between september and october , about samples were taken from several patients presenting with respiratory symptoms to king abdulaziz university hospital, jeddah, saudi arabia. however, during this study conducted to detect the susceptibility to the influenza viruses circulating in the western part of saudi arabia, out of all the tested samples, ( . %) respiratory samples were positive for influenza h n virus, ( . %) was positive for influenza h n virus, and ( . %) were positive for influenza b virus [ ] . furthermore, h n , now in the post-pandemic period, has become a seasonal influenza virus that continues to circulate with localized outbreaks of varying magnitude [ ] . interestingly, the presentation of influenza virus infections in humans usually vary from mild, self-limiting respiratory-like illness, to severe cases that may result in death [ , ] . nevertheless, a recent study has shown that subclinical infection in human exists, as revealed by the serological surveillance [ , ] . therefore, the epidemiological surveillance of influenza in saudi arabia is highly important especially with the fact that influenza cases have also been highly reported can spread globally [ ] . thus, geographic influences on influenza virus infection in saudi arabia must be of concern [ ] . this is relevant because a remarkably high number of egyptian muslims visit saudi arabia yearly to participate in the umrah and/or the hajj pilgrimage; in addition, the impact of the poultry industry in egypt is also worth considering, with an estimated billion birds and several millions of engaged laborers with/without surveillance [ ] . it is well-known that the influenza drugs, antiviral agents, and the current seasonal influenza vaccines are effective in reducing the incidence and severity of the disease, sickness, and/or complications. however, the important strategy for influenza management includes the provision of prophylaxis and treatment [ ] . however, it is possible for widespread drug resistance against antiviral agents or vaccines to emerge in patients who extensively abused the drugs, in addition to those who have never received such treatment, globally [ ] [ ] [ ] . furthermore, influenza viruses pose a challenge to vaccine developers and manufacturers due to the fact that these viruses are continually changing in nature, including hemagglutinin and neuraminidase [ , ] . moreover, while resistance to neuraminidase inhibitors (e.g., oseltamivir and zanamivir) have been reported to sporadically occur, the resistance to oseltamivir has been widely reported since , with a worldwide spread [ ] . this highlights why there is an urgent need for the public health system to monitor continuously via globally active influenza surveillance programs. furthermore, there is need to monitor the circulating influenza viruses strains, as well as the occurrence of any resistance, using appropriate diagnostic methods. this is considered highly essential in saudi arabia. interestingly, survey data has shown an increasing report of the viral infection from egypt, since hajj egyptians has ranked in the top list of countries with the highest number of mecca pilgrims in the last years [ ] . influenza is highly susceptible to antiviral drugs such as oseltamivir, according to a more recent epidemiological study [ ] . although millions of muslims, globally, travel annually to saudi arabia to perform hajj and/or umrah in the holy places including both makkah and al-madinah for very limited period (~ days), this gathering could play a major role in the introduction of new influenza viruses, not only to saudi arabia but also to the rest of the world [ , ] . unfortunately, there is no such influenza surveillance program in saudi arabia, thus this pose a serious public health concern. recently, in a study of pilgrims screened on arrival at the hajj season in saudi arabia, ( . %) had influenza a virus ( out of the had h n virus) [ ] . additionally, the epidemiological data showed that the pilgrims had the potential not only to introduce these viruses to saudi arabia, but also to export the influenza virus back to their home countries [ , ] . this can occur in saudi arabia, despite the availability of a tiv containing inactivated antigens for influenza virus types a and b, which can protect against the influenza virus infection [ ] . importation of resistant and highly pathogenic viruses including influenza viruses can occur worldwide. despite this, there is lack of studies and data on drug susceptibility, and a very limited number of studies and reports on viral isolates, except for one study conducted in jeddah, according to the best of our knowledge [ ] . most importantly, this highlights the importance of circulating influenza viruses in saudi arabia, hence there is need to ensure effective use of antivirals for prophylaxis and treatment of influenza. furthermore, the rate of vaccination against influenza is very low among pilgrims and healthcare workers [ , ] . moreover, studies are needed to provide a clear picture on the impact of drug resistance on saudi arabia's endemic pathogens, including the influenza viruses. in a recent communique, the ministry of environment, water, and agriculture for saudi arabia reported two cases of h n avian influenza in the kharj governorate [ ] . the latest update by the ministry revealed that the number of samples collected from saudi regions since the start of the influenza outbreak had reached , . positive results from samples and laboratory tests indicate positive cases, and the saudi authorities have taken action by culling as many as , birds within a -h period [ ] . in contrast, several epidemic zoonotic cases of influenza h n have been reported in domestic cats in several countries in asia, europe, the usa, and italy [ ] . moreover, the epidemic of influenza in dogs might be related to a serious public health issue and could be shown to have resulted from zoonotic diseases from pets, similar to the avian influenza h n outbreak reported in pet dogs in south korea in [ ] . nevertheless, a recent study has shown that the role of pets, particularly cats and dogs in the epidemic of influenza as a source of human infection seems limited. however, cats were shown to be fully susceptible to experimental infection, and infected cats were able to infect naive cats [ ] . in , pandemic h n infection in a domestic cat in the usa from iowa was diagnosed by a novel pcr assay; thus, human-to-cat transmission was presumed [ ] . despite this prior evidence, the role of pets including cats and dogs seem even more limited in the dispersal of avian influenza to humans. rather, humans may be the source of pet infection, as suggested for influenza h n and/or h n virus infections [ , [ ] [ ] [ ] . most importantly, epidemic zoonotic cases of influenza among pets has highlighted the importance of circulating influenza viruses globally; especially, to ensure the effective use of antivirals for the prophylaxis and treatment of influenza, in particular, with the increase in the number of pets stores in saudi arabia, especially in riyadh [ , ] . surprisingly, previous data focused on the occurrence of zoonotic infection of different influenza virus types, and particularly, the transmission of avian influenza virus h n to domestic dogs [ ] . several studies have examined and confirmed the occurrence of zoonotic infection of the influenza a virus h n pandemic, especially in domestic cats [ , ] . nevertheless, epidemiological studies on different zoonotic infections among the pets in saudi arabia including cats, dogs, and/or baboons are very rare. however, a previous case report confirmed a relationship between some zoonotic diseases causing respiratory symptoms, such as influenza, among pets [ ] . this study suggests that severe lung infection with dry cough and severe anemia should lead to the suspicion of a secondary infection with zoonotic balantidiasis, which infected a hamadryas baboon from saudi arabia in a research center for pets in riyadh [ ] . furthermore, two other epidemiological zoonotic study on balantidium coli protozoan zoonotic infection in camel was reported from riyadh [ ] . in addition, another previous data confirmed the occurrence of toxocara canis zoonotic infection based on respiratory symptoms reported at the pet clinics in saudi arabia (and also in riyadh where the symptoms occurred in dogs) [ ] . still, more such studies are needed to highlight the important issues and/or provide clearer pictures of the zoonotic pathogens among pets in saudi arabia; however, pet ownership has been growing rapidly as well as the number of pet stores among the saudi population. alkhurma hemorrhagic fever virus (ahfv) in humans was discovered in [ ] . the first case reported in a butcher from the city of alkhurma, a district south of jeddah in saudi arabia, died of hemorrhagic fever after slaughtering a sheep. the viral infection has a reported fatality rate of up to % [ ] . interestingly, one of the previous reports regarding this disease showed a misunderstanding of the real name of this infection, called alkhurma, not alkhumra [ , ] . because subsequent cases were diagnosed in patients from the small town known as alkhurma in jeddah from where the virus got its scientific name; the name was accepted by the international committee on taxonomy of viruses [ ] . thus, based on evidence, the first case was confirmed to be the butcher, following the slaughtered sheep [ ] . therefore, a study was conducted among affected patients to address this disease as a public health issue. blood samples were collected from household contacts of patients with laboratory-confirmed virus for follow-up testing by enzyme-linked immunosorbent serologic assay (elisa) for ahfv-specific immunoglobulin (ig) g. samples from persons seeking medical care were tested by elisa for ahfv-specific igm and igg using ahfv antigen. viral-specific sequence was performed by reverse transcription pcr (tibmolbiol, lightmix kit; roche applied science, basel, switzerland). a total of cases were identified through persons seeking medical care, whose illnesses met the case definition for ahfv, and another cases were identified through follow-up testing of household contacts [ ] . subsequently, the virus was isolated from six other butchers of different ages (between and years) from the city of jeddah, with two deaths. the diagnosis was established from their blood sample tests. the serological tests later confirmed four other patients with the disease [ ] . from to , the study on the virus initial identification in the city of alkhurma again identified other suspected cases; with laboratory confirmation of the disease in (~ %) of them. among the , ( %) had hemorrhagic manifestations and ( %) died [ ] . the virus was later identified in three other locations: from the western province of saudi arabia (ornithodoros savignyi and hyalomma dromedarii were found by reverse transcription in ticks) and from samples collected from camels in najran [ , ] . ahfv virus was considered as one of the zoonotic diseases; however, the mode of transmission is not yet clear. recently, it was suggested that the disease reservoir hosts may include both camels and sheep. the virus might also be transmitted as a result of skin wounds contaminated with the blood or body fluids of an infected sheep; through the bite of an infected tick, and through drinking of unpasteurized or contaminated milk from camels [ , ] . in humans, this zoonotic disease may present with clinical features ranging from subclinical or asymptomatic features to severe complications. it is related to kyasanur forest disease virus, which is localized in karnataka, india [ , ] . however, epidemiologic findings suggest another wider geographic location for the disease in western (including jeddah and makkah) and southern (najran) parts of saudi arabia, and the virus infections mostly occur in humans [ , , ] . a study was conducted by alzahrani et al. in the southern part of saudi arabia particularly in the city of najran (with populations of~ , ), an agricultural city in saudi arabia, where domestic animals are reared at the backyard of owners. after the initial virus identification, from january through april , persons with positive serologic test results were identified. infections were suspected if a patient had an acute febrile illness for at least two days; when all other causes of fever have been ruled out [ ] . additionally, data analysis indicated that patients infected with the virus were either in contact with their domestic animals, involved in slaughtering of the animals, handling of meat products, drinking of unpasteurized milk, and/or were bitten by ticks or mosquitoes. symptoms consistent with ahfv infection-including fever, bleeding, rash, urine, color change of the feces, gum bleeding, or neurologic signs-then develop [ ] . fortunately, infected patients responded to supportive care (including intravenous fluid administration and antimicrobial drugs when indicated), with no fatal cases. in summary, ahfv is a zoonotic disease with clinical features ranging from subclinical or asymptomatic features to severe complications. another study highlighted different characteristics of the exposure to the blood or tissue of infected animals in the transmission of ahfv to humans. of the patients confirmed with infections, % were butchers, shepherds, and abattoir workers, or were involved in the livestock industry [ ] . more recently, a study on infection using c bl/ j mice cells showed that the clinical symptoms of the disease were similar to the presentations in humans [ ] . however, alkhurma disease resulted in meningoencephalitis and death in wistar rats, when high titers to the infection occurred [ ] . in addition, exposures to mosquito bites are regarded as potential sources of transmissions of the infection; however, very few available data support this [ ] . although, available data shows that alkhurma virus has been isolated following mosquito bites [ ] . however, another study suggested that mosquitoes may play a role only as a vector in the transmission of the disease [ ]. cchf is a zoonotic viral disease from the bunyaviridae family, and the principal vector for the disease is ticks of the genus hyalomma. it is most commonly endemic in africa, middle east, asia, and eastern europe [ , ] . it is an acute, highly-contagious, and life-threatening vector-borne disease responsible for severe hemorrhagic fever during outbreaks, and a fatality rate of up to % [ , ] . the infectious disease was recognized first in the crimean peninsula in , and it was named crimean hemorrhagic fever virus because the virus was isolated for the first time from a febrile child in from stanleyville (now kisangani), democratic republic of congo [ ] . currently, the virus infects both humans and animals following tick bites [ ] . however, a human can be infected by the animal through contact with the blood or tissues of the infected animal, in particular, exposures at the abattoirs are common. therefore, workers in contact with animals (e.g., veterinarians, farmers' and workers in slaughterhouses) form a high percentage of those affected [ ] . also, different species of infected animals-such as camels, cattle, sheep, goats, and ostriches-might be infected with no clinical signs [ ] . in addition, human-to-human transmission is also documented, mostly through a form of nosocomial or in-house infection [ , [ ] [ ] [ ] . lately, antibodies to the virus have been detected in different animal species, as reported in , in egyptians animals' sera [ ] . the preliminary seroepidemiological survey detected antibodies to the virus in . % of camels' sera and . % of sheep sera, but no antibody was detected against the virus in the sera of other animals such as donkeys, horses and mules, pigs, cows, and buffaloes [ ] . the epidemiology and distribution of cchf in saudi arabia are unclear, but there are reports of cchf as a result of the trading and importing of infected livestock from neighboring countries to saudi arabia [ ] . in , the cchf virus (cchfv) caused an outbreak involving seven individuals in makkah, although the virus had not been reported previously in saudi arabia. therefore, a study on the epidemiology of this virus was carried out in makkah, jeddah, and taif from - . about out of different species of ticks that were capable of transmitting the disease were collected from camels, cattle, sheep, and goats, but camels had the highest rate of tick infestation ( %), and h. dromedarii was the commonest tick ( %). an investigation in makkah between and , which included a serological survey of abattoir workers in contact with sheep blood or tissue, identified human cases of confirmed or suspected cchf with fatalities [ ] . the report from the investigation stated that the virus might have been introduced to saudi arabia through the jeddah seaport via infected ticks on imported sheep; since then, it has been endemic in the western province of saudi [ , ] . in addition, another previous study confirmed that the highest seropositivity rate of the virus in saudi arabia localities was associated with animals imported from sudan [ ] . furthermore, the who reported countries with cchf including saudi arabia; however, all the remaining countries are either close to saudi arabia or are islamic countries with high numbers of muslims who travel annually to saudi arabia for hajj pilgrimage. the same who epidemiological data suggest that in these countries including saudi arabia, in recent years, there has been report of steadily increasing number of sporadic human cases, incidence, and outbreaks of the virus [ ] . furthermore, another study by who investigating cchfv in the eastern mediterranean region (emr) stated that cchf is a clear and growing health threat in the who emr. cases are being reported in new areas, showing a geographical extension of the disease that is probably linked to the livestock trade and the spread of infected ticks by migratory birds. according to ecological models, the increase in temperature and decreased rainfall in the who emr could have resulted in the sharp increase in distribution of suitable habitats for hyalomma ticks and the subsequent drive of cchfv infection northwards [ ] . jazan province, the red sea port city on saudi arabia's southern border with yemen, serves as the east-west portal from sub-saharan africa at djibouti and the south-north route across the yemeni frontier. it is a heavily traveled corridor for humans and animals entering saudi arabia, particularly during the annual hajj pilgrimage. in november , a total of ( %) enrolled soldiers reported symptomatic illness during deployment, ( %) of whom were hospitalized. reported signs and symptoms included fever (n = ), rash (n = ), and musculoskeletal complaints (n = ). a surveillance study was conducted to detect the causes of the several outbreaks through that area, which was reported as endemic over a wide geographic range. from the surveillance, serologic testing for cchfv, ahfv, denv, and rvf was completed for saudi military units from several saudi arabian provinces. these units were previously stationed in other parts of the country, and were deployed to jazan province; the initial screening for igg of each of these viruses was conducted by igm testing for all igg-reactive samples. among the samples from all military forces, the study identified reactive serum samples with a combined seroprevalence of . cases/ soldiers tested. a confirmed serologic status of soldiers who were evaluated for igg and igm elisa reactivity against cchfv, rvf, ahfv, and denv infections were positive for , , , and sample, respectively [ ] . rvf is a common arbovirus zoonotic disease caused by the rvf virus. the virus belongs to the genus phlebovirus and family bunyaviridae. it is most common in domestic animals, and causes mild to life-threatening infections in humans. the name of the disease was derived from the great rift valley of kenya, when the disease was described for the first time in [ ] . epidemiological tests have since been described after a highly fatal epizootic occurred there in [ ] . rvf is a viral zoonosis with evidence of widespread occurrence in humans and animals in africa and the arabian peninsula. the epidemiology of this virus in saudi arabia might be closely related to the ecological factors that are prevalent, as shown from another area, along the great rift valley, which traverses ethiopia and kenya to northern tanzania with the drainage ecosystems [ ] . saudi arabia has many of the world's mosquito vectors of parasitic and arboviral diseases. however, few studies have addressed their geographic distribution and larval habitat characteristics [ ] . there are complex interactions between these factors that significantly impact mosquitoes ecological fitness and vectorial capacity for disease transmission, with important implications for vector management and control at the local and regional levels [ , ] . therefore, studying these factors for different mosquito fauna will help in monitoring potential modifications of larval habitats due to rains, global climate change, or man-made activities. previous studies on the ecology, distribution, and abundance of mosquito species in kingdom of saudi arabia are generally few and sporadic; and most of these studies were conducted in the western and southern regions. these studies were conducted in the asir province in - and - [ , ] [ , ] . these studies reported the presence of many species from many genera, the most important of which are anopheles, aedes, and culex. among these studies, only a few provided the description of habitats of the larvae of these vectors. even fewer studies provided evidence on the active role of some species on disease transmission; the existing ones were mainly for anopheles vectors of malaria [ , , ] , as well as aedes and culex vectors of arboviruses such as sindbis and dengue fever [ , , ] . rvf is not considered a major type in the arboviruses family, which mostly are adapted to a narrow range of vectors; however, among this family, the rvf infection has a very wide range of vector including mosquitoes such as aedes and culex, flies, and often, ticks [ ] . interestingly, for different rvf species, rvf vectors have special roles about how they sustain the transmission of the disease ecologically to humans [ ] . in some cases, the impact of rainfall, soil type, water, the persistence of breeding, and often wind, have significant effect on vector distribution [ ] . epizootics studies indicate that rvf disease follows unusually severe rainy seasons, a situation that may likely favor the breeding of a very large insect population, needed as a vector prerequisite. globally, rvf epidemiology was first reported in africa with the rvf epizootics in kenya when laboratory test reports confirmed virus isolation [ ] [ ] [ ] . in , the disease, for the first time, affected humans and livestock outside africa, with the larger rvf disease incidence following outbreaks, reported in saudi arabia [ ] and yemen. lately, rvf infections have been associated with minimal genetic diversity, epidemiologically; which has lately been considered to be a newly introduced single lineage of rvf viral disease [ ] . epidemiological reports from both saudi arabia and yemen showed that the outbreak, which occurred in , resulted in about human infections, and deaths [ ] . furthermore, the fatality rate reported in southern saudi arabia then, reached %, and was considered the most severe epidemic in that area ever since [ ] . moreover, the disease outbreak was thought to have been transmitted in countries such as saudi arabia by infected imported ruminants from east africa via the port of djibouti and probably from kenya and/or sudan [ ] . however, the fact remains that the rvf epidemic has been around for more than years, with infections occurring at prolonged intervals in eastern and southern africa [ , ] . consistent with this, another report showed that the same virus strain was implicated in the - rvf outbreaks in kenya and the outbreaks in saudi arabia and yemen [ ] . the outbreaks in kenya later resulted in about , human infected with about patients deaths [ , ] . surprisingly, in , jup et al. found the mosquito species that was identified as a potential vector, which led to the assumption that the zoonotic viral disease in saudi arabia was transmitted by culex tritaeniorhynchus [ ] . other species of mosquitoes were implicated in the transmission of this viral disease in other countries closer to saudi arabia [ ] [ ] [ ] . furthermore, another study reported the unexplained rvf virus infection among people from saudi arabia, with isolation and genetic virus characterization associated with illness in livestock, along the southwestern border of saudi arabia in september [ ] . the study reported that vertical transmission of the virus in the epidemic mosquito vector occurred in saudi arabia. in addition, the study stated that the most abundant culicine mosquitoes collected were aedes vexans arabiensis, culex pipiens complex, and culex tritaeniorhynchus, which were considered to be the most important epidemic and epizootic vectors of rvf virus in saudi arabia [ , ] . however, the same study, focusing on a very important issue which occurred during the rainy seasons; suggested that aedes vexans arabiensis has the potential to be an important epidemic and epizootic vector because of the tremendous numbers of individual mosquitoes produced after a flood [ ] . characteristically, once the virus is introduced into permissive ecologies, it becomes zoonotic; thus, they are able to enhance vulnerability of the area to periodic outbreaks, with the potential to spread further into non-endemic environments with favorable conditions [ , ] . saudi arabia is considered a region where rvf virus has circulated actively. noticeable data regarding zoonotic infection from animal to human from the arabian peninsula including saudi arabia has recently showed that it may be due to the consumption of unpasteurized camel milk [ , , ] . wernery reported camelus dromedarius as the animal host and/or reservoir of rvf zoonotic infection, which was diagnosed in the arabian peninsula [ ] . due to the scientific data regarding rvf disease, it is quite clear that globalization of trade and altered weather patterns are a concern for the future spread of more infections, since the causative agent of this viral disease is capable of utilizing a wide range of vectors for its transmission. thus, this poses a significant challenge to outbreak prediction, with inherently complex methods of infection control; therefore, mitigation and management of the virus will require concerted efforts [ , , ] . dengue hemorrhagic fever (dhf) viral disease is a serious global mosquito-borne infection. the clinical manifestation ranges from mild febrile illness to severe sickness which may include dengue shock syndrome [ ] . the dhf virus belongs to the genus flavivirus in the flaviviridae family, which can usually be spread by mosquitoes of the genus aedes aegypti, but less often through the genus aedes albopictus [ , ] . also, this virus is a single-stranded positive-sense rna virus that exists as four different serotypes (den- , den- , den- , and den- ) [ ] . in saudi arabia, the disease is limited to the western and southwestern regions, such as jeddah and makkah where aedes aegypti exists. however, all dhf cases in saudi arabia presented as a mild disease [ , ] . in fact, the first experience of dhf virus isolation from saudi arabia was recorded during an outbreak of the virus in [ ] , where the confirmed cases reported in jeddah were caused by denv- [ ] [ ] [ ] . however, during this first outbreak, in both summer and rainy season, at the end of the year, both denv- and denv- were isolated. in , during the rainy season in jeddah, there was an emergence of the denv- virus [ ] . in subsequent years, from - ; the emergence of dhf occurred with the three identified serotypes (denv- , denv- , and denv- ) isolated in jeddah [ ] . khan [ , ] . however, egger suggested that the reemergence of the disease in saudi arabia might be explained by the growing levels of urbanization, international trade, and travels [ ] . in keeping with the findings of most previous studies, the epidemiological occurrence of dhf infection using the saudi's national data indicated that the majority ( %) of patients with dengue virus infection were saudi nationals [ ] . on the contrary, from the epidemiological report based on saudi's national data in previous publications, an estimated % of patients with dhf presented in jeddah [ ] . kholedi [ ] . in yet another recent study, the virus was reported as % in saudi patients [ ] . all of these saudi studies were conducted in jeddah. from makkah city, the reported epidemiological study identified . % of dhf infection cases among saudi nationals [ ] . similarly, a later study puts the estimate at more than % of saudi nationals [ ] . these previously published studies suggest that differences in proportions may exist between saudi nationals infected with dhf virus in jeddah and makkah city. contrary to previous data from jeddah, in makkah, it was clear that the majority of patients presenting with clinically significant dhf were saudi nationals. therefore, these results emphasized the fact that saudi nationals are at greater risk of dhf infection. the awareness of these results is considered a cornerstone to enhancing the ability of healthcare professionals' identification of the disease; and this might play an important role in the development of effective eradication strategies for the disease in saudi arabia localities. furthermore, the first cases of the virus, confirmed in al-madinah in , showed that the isolated virus serotypes were denv- and denv- [ ] . in , the moh in saudi arabia reported a total of cases of the dhf infection, with an estimated case fatality rate of about . per thousand in saudi arabia [ ] . in august , several countries in asia, including malaysia, singapore, and pakistan reported about , , , and dengue cases including deaths, respectively. in the same period ( ), saudi arabia reported confirmed dengue cases in makkah, of which occurred in august , suspected cases, and cases pending laboratory confirmations. from these epidemic data indicating the reemergence of dhf infection in saudi arabia; jeddah, makkah, and al-madinah were shown to be the more susceptible areas, for this infectious disease, and this could be due to the fact that these cities are the sites of both the annual hajj pilgrimage and/or the minor umrah pilgrimage, which draw millions of muslims to saudi arabia [ , ] . currently, there are few epidemiological studies on dhf virus infection in saudi arabia. a study by al-azraqi et al. was conducted in hospitals and primary healthcare centers in two cities in the southern province of saudi arabia, particularly in jizan, and aseer. the study, which was limited to the seroprevalence among clinically suspected hospital-based patients, detected about . % positive cases of dengue virus igg among randomly selected patients attending the outpatient clinics for any reason. the associated risk factors were male gender, younger age ( - years) , lack of electricity, and having water basins in the house [ ] . the authors suggested that the virus may occur in sporadic cases in jizan, due to the nature of the city. jizan is relatively flat and located at sea level [ ] ; thus the likelihood of the formation of small stagnant water following the rainfall in the city is high [ ] . interestingly, a retrospective cross-sectional study, which compared the clinical findings and/or the diagnostic laboratory results in uncomplicated patients, and patients who developed dhf, was conducted at dr. soliman fakeeh hospital in jeddah, between january and june . about patients with a discharge diagnosis of dhf or dengue shock syndrome were identified [ ] . of these, ( %) were adult patients within the age range - years, and % were children with age ranging from months to years. however, among all these patients, % of the adults and % of the pediatric cases were males. the clinical data from the hospital showed that in the adult patients, about % made a full recovery without complications while two patients died [ ] . more recently in january , the moh began an intensive campaign to eradicate the dhf virus from saudi arabian cities, to enhance public health awareness, and facilitate a change in hygiene behavior of citizens and residents. this resulted in a . % reduction in the number of dhf infection among inpatient cases in jeddah when compared to the same period in the previous year. however, the overall drop in dhf cases reached % in , compared to the previous year [ ] . furthermore, recently, it is well-known that in saudi arabia, the dhf infection has been limited to the western and southwestern regions such as jeddah and makkah where aedes aegypti exists. however, all dhf cases in jeddah, saudi arabia, were mostly mild cases [ , , ] and the prospect of dengue virus control lies with vector control, health education, and possibly vaccine use. west nile fever is one of the emerging zoonotic infections, which is caused by an arthropod-borne virus belonging to the genus flavivirus, of the rna family flaviviridae. the virus' main reservoir, which is responsible for the transmission of the disease, is the genus culex mosquitoes [ , ] . the west nile virus (wnv) derived the name from the site where the first case was isolated in , from the blood of a woman with mild febrile illness living in the west nile district of uganda [ ] . the first outbreak, in - , was reported in israel [ ] . this constituted a turning point in the epidemiology of the virus, because it was thought to have originated from israel following the introduction from africa, and later introduced to the usa in [ , ] . subsequently, the infection was documented across the globe [ ] , with the exception of antarctica [ ] , in various species of vertebrates, including humans, mammals, non-human primates, birds, rodents, reptiles, and amphibians [ ] . however, birds are considered as one of the main reservoirs of the virus [ ] . saudi arabia is geographically close to several of the countries where wnv had circulated actively or had been reported; thus, there is a high risk of the disease being introduced into saudi arabia. wnv is known to cause neurological disease in both humans and horses. however, the clinical manifestations of the disease in horses include ataxia, paralysis of the limbs, recumbency, hyperexcitability, and hyperesthesia. in al-ahsa, saudi arabia, a study was performed on horses to test the incidence of the virus using the clinical examination and serologic elisa test. however, from this previous study, while clinical examination for neurologic signs detected no significant findings, wnv antibodies were positively identified at serology among . % of the tested population [ ] . in , lanciotti et al. found this virus to be responsible for an outbreak of encephalitis in two fatal human cases from northeastern usa in late summer; and suggest a closely relation between this outbreak in the usa to a wnv infection which was isolated from a dead goose in israel in [ ] . the first cases of wnv in horses was identified in egypt and france in the s [ ] ; ever since, wnv has had significant public health impact worldwide due to its resurgence and dynamic epidemiologic features in humans and animals. between and , a study in iran identified wnv antibodies in horses, and the results confirmed the highest activity of the virus reported in the western and southern provinces with seroprevalences of up to % in some areas of iran [ ] . although human cases and/or animal infections with wnv including horses have also been reported in jordan and lebanon (direct and close neighbors of saudi arabia) between and [ ] [ ] [ ] ; however, the reported wnv in patients or horses in these areas might have circulated in natural transmission cycles with close relationship to the wnv isolated from human and horses in jordan, lebanon, and iran in , , and , respectively. humans and horses (incidental hosts), are unable to develop sufficient viremia to infect mosquitoes, hence, they are not included in the wnv lifecycle [ ] . more recently, in , using standard procedures, the central veterinary research laboratory in dubai, the united arab emirates, described the first wnv isolation in a dromedary calf; and this supports the conclusion that wnv is present in the country [ ] . the wnv zoonotic infection was probably transmitted through the human-animal interface; that is through the well-known contact with infected arabian camels in saudi arabia. interestingly, dromedary are exported from the united arab emirates to saudi arabia and vice versa; due to the closely related wnvs genes and their circulation through the natural transmission cycles worldwide, a complete genome sequencing for more wnvs strains, as well as comparative genomic and phylogenetic studies in saudi arabia, are needed to ascertain whether the dromedary infection with wnv exists in the country or not. however, the same facts have been suggested recently ( ), when it was suggested that wnv infection was introduced into turkey at the time of the outbreaks in saudi arabia and yemen. it was further suggested that the virus may have been introduced via unlawful entrance of viremic domestic or wild animals through the borders or through vectors that carry the virus into turkey [ ] . camels play an important role in public health issues regarding zoonosis and they have been involved in most of the zoonotic infections which occurred in saudi arabia in the last three decades. they are reported as sources of infections-including rabies, mers-cov, alkhurma virus, cchfv, and rvf virus [ , , , , , , ] -via direct physical contacts with camels and/or indirectly by having camels within or near the household in saudi arabia. however, some zoonotic infections among camels are sometimes asymptomatic; thus, they play a vital role in the mechanism of transmission of various diseases [ ] . furthermore, wernery et al. reported that wnv can be transmitted by mosquito bites in different species including to humans, horses, camelids, and many other mammalian species as well as reptiles and birds [ , , ] . to the best of our knowledge, there is still no extensive surveillance data regarding this disease among wildlife animals in saudi arabia. strikingly, several of the human zoonotic cases that involve camels-which included different viral, bacterial, and parasitic infections on the arabian peninsula-have recently been highlighted as being caused by the consumption of unpasteurized camel milk [ ] . currently, in this review, some aspects of the most common viral diseases of zoonotic importance in saudi arabia were summarized; these are presented in table . however, data regarding emerging and reemerging zoonotic viral diseases are reported as they occur from time to time from the same, new, and/or different localities from saudi arabia. while other viral zoonotic infections occur in other countries, which are considered to be close to saudi arabia, some infections spread to some localities within saudi arabia because of the geographical proximity as shown in figure . interestingly, some of these zoonotic viral pathogens were first exotic to saudi arabia (e.g., mers-cov and ahfv) and should be of more concern when reported in prevalence studies, and whenever they are detected by saudi authorities. epidemiological data should be focused more on both the trade routes and wildlife migration across the region, since these are potential risks for saudi arabia (e.g., from yemen, egypt, gulf areas, and sudan). fortunately, there are many ways and/or approaches to improve the control of such different zoonotic pathogens in animals and humans in saudi arabia. however, the control measures of these viral zoonotic pathogens will not only benefit saudi arabia or arabian peninsula but will also be of high benefit to other countries, especially those with low prevalence, by stopping or controlling the spread of the epidemic worldwide. prevention, control, and management of several zoonotic diseases usually require several important measures including the following. having vaccination protocols for all suspected animal species by the use of up to date vaccines and compliance with the standards needed for all animals. taking into account the highly needed and important investigation for these zoonotic viral diseases vectors, including vector breeding control (including vectors, hosts, and arthropods), and control of the animals (livestock) movements, with respect to trade and export [ , ] . because an intensive livestock trade exists between saudi arabia and its neighboring countries, there may be increased risk of reemerging viral diseases of all kinds [ , ] . this is supported by several previous studies concerned with the route of livestock trade between saudi arabia and the neighboring countries (e.g., rabies through yemen and/or oman [ , ] interestingly, some of these zoonotic viral pathogens were first exotic to saudi arabia (e.g., mers-cov and ahfv) and should be of more concern when reported in prevalence studies, and whenever they are detected by saudi authorities. epidemiological data should be focused more on both the trade routes and wildlife migration across the region, since these are potential risks for saudi arabia (e.g., from yemen, egypt, gulf areas, and sudan). fortunately, there are many ways and/or approaches to improve the control of such different zoonotic pathogens in animals and humans in saudi arabia. however, the control measures of these viral zoonotic pathogens will not only benefit saudi arabia or arabian peninsula but will also be of high benefit to other countries, especially those with low prevalence, by stopping or controlling the spread of the epidemic worldwide. prevention, control, and management of several zoonotic diseases usually require several important measures including the following. having vaccination protocols for all suspected animal species by the use of up to date vaccines and compliance with the standards needed for all animals. taking into account the highly needed and important investigation for these zoonotic viral diseases vectors, including vector breeding control (including vectors, hosts, and arthropods), and control of the animals (livestock) movements, with respect to trade and export [ , ] . because an intensive livestock trade exists between saudi arabia and its neighboring countries, there may be increased risk of reemerging viral diseases of all kinds [ , ] . this is supported by several previous studies concerned with the route of livestock trade between saudi arabia and the neighboring countries (e.g., rabies through yemen and/or oman [ , ] ; rvf through kenya, djibouti, and/or egypt [ , , ] ; cchf through sudan [ ] ; influenza through oman and egypt [ , , , [ ] [ ] [ ] ; wnv through emirates, egypt, jordan, and israel [ , , , , ] ; and dhfv through egypt [ ] ; as well as mers-cov and ahfv viral infections, which originated and are transmitted globally from saudi arabia) [ , , , , ] . therefore, it is clear that a huge gap still exists in the sharing of published data about the acknowledged epidemiology of zoonotic diseases in saudi arabia, which rigorously prohibits speculations about the health burden of people. currently, there are surveillance activities for some viral diseases-such as rabies, mers-cov, and influenza-but these are still being weakly addressed or neglected, especially at the human-animal interface. the important role of vaccination both in the prevention and control of animal diseases and the need to check the human sources in food or water must not be neglected. also, management of animals, both outdoors and indoors must be taken seriously. however, owners of pets clinics and pets stores should be held responsible in ensuring that they keep their pets' vaccination protocols up to date, and prevent any kind of animal behavior that might result in zoonotic risks to humans through bites or scratches by pets. therefore, pet clinics and/or pets stores should be always considered a serious public health issue and vaccination should be obligatory. therefore, the importance of the annual vaccination routine programs for all stray dogs against rabies, and regular investigation of other animals, should be considered. in addition to this, pet clinics and stores should monitor pets' health records, and their owners should be held fully responsible in ensuring that their animals remain healthy and fully vaccinated. this will guarantee for them and their neighbors a zoonotic disease-free environment (e.g., against rabies virus particularly in dogs). this is particularly important in view of the case of human rabies reported in march from a makkah hospital. this involved a -year-old saudi man who was admitted to the hospital with a history of an unprovoked scratch on his face by a dog. a month after his admission, his saliva pcr test confirmed rabies virus [ ] . nevertheless, rabies is endemic in animals in the arabian peninsula, with increasing numbers of reported cases form certain countries in the area including saudi arabia, yemen, and oman [ , ] . kuwait, qatar, and the united arab emirates are considered to be rabies-free, whereas there is no available information about bahrain [ , ] . furthermore, animal rabies cycle and cases reported in these endemic countries including saudi arabia are characterized by different animal species such as camels, cattle, goats, and sheep; however, the majority of cases are reported in feral dogs [ , ] . fortunately, studies about pets with different zoonotic infections from pet clinics and/or pet stores in saudi arabia have been rarely detected among cats, dogs, and baboons. however, there was a previous study, which reported the occurrence of toxocara canis infection in pets (dogs) in riyadh, saudi arabia [ ] . there were also two previous reports regarding a protozoan zoonotic infection of some pets with clinical manifestation, particularly in papio hamadryas baboon in riyadh [ , ] . in addition to this, another report highlighted the protozoan zoonotic infection in camels, in riyadh [ ] ; however, more of these kind of studies are needed, because, they provide important opportunities to present a clear picture about indoor and outdoor animals and zoonotic pathogens such as viral, bacterial, fungal, etc. which involved, in saudi arabia. by enhancing biosecurity and management in animal farms, the risk of reemerging pathogens particularly responsible for zoonotic diseases caused by viruses, can be reduced. this is a matter of economic importance; in view of the large livestock trade existing or that existed between countries in the indian ocean and eastern africa countries where several zoonotic diseases are endemic. however, a phylogenetic study strongly suggests that some zoonotic infections have been introduced into saudi arabia through ruminant trade [ , ] . furthermore, following the adoption of the recommended guidelines of the world organization for animal health through its office international des epizooties (oie) code, if such policies regarding the exportation and/or importation of animals are exactly followed, these would greatly limit the extent of this risk [ ] . furthermore, an emphasis should be made on surveillance to detect any sign of zoonotic disease that might occur in any animal kept directly in a quarantine station in any country of origin for days prior to shipment to another country to ensure no clinical sign develops during that period. in addition, the longer quarantine periods or restriction of imported animals-particularly pets (e.g., dogs, cats, rodents, and monkeys) or goats, sheep, and camels-from endemic countries may be effective in reducing the introduction of zoonotic viruses. of such measures, the control of vectors (e.g., ticks and mosquitoes), particularly the intermediate hosts and animal reservoirs, should be key components in the intervention strategy for zoonoses in saudi arabia. while the improving, enhancing, providing, and upgrading of laboratory techniques and/or testing in both veterinary and human medicines are fundamental to early detection and containing of any zoonotic disease or transmitted infection. indeed, epidemiologic evidence should be linked with the seasonal time during the year for different zoonoses, and/or with any symptoms related to zoonotic infections that occur on the mainland a few years earlier. up to date ecological factors on evolutionary issues, social movements, economic, and epidemiological mechanisms affecting zoonotic pathogens' or their persistence and emergence, are not yet well understood. however, studies on the ecological, socioeconomic, and health issues are needed to assess the sustainability and acceptability of measures by breeders, as well as information that ensures appropriate slaughtering or consumption practices, which will decrease the risk of infection to humans [ ] . due to these facts about the ecological cascade and evolutionary perspectives, authorities can provide valuable insights into pathogen ecology and can inform zoonotic disease control programs; and thus evaluate their global effect in terms of actual disease and its socioeconomic correlations. enhancing biosecurity and management in the treatments of various zoonotic infections may result in appropriate use of vaccinations, drugs, and antibiotics, however, the overuse of these agents result in various types of resistance. furthermore, regardless of the influenza virus resistance level to treatment, according to a serosurveillance, the enzootic influenza virus h n in egypt is endemic [ ] . the same result to oseltamivir-resistant influenza viruses are reported globally, with a high susceptibility to these antiviral drugs among all reported cases of the virus from egypt. resistance was also found in most infected viral cases that are usually acquired in humans through intensive contacts, particularly with backyard birds, among women and children [ , , , ] . therefore, drug regimens in saudi must include vaccines against this virus during hajj and umrah seasons, for egyptians. most importantly, epidemic zoonotic cases of influenza among pets has highlighted the importance of circulating influenza viruses globally, and the importance of ensuring the effective use of antivirals for the prophylaxis and treatment of influenza, especially because of the increased number of new pets stores in saudi arabia, particularly in riyadh [ , ] . thus, studies on drug resistance are considered to be of a high public health importance, although, this might demonstrate the best scenario of how drug resistance in saudi arabia can pave its own way and/or role into the reemerging of different zoonotic pathogens. on the other hand, few studies have been done in this area to identify the relationship between different gatherings and the occurrence of signs and clinical symptoms of viral infections, especially among humans of different ages and gender. however, there are several suggestions and information regarding zoonoses (e.g., influenza and mers-cov infections) in saudi arabia among the elderly, based on age and gender [ , ] . more recently, increased availability of limited public health data on the prevalence of some zoonotic diseases and associated risk factors or data that identifies the relationship between different zoonotic pathogen antibodies in pregnant women, are of importance [ , ] . central to the profound worldwide changes in religious beliefs and activities is the birth of a new era of both emerging and reemerging diseases that could be arranged under the umbrella of social movements, along with its own role in the spread of zoonotic diseases. thus, any prevention and/or control strategies against any zoonotic pathogen have to take this point of view into account. furthermore, annually, saudi arabia hosts the largest international gathering of hajj where many millions gather in a small geographical area. this puts saudi arabia in the front line of threats of pandemic diseases [ ] . thus, saudi arabia must keep a high level of alertness in monitoring the situation of these pathogens, particularly in view of the potential for global spread of pandemic viruses especially during winter and around the hajj season (e.g., mers-cov infections, ahfv, and influenza viruses). therefore, there is need to prevent further spread of the virus locally, regionally, and internationally. interestingly, with wnv outbreaks, the israeli-like wnv that was isolated in white storks in egypt in - suggests that migrating birds do play a crucial role in the geographical spread of the virus [ ] . recently, the same fact was again suggested in , when the same infection by this virus was introduced into turkey at the time of the outbreaks in saudi arabia and yemen; it was stated that the wnv virus might have been introduced via unlawful entry of the viremic domestic or wild animals through the borders, or by vectors carrying the virus to turkey [ ] . more recently, epidemiological data of zoonotic viral pathogens from saudi arabia and/or from other neighboring countries after it was confirmed through laboratory test isolation from dromedaries (e.g., rabies, mers-cov, rvfv, and wnv) may enhance a high interest in the search for other novel zoonotic viruses in dromedaries [ , , , , , ] . furthermore, the habits of ingestion off unpasteurized milk from camels as a rare delicacy by saudi people need to be checked. moreover, viral pathogens such as rvfv are acquired through the importation of camels, while the remaining pathogens (e.g., rabies and influenza viruses) are endemic worldwide. of these (e.g., influenza virus), there is need for a highly preventive zoonotic control in saudi, due to fact that the isolation and genetic characterization of h n was reported in among vaccinated meat-turkeys flock in egypt, a neighboring country, that was previously reported to have more than , travelers to saudi arabia during hajj pilgrimage seasons, annually [ ] . this might be considered as one of such important risk factor of possible introduction or spread of influenza pathogen in saudi arabia [ , ] . lastly, increased zoonotic pathogens surveillance, particularly influenza, during the hajj season, increased infection control interventions, screening, and quarantine of suspected cases, provision of adequate medical treatment, sustainable awareness, increased education and training of target groups at high risk (e.g., doctors, nurses, veterinarians, and animal workers such as farmers and abattoir workers, etc.) are of great importance to reduce the burden of zoonoses among saudi arabian localities. fortunately, in collaboration with three organizations-including the moh in saudi arabia, the usa centers for disease control and prevention, and the who-a successful preparedness plan during the hajj season was put in place to vaccinate all pilgrims before leaving their home countries [ ] . altogether, there is an urgent need for collaborative surveillance and intervention plans for the control of zoonotic pathogens in saudi arabia. with saudi arabia, the focal point of the ongoing zoonotic pathogens outbreak could be due to the large number of religious pilgrims congregating annually particularly in makkah, jeddah, and al-madinah, the main three cities for hajj and umrah, which drastically increased the potential for uncontrolled global spread of zoonotic infections [ ] . a zoonotic pathogen outbreak could be dramatically decreased among the annual saudi pilgrims if we take into account the fact that: jeddah governorate, the main seaport in saudi arabia is considered to be the main entry point for over million pilgrims coming for hajj or umrah annually. all these numbers of pilgrims arrive through the jeddah islamic port before going on to makkah, for the start of their umrah and/or hajj. surprisingly, the current review showed that during an outbreak, each of these eight most zoonotic viruses (rabies, mers-cov, influenza, ahfv, cchfv, rvfv, dhfv, and wnv) which occurred and/or cases confirmed in saudi arabia particularly from (jeddah and/or makkah) areas with at least one or all of these eight zoonotic viral pathogenic diseases [ , , , , [ ] [ ] [ ] [ ] , , , ] . the spread could also have been due to the fact that jeddah is the main port for animal importation to saudi arabia. at the same time, it is the closest area to several countries where some zoonotic outbreaks were reported. to enhance this spread, the role of the active circulation of zoonotic viruses, during their natural transmission cycle, has been reported, however, an importation might increase risk of disease introduction to saudi arabia. • almost annually, from the more than million pilgrims who come to makkah and madinah from different countries worldwide during hajj and umrah, the kingdom's revenue in was put at more than billion saudi riyals (~about . billion us dollars), % up from the figures. this hajj revenue accounted for % of the gross domestic product for the kingdom of saudi arabia. to avert all that number of health hazards from zoonotic diseases in view of economic facts, the global community and particularly the pilgrims need more gift items made in saudi arabia to control and prevent the spread of zoonotic diseases which could be transmitted among hajj and umrah pilgrims. therefore, the following recommendations are suggested in order to improve public awareness and/or health education of zoonotic viral diseases in saudi arabia: based on findings of previous studies, health education strategies could enhance the awareness of the saudi population regarding viral zoonotic diseases through health education program experiences of other countries, particularly during hajj and umrah seasons. this response can draw on the availability of several studies on how to improve, control, and prevent the spread of several zoonoses in both animals and humans, worldwide [ , [ ] [ ] [ ] [ ] . public health authorities must highlight the importance of promoting health education and facilitate the outcomes of studies for reducing patient cases in saudi arabia. the saudi authorities and government bodies such as the moh should also launch different programs and workshops to increase public awareness about these zoonotic infections. this should involve the cooperation of the saudi regime, and the private and public sectors. different activities may be needed in saudi arabia-such as the practice of self-protection against these diseases, adult control strategies, control activities, and regular workshops-to achieve control and prevention. enhancing of self-awareness among people through health education programs or other strategies for the prevention of viral zoonotic diseases, which require vectors (such as mosquitoes, ticks, and fleas) for their transmission; are important issues on which the saudi population should be educated. they should also be educated about the adverse effects of arbitrary application of insecticides without prior knowledge on dose, resistance, and side effects. increasing the knowledge about the biology and ecology of the animal vectors in society is also crucial. furthermore, the saudi ministry of culture and information should establish intensive health education programs on television channels, radio, and newspapers to increase public awareness and to maintain hygiene conditions within the kingdom and in saudi houses. the saudi ministry of agriculture could play a big role by regularly controlling the application of vaccinations and/or antibiotics on animals which used in the veterinary sector, and also accounting the misuse of such agents following other developed and developing countries on controlling and/or accounting drug strategies [ , , ] . thus, veterinary regulations of animal antibiotics-including overuse of drugs and their application-must be enforced to alleviate the serious public health problems. funding: this research received no external funding. the authors thank the dental oral rehabilitation (dor) research center at king saud university, college of dentistry, kingdom of saudi arabia, riyadh for their support. also the authors wish to appreciate all the researchers whose articles were used in the present research. the authors declare no conflict of interest. the life and work of rudolf virchow - : cell theory, thrombosis and the sausage duel joint who/fao expert committee on zoonoses viral zoonosis: a comprehensive review zoonotic disease programs for enhancing global health security risk factors for human disease emergence anthropogenic environmental change and the emergence of infectious diseases in wildlife host range and emerging and reemerging pathogens emerging of new viral zoonoses emerging pathogens: the epidemiology and evolution of species jumps global trends in emerging infectious diseases perspectives on emerging 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years, a number of reports have appeared describing the successful treatment of patients with sepsis. moreover, novel data on the anti-viral potential of phages may be interpreted as suggesting that phages could be used as an adjunct therapy in severe covid- . thus, clinical trials assessing the value of phage therapy in sepsis, including viral sepsis, are urgently needed. the world health organization (who) considers sepsis to be a global health priority. recently, it has been emphasized that, despite some success in preclinical studies on experimental sepsis, no significant progress has been achieved in clinical therapy [ ] . although more than a hundred clinical trials have focused on sepsis, none of them have provided data that could be used for improvements or a cure [ ] . furthermore, the most recent data indicate that, despite best efforts to provide protocol-based care pathways, mortality from sepsis may reach nearly % [ ] . gaidelyte et al. showed that most of the sepsis-causing bacteria carry functional phages that are released and circulate in the blood of septicemic patients [ ] . those phages can lyse other isolates of the same bacterial strain but not the pathogenic strain in sepsis. thus, those prophages play a role in clonal selection of pathogens in this disorder. at the same time, it appears reasonable that phage application may potentially be used in the treatment of sepsis based on both well-known anti-bacterial as well as non-bacterial activities of phages, especially those related to their anti-inflammatory and immunomodulating effects [ , ] . in fact, in addition to data obtained in experimental animals, there are already reports of successful phage therapy in patients with sepsis [ ] . in this review, we summarize the progress in treating sepsis with phage therapy over the last three years. leshkasheli et al. demonstrated the therapeutic efficacy of phage therapy in galleria mallonella larvae and in a mouse model of sepsis caused by acinobacter baumanii [ ] . similar data were obtained by wu et al., who showed that a phage effective against that pathogen can rescue lethal sepsis mice [ ] . interestingly, the effect of a single phage treatment ( ml ip at a dose of ) was comparable to the effect of a phage cocktail containing phages. in experiments performed by wang et al., phage therapy applied concurrently with the inoculation of the pathogen rescued % of mice, whilst phage administration applied h after inoculation with a. baumanii reduced survival to approx. % [ ] . phage therapy efficacy has also been studied in a mouse model of neonatal sepsis caused by escherichia coli, klebsiella pneumoniae, haemophilus influenzae, pseudomonas aeruginosa, citrobacter freundii and moraxella catarrhalis. a single intraperitoneal (ip) phage dose rescued - % of mice depending on the phage dose. more detailed studies have revealed that a concentration as low as . moi (multiplicity of infection) was effective in rescuing % of mice, whilst a . moi dose rescued only % of mice. interestingly, the mice could be rescued even when phage administration was delayed for h after pathogen inoculation. while both a single phage and phage cocktails were effective, optimal results have been achieved with cocktails [ ] . high effectiveness of phage therapy in the treatment of experimental sepsis induced by multidrug resistant p. aeruginosa was also confirmed by alvi et al. [ ] . phage-treated bacteremic mice had a survival rate of almost %, and no viable pathogen could be detected at h post inoculation. the authors estimate that, in order for therapy of sepsis to be efficient, phages should persist in the blood for at least - h. a native agarose gel electrophoresis was applied to assess phage surface associated with high blood persistence [ ] . phages have been shown to upregulate gene expression of an anti-inflammatory cytokine il-r antagonist [ ] and downregulate nf-kappa b signaling [ ] . interestingly, suppressing nf-kappa b signaling may be beneficial in an experimental mouse model of sepsis [ ] . a -year-old boy with digeorge syndrome, recalcitrant p. aeruginosa bacteremia, and an allergy to antibiotics received a cocktail of two phages every h intravenously (iv) for h. treatment with antibiotics, including meropenem, tobramycin, and polymyxin b, was also continued. blood cultures turned negative and then positive again after cessation of phage therapy. the therapy was subsequently resumed, and blood cultures were reverted to negative again. thus, in this patient, phage therapy resulted in sterilization of the blood [ ] . jennes et al. described a patient with acute kidney injury complicated by septicemia caused by colistin-only sensitive p. aeruginosa and acute kidney failure. the patient received a ml cocktail of two phages in a h iv infusion for days. blood cultures turned negative immediately, c-reactive protein (crp) dropped, fever disappeared, and renal function recovered [ ] . recently, australian authors have reported results of adjunctive phage therapy (combined with antibiotics) of s. aureus bacteremia in patients, most of whom suffered from infective endocarditis. a phage cocktail composed of three phages was administered twice daily for days. a pfu infused phage dose yielded approx. × pfu/ml of blood, which suggests that such a dose may be adequate to achieve a desired therapeutic effect. the iv infusions were well tolerated; no fever, rashes, hypotension or other adverse reactions were observed. clinical improvement was evident in eight of the patients, and inflammation markers declined during or soon after the therapy [ ] . further progress in phage therapy of sepsis has recently been achieved by introducing engineered phages used to treat a patient with a disseminated drug resistant mycobacterial infection. genome engineering and forward genetics were applied to obtain lytic phage derivatives that were infused iv as a three-phage cocktail ( pfu per dose of each phage) twice daily for weeks. phage infusion was well tolerated, without significant side effects. serum phage titers reached levels exceeding /ml. weak anti-phage protein antibody responses were noted, but no evidence of phage neutralization was observed. the clinical condition of the patient improved while sterilization of the blood and sputum was achieved [ ] . sepsis is one of the principal causes of morbidity and mortality in neonates and young children in low and middle-income countries. recently, scientists in iraq have developed a cocktail of phages against pathogens implicated in neonatal sepsis in a bagdad teaching hospital (e. coli, k. pneumoniae, h. influenzae, p. aeruginosa, c. freundii, and m. catarrhalis). the cocktail containing phages showed activity against all bacterial hosts in vitro and should be further examined for its in vivo efficacy in experimental and clinical sepsis [ ] . in summary, in animal studies, phages were administered ip at a dose of - , and the outcome was assessed as animal rescue and reduction of bacterial burden in organs or blood. in human studies, the outcome was assessed as a clinical improvement combined with laboratory signs (e.g., disappearance of fever, improvement in general patient well-being, improvement in organ function (e.g., renal function), drop of crp, negative blood cultures). no significant adverse effects were observed in treated patients, which is in line with our data on large cohorts of patients [ ] . phage treatments elicit antibody responses; however, those serum antibodies do not appear to significantly influence the outcome of therapy [ ] . on the other hand, it cannot be excluded that phage therapy also induces the formation of a phage-antibody immune complex. therefore, the clinical significance of a humoral response to phages during the therapy requires further studies. furthermore, more detailed studies on phage pharmacokinetics would be helpful for the advancement of phage therapy. recent data suggest that disturbances in the microbiome can enhance susceptibility to sepsis [ ] . interestingly, fecal microbiota transplantation (fmt) may rescue mice from human pathogen-mediated sepsis [ ] . additionally, fmt improves survival in sepsis induced in rats [ ] . notably, it has been suggested that transfer of phages may play a role in the efficacy of fmt [ , ] . interestingly, a clinical trial of fmt for patients with covid- is ongoing [ ] . moreover, promising results have been achieved in subgroups of septic patients treated with the il-r antagonist ( day mortality . % vs. . % placebo) [ ] . in addition to the data presented earlier [ ] , these findings seem to strengthen arguments for the potential application of phages in the treatment of sepsis. in addition to phages, phage-derived lytic enzymes (lysins) have also been studied as a potential weapon against multi-drug resistant bacteria. recently, the results of a first placebo-controlled clinical trial involving an antistaphylococcal lysin (exebacase) administered in conjunction with antibiotics in patients with s. aureus septicemia (most with infectious endocarditis) have been published. in comparison to the control (antibiotics only), the exebacase group showed higher responder rates as well as a reduction in length of stay and readmission rates. no hypersensitivity reactions to exebacase were reported. although preexisting anti-lysin antibodies were detectable in some patients, this did not affect the efficacy and the safety of treatment. these results offer the first tangible opportunity to improve clinical results and reduce mortality in staphylococcal sepsis using phage-derived lysin [ ] . table summarizes the recent progress in successfully applying phages and lysin in experimental and clinical sepsis. severe acute respiratory syndrome coronavirus (sars-cov- ) probably originated from a virus that has been circulating in horseshoe bats for several decades. it was first detected in wuhan, china, which suggests the existence of an intermediary (pangolin?) that facilitated transmission to humans. there were also hints that the virus escaped or was deliberately released from a local institute [ ] . as of september there have been almost million covid- cases worldwide with > million deaths, and > million patients have recovered [ ] . it has been noted that severe covid- patients may develop typical manifestations of septic shock with blood and respiratory tract cultures testing negative for bacteria and fungus. therefore, viral sepsis could be responsible for clinical manifestations in those patients in whom systemic cytokine storm, lymphopenia, and thrombotic complications are usually detectable. effective antiviral therapy combined with attempts to modulate the innate immune response and upgrade the adaptive immune response are recommended to improve the outcome [ ] [ ] [ ] . in fact, antiviral and anti-inflammatory treatments may be effective early in the disease [ , ] . the lungs-the primary target organ of the sars-cov- virus-are relatively accessible to phages delivered by different routes, including oral administration. however, nasal or tracheal delivery are preferred to achieve sufficient in situ concentrations. aerosol phage preparations as well as nebulized preparations may serve as the most efficient therapeutic applications [ ] . growing data suggests that phages may interfere with the pathogenic action of eukaryotic viruses [ ] . this historical data has been supported by new findings indicating a protective action of the t phage on human lung epithelial cells infected with human adenovirus (adv); furthermore, adsorption of adv to human lung and kidney epithelial cells as well as viral replication was also inhibited [ ] . new data suggest that cell layers of the body may be the major sink for administered phages; interestingly, lung epithelial cells show the highest accumulation of phages [ ] . moreover, the expression of adv genes and synthesis of adv dna may also be downregulated by t and staphylococcal phages [ ] . we hypothesized that phage therapy might be helpful in combatting covid- [ ] . thus, it is known that cov-expressing cells display markedly upregulated levels of reactive oxygen species (ros), and high levels of ros are observed in the lungs of patients with covid- [ , ] . phages downregulate ros production induced by bacteria and endotoxins [ ] . lymphocytopenia is frequently found in covid- , while the virus is known to induce apoptosis [ , ] . moreover, autopsies have revealed atrophy of the spleen and the lymph nodes [ ] . interestingly, phages may reduce apoptosis of human airway epithelial cells when cultured in vitro [ ] . in addition, sweere et al. demonstrated that pf phages cause upregulation of interferon (ifn) alpha and il- , thus promoting an antiviral signature in the lungs of mice [ ] ; gogokhia et al. found that phages of lactobacillus, e. coli and bacteroides stimulate production of another potent antiviral cytokine, ifn gamma [ ] . recently, we showed that the t phage induces upregulation of the human defensin gene (hbd ) [ ] . this peptide, which is exposed primarily by epithelial cells, reduces viral replication and may enhance pathways responsible for other anti-microbial effects, both anti-bacterial and anti-viral. hbd activates primary anti-viral innate immune responses [ ] . it suppresses hiv infection of hela cells in vitro [ ] , drastically reduces human respiratory syncytial virus (hrsv) infection of human lung epithelial cells [ ] , and inhibits the infectivity of hiv virions of human tonsil epithelial cells [ ] . thus, defensins have been shown to participate in antimicrobial defenses in the human respiratory tract, and the up-regulation of hbd may enhance those defenses [ ] . therefore, t -induced hbd could also be engaged in mediating anti-sars-cov- defenses, which requires experimental confirmation. an inflammatory response causes activation of the hemostatic system (endothelial and platelet activation and coagulation promoting thrombosis)-a syndrome also referred to as thromboinflammation which is relevant in covid- [ ] . in fact, thrombocytopenia is associated with increased risk of severe disease [ ] . platelets are known to interact with viruses and have recently been shown to be transient carriers of hiv, thus contributing to hiv dissemination by propagating the virus to macrophages. this process could be prevented by the anti-integrin alphaiib/beta antibody [ ] . coronaviruses may also infect bone marrow cells [ ] . sars-cov rna may be present in platelets of covid- patients, which suggests that platelets can participate in the dissemination of the virus [ ] . it would be of interest to determine if this phenomenon could also be blocked by the anti-alphaiib/beta antibody, which has been effective in preventing hiv dissemination. the alphaiib/beta integrin binds specifically to a kgd (lys-gly-asp) sequence motif exposed on the gp capsid protein of t phages. therefore, t phages could interfere with platelet-dependent sars-cov dissemination, mimicking the effect of the anti-alphaiib/beta antibody. in fact, it has been demonstrated that such interference could enable t phages to reduce the adhesion of platelets to fibrinogen [ ] . interestingly, there appears to be another target for anti-covid- effects of phages. sars-cov- binds to its receptor, angiotensin-converting enzyme (ace ), through the receptor-binding domain (rbd) present in its major structural protein spike. recently, an exposed kgd motif has been identified in ace , thus enabling it to interact with integrin alphaiib/beta [ ] . therefore, platelets could associate with the sars-cov- -ace complex using their alphaiib/beta integrin receptor targeting the kgd sequence present within the ace molecule. this phenomenon could contribute to sars-cov- dissemination and upregulate platelet-mediated coagulopathy and tissue injury. the presence of platelet-fibrin thrombi is common in lung lesions in patients with covid- and is considered to be the main target of therapy [ ] . occupation of the platelet alphaiib/beta integrin receptor by the phage kgd could inhibit platelet engagement with the complex formed by the virus and its receptor and prevent the ensuing pathology. in recent years, a number of reports derived from experimental studies in animals and human clinics have suggested the potential value of phage therapy in the treatment of sepsis. the activity of both anti-bacterial and non-bacterial phages is relevant for successful phage therapy. the anti-inflammatory and the immunomodulating properties of phages could also be useful in the treatment of severe covid- syndrome including viral sepsis (table ) . a recent article from china concludes that phage therapy in sepsis treatment can be expected in the near future [ ] . the data discussed in this review support this assumption. as pointed out, relevant clinical trials assessing the therapeutic value of phage therapy in those clinical settings are urgently needed [ ] . . phages can interact with epithelial cells and protect those cells from virus-induced damage and apoptosis; this could be especially relevant for lung epithelial cells. . phages may prevent viral adsorption to epithelial cells and downregulate viral replication in those cells. . phages may induce production of cellular chaperones protecting cells from viral injury (e.g., induction of hsp in human alveolar cells). . phages inhibit inflammation (downregulation of nuclear factor (nf) kappa b and reactive oxygen species (ros) production). . phages induce anti-viral immunity (e.g., induction of interferon (ifn)-alpha and ifn-gamma, defensin and inhibition of hsp ). . phages may interfere with severe acute respiratory syndrome coronavirus (sars-cov)-b binding to angiotensin-converting enzyme (ace ). author contributions: a.g. wrote the major part of the manuscript. a.g., j.b., and r.m. contributed to the conceptualization and reviewed the manuscript. all authors have read and agreed to the published version of the manuscript. funding: this work was supported by statutory funds of the institute of immunology and experimental therapy, wrocław, poland ( - ). a.g., r.m., and j.b. are co-inventors of patents owned by the institute and covering phage preparations. sepsis therapies: learning from years of failure of 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a primary epithelial cell screening tool to investigate phage therapy in cystic fibrosis bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection expansion of bacteriophages is linked to aggravated intestinal inflammation and colitis the effects of t and a / phages on the expression of immunologically important genes in differentiated caco- cells human β-defensin plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity human beta-defensins suppress human immunodeficiency virus infection: potential role in mucosal protection role of human beta-defensin- during tumor necrosis factor-alpha/nf-kappab-mediated innate antiviral response against human respiratory syncytial virus human beta-defensins and - cointernalize with human immunodeficiency virus via heparan sulfate proteoglycans and reduce infectivity of intracellular virions in tonsil epithelial cells farming the microbiome for homeostasis and health inflammatory response in relation to covid- and other prothrombotic phenotypes thrombocytopenia is associated with severe coronavirus disease (covid- ) infections: a meta-analysis platelets from hiv-infected individuals on antiretroviral drug therapy with poor cd + t cell recovery can harbor replication-competent hiv despite viral suppression platelet gene expression and function in covid- patients a potential inhibitory role for integrin in the receptor targeting of sars-cov- pulmonary post-mortem findings in a series of covid- cases from northern italy: a two-centre descriptive study effects of phage therapy on sepsis pathogens , , key: cord- - d o xk authors: choi, won hyung; lee, in ah title: the mechanism of action of ursolic acid as a potential anti-toxoplasmosis agent, and its immunomodulatory effects date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: d o xk this study was performed to investigate the mechanism of action of ursolic acid in terms of anti-toxoplasma gondii effects, including immunomodulatory effects. we evaluated the anti-t. gondii effects of ursolic acid, and analyzed the production of nitric oxide (no), reactive oxygen species (ros), and cytokines through co-cultured immune cells, as well as the expression of intracellular organelles of t. gondii. the subcellular organelles and granules of t. gondii, particularly rhoptry protein , microneme protein , and inner membrane complex sub-compartment protein , were markedly decreased when t. gondii was treated with ursolic acid, and their expressions were effectively inhibited. furthermore, ursolic acid effectively increased the production of no, ros, interleukin (il)- , il- , granulocyte macrophage colony stimulating factor (gm-csf), and interferon-β, while reducing the expression of il- β, il- , tumor necrosis factor alpha (tnf-α), and transforming growth factor beta (tgf-β ) in t. gondii-infected immune cells. these results demonstrate that ursolic acid not only causes anti-t. gondii activity/action by effectively inhibiting the survival of t. gondii and the subcellular organelles of t. gondii, but also induces specific immunomodulatory effects in t. gondii-infected immune cells. therefore, this study indicates that ursolic acid can be effectively utilized as a potential candidate agent for developing novel anti-toxoplasmosis drugs, and has immunomodulatory activity. recently, zoonotic diseases have been consistently occurring in different countries worldwide, causing serious threats in many countries and to humans globally. in this respect, the prevention and education of various zoonotic diseases are becoming a major issue. toxoplasmosis is a parasitic disease that is induced through the infection of toxoplasma gondii in many countries worldwide as a zoonotic parasitosis, causing serious diseases and chronic infection in various sites of the human body in all age groups, including adults and young children as well as pregnant women. in particular, because toxoplasmosis is a zoonotic disease that infects humans through cats, many people that have cats as pets, particularly pregnant women, need to take caution in this regard. in the biological aspect, t. gondii not only has intracellular organelles such as the golgi, endoplasmic reticulum, and mitochondrion, but also unique subcellular organelles such as the conoid, apicoplast, surface antigens (sags), dense granule proteins (gras), rhoptries, and micronemes [ ] . t. gondii has an inner membrane complex (imc) involving the plasma membrane, consisting of a unique double membrane structure which is combined with a cytoskeletal network. the imc acts as a major factor in the proliferation and growth for the survival of t. gondii, as well as in motility and cell division, which has been reported to include the t. gondii-infected cells treated with sf ( figure ). furthermore, it was clearly shown that there were significant differences between the untreated t. gondii-group and the experimental groups. in these aspects, it was demonstrated that ua effectively induced the selective anti-parasitic effect/action compared to the sf-treated group and the untreated group through a direct inhibitory effect on the viability of t. gondii. this shows that ua has the potential to be utilized as an anti-t. gondii candidate agent and/or a synergic compound with the existing drugs for developing novel anti-toxoplasmosis agents. therefore, these results indicate that ua strongly inhibits or blocks the survival of t. gondii by effectively inducing anti-t. gondii activity through the selective inhibitory activity and/or action against the viability of t. gondii. the t. gondii-infected cells treated with sf ( figure ). furthermore, it was clearly shown that there were significant differences between the untreated t. gondii-group and the experimental groups. in these aspects, it was demonstrated that ua effectively induced the selective anti-parasitic effect/action compared to the sf-treated group and the untreated group through a direct inhibitory effect on the viability of t. gondii. this shows that ua has the potential to be utilized as an anti-t. gondii candidate agent and/or a synergic compound with the existing drugs for developing novel anti-toxoplasmosis agents. therefore, these results indicate that ua strongly inhibits or blocks the survival of t. gondii by effectively inducing anti-t. gondii activity through the selective inhibitory activity and/or action against the viability of t. gondii. the normal lung cells were infected with t. gondii (cells: t. gondii = : ), and t. gondii-infected cells were treated with different concentrations ( . - µg/ml) of ursolic acid (ua) and sulfadiazine (sf) at • c for h, respectively; their survival rates were inhibited a dose-dependent manner. the results are presented as a percentage of the untreated normal group. * p < . was considered to be significant. t. gondii causes unique anti-apoptotic features and parasitic survival cycles or stages, such as the inactivation of apoptotic proteins during the parasitic life-cycle in host cells after infection, showing critical roles during the proliferation and survival of t. gondii. in particular, the pvm, that functions as a critical indicator during the proliferation stage of t. gondii, is activated in a time-dependent manner after cell invasion of t. gondii, which causes finally the proliferation and the growth of t. gondii. in this aspect, we investigated the inhibitory action of ua against subcellular organelles such as rhoptries, micronemes, and inner membrane complex that form its intracellular metabolic network as well as the homeostasis of t. gondii. as shown in figure , when t. gondii was treated with different concentrations ( and µg/ml) of ua and sf, respectively, the intracellular organelles (rhoptry protein (rop ), microneme protein (mic ), and inner membrane complex sub-compartment protein (imc sub- )) of t. gondii were markedly decreased in a dose-dependent manner, and their changes were clearly observed under electrophoresis. in particular, it is known that rhoptry plays as a critical factor when t. gondii enters into host cells during invasion stage [ ] [ ] [ ] [ ] [ ] . in these aspects, this result shows clearly that ua not only has the direct inhibitory effect against the survival of t. gondii but also induces the anti-t. gondii effect by strongly blocking or inhibiting the homeostasis and network structure of the intracellular organelles of t. gondii. therefore, these results demonstrate substantial evidence that ua consistently causes anti-t. gondii activity/action by effectively inhibiting the intracellular organelles of t. gondii. °c for h, respectively; their survival rates were inhibited a dose-dependent manner. the results are presented as a percentage of the untreated normal group. * p < . was considered to be significant. organelles of t. gondii t. gondii causes unique anti-apoptotic features and parasitic survival cycles or stages, such as the inactivation of apoptotic proteins during the parasitic life-cycle in host cells after infection, showing critical roles during the proliferation and survival of t. gondii. in particular, the pvm, that functions as a critical indicator during the proliferation stage of t. gondii, is activated in a time-dependent manner after cell invasion of t. gondii, which causes finally the proliferation and the growth of t. gondii. in this aspect, we investigated the inhibitory action of ua against subcellular organelles such as rhoptries, micronemes, and inner membrane complex that form its intracellular metabolic network as well as the homeostasis of t. gondii. as shown in figure , when t. gondii was treated with different concentrations ( and μg/ml) of ua and sf, respectively, the intracellular organelles (rhoptry protein (rop ), microneme protein (mic ), and inner membrane complex sub-compartment protein (imc sub- )) of t. gondii were markedly decreased in a dose-dependent manner, and their changes were clearly observed under electrophoresis. in particular, it is known that rhoptry plays as a critical factor when t. gondii enters into host cells during invasion stage [ ] [ ] [ ] [ ] [ ] . in these aspects, this result shows clearly that ua not only has the direct inhibitory effect against the survival of t. gondii but also induces the anti-t. gondii effect by strongly blocking or inhibiting the homeostasis and network structure of the intracellular organelles of t. gondii. therefore, these results demonstrate substantial evidence that ua consistently causes anti-t. gondii activity/action by effectively inhibiting the intracellular organelles of t. gondii. we evaluated the effect of ros and no production induced by ursolic acid in immune cells infected with t. gondii. the production of no and ros was measured in a dose-dependent manner when t. gondii-infected co-cultured immune cells were incubated with various concentrations ( - µg/ml) of ua and sf for h, respectively. in particular, the production of ros was increased in the t. gondii-infected group treated with ua compared with t. gondii-infected group. however, in µg/ml of ua, ros activity gradually indicated lower concentrations than other ua-treated groups (figure ), and these changes of ros activity show that ua acts as a modulator which consistently inhibits the proliferation and the growth of t. gondii by promoting the production of ros in immune cells after the parasite infection. even though sf-treated groups significantly increased ros production compared to the t. gondii-infected group, there were low ros-productive rates compared to ua-treated groups. furthermore, the rate of no production was strongly increased in t. gondii-infected group treated with ua compared with normal and t. gondii-infected groups, but in the range of µg/ml of ua, the rate of no production were low concentrations compared with other ua-treated groups ( figure ). however, the activities of both ros and no induced by ua were significantly increased more than normal and infection groups. interestingly, the activities of ros and no in t. gondii-infected cells treated with sf were lower than t. gondii-infected groups treated with ua. the activities of ros and no were significantly increased in t. gondii-infected groups treated with ua and sf, respectively, whereas their activities were gradually reduced in high concentrations. these results show that ua not only consistently induces the production of ros and no through the interaction between it and immune cells in t. gondii-infected immune cells, but may also be used as a synergic compound for the immunomodulation after parasitic infection. we evaluated the effect of ros and no production induced by ursolic acid in immune cells infected with t. gondii. the production of no and ros was measured in a dose-dependent manner when t. gondii-infected co-cultured immune cells were incubated with various concentrations ( - μg/ml) of ua and sf for h, respectively. in particular, the production of ros was increased in the t. gondii-infected group treated with ua compared with t. gondii-infected group. however, in μg/ml of ua, ros activity gradually indicated lower concentrations than other ua-treated groups (figure ), and these changes of ros activity show that ua acts as a modulator which consistently inhibits the proliferation and the growth of t. gondii by promoting the production of ros in immune cells after the parasite infection. even though sf-treated groups significantly increased ros production compared to the t. gondii-infected group, there were low ros-productive rates compared to ua-treated groups. furthermore, the rate of no production was strongly increased in t. gondii-infected group treated with ua compared with normal and t. gondii-infected groups, but in the range of μg/ml of ua, the rate of no production were low concentrations compared with other ua-treated groups ( figure ). however, the activities of both ros and no induced by ua were significantly increased more than normal and infection groups. interestingly, the activities of ros and no in t. gondii-infected cells treated with sf were lower than t. gondii-infected groups treated with ua. the activities of ros and no were significantly increased in t. gondii-infected groups treated with ua and sf, respectively, whereas their activities were gradually reduced in high concentrations. these results show that ua not only consistently induces the production of ros and no through the interaction between it and immune cells in t. gondii-infected immune cells, but may also be used as a synergic compound for the immunomodulation after parasitic infection. t. gondii proliferates rapidly in host cells through parasitic interactions and signals for its survival after cell invasion, which finally induces destruction of host cells. we evaluated the synergic effect and the activation of cytokines caused by ursolic acid in co-cultured immune cells (macrophages, t cells, b cells and basophil cells) infected with t. gondii. the production of the cytokines (interferon-β, granulocyte macrophage colony stimulating factor (gm-csf), transforming growth factor beta (tgf-β ), and tumor necrosis factor alpha (tnf-α)) in t. gondii-infected immune cells was notably changed in a concentration-dependent manner when the cells were incubated with different concentrations ( - μg/ml) of ua, and their changes were measured under an elisa reader. in particular, the production of interferon-β and gm-csf was effectively increased in t. gondii-infected cells treated with ua compared with t. gondii-infected cells, and these changes show that ua accelerated the production and release of interferon-β and gm-csf by stimulating the macrophage, b cells, and basophil cells ( figure ). moreover, the production of interferon-β was not changed in sf-treated groups, and the production of gm-csf was gradually decreased. furthermore, the expression of tgf-β and tnf-α was markedly reduced in t. gondii-infected cells treated with ua, whereas t. gondii-infected cells treated with sf did not cause the production of tgf-β and tnfα compared with other groups (figure ) . interestingly, t. gondii-infected cells treated with sf did not significantly produce the expression of interferon-β, tgf-β and tnf-α compared to the infected group. this shows that sf is not involved in immune response for expressing tnf-α, tgf-β , and interferon-β in immune cells. furthermore, the production of tgf-β was decreased in t. gondiiinfected cells treated with ua, but the result shows that ua reduced the expression of tgf-β in immune cells by directly inhibiting t. gondii. these results demonstrate that ua not only promotes defense against parasitic infection by effectively inducing the production of interferon-β and gm-csf from immune cells when the cells are infected with t. gondii, but also inhibits the overexpression of inflammatory cytokine by reducing the expression of tnf-α caused by infection. t. gondii proliferates rapidly in host cells through parasitic interactions and signals for its survival after cell invasion, which finally induces destruction of host cells. we evaluated the synergic effect and the activation of cytokines caused by ursolic acid in co-cultured immune cells (macrophages, t cells, b cells and basophil cells) infected with t. gondii. the production of the cytokines (interferon-β, granulocyte macrophage colony stimulating factor (gm-csf), transforming growth factor beta (tgf-β ), and tumor necrosis factor alpha (tnf-α)) in t. gondii-infected immune cells was notably changed in a concentration-dependent manner when the cells were incubated with different concentrations ( - µg/ml) of ua, and their changes were measured under an elisa reader. in particular, the production of interferon-β and gm-csf was effectively increased in t. gondii-infected cells treated with ua compared with t. gondii-infected cells, and these changes show that ua accelerated the production and release of interferon-β and gm-csf by stimulating the macrophage, b cells, and basophil cells ( figure ). moreover, the production of interferon-β was not changed in sf-treated groups, and the production of gm-csf was gradually decreased. furthermore, the expression of tgf-β and tnf-α was markedly reduced in t. gondii-infected cells treated with ua, whereas t. gondii-infected cells treated with sf did not cause the production of tgf-β and tnf-α compared with other groups (figure ) . interestingly, t. gondii-infected cells treated with sf did not significantly produce the expression of interferon-β, tgf-β and tnf-α compared to the infected group. this shows that sf is not involved in immune response for expressing tnf-α, tgf-β , and interferon-β in immune cells. furthermore, the production of tgf-β was decreased in t. gondii-infected cells treated with ua, but the result shows that ua reduced the expression of tgf-β in immune cells by directly inhibiting t. gondii. these results demonstrate that ua not only promotes defense against parasitic infection by effectively inducing the production of interferon-β and gm-csf from immune cells when the cells are infected with t. gondii, but also inhibits the overexpression of inflammatory cytokine by reducing the expression of tnf-α caused by infection. we evaluated the activation and interaction of various cytokines induced by ursolic acid through co-cultured immune cells (macrophage, t cells, b cells and basophil cells) infected with t. gondii. the changes of the cytokines (interleukin (il)- β, il- , il- , and il- ) in t. gondii-infected immune cells were measured in a dose-dependent manner when the cells were incubated with different concentrations ( - μg/ml) of ua, and their changes were evaluated under an elisa reader. in particular, the production of il- β and il- was reduced in the groups treated with ua compared with the infection group, and the expression of il- and il- was significantly increased in t. the changes of the cytokines (interleukin (il)- β, il- , il- , and il- ) in t. gondii-infected immune cells were measured in a dose-dependent manner when the cells were incubated with different concentrations ( - µg/ml) of ua, and their changes were evaluated under an elisa reader. in particular, the production of il- β and il- was reduced in the groups treated with ua compared with the infection group, and the expression of il- and il- was significantly increased in t. gondii-infected cells treated with ua (figures and ). these changes demonstrate that ua promotes the production and the expression of il- and il- by consistently stimulating the immune cells as well as inhibit the expressions of il- β and il- induced by t. gondii infection. interestingly, the production of il- β in t. gondii-infected cells treated with sf was significantly increased and the expression of il- was gradually decreased compared with the infection group. however, there were no significant changes of the production of il- and il- in t. gondii-infected cells treated with sf. in particular, significant differences in the cytokines were clearly measured between the t. gondii-infected group and ua-treated group, and these changes of cytokines were confirmed in all the groups. furthermore, the results show that this co-culture system through immune cells can be usefully utilized as a co-culture method for measuring various cytokines, and as an effective alternative to in vivo experiments. these results indicate that ua not only effectively induces the production of cytokines between the immune cells by increasing the expression of il- and il- in macrophage and b cells after t. gondii infection, but also suppresses or strongly reduces the expression of the inflammatory cytokines from the immune cells. gondii-infected cells treated with ua (figure and ). these changes demonstrate that ua promotes the production and the expression of il- and il- by consistently stimulating the immune cells as well as inhibit the expressions of il- β and il- induced by t. gondii infection. interestingly, the production of il- β in t. gondii-infected cells treated with sf was significantly increased and the expression of il- was gradually decreased compared with the infection group. however, there were no significant changes of the production of il- and il- in t. gondii-infected cells treated with sf. in particular, significant differences in the cytokines were clearly measured between the t. gondiiinfected group and ua-treated group, and these changes of cytokines were confirmed in all the groups. furthermore, the results show that this co-culture system through immune cells can be usefully utilized as a co-culture method for measuring various cytokines, and as an effective alternative to in vivo experiments. these results indicate that ua not only effectively induces the production of cytokines between the immune cells by increasing the expression of il- and il- in macrophage and b cells after t. gondii infection, but also suppresses or strongly reduces the expression of the inflammatory cytokines from the immune cells. in spite of constant public health efforts worldwide for the past decades, infectious diseases such as malaria, influenza, cholera, yellow fever, mers, sars, and tuberculosis have been consistently causing crises in public health as well as global concerns due to their pandemic potential. these serious infectious pathogens have enhanced adaptability to environmental variation and infectivity to host as well as resistance to existing drugs for their viability, evolving in a time-dependent manner. in these aspects, global nonprofit organizations and/or profit companies such as who and the bill & melinda gates foundation as well as various global leading pharmaceutical companies have continuously supported the study for blocking or treating chronic infectious diseases such as zika, aids, and tuberculosis as well as acute infectious diseases including influenza, malaria, cholera, yellow fever, and ebola, focusing on the development of novel drugs and effective vaccine against pathogens. furthermore, in spite of various efforts for developing anti-parasitic drugs against zoonotic parasitosis, effective novel drugs such as anti-malaria drugs have not yet been developed or launched. many people are living with animals such as cats and dogs. in particular, toxoplasmosis, a zoonotic parasitosis, is transmitted via physical contact with pets and companion animals, including cat and dogs. with respect to these aspects, we carefully focused on the parasitic infectious diseases that are consistently caused through various infection pathways and factors. among various parasites, t. gondii induces serious symptoms such as cerebral calcification and meningoencephalitis in the brain, while causing fetal infection through mother during pregnancy, as it is a zoonotic parasite which affects both humans and animals, resulting in parasitic diseases such as toxoplasmosis [ , ] . in spite of constant public health efforts worldwide for the past decades, infectious diseases such as malaria, influenza, cholera, yellow fever, mers, sars, and tuberculosis have been consistently causing crises in public health as well as global concerns due to their pandemic potential. these serious infectious pathogens have enhanced adaptability to environmental variation and infectivity to host as well as resistance to existing drugs for their viability, evolving in a time-dependent manner. in these aspects, global nonprofit organizations and/or profit companies such as who and the bill & melinda gates foundation as well as various global leading pharmaceutical companies have continuously supported the study for blocking or treating chronic infectious diseases such as zika, aids, and tuberculosis as well as acute infectious diseases including influenza, malaria, cholera, yellow fever, and ebola, focusing on the development of novel drugs and effective vaccine against pathogens. furthermore, in spite of various efforts for developing anti-parasitic drugs against zoonotic parasitosis, effective novel drugs such as anti-malaria drugs have not yet been developed or launched. many people are living with animals such as cats and dogs. in particular, toxoplasmosis, a zoonotic parasitosis, is transmitted via physical contact with pets and companion animals, including cat and dogs. with respect to these aspects, we carefully focused on the parasitic infectious diseases that are consistently caused through various infection pathways and factors. among various parasites, t. gondii induces serious symptoms such as cerebral calcification and meningoencephalitis in the brain, while causing fetal infection through mother during pregnancy, as it is a zoonotic parasite which affects both humans and animals, resulting in parasitic diseases such as toxoplasmosis [ , ] . furthermore, it induces serious complications in immune-deficient patients and hiv patients, which attenuates the interaction of the immune system that specifically responds to pathogens in the host [ , ] . t. gondii inhibits the interaction of cytokines such as il- and ifn-γ that are activated through the immune system, attenuating the production of cytokines from host cells. in addition, it is known that t. gondii blocks parasitic inhibitory system while inhibiting the signal pathways of cell cycle initiators or apoptotic mediators for the apoptotic stage in host cells after invasion [ , ] . t. gondii infection induces an imbalance of immune response by causing changes in cytokines such as tnf-α and tgf-β in the host, resulting in immune deficiency by breaking homeostasis and interaction of the immune system in the host. moreover, t. gondii induces the proliferative and growth cycle of t. gondii by rapidly forming pvm and by regulating cell cycle factors for its survival in host cells, maintaining the survival by accelerating the proliferation of t. gondii in host [ , ] . the subcellular organelles and granules of t. gondii, such as the conoid, gras, sags, the rhoptry, microneme proteins, and the inner membrane complex, were focused upon as major targets for blocking t. gondii infection, which was used as major recombinant proteins or factors for protective immunity including vaccination [ ] [ ] [ ] [ ] . for these reasons, in this study we not only evaluated the anti-parasitic effect of ua which inhibits the survival of parasite after t. gondii infection through a t. gondii-infected co-cultured immune system, but also confirmed its mechanism of action with respect to anti-t. gondii effects and immunomodulatory activity. in the present study, the survival rates of t. gondii were markedly inhibited when the parasites were treated with ua and sf compared with the non-treated t. gondii group, and the expression of subcellular organelles and granules of t. gondii in the infected cells was effectively decreased in t. gondii-infected group treated with ua compared with other groups. furthermore, the production of ros and no was significantly increased in the t. gondii-infected group treated with ua compared with both sf-treated and infection groups. interestingly, although ros production was activated in both t. gondii-infected cells treated with ua and sf, its rate was gradually decreased in the range of high concentrations of the compounds. in these aspects, no and ros production not only activates immune defense systems against various pathogens such as bacteria and viruses but also maintains effectively the changes of the homeostasis in the organism, promoting the production of cytokines induced by the interaction of immune cells. cytokines such as il- β, il- , and tnf-α were increased in the infected cells after t. gondii infection, and their production was significantly suppressed after ua treatment. in particular, although the production of tgf-β was reduced in t. gondii-infected immune cells treated with ua, it was shown that ua suppressed the expression of tgf-β in immune cells by inhibiting the survival of t. gondii. in addition, ua not only effectively increased the production of anti-inflammatory cytokines such as il- and interferon-β in t. gondii-infected cells but also activated the expression of il- and gm-csf, promoting the development of th cells and the production of interferon-γ that is induced by nk cells. taken together, the results of this study showed the anti-parasitic effects of ua against the survival of t. gondii as well as demonstrated its mechanism of action causing anti-t. gondii effect in t. gondii-infected co-cultured cells. furthermore, these results indicated that the expression of subcellular organelles and granules of t. gondii was specifically inhibited or blocked in a concentration-dependent manner after treatment with ua. this study provides evidence that ua not only decreases the inflammatory cytokines (il- β, il- and tnf-α) expressed from the immune cells after t. gondii infection, but also effectively increases the production of anti-inflammatory mediators such as il- , il- , gm-csf, and interferon-β, including the activation of ros and no production. interestingly, it was confirmed in this study that macrophage and other immune cells did not directly inhibit or rapidly decrease the survival of parasite as well as the proliferation of t. gondii through the bactericidal action such as phagocytosis of macrophage after t. gondii infection. this may be the result of the differences between the in vitro system and the in vivo experiment including rats and mice. however, the parasite may not be eliminated by the bactericidal action of macrophages and other immune cells in the infected body because t. gondii is found in various body sites of patients in the clinic. moreover, it may be changed or maintained to the parasite type that indicates characteristics of hibernation like t. gondii me strain (type ii) in the body. in summary, the results of this study demonstrate that ua induces anti-t. gondii effects/action by effectively blocking or inhibiting the survival of t. gondii, and also has anti-parasitic activity that consistently inhibits the proliferation/growth of t. gondii by strongly inhibiting the expression of subcellular organelles and granules of t. gondii, such as rop , mic , and imc sub in t. gondii-infected cells. in addition, these results show clearly that ua has the immunomodulatory action/activity which causes the change of cytokines in immune cells by decreasing the inflammatory cytokines (il- β, il- , and tnf-α) and/or by activating anti-inflammatory cytokines including il- , il- , gm-csf, or interferon-β through the interaction between ua and immune cells. furthermore, the results indicate that ua induces the death of t. gondii by promoting the production of ros and no, such as super oxides and hydroxyl radicals in t. gondii-infected immune cells. therefore, this study indicates that ua can be used effectively as a potent candidate agent or a synergic compound with existing drugs in the development of novel anti-t. gondii drugs of the next generation against toxoplasmosis, with potential as a modulatory substance against the immune response of the parasite in parasite-infected immune cells. moreover, ua requires further study for efficacy and safety against toxoplasmosis in preclinical stages through in vivo animal models in the near future. the anti-toxoplasmosis drug, sulfadiazine (sf), was dissolved in dmso, and ursolic acid (ua) was also dissolved in dmso to a concentration of mg/ml according to the manufacturer's instructions. sulfadiazine was used as a standard drug to evaluate whether or not ursolic acid has an anti-parasitic effect and activity against t. gondii. the compounds were filtered using . -µm membrane syringe filters (roshi kaisha, ltd., tokyo, japan) before use, and were stored at − • c deep-freezer until use. macrophages (raw . ), t cells (el ), b cells (fb ), basophil cells (rbl- h ), and normal lung epithelial cells (l ) were purchased from korean cell line bank at seoul national university and american type culture collection (manassas, va, usa). the cells were cultured in rpmi medium and dmem containing mm l-glutamine, supplemented with % decomplemented fetal bovine serum (fbs), penicillin ( units/ml), and streptomycin ( µg/ml) in a humidified atmosphere containing % co in air at • c. the t. gondii rh was suspended with × pbs, which was injected in the abdominal cavity of each balb-c/mouse. five days after the injection, t. gondii was collected from the peritoneal fluids of mouse kept in the abdominal cavities of the mice before use. in the in vitro study, host cells were infected with tachyzoite of t. gondii at a cell-to-parasite ratio of : . the inhibitory effect of ursolic acid against t. gondii was confirmed by directly evaluating the viability of t. gondii exposed to ua and sf through the mtt assay. briefly, after t. gondii was seeded in a -well plate ( × /well), t. gondii was incubated with different concentrations ( . - µg/ml) of ua and sf for h, respectively. the normal lung cells were infected with t. gondii (cells: t. gondii = : ), and t. gondii-infected cells were treated with different concentrations ( . - µg/ml) of ursolic acid (ua) and sulfadiazine (sf) at • c for h, respectively; then their viabilities were evaluated by mtt assay. it was to be determined whether or not ua has anti-parasitic activity and/or effect against t. gondii. the cells were divided into normal and experimental groups, and the survival rate (sr) of t. gondii was calculated as follows: % of sr = (od drug-tested wells − od blank )/(od control − od blank ) × . the optical density (od) was measured at a wavelength of nm using an elisa leader. the inhibitory effect of ursolic acid against the viability of t. gondii was evaluated by measuring the expression of subcellular organelles of t. gondii in t. gondii-infected cells exposed to ua and sf. after normal lung cells were seeded in a -well plate ( × /well), the cells were infected with t. gondii ( : , cells/parasite ratio), which was incubated with different concentrations ( - µg/ml) of ua and sf for h respectively, and their total rna was isolated from t. gondii strain using rneasy mini kit (qiagen, hilden, germany). total rna was obtained from untreated control and treated groups following the manufacturer's recommended procedure, and the total rna was quantified in an eppendorf biospectrometer (eppendorf, seoul, korea). one pair of primers was designed using the primer (version . ) and ncbi primer-blast according to the sequence of the selected specific gene in ncbi genbank as follows: t. gondii mic (accession number: af , bp), rop (accession number: am , bp), imc sub- (accession number: hq , bp), and β-actin (accession number: nm , bp). the specific genes of t. gondii were synthesized to complementary dna (cdna) through one-step rt-pcr kit (bioneer, daejeon, korea). the cdna was analyzed by electrophoresis in a . % (w/v) agarose gel containing µg/ml ethidium bromide at v for h, which was visualized under ultraviolet illumination. the β-actin was used as an internal standard for the amount of the expression of cdna present in each sample. the groups were divided into the untreated positive group (t. gondii-infected group) and experimental groups (ua and sf-treated groups). the effect of nitric oxide (no) and reactive oxygen species (ros) production of ursolic acid was confirmed by measuring no and ros production in t. gondii-infected co-cultured cells (macrophage and basophil cells) exposed to the compounds. the cells were divided into normal and experimental groups as described above. briefly, after the co-culture cells were seeded in a -well plate ( × /well), the cells were infected with t. gondii ( : , cells/parasite ratio), which was incubated with different concentrations ( - µg/ml) of ua and sf for h, respectively. the no and ros production were measured in the control and treated groups according to the manufacturer's instructions through no and ros production kits (cell biolabs, inc. san diego, ca, usa) respectively. the rates of no and ros production were measured using an elisa microplate leader (biotek instruments, inc., winooski, vt, usa). the effect of ursolic acid regarding immunomodulatory action was evaluated by measuring the expression of cytokines in t. gondii-infected co-culture immune cells (macrophage, t cells, b cells, and basophil cells) exposed to ua and sf, respectively. the cells were divided into normal and experimental groups. first, the macrophage and basophil cells were sequentially seeded in a -well plate ( × /well) in a humidified atmosphere containing % co in air at • c. second, t cells and b cells were additionally seeded in the plate ( × /well) after h. namely, the co-cultured immune cells were seeded in a -well plate ( × /well) and the cells were finally infected with t. gondii ( : , cells/parasite ratio) after h, and incubated with different concentrations ( - µg/ml) of ua and sf for h, respectively. the cytokines (tnf-α, tgf-β , interferon-β, gm-csf, il- β, il- , il- , and il- ) were respectively measured in normal, infected, and treated groups according to the manufacturer's instruction through cytokine elisa kits (thermofisher scientific, san diego, ca, usa) and inflammatory multi-cytokine kits (qiagen, hilden, germany), and the untreated t. gondii-infected cells were used as an positive group. all the results were expressed as mean ± standard deviation (sd) of three independent experiments. statistical analysis of the data was performed using student's t-test and analysis of variance (anova). a value of * p < . was considered to be statistically significant. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: 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surface antigen (sag ) of toxoplasma gondii targeted delivery of toxoplasma gondii antigens to dendritic cells promote immunogenicity and protective efficiency against this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - sonr or authors: lechien, jerome r.; ducarme, morgane; place, sammy; chiesa-estomba, carlos m.; khalife, mohamad; de riu, giacomo; vaira, luigi angelo; de terwangne, christophe; machayekhi, shahram; marchant, arnaud; journe, fabrice; saussez, sven title: objective olfactory findings in hospitalized severe covid- patients date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: sonr or objective: we investigate the prevalence of the self-reported and objective sudden loss of smell (sls) in patients with severe coronavirus disease (covid- ). methods: severe covid- patients with self-reported sls were recruited at hospitalization discharge. epidemiological and clinical data were collected. the sino-nasal outcome test- (snot- ) was used to evaluate rhinological complaints. subjective olfactory and gustatory functions were assessed with the national health and nutrition examination survey (nhnes). objective sls was evaluated using psychophysical tests. potential associations between olfactory evaluation and the clinical outcomes (duration of hospitalization; admission biology; one month serology (igg), and chest computed tomography findings) were studied. results: forty-seven patients completed the study ( females). subjectively, eighteen ( . %) individuals self-reported subjective partial or total sls. among them, only three and four were anosmic and hyposmic, respectively ( . %). considering the objective evaluation in the entire cohort, the prevalence of sls was . %. elderly patients and those with diabetes had lower objective olfactory evaluation results than young and non-diabetic individuals. conclusions: the prevalence of sls in severe covid- patients appears to be lower than previously estimated in mild-to-moderate covid- forms. future comparative studies are needed to explore the predictive value of sls for covid- severity. since the onset of the coronavirus disease (covid- ) pandemic, many patients reported sudden loss of smell (sls) [ ] . however, due to the health emergency, only a few studies investigated sls with objective testing, which remains essential to confirm the olfactory dysfunction [ ] [ ] [ ] . all these studies involved outpatients with mild covid- forms. the mean age and the prevalence of comorbidities were low [ ] [ ] [ ] , leading some authors to suspect that sls could be more specific to mild covid- forms [ ] . in this study, we investigated the prevalence of self-reported and objective sls in severe covid- patients. adults ( - years old) with severe covid- were recruited from the department of medicine of the epicura hospital (hornu, belgium, ethics committees: epicura - , institut jules bordet cr ). the disease was confirmed through nasopharyngeal swab (rt-pcr). patients were defined as severe covid- if they required continuous care (oxygenotherapy, blood pressure monitoring) in internal medicine or intensive care units. patients with a neurological disorder, chronic rhinosinusitis, or a history of nasal surgery prior to the pandemic were excluded. the lethal cases were not included because investigators assessed olfaction once the patient condition improved. epidemiological and clinical data were collected at the hospital discharge. details of the patient-reported outcome questionnaire used for data collection were reported in a previous study [ ] . briefly: ( ) olfactory and gustatory questions were based on the smell and taste component of the national health and nutrition examination survey; ( ) symptoms were evaluated through a point scale ranging from (no symptoms) to (severe symptoms) [ ] ; ( ) nasosinusal symptoms were evaluated through the french sino-nasal outcome test- (snot- ) [ ] . patients benefited from psychophysical olfactory evaluation through sniffin'stick tests (medisense, groningen, the netherlands): sixteen pens were presented to patients every s. the patient had to choose the adequate term describing the smell among given options. the test was scored on a total of points and allowed categorization into groups: normosmia (score between and ), hyposmia (score between and ), and anosmia (score < ) [ ] . moreover, the following hospitalization outcomes were recorded: duration of hospitalization (days); admission biology (d-dimer; hemoglobin; leucocyte count; lymphocyte count; crp; creatitin; bilirubin; platelet count; ldh; na + ; k + ; cl − ); month serology (igg); and chest computed tomography findings. meanwhile, subjective and objective evaluations were made. the relationship between clinical and olfactory outcomes was analyzed through multiple linear regression between scale variables and through the mann-whitney test and boxplot representation for groups versus scale variables (spss, v , ; ibm-corp., armonk, ny, usa). the local ethics committee approved the study (ijb:ce ). complete evaluation was performed in patients, including females. patients were hospitalized in epicura hospital from march , to april . evaluations were conducted . ± . days after the onset of symptoms, corresponding to - weeks after the end of the hospitalization. clinical outcomes are reported in table . the most prevalent symptoms were: fever, asthenia, and anorexia. the mean duration of symptoms before hospitalization was . ± . days. eight patients were hospitalized in the intensive care unit (icu) for a mean duration of . ± . days. no patients received drugs for olfactory dysfunction. the ct scan and blood test features are reported in table . abbreviations: crp = c-reactive protein; ct = computed tomography; gerd = gastroesophageal reflux disease; sd = standard deviation. psychophysical olfactory evaluations indicated that four ( . %) and nine ( . %) patients reported anosmia and hyposmia (in the entire cohort), respectively ( table ). note that three hyposmic patients reported in the patient-reported outcome questionnaire that they had hyposmia prior to the infection. excluding these three patients, the prevalence of objective sls in our cohort was . %. eight and patients experienced (subjective) total and partial loss of smell, respectively, over the clinical course of the disease; accounting for . % of individuals. among them, only three and four were anosmic and hyposmic ( . %), respectively. the three patients who experienced hyposmia prior to the pandemic were not included in the subjective sls patients. according to subjective evaluations of olfaction, thirty-eight-point-three percent of patients complained of sls. additional olfactory outcomes are reported in table . patients with diabetes had lower sniffin'stick test results compared with those without diabetes (mann-whitney u test; p = . ). the linear regression analyses revealed significant negative associations between the sniffin'stick test and age (r s = − . ; p = . ). symptom duration was significantly correlated with the severity of fever (r s = . ; p = . ) and dysphonia (r s = . ; p = . ). duration of hospitalization was significantly correlated with age (r s = . ; p = . ). serum igg concentration measured by the sars-cov- liaison ® test (diasorin, centralino, italy) was negatively correlated with the severity of nasal burning (r s = − . ; p = . ). the number in parentheses represents the percentage. abbreviations: sd = standard deviation; snot- = sino-nasal outcome test- questionnaire. olfactory disorder is undoubtedly a key symptom of mild covid- patients, affecting more than % of patients [ , ] . however, its prevalence remains uninvestigated in severe forms of the disease. in this study, we found that . % of patients with severe disease experience sls. among them, thirty-eight-point-nine percent had abnormal objective tests one month after the onset of the infection. irrespective of the method used to evaluate the prevalence of sls (patient-reported outcome questionnaire versus objective tests), these data indicate that sls could be more prevalent in mild-to-moderate forms of the infection. according to a previous study conducted in the same population and with the same methods, self-reported sls concerned more than % of mild covid- patients, and among them, sixty-two percent had abnormal objective evaluations [ ] . the higher incidence of sls in mild forms of covid- suggests a relative compartmentalization of the disease. such compartmentalization may involve differences in immune responses to sars-cov- at the level of nasal and olfactory mucosa. in patients with potent mucosal immune responses, viral replication and dissemination to the lower respiratory tract may be better controlled, and this could be at the expense of local inflammation and symptoms involving nasal and bulb regions. in patients with less potent mucosal immune responses, viral replication could spread to the lower respiratory tract and lead to systemic immune response and inflammation. this hypothesis is supported by our observation that nasal burning was inversely correlated with sars-cov- serum igg, whereas severe forms of the disease have been positively correlated with sars-cov- igg responses [ ] . further studies are needed to test this hypothesis. both age and diabetes could be favoring factors in the development of sls, which is well known in other olfactory diseases [ , ] . the mechanisms underlying the development of olfactory dysfunction in patients with diabetes may involve neuropathy and damage in the olfactory nerves. the main limitations of the present study are the low number of patients, the lack of a control group, and the performance of olfactory tests one month after the onset of symptoms. performing the tests during hospitalization was difficult due to the sanitary situation, the patient clinical state, and the difficulties in correctly sensing the pens with transnasal oxygenation. although this possibility is not supported by patient-reported symptoms, the delay between the onset of symptoms and the objective olfactory testing may underestimate the incidence of olfactory dysfunction because of olfactory mucosa recovery. the prevalence of sls in severe covid- patients appears to be lower than previously estimated for mild-to-moderate covid- forms. future comparative studies are needed to explore the predictive value of sls for covid- severity. olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid- ): a multicenter european study smell dysfunction: a biomarker for covid- objective olfactory evaluation of self-reported loss of smell in a case series of covid- patients olfactory and gustatory function impairment in covid- patients: italian objective multicenter-study loss of smell and taste in european mild-to-moderation covid- patients french adaptation and validation of the sino-nasal outcome test- : a prospective cohort study on quality of life among subjects immunology of covid- : current state of the science association between diabetes mellitus and olfactory dysfunction: current perspectives and future directions age-related deficits in taste and smell we thank the workers of the anatomy lab of umons who helped to collect the data. the authors have no conflict of interest. key: cord- - bijio authors: eltom, kamal h.; samy, abdallah m.; abd el wahed, ahmed; czerny, claus-peter title: buffalopox virus: an emerging virus in livestock and humans date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: bijio buffalopox virus (bpxv) is the cause of buffalopox, which was recognized by the fao/who joint expert committee on zoonosis as an important zoonotic disease. buffalopox was first described in india, later in other countries, and has become an emerging contagious viral zoonotic disease infecting milkers with high morbidity among affected domestic buffalo and cattle. bpxv is a member of the genus orthopoxvirus and a close variant of the vaccinia virus (vacv). recent genome data show that bpxv shares a most recent common ancestor of vacv lister strain, which had been used for inoculating buffalo calves to produce a smallpox vaccine. over time, vacv evolved into bpxv by establishing itself in buffaloes to be increasingly pathogenic to this host and to make infections in cattle and humans. together with the current pandemic of sars-cov /covid , bpxv infections illustrate how vulnerable the human population is to the emergence and re-emergence of viral pathogens from unsuspected sources. in view that majority of the world population are not vaccinated against smallpox and are most vulnerable in the event of its re-emergence, reviewing and understanding the biology of vaccinia-like viruses are necessary for developing a new generation of safer smallpox vaccines in the smallpox-free world. buffalopox virus (bpxv)-the etiological agent of buffalopox-is member of the genus orthopoxvirus, subfamily chordopoxvirinae, family poxviridae https://talk.ictvonline.org/ictv-reports/ ictv_ th_report/ [ ] . bpxv is a close variant of the vaccinia virus (vacv), the type-species of the genus orthopoxvirus (opxv). buffalopox was first described in india [ ] [ ] [ ] and further reports on the disease came from other countries [ ] [ ] [ ] . discovery of the virus was achieved around the time of smallpox epidemics and the beginning of vaccination programs with vacv. the first isolation of the virus was made in northern india in the year and the virus continued to cause sporadic outbreaks in asian buffaloes (bubalus bubalis) in bangladesh, india, indonesia, pakistan, egypt, russia, and italy [ ] , figure . in the same year of the first isolation of the virus, the disease was recognized by the fao/who joint expert committee on zoonosis as an important zoonotic disease [ ] . forty years buffalopox virus resembles vacv in terms of its size, shape, structure, physico-chemical properties, and autonomous replication in "viroplasm zones" [ , ] . buffalopox virus replicates in a wide range of cells [ ] [ ] [ ] [ ] [ ] . cell cultures of bovine and monkey origins are most frequently used for virus propagation, accompanied by a cytopathic effect (cpe). whole-genome restriction fragment length polymorphism (rflp) studies have indicated genetic similarity between vacv and bpxv [ ] . phylogenetic analysis based on some genes sequences [ , ] and complete genomes [ , ] revealed that bpxvs clustered closely with vacv rather than with other opxvs. furthermore, phylogenomics data support the hypothesis that vacv lister and allied vaccine strains (western reserve, copenhagen, etc.) share a most recent common ancestor with bpxv to the exclusion of other vacv strains [ ] . analysis of the available complete genomes of four isolates (three from india and one from pakistan) confirmed the monophyly bpxvs [ ] . epidemiology of buffalopox should be reconsidered more than years after cessation of smallpox eradication campaigns [ ] . bpxv resembles vacv in its pathogenesis, pathology, and histology. after recovery from infection, animals and humans are protected by both cell and antibody-mediated immunity. neutralizing, hemagglutination-inhibiting, and precipitating antibodies are important for protection; they appear after days approximately following experimental infection and maternal antibodies are transferred to the newborn animals via colostrum [ ] . many authors guessed that bpxv is likely to have emerged from the lister vaccine strain of vacv, the strain that had been used in buffalo calves in india to produce smallpox vaccine [ , , ] . support for this has been provided by analyzing the complete genomes of bpxvs sequenced so far [ , ] . the emergence of bpxv occurred by gradual adaptation of the vaccine strain in buffaloes [ ] until it converted to pathogenic, leading to outbreaks in this new host. consequently, buffalopox outbreaks occurred frequently in many parts of india, affecting both buffaloes [ , , ] and humans [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . reports on zoonotic outbreaks of buffalopox have been made from pakistan as well [ , ] . subsequently, reports on bpxv cases in other hosts, such as cattle, have also been made [ ] . transmission of the virus to cattle and humans is alarming as it might have serious public health implications, in the view that no more vaccination against smallpox is practiced since after its eradication [ ] . the products of host-range genes have been demonstrated to affect the infecting ability of the virus for cells by subverting immune responses of the host [ ] . the most important host-range genes of opxvs that have been sequenced for bpxv strains are e l, k l, c l, and b r, which are implicated in altering the antiviral defense mechanism of the host cell. the full-length sequences of these four genes of bpxvs-obtained from outbreaks in buffaloes, cattle, and humans in india-were analyzed, to investigate their evolutionary relationship to other opxvs circulating in the world vis-à- buffalopox virus resembles vacv in terms of its size, shape, structure, physico-chemical properties, and autonomous replication in "viroplasm zones" [ , ] . buffalopox virus replicates in a wide range of cells [ ] [ ] [ ] [ ] [ ] . cell cultures of bovine and monkey origins are most frequently used for virus propagation, accompanied by a cytopathic effect (cpe). whole-genome restriction fragment length polymorphism (rflp) studies have indicated genetic similarity between vacv and bpxv [ ] . phylogenetic analysis based on some genes sequences [ , ] and complete genomes [ , ] revealed that bpxvs clustered closely with vacv rather than with other opxvs. furthermore, phylogenomics data support the hypothesis that vacv lister and allied vaccine strains (western reserve, copenhagen, etc.) share a most recent common ancestor with bpxv to the exclusion of other vacv strains [ ] . analysis of the available complete genomes of four isolates (three from india and one from pakistan) confirmed the monophyly bpxvs [ ] . epidemiology of buffalopox should be reconsidered more than years after cessation of smallpox eradication campaigns [ ] . bpxv resembles vacv in its pathogenesis, pathology, and histology. after recovery from infection, animals and humans are protected by both cell and antibody-mediated immunity. neutralizing, hemagglutination-inhibiting, and precipitating antibodies are important for protection; they appear after days approximately following experimental infection and maternal antibodies are transferred to the newborn animals via colostrum [ ] . many authors guessed that bpxv is likely to have emerged from the lister vaccine strain of vacv, the strain that had been used in buffalo calves in india to produce smallpox vaccine [ , , ] . support for this has been provided by analyzing the complete genomes of bpxvs sequenced so far [ , ] . the emergence of bpxv occurred by gradual adaptation of the vaccine strain in buffaloes [ ] until it converted to pathogenic, leading to outbreaks in this new host. consequently, buffalopox outbreaks occurred frequently in many parts of india, affecting both buffaloes [ , , ] and humans [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . reports on zoonotic outbreaks of buffalopox have been made from pakistan as well [ , ] . subsequently, reports on bpxv cases in other hosts, such as cattle, have also been made [ ] . transmission of the virus to cattle and humans is alarming as it might have serious public health implications, in the view that no more vaccination against smallpox is practiced since after its eradication [ ] . the products of host-range genes have been demonstrated to affect the infecting ability of the virus for cells by subverting immune responses of the host [ ] . the most important host-range genes of opxvs that have been sequenced for bpxv strains are e l, k l, c l, and b r, which are implicated in altering the antiviral defense mechanism of the host cell. the full-length sequences of these four genes of bpxvs-obtained from outbreaks in buffaloes, cattle, and humans in india-were analyzed, to investigate their evolutionary relationship to other opxvs circulating in the world vis-à-vis the vaccine strains. sequences of these genes revealed a higher degree of similarity to those of vacv strains [ ] . the functions of the vacv e l gene were studied extensively in deletion mutants, which caused abortive replication and expression of only a subset of viral genes in most mammalian cell lines [ , ] . e l encodes a -kda and a -kda protein that suppresses the antiviral response of the host cell by inhibiting both protein kinase and rnasel [ , ] . the k l gene confers interferon resistance and was shown to repress activation of the protein kinase (pkr) and phosphorylation of eif α in mammalian cells [ ] , which can result in inhibiting the antiviral defense mechanism. the c l gene is conserved in all opxv genomes [ ] and causes inhibition of apoptosis [ ] and blockage of antiviral effects by antagonizing interferons (ifn) [ ] . the b r gene is essential for the formation of extracellular virus particles (ev) [ ] and is involved in viral evasion from the immune response of the host [ ] . when this gene was deleted from vacv strain wr, it resulted in a decrease in ev production, reduction in the plaque size in vitro and in high attenuation of the virus in vivo in comparison with the parental strain [ ] . point mutation of at least one amino acid was observed within this gene in cattle and human isolates of bpxv [ ] , which also occurred when a bpxv isolate was passaged times [ ] . some of these mutations might be critical for the virus to adapt to new hosts and can be implicated in the zoonotic nature of this virus [ ] . clinical signs of buffalopox resemble those of vacv infections. characteristic signs in buffaloes include a local pox exanthema (pustulation with central necrosis) and localized pock-lesions on the muzzle, udder, teats, inside of the thighs, scrotum, base of the ears, inner surface of earflap, and eyes in the mild form [ ] . the disease may also proceed to severe systemic disease of a cyclical pattern with generalized lesions in individual cases [ ] . although buffalopox does not occur very frequently, the disease is economically important in countries where buffaloes are reared. the disease has a negative impact on the dairy industry as a consequence of reduced productivity ( - % reduction) of affected milking animals when severe local pocks affect the udder and teats, which in turn may lead to mastitis [ , ] . humans in close contact with affected animals can get infected by the virus. in humans, infection with bpxv was manifested as pox lesions in the flexor aspect of distal forearms, in the hand, dorsae of hands, wrist fingers, and thumbs, right preauricular area, right angle of mandible, right ala of nose, and forehead with or without swelling of the regional lymph nodes, general malaise, and fever [ , , , , , ] . several reports on bxpv outbreaks involving both animals and humans or infections of individual cases were made from india. an outbreak of buffalopox in animals and humans in maharashtra state of india in was reported. it involved herds and resulted in % overall morbidity; some animals also exhibited lesions on their hindquarters, suggesting secondary or even a generalized infection. milkers suffered pox-like local lesions on their hands, forearms, and forehead, presented with pyrexia, axillary lymphadenopathy, and general malaise [ , ] . further similar outbreaks had also been described also in animals and humans [ , ] and a recent case report on human infection with bpxv was made on an indian milkman and owner [ ] . manual milking with bare hands exposed these individuals to the infection. a report on laboratory-acquired bpxv infection in humans was also made in india, highlighting the need for observance and enforcement of strict biosafety measures within laboratories [ ] . in pakistan in - , reports were made describing a nosocomial outbreak of bpxv in humans in the five major burn units in karachi. here, patients developed pox lesions at burn wounds and the intact skin surrounding them. the source of infection was vacv-contaminated buffalo fat, which had been used as a first-aid medication for dressing the burns. this event showed an indirect mode of transmission of an opxv [ , ] . although outbreaks of orthopoxvirus infections-unique to the indian subcontinent region-were repeatedly described and significant veterinary research work was conducted in this respect, limited diagnostic tools were developed [ ] . clinical examination and collection of specimens (swab and serum) from both animals (buffaloes, cattle) and humans are the first steps of diagnosis. these samples are then subjected to electron microscopy examination, inoculation in cell culture for isolation of the virus, plaque reduction and neutralization test, pcr, and partial genome sequencing [ , ] . like other opxvs, bpxv can be isolated from the lesion scabs of animals and humans by inoculation in embryonated chicken eggs as well as in a number of cell lines including chick embryo fibroblast cells, pup kidney cells, vero cells and baby hamster kidney cells [ ] [ ] [ ] [ ] [ ] . cytopathic effects can be observed in - days. bpxv is serologically uniform and cross-reacts with both vacv and cpxv, as well as with other opxvs. therefore, serological assays are not advantageous for virus differentiation unless a targeted monoclonal antibody is used. classical assays used to distinguish bpxv from other opxvs would be double immunodiffusion (id), complement fixation (cf), and immunoelectrophoresis (ie). today, enzyme-linked immunosorbent assay (elisa), immunofluorescence assays (if), and western blotting (wb), together with neutralization and plaque reduction tests (nt/prt) are more commonly used. earlier investigations with monoclonal antibodies have shown that bpxv is serologically more closely related to vacv than to other opxvs [ , ] . recent attempts were made for specific detection of bpxv and differentiation from other opxvs. monoclonal antibodies against the bp strain of bpxv were produced and used in antigen capture elisa. although these monoclonal antibodies differentiated the bpxv from other opxvs, only two of them significantly bound different bpxv strains; none of them had virus-neutralizing abilities; furthermore, they did not bind the polypeptides shared by other bpxv strains in western blotting [ ] . therefore, they are of no use for serodiagnosis of bpxv infections. some recombinant proteins antigens were evaluated for the specific detection of bpxv [ , ] ; they cross-reacted with other opxv as they were based on conserved proteins (a l and h l) in opxv. however, their potential use in diagnostic assays of bpx infections was not evaluated. primers for the c l gene of opxv were used in conventional pcr, duplex pcr, and real-time pcr [ ] for the detection of bpxv. the primers amplify a bp pcr product unique for bpxvs. in duplex pcr, using these primers together with those for the dna polymerase (pol dna), opxv species, as well as capripox and parapox viruses, amplified only a bp amplicon of the pol dna, whereas bpxv amplified both the bp and pb pcr products. the sensitivity of real-time pcr, however, was times more than the conventional pcr [ ] . currently, no licensed specific antivirals are available for the treatment of bpxv infections in humans and animals. if possible, acute infections can be curtailed with immune sera; but, in the case of immunosuppressed individuals, serum therapy does not prevent the local infection from developing into a generalized systemic disease [ , , ] . however, reservations against polyclonal human immune sera arise due to safety reasons, as their biological compounds are not characterized very well [ ] . for the prevention of secondary bacterial infections, symptomatic treatment is provided. because bpxv is closely related to vacv, the antivirals cidofovir and st may be effective for local and systemic treatment in humans and animals. recently, a new series of thiazolo [ , -a] pyrimidine- -carboxylate derivatives a-f and a-f were synthesized and characterized [ ] . the compounds were tested for in vitro antimicrobial and antiviral activities. the probable mode of action of these active compounds was determined through in silico docking study by docking the receptor methionyl-trna synthetase and human inosine- -monophosphate dehydrogenase (impdh) for antibacterial and antiviral activities, respectively. of these compounds, c elicited excellent in vitro antimicrobial activity against all tested strains. on the other hand, compound a elicited . % and . % inhibition of the camelpox virus (cmlv) and bpxv, respectively. moreover, this compound exhibited minimum docking and binding energy along with the maximum hydrogen/hydrophobic interaction with impdh [ ] . recently, some protein kinase inhibitors were tested for antiviral activity. among these, the kinase inhibitor cgp , which blocks the mitogen-activated protein kinase (mapk) interacting kinase (mnk ), was found to be promising as an antiviral agent against bpxv. in in vivo studies, this compound was found to decrease the synthesis of the viral genome and to reduce synthesis of viral proteins, whereas in in ovo studies it prevented the formation of pock lesions on the chorioallantoic membrane (cam) as well as associated mortality of the chick embryos [ ] . no specific vaccine against bpxv infection is available. however, prophylactic control and protection of animals in an infected herd is possible with a live vaccine based on an attenuated vacv strain. vaccinia virus vaccines were initially produced in buffaloes (dermo-vaccine) in india [ ] . later cell culture adapted strains were used. the program resembles that of vaccinia and cowpox prophylaxis. however, the protective efficacy of the third generation vaccinia based vaccines has been tested in animals (mice, rabbit, and monkeys) [ ] . in addition to the safety of these vaccines, the elicited immune responses provided protection against challenge with the respective virus. the use of these vaccines would be advantageous for use in buffalo and humans in contact with buffalo, as no data is available about prophylaxis in humans at risk of bpxv infections. recombinant dna vaccines from the envelope proteins (a l and hl) of opxv-derived from a bpxv strain-were tested in animal models [ , ] . an increase in antigen-specific serum igg level as well as in neutralizing antibody titers was observed in the recombinant vaccines. in passive protection experiments in suckling mice, hyperimmune sera of the recombinant a l vaccine conferred % protection [ ] , while % protection was reached with anisera of the h l [ ] . a combined vaccine containing both a l and h l recombinant proteins elicited a high immune response in mice measure by specific igg titers in elisa and neutralizing antibody titers. in addition, complete protection of mice vaccinated with this combination was seen when they were challenged by virulent virus strain [ ] . over time, bpxv evolved from vacv and established itself in buffaloes to be increasingly pathogenic to the new host, i.e., buffaloes, and further to make infections in cattle and humans [ ] . the emergence of a pathogenic opxv, which can spread efficiently from human-to-human, should be considered an immediate public health risk [ ] [ ] [ ] . together with the current pandemic of sars-cov /covid , bpxv infections in india [ , , ] illustrate how vulnerable the human population is to the emergence and re-emergence of viral pathogens from unsuspected sources. a deep understanding of the underlying molecular mechanisms, that control the species tropism of poxviruses in non-evolutionary hosts is of utmost importance [ ] . as more information becomes available on tropism determinants of poxviruses, new strategies will likely be developed to control zoonotic infections. raising awareness, improvement of diagnostic techniques, education, and preparedness for early intervention, and development of disaster guidelines are necessary given the potential disease outbreak, in case it happens in the future [ ] . like most opxvs and other zoonotic poxviruses reservoir host(s) that maintain bpxv in the environment did receive attention and yet is unknown-although it is thought to be most likely rodents [ ] ; knowledge on this respect is essential for the prevention of introduction of the virus in naïve buffalo and cattle populations and for designing eradication and control programs. increasing incidences of opxv infections are being reported across the world: bpxv in asia, vacv and vacv-like viruses (vlvs) in brazil [ ] , mpxv in east and central africa and the usa [ ] and novel oxpvs (akmv [ ] , akpv [ ] , ectv-like opxv [ , ] )-which are being described at an increasing rate. emergence and re-emergence of these opxvs are alarming in view that about % of the world population > years are not vaccinated against smallpox and are most vulnerable in the event re-emergence of this disease [ ] . in addition, the rise in global bioterrorism necessitates the use of third generation smallpox vaccines, as they have been shown to be safer than preceding generations [ ] , in the most vulnerable populations. furthermore, efforts should be increased for searching a new generation of safer smallpox vaccines, a necessary step towards which research should be directed for reviewing and understanding the biology of vlvs in the smallpox-free world. virus taxonomy. ninth report of the international committee on taxonomy of viruses family poxviridae 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virus may derive from brazilian smallpox vaccine passatempo virus, a vaccinia virus strain aracatuba virus: a vaccinialike virus associated with infection in humans and cattle novel orthopoxvirus infection in an alaska resident human monkeypox: current state of knowledge and implications for the future human infection with a zoonotic orthopoxvirus in the country of georgia fatal outbreak in tonkean macaques caused by possibly novel orthopoxvirus novel orthopoxvirus and lethal disease in cat we acknowledge the support of the open access publication funds of the university of goettingen. the authors declare no conflict of interest. key: cord- -c ikb g authors: nadeem, muhammad shahid; zamzami, mazin a.; choudhry, hani; murtaza, bibi nazia; kazmi, imran; ahmad, habib; shakoori, abdul rauf title: origin, potential therapeutic targets and treatment for coronavirus disease (covid- ) date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: c ikb g the ongoing episode of coronavirus disease (covid- ) has imposed a serious threat to global health and the world economy. the disease has rapidly acquired a pandemic status affecting almost all populated areas of the planet. the causative agent of covid- is a novel coronavirus known as sars-cov- . the virus has an approximate kb single-stranded positive-sense rna genome, which is . % to % identical to that of sars-cov, cov-pangolin, and the coronavirus the from horseshoe bat. according to available information, sars-cov- is inferred to be a recombinant virus that originated from bats and was transmitted to humans, possibly using the pangolin as the intermediate host. the interaction of the sars-cov- spike protein with the human ace (angiotensin-converting enzyme ) receptor, and its subsequent cleavage by serine protease and fusion, are the main events in the pathophysiology. the serine protease inhibitors, spike protein-based vaccines, or ace blockers may have therapeutic potential in the near future. at present, no vaccine is available against covid- . the disease is being treated with antiviral, antimalarial, anti-inflammatory, herbal medicines, and active plasma antibodies. in this context, the present review article provides a cumulative account of the recent information regarding the viral characteristics, potential therapeutic targets, treatment options, and prospective research questions. the outbreak of a novel respiratory syndrome, referred to as coronavirus disease (covid- ) , was first recognized in wuhan, china, in december . the causative agent for this deadly condition is a coronavirus known as sars-cov- . covid- is demonstrated by fever, dry cough, persistent pressure in the chest, and shortness of breath [ , ] . sneezing, runny nose, and symptoms similar to the common cold are observed in only % of patients. about % to % of patients have shown diarrhea like symptoms [ , ] . the mortality rate of covid- is . % to %, which is less than that of sars (severe acute respiratory syndrome), which has a mortality rate of . %, and less than that of mers (middle east respiratory syndrome), up to . % deaths. individuals already suffering from cardiovascular disease, hypertension, respiratory disease, or diabetes are at a high risk of mortality. age and gender-specific variations in the death rate have also been reported [ ] . the disease became pandemic within a few months of its emergence, indicating a high transmission ability as coronaviruses, first discovered in the s, are found in birds and mammals, especially in bats, cats, camels, and rats [ ] . the causative agent of covid- (sars-cov- ) belongs to the genus β-coronavirus, family coronaviridae, and order nidovirales. a similar human coronavirus was found to be responsible for sars in and . the virus responsible for covid- has a single-stranded positive-sense rna genome of about kb, which has % to % identity with that of the coronavirus from the pangolin (manis javanica) and horseshoe bat (rhinolophus sinicus) (bat-covratg ), respectively [ , [ ] [ ] [ ] [ ] . bats have been reported as being the rich source of coronaviruses [ , ] , although only a few of these coronaviruses can infect humans [ , ] . according to the literature, the sars and mers viruses have zoonotic transmission, originating from bats using palm civets and camels, respectively, as the intermediate hosts [ ] [ ] [ ] [ ] [ ] . the recent reports have suggested that sars-cov- is a modified coronavirus of bat origin [ , ] , which came to humans as a result of zoonotic transmission [ , ] . a coronavirus identified from the malayan pangolin has been shown to have a % similarity with sars-cov- . the receptor-binding domain (rbd) of pangolin-cov has only a one amino acid difference with that of sars-cov- ; the infected pangolins exhibit pathological symptoms similar to humans suffering from covid- , and their blood circulating antibodies can react with the spike protein of sars-cov- [ , ] . although the ratg coronavirus isolated from bat has about % identity with sars-cov- , its rbd is different from that of the later, exhibiting a low binding ability to the human ace [ ] . however, the rbd of the s-protein from pangolin-cov is highly similar to that of sars-cov- , six residues critical for receptor binding being identical in both [ ] . a comparative analysis of genetic data available to date has suggested that sars-cov- originated by the recombination of pangolin-cov and the bat-cov-ratg -like virus [ , [ ] [ ] [ ] . based on this information, the pangolin is considered to be one of the possible intermediate hosts between bat and human. snakes, minks, and turtles are also being investigated as the potential intermediate hosts [ , ] (figure ). five out of the six critical amino acid residues comprising the rbd of the s-protein from sars-cov and sars-cov- are different, contradicting the theories about the laboratory origin of sars-cov- by the manipulation of sars or mers like viruses [ ] . studies based on the analysis of n, s, and orf a/ b genes have shown conserved sequences suggesting that sars-cov- is an animal virus, which was transmitted to humans by undergoing evolutionary adaptations [ , ] . the sars infection from , also involved zoonotic transmission of the virus to humans. hence, further studies are required to confirm the intermediate hosts of coronaviruses to control zoonotic transmission and avoid the outbreak of such viral infections in the future [ ] . on march , , who published a pcr based detection method for sars-cov- . the procedure could detect the virus in the blood, sputum, and nasopharyngeal swab [ , ] . noncontrast chest ct (computed tomography) can also be used for the diagnosis of viral pneumonia. however, ct scans can be negative in the case of covid- [ ] . on the other hand, patients with negative rt-pcr test results can show pneumonia-like symptoms on a ct scan [ ] . in a comparative study, the sensitivity of a chest ct was found to be %, whereas the sensitivity of the pcr test was only % [ , ] . rt-pcr based diagnosis also gave false-positive results [ ] . low viral load, inefficient sampling, poor sample storage or processing conditions, along with a lack of specific primers due to the high rate of mutations in rna viruses, are some of the apparent factors for the poor sensitivity of pcr based diagnoses. recently, some parallel procedures have also been reported for the diagnosis of covid- . one of these procedures is loop-mediated isothermal amplification (lamp), which is a faster single-step procedure, having > % sensitivity [ , ] . further modifications of lamp-based procedures have been reported, which can reduce the testing time with minimum equipment requirements [ , ] . to develop serological procedures, iga and igm have been evaluated against sars-cov- by immunofluorescence assays [ ] [ ] [ ] . however, further refining of rt-pcr and the serological procedures are required to improve the sensitivity and specificity. on march , who published a pcr based detection method for sars-cov- . the procedure could detect the virus in the blood, sputum, and nasopharyngeal swab [ , ] . noncontrast chest ct (computed tomography) can also be used for the diagnosis of viral pneumonia. however, ct scans can be negative in the case of covid- [ ] . on the other hand, patients with negative rt-pcr test results can show pneumonia-like symptoms on a ct scan [ ] . in a comparative study, the sensitivity of a chest ct was found to be %, whereas the sensitivity of the pcr test was only % [ , ] . rt-pcr based diagnosis also gave false-positive results [ ] . low viral load, inefficient sampling, poor sample storage or processing conditions, along with a lack of specific primers due to the high rate of mutations in rna viruses, are some of the apparent factors for the poor sensitivity of pcr based diagnoses. recently, some parallel procedures have also been reported for the diagnosis of covid- . one of these procedures is loop-mediated isothermal amplification (lamp), which is a faster single-step procedure, having > % sensitivity [ , ] . further modifications of lamp-based procedures have been reported, which can reduce the testing time with minimum equipment requirements [ , ] . to develop serological procedures, iga and igm have been evaluated against sars-cov- by immunofluorescence assays [ ] [ ] [ ] . however, further refining of rt-pcr and the serological procedures are required to improve the sensitivity and specificity. sars became epidemic in many countries around the world in and . the disease had many symptoms similar to those of covid- . however, sars-cov- and sars-cov have shown differences, as well as similarities, in their genomic composition, incubation time, and infection mechanisms. a set of affinities has been tabulated that can help us to establish the correlation between the two viruses (table ) . to date, the mortality rate of covid- is . % to . %. there are more than million reported infections and , deaths (as recorded on april ). the mortality rate was between . % to %. it was restricted to individuals and deaths. [ , , ] the virus needs a longer incubation time (average days) to represent covid- symptoms. the virus needed a relatively short incubation time ( - days) to exhibit symptoms. [ , ] in covid- , the infection ratio between males and females is . : , indicating that the disease is more prevalent among males. old aged people also have a high mortality rate. the male to female ratio was : . ; more prevalent in females. there was a higher death rate in old aged patients. the intra-or inter-species transmission of β-coronaviruses (covs) requires a viral interaction with the host cell receptors, and the subsequent invasion of the host cells [ ] . the genome of the coronavirus codes for a surface glycoprotein, known as a "spike" protein (s-protein), that specifically binds to the host cellular receptors to initiate the infection process. in fact, the spike protein performs a "key" like function to "unlock" the door and facilitate the cellular entry of a coronavirus. studies based on d models of spike proteins from the sars-cov and sars-cov- viruses, have shown a considerable overall similarity [ , ] . the cryo-em structure of the sars-cov- s-protein has also been reported [ ] (figure ) . the overall structure of the s-protein consists of several functional domains. the rbd, fusion domain (fd), and the s cleavage site could be critical for future studies to develop therapeutic strategies [ ] . the protein exhibits a high binding affinity with ace , as represented by a low dissociation constant value (kd~ nm). the receptor binding affinity of the sars-cov- s-protein is times higher than that of the sars-cov s-protein [ , [ ] [ ] [ ] [ ] . furthermore, studies using human, pig, and civet cell lines have allowed sars-cov- infection and replication, indicating that the virus makes use of the ace receptor for infection [ , [ ] [ ] [ ] . ace is cleaved by a protease (tmprss ) in order to activate virus entry. this can be inhibited by protease inhibitors such as camostat mesylate [ ] . ace is highly expressed in the lungs; a vast surface area makes the lung tissue highly susceptible to sars-cov- infection [ ] . in addition to the lungs, the ace receptor is also expressed in the endothelial cells of intestine, kidney, and heart cells [ ] . pathogens , , x for peer review of of six amino acids are different to that of sars-cov- . the intra-or inter-species transmission of β-coronaviruses (covs) requires a viral interaction with the host cell receptors, and the subsequent invasion of the host cells [ ] . the genome of the coronavirus codes for a surface glycoprotein, known as a "spike" protein (s-protein), that specifically binds to the host cellular receptors to initiate the infection process. in fact, the spike protein performs a "key" like function to "unlock" the door and facilitate the cellular entry of a coronavirus. studies based on d models of spike proteins from the sars-cov and sars-cov- viruses, have shown a considerable overall similarity [ , ] . the cryo-em structure of the sars-cov- s-protein has also been reported [ ] (figure ) . the overall structure of the s-protein consists of several functional domains. the rbd, fusion domain (fd), and the s cleavage site could be critical for future studies to develop therapeutic strategies [ ] . the protein exhibits a high binding affinity with ace , as represented by a low dissociation constant value (kd ⁓ nm). the receptor binding affinity of the sars-cov- s-protein is times higher than that of the sars-cov s-protein [ , [ ] [ ] [ ] [ ] . furthermore, studies using human, pig, and civet cell lines have allowed sars-cov- infection and replication, indicating that the virus makes use of the ace receptor for infection [ , [ ] [ ] [ ] . ace is cleaved by a protease (tmprss ) in order to activate virus entry. this can be inhibited by protease inhibitors such as camostat mesylate [ ] . ace is highly expressed in the lungs; a vast surface area makes the lung tissue highly susceptible to sars-cov- infection [ ] . in addition to the lungs, the ace receptor is also expressed in the endothelial cells of intestine, kidney, and heart cells [ ] . the interaction of the viral s-protein with ace , its subsequent activation by protease (tmprss ), and its viral entry into the cell. the schematic primary structure of the sars-cov- spike protein is elaborated indicating the major domains. ss-signal sequence, rbd-receptor binding domain, rbd subdomains and -sd and sd , s /s -the protease cleavage site, s ′-the protease restriction site indicated by the arrows, fp-fusion peptide, hr and hr -heptad repeats and , figure . the interaction of the viral s-protein with ace , its subsequent activation by protease (tmprss ), and its viral entry into the cell. the schematic primary structure of the sars-cov- spike protein is elaborated indicating the major domains. ss-signal sequence, rbd-receptor binding domain, rbd subdomains and -sd and sd , s /s -the protease cleavage site, s -the protease restriction site indicated by the arrows, fp-fusion peptide, hr and hr -heptad repeats and , ch-central helix, cd-connector domain, tm-transmembrane domain, and ct-cytoplasmic tail. (the schematic was adopted and modified from [ , ] .) according to recent information, the glutamine at the amino acid position in the receptor-binding protein of sars-cov- that corresponds to the residue in sars-cov, is recognized by lysine residue in the human ace receptor [ ] . an interaction of polar residues in the ectodomain of ace with the receptor binding domain of sars-cov- spike protein has been reported [ ] . downstream interaction and the invasion process include the cleavage of the spike by a serine protease at the "s " domain ( figure ) [ ] , followed by the interaction of the virus s-protein fusion domain with the host cell plasma membrane. virus entry takes place either by fusion with the plasma membrane or by endocytosis, and the subsequent fusion of membranes in endosomes [ ] . in addition to virus-plasma membrane fusion, sars-cov can adopt clathrin-dependent endocytosis [ ] . once inside the host cell, the viral rna is translated in the cytoplasm producing polyproteins and structural proteins. after translation, the replication of the genome occurs [ ] . new virus particles are formed in the membranes of the golgi apparatus and the endoplasmic reticulum, after which the vesicles harboring the viral particles are fused with plasma membranes for the release of the virus [ , ] . because sars-cov- , in binding with ace , is dependent on an interaction and the cleavage of the spike protein by a serine protease, a spike protein-based vaccine or serine protease specific inhibitors could be potential therapies against sars-cov- infection [ ] . the comparative homology studies of sars-cov- proteins (s, n, m, and e proteins) with those from sars and mers viruses, have suggested some targets for vaccine development [ ] . polyclonal antibodies raised against sars-cov are found to prevent a spike-mediated host cell invasion of sars-cov- [ ] . recently, . billion potential protease blockers have been investigated by molecular docking studies. many of these molecules can be evaluated in wet labs in the near future [ ] . ace blockers can be another option to avoid the infection [ ] . similarly, there are some molecules including gsk a, kt , kt , and bms that have strong binding affinities with rbd of the viral s-protein [ ] . these molecules can help to control rapid infections by engaging the virus at entry points [ ] . currently, a tremendous amount of research is in progress to develop a vaccine against covid- . however, vaccine development is time consuming process, and the newly introduced vaccine will require several safety evaluations [ ] . according to estimates, a vaccine against covid- may take more than a year to become available [ ] . even after the preparation of an effective vaccine, under the present pandemic situation, human trials will be a big challenge for researchers. at present covid- is being treated with some broad-spectrum antiviral drugs including remdesivir, favipiravir, and chinese herbal medicine [ , ] . in vitro studies have shown that chloroquine and remdesivir are effective against sars-cov- [ ] [ ] [ ] . chloroquine phosphate has shown treatment efficacy and safety against sars-cov- associated pneumonia; these findings are based on multiple trials in hospitals in china [ ] . application of an anti-inflammatory drug such as baricitinib, together with an antiviral drug, has also been recommended to treat covid- [ ] . high doses of ascorbic acid (vitamin c) are suggested for the prevention of the covid- disease [ ] . type i interferon can inhibit viral replication. according to studies, interferon β could inhibit the replication of sars-cov [ , ] . however, its efficacy against sars-cov- needs further investigation. the use of convalescent plasma for the treatment of covid- has been suggested. however, the absence of multiple trials on a large scale, and the concern that antibodies may demonstrate donor dependent titters and specificities, are the main deficits of convalescent plasma therapy [ ] . there are several challenges in the management and control of coronaviruses. a wide range of coronaviruses with a highly mutable single-stranded rna genome are found [ , ] in many mammalian and avian sources [ ] that closely interact with each other, as well as with humans. the long-term viability of coronaviruses in airborne aerosols, and on daily utensils composed of plastics, stainless steel, and cardboard, enhance the chances of transmission of infection between individuals and species [ , , ] . the recurrence of sars-cov- has been reported in convalescence times [ ] , which can make the treatment more difficult and increase the chance of complications. the interventions into the ace binding abilities of sars-cov- and similar viruses, by the inhibition of the corresponding serine protease [ ] , can be an area of investigation. easy, highly reliable, and early-stage diagnosis procedures are still required as a challenge for biomedical researchers [ , ] . the presence of sars-cov- in stool samples of infected individuals raises the question about the fecal-oral transmission of the disease [ ] . recently, the viability of sars-cov and sars-cov- has been described ( ); however, the genetic factors behind the long-term survival of these coronaviruses need further investigation [ ] . sars-cov has shown low stability at higher temperatures and at specific air humidity levels [ , ] ; the effect of temperature and other environmental factors on the viability of sars-cov- are unclear. inactivation of coronaviruses by disinfectants, such as % to % ethanol or . % sodium hypochlorite, is well established [ ] . the efficacy and specificity of antiviral and antimalarial drugs being used in the treatment of covid- need further clinical trials. the ongoing covid- outbreak that emerged from wuhan, china, has acquired pandemic status. the causative agent of covid- is a modified coronavirus, known as sars-cov- that has similarities with the coronaviruses responsible 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interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines convalescent plasma to treat covid- : possibilities and challenges the authors are grateful to the doctors, medical staff, and volunteers contributing to overcome the coronavirus disease (covid- ) . the authors have no conflict of interest. key: cord- -weiwksh authors: ramírez-castillo, flor yazmín; loera-muro, abraham; jacques, mario; garneau, philippe; avelar-gonzález, francisco javier; harel, josée; guerrero-barrera, alma lilián title: waterborne pathogens: detection methods and challenges date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: weiwksh waterborne pathogens and related diseases are a major public health concern worldwide, not only by the morbidity and mortality that they cause, but by the high cost that represents their prevention and treatment. these diseases are directly related to environmental deterioration and pollution. despite the continued efforts to maintain water safety, waterborne outbreaks are still reported globally. proper assessment of pathogens on water and water quality monitoring are key factors for decision-making regarding water distribution systems’ infrastructure, the choice of best water treatment and prevention waterborne outbreaks. powerful, sensitive and reproducible diagnostic tools are developed to monitor pathogen contamination in water and be able to detect not only cultivable pathogens but also to detect the occurrence of viable but non-culturable microorganisms as well as the presence of pathogens on biofilms. quantitative microbial risk assessment (qmra) is a helpful tool to evaluate the scenarios for pathogen contamination that involve surveillance, detection methods, analysis and decision-making. this review aims to present a research outlook on waterborne outbreaks that have occurred in recent years. this review also focuses in the main molecular techniques for detection of waterborne pathogens and the use of qmra approach to protect public health. tracking [ , , ] . in developing countries, the lack of financial and technological resources contributes to wbdos [ ] . currently, it is estimated that there are species of pathogens infecting humans, which includes bacteria ( species), viruses ( types), parasitic protozoa ( species), and several fungi and helminths species [ , ] . the development of a disease, when and if infection to the host is produced, depends on factors such as minimal infectious dose (mid), pathogenicity, host susceptibility and environmental characteristics. enteric bacteria have a mid in the range of to cells but it is much lower with some species, such as shigella spp., ( - ), campylobacter spp., (about ), e. coli o :h ( - ), and v. cholera ( ) [ , ] . moreover, protozoan only need a few oocysts ( - ) to produce the disease as well as the viruses which a small number of these are enough to develop a disease [ ] . the protozoa microsporidia, as the bacteria mycobacterium avium intracellulare, helicobacter pylori, tsukamurella, cystoisospora belli and viruses such as adenoviruses, parvoviruses, coronaviruses (sars), and polyomavirus are some examples of the emerging potential waterborne pathogens [ , ] . furthermore, most of these organisms appear to have certain resistance against chlorine such as the microsporidia, enterocytozoon bienusi, encephalitozoon hellem and e. intestinales. m. avium and several viruses have resistance to common disinfectants for drinking water and to inactivation by uv light and heat [ ] , which represents a higher challenge in the treatment to remove these pathogens from water sources. the major pathogens microorganisms in drinking water systems and their related diseases are listed in table . presently, there is no unified method to encompass the collection and analysis of a water sample for all pathogenic microorganisms of interest [ ] . the challenges of the detection methods are the physical differences between the major pathogen groups, low concentration of pathogens in a large volume of water which usually requires enrichment and concentration of the samples prior to detection processing, the presence of inhibitors from the sample (especially if it comes from polluted water), established general protocols for sample collection, culture-independent detection method, as well as detection of the host origin of pathogens [ ] . the most important requirements for reliable analysis include: specificity, sensitivity, reproducibility of results, speed, automation and low cost [ ] . even though culture dependent methods are extensively used for pathogens detection in water, these methods are limited by their low sensitivity and the excessive time needed to obtain reliable results. furthermore, since there is a broad environmental distribution of human pathogens that exist in a viable but non-culturable (vbnc) state such as e. coli, helicobacter pylori and v. cholerae, false negative results may arise from culture dependent methods [ , ] . in both culture and molecular methods, index pathogens for monitoring water quality have been selected in order to indicate the presence of a large amount of pathogens in water. among these, e. coli ( figure ) has been extensively used due to the fact that detection methods for these pathogens are relatively easy and inexpensive; nonetheless, they may have the disadvantage of not providing information on their host origin and, sometimes, they do not correlate with other pathogens present in the water, such as the viruses and protozoa. thus, water characterized as pathogen-free by monitoring e. coli, for example, may be contaminated with viruses or protozoa [ ] . molecular methods can be very specific for particular species and provide further phylogenetic information about pathogens [ ] . these methods allow the use of alternatives indicators which easily relate with the host source. this permits the discrimination between human and animal pathogens and tracking the source of pollution [ , ] . it has been suggested that host-origin libraries, based on a phenotypic methods, may be useful for tracking the pathogen source but the development of such libraries may incur a significant cost [ ] . therefore, molecular methods seem to better suited for health risk assessments. nowadays, there are a number of different molecular methods to detect diverse pathogens. they are used to evaluate the microbiological quality of water, the efficiency of pathogens removal in drinking and wastewater treatment plants, and as microbial source-tracking (mst) tools [ ] . several examples of detection methods and their limits of detection are listed in table . molecular techniques, such as nucleic acid amplification procedures, offer sensitive and analytical tools for detecting a variety of pathogens, including new emerging strains, present the possibility of automation, and real-time analysis to provide information for microbial risk assessment purposes [ ] . immunology-based methods e. coli o :h and s. enterica typhimurium. . × cfu/ml of e. coli and . × cfu/ml of salmonella contaminated food. [ , ] * gc logs are mean values of genome copy logs. polymerase chain reaction (pcr) is one of the most commonly used molecular-based methods for detection of waterborne pathogens [ ] . pcr operates by amplifying a specific target dna sequence in a cyclic three-step process-denaturalization, annealing and extension-in order to achieve exponential amplification of the target sequence [ ] . several variations of pcr such as multiplex pcr (mpcr) allow simultaneous detection of several target organisms by coding specific genes of diverse pathogens in the sample in a single reaction. pcr method has the advantage of quick analysis. however, it necessitates accurate primers and optimal reaction mixture to avoid the risk of false positive and negative results [ , ] . limitations of dna based methods such as pcr include the inability to discriminate between viable from non-viable cells that both contain dna, the low concentration of several pathogens in water such as cryptosporidium, giardia and viruses, and the lack of data to indicate the real infectious risk to a population. challenges of molecular techniques include: the need for water concentration methods (i.e., for virus, ultrafiltration and direct flocculation protocols are used as concentration methods), the presence of inhibitors in water samples including humid acids and metals, to which pcr is sensitive, and the reproducible purification techniques by dna or rna from heterogeneous samples. in addition, result validation is required, since as indicated by hartman and co-workers [ ] ; also, high sensitivity in molecular techniques introduces a high risk of false positive results. an example of mpcr includes the technique developed by omar and barnard [ ] to detect the pathogenic and commensal e. coli from clinical and environmental water sources. to distinguish pathogenic e. coli from commensal, the presence of genes was evaluated in environmental waters. in addition, as control to evaluate the sensitivity of the technique and the false negative due to pcr, inhibitors were controlled using mdh gene (malate dehydrogenase) and gapdh gene (glyceraldehyde- phosphate dehydrogenase). another variation of the pcr is quantitative real-time pcr (qpcr) which enables quantification of dna targets by monitoring amplified products during cycling as indicated by increasing fluorescence [ ] . this method provides high sensitivity and specificity, faster rate of detection, minimizes the risk of cross-contamination, and there is no need for a post-pcr analysis [ ] . the dual-labeled fluorescent probes such as taqman probe and the fluorescent dye sybr green are the most used techniques for detecting pathogens [ ] . qpcr approach has been used to quantify human pathogens, such as e. coli o :h [ ] , and campylobacter spp., [ ] . these qpcr systems can specifically detect and quantify pathogens at concentrations as low as one target molecule per reaction. however, most of these methods can only detect and quantify one pathogen in a single reaction [ ] . ishii and co-workers [ , ] designed a microfluidic qpcr by using taqman probes (hydrolysis probe-based qpcr) labeled with different fluorophores that can detect l. monocytogenes, v. cholerae, v. parahaemolyticus, pseudogulbenkiana sp., s. typhimurium, s. flexneri, c. perfringens and pathogenic e. coli at levels of detection of cells/l. other advances in pcr include the microfluidics and nanobiotechnology field, allowing the construction of high-density and low-volume qpcr platforms, such as the openarray system that accommodates reactions per array [ ] . qpcr protocols have also been developed for the detection and identification of cryptosporidium spp., in river water with a lower quantitation limit of . oocysts/sample [ ] . for detection of g. lamblia and g. ardeae in wastewater samples, qpcr reached a sensitivity of . cysts per reaction [ ] . moreover, because of the importance of biofilm coating pipes in drinking water systems, the detection of pathogens in microbial communities is important. l. monocytogenes has been investigated in biofilms by using qpcr techniques with a number of l. monocytogenes detected growing in biofilm of × cfu/cm [ ] . for rna virus detection, quantitative reverse-transcriptase (qrt)-pcr was developed in order to provide quantitative estimation of the concentration of pathogens in water [ ] . this technique has the advantages of detecting viable cells due to detect messenger rna (mrna), which is present only in viable organisms. however, damaged genomes may fail to be detected with this technique [ , ] . oligonucleotide microarrays are a powerful genomic technology that is widely utilized to monitor gene expression under different cell growth conditions, detecting specific mutations in dna sequences and characterizing microorganisms in environmental samples [ ] . dna microarrays are arrays containing high density immobilized nucleic acids (genomic dna, cdna or oligonucleotides) in an ordered two-dimensional matrix that enables the simultaneous detection of hundreds of genes in a single assay via nucleic acid hybridization [ ] . microarrays are made up of glass slides or chips coated with specific oligonucleotide probes which are chemically synthesized short sequences ( to bp) [ , ] . microarray technology allows the rapid detection of multiple genes of multiple organisms simultaneously in the sample due its capacity for screening large numbers of sequences [ ] , has high throughput capacity and is able to be automated. therefore, large-scale and data-intensive experiments are performed in microarrays [ , ] . microarrays have also the ability to detect antimicrobial resistance to different antibiotics, and the probes could be designed for detecting the host origin of contaminants, which represents another advantage for characterization of contamination in water. however, advanced molecular technologies such as microarrays could experience difficulties distinguishing between viable and non-viable cells, have a relatively high cost, and may have non-specific hybridization resulting in a lower specificity and low sensitivity [ ] . dna microarray coupled with pcr amplification of target genes has been developed for higher sensitivity. through these steps, signal sensitivity increased around -fold [ ] . nonetheless, the pcr-microarray technique might lack sensitivity when the amount of sample is limited, since the technique requires splitting the samples into several pcr reactions to amplify [ ] . dna microarray in a two-step pcr-dna microarray assay that first amplifies the s or s rrna gene, respectively, followed by hybridization of these products onto a low-density dna microarray was developed to detect most of the common waterborne protozoan pathogens including c. parvum, g. intestinalis, acanthamoeba spp., entamoeba histolytica, and naegleria spp. [ , ] . in , wilson and co-workers [ ] were able to identify pathogenic bacteria, eukaryotes and viruses by using species-specific primer sets to amplify multiple diagnostic regions unique to each individual pathogen in the microarray. inoue and co-workers [ ] studied the occurrence of pathogenic bacterial species in groundwater and were able to distinguish between human and animal sources. microarrays for detection of human enteric viruses in community wastewaters using cell culture coupled with multiple targets including dna and rna viruses have also been developed [ ] . the virochip, a pan-virus dna microarray containing thousands of -mer oligonucleotide probes to target all viral families to infect humans, has been described by wang and co-workers [ ] . leski et al. [ ] developed a high-density re-sequencing microarray that has the capability of detecting different types of pathogens ranging from bacteria, protozoa, and viruses, including b. anthracis, francisella tularensis and ebola virus with limits of detection of to copies per test for nearly all the pathogens with high specificity. at present, standard and custom-made dna microarrays are commercially available from companies such as affymetrix, corning and agilent technologies [ , ] . one example of these is the phylogenetic microarray "phylochip" by affymetrix which consists of , oligonucleotide probes capable of identifying strains of bacteria and archaea [ , ] . this technology is very powerful because most known bacteria can be detected in samples without culturing, and the sensitivity allows also the detection of lower-abundance organisms (detection limit of ~ . % of microbial communities) [ ] . pyrosequencing is a dna sequencing technology for short-read dna sequencing that uses enzyme-couple reaction and bioluminescence to monitor the pyrophosphate release accompanying nucleotide incorporation, in real time [ ] . pyrosequencing technology provides a large number of sequences read in a single run [ ] . in the pyrosequencing reaction, the enzymes dna polymerase, atp sulfurylase, luciferase and apyrase are needed. the nucleotides are added to form the complementary strand of the single-stranded template, to which a sequencing primer is annealed. during the nucleotide incorporation, the pyrophosphate (ppi) is released. atp sulfurylase converts the ppi into atp and the atp is then converted to visible light by luciferase and the produced light signal is detected [ , ] . the methodology includes a primary step of concentrating the water pathogens followed by a secondary concentration depending of the type of the pathogen. the third step involves dna extraction (for bacteria and protozoa) and nuclease treatment (viral dna/rna extraction). then, the dna is amplified, the pyrosequencing is taking place and finally the analysis and bioinformatics can be performed [ , ] . in recent years, a new whole genome amplification and sequencing approach called "single virus genomics", which enables the isolation and complete genome sequencing by pyrosequencing of the single virus particle, has been described [ ] . roche pyrosequencing and other commercially available high-throughput sequencing platforms such as solexa/illumina genome analyzer, applied biosystem solid sequencing as well as the most recent, the ion torrent system, have revolutionized the study of microbial diversity [ ] . pyrosequencing could identify novel pathogens associated with water and address multiple etiologies. however, the dna amount present in wastewater samples could limit the sensitivity of this tool as it requires dna templates at picomole level but a much lower amount of dna may be available in water samples. this technology is also limited by the cost, the complexity of analysis, the need for increasing availability of massive computing power and the efficiency of data generation [ ] . pyrosequencing has been used to the analysis of bacterial biofilm communities in water meters of a drinking water distribution system [ ] , mixed urban watershed [ ] , characterization of nontuberculous mycobacterium communities in unchlorinated drinking water [ ] , and the detection of bacteriophages in reclaimed and potable and lake waters [ ] . fish or fluorescence in situ hybridization is based on hybridization of the sample with rrna oligonucleotide probes labeled covalently at one end with fluorescent dye. this technique allows an enumeration of particular microbial cells by the use of confocal microscopy, fluorescence microscopy or flow cytometry in order to obtain qualitative and quantitative results [ ] . fish is commonly used for the detection and identification of different microorganism in mixed populations such as in biofilms ( figure ) and has been used to produce a quantitative description of the microbial community structure in activated sludge and wastewater [ , ] , to study mechanisms of survival, infection at cellular level and detection of emerging pathogens from water, sewage and sludge [ ] . one example is the two-color fish assay, based on species-specific probes for c. parvum (cpar probe) and c. hominis (chom probe), and has been shown to distinguish between the two species of concern to public health [ , ] . several kits of fish are now available in the market, one example of these are the commercial kit label it ® fluorescence in situ hybridization kit cy ® , fluorescein and tm-rhodamine, which optimized the preparation and hybridization of fluorescently labeled dna probes [ ] . viable but non-culturable (vbnc) cells could detected by fish couple with direct viable count (dvc) assay [ ] [ ] [ ] . in the assay, the cells are cultured in reach media with antibiotics which prevent cellular division, increases of intracellular rrna and allow the elongations of viable cells. fish is made with specific probes that target rrna labeled with fluorescent dye. the sample is stained by ,-diamino- -pheynyl indole (dapi) that is bound to double-stranded dna to count the total amount of cells in the samples, and the results are compared to culture methods to measure the proportion of vbnc cells [ ] . members of the family enterobacteriaceae and e. coli in drinking water systems, freshwater and river water have been detected by this tool [ ] . this assay is able to detect viable e. coli/ ml in more than non-e. coli/ ml [ ] . however, the technology is limited by a low sensitivity, and the necessity of pre-enrichment and concentration steps [ ] that also may increase inhibitor concentrations and lead to false negative results [ ] . vbnc cells are living cells, metabolically active, that may still retain or regain their virulence potential upon the resuscitation (return to culturable state) under favourable environmental conditions or when the stress is removed [ ] [ ] [ ] [ ] . due to the fact that vbnc cells cannot be identified by culturable methods, the number of pathogens in this state could be underestimated and, if all bacteria in the sample are in vbnc state, the sample may be regarded as pathogen-free due to non-detection [ ] . the conversion to the vbnc state is supposed to be a response to adverse environmental conditions [ ] [ ] [ ] . resuscitation of these species could be triggered by a variety of stimuli, such as rich medium, an increase in temperature, and the presence of host cells. factors as the strain used, the age of vbnc cells, and the conditions that induced the vbnc state also affect the resuscitation [ , , ] . besides fish, other tools to detect vbnc bacteria includes: immunological techniques; rt-pcr, which target the mrna that indicates the presence of viable cells that carry out transcription; dnase i protection assay, since only the viable cells have intact membranes to protect genomic dna from digestion by exogenous nucleases; enzymatic activity, as viable cells carry out metabolic reactions and respiration; bacteriophage-based assay, using a combination of fluorescence intensity and nutrient uptake analysis; as well as the commercial kit live/dead ® baclight tm assay to distinguish viable cells from dead cells based on the membrane integrity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . immunological detection with antibodies is a technology that has been employed for the detection of multiple pathogens [ , ] . these methods are based on antibody-antigen interaction, whereby a particular antibody will bind to specific antigen which comprise the use of polyclonal and monoclonal antibodies [ , ] . immunological detection includes, for example, serum neutralization tests (snt), immunofluorescence, and enzyme-linked immunosorbent assays (elisa). snt test has been used for the serotyping of viruses and involves mixing a sample, extracted from a plaque assay, with antiserum and then assessing the decrease of infectivity by plaque assay [ ] . assays based on immunofluorescence and immunomagnetic separation (ims) are used to detect protozoan parasites. the us environment protection agent (epa) has established method . for the combined detection of cryptosporidium spp. and giardia spp., or method for cryptosporidium spp., both of which use ims followed by immunostaining and fluorescent microscopy [ , ] . nevertheless, limitation such as low sensitivity, false negative results, cross-reactivity with closely related antigens and the need for a pre-enrichment in order to reduce the cell surface antigens, are present in these techniques [ ] . biosensor based methods have flourished in recent years. biosensor is an analytical device that consists of a bioreceptor that recognizes the target analyte (e.g., enzymes, antibodies, nucleic acids, cell receptors, aptameters, recombinant antibodies, imprinted polymers and synthetic catalyst) and a transducer that converts the biological interactions into a measurable electrical signal (optical, electrochemical, mass-based, thermometric, or micromechanical) [ , , ] , thereby providing selective quantitative or semiquantitative analytical information [ ] . optical biosensors rely on a change in the optical properties of the surface caused by the binding of the analytes used for detection [ ] . electrochemical biosensors are based on measuring changes in conductance, resistance or capacitance of the active surface. in these devices, one of the electrodes is immobilized with a recognition molecule. when analytes bind, a change in electrical properties occurs providing the sensor signal [ , ] . mass-sensitive biosensors include quartz crystal microbalance biosensor, which uses a quartz crystal inserted in two electrodes. as quartz is piezoelectric (used for wave propagation), the crystal can be excited by applying a voltage across the electrodes and will exhibit a resonance frequency [ , ] . biosensor methods have the advantages of automation and miniaturization of biological analytical techniques, have short analysis times, portability, real-time measurements and do not require sample pre-enrichment [ ] . nevertheless, problems such as great sensitivity to ph, change of mass, temperature, etc., represent a great challenge to the use of these methods [ ] . in , taylor and co-workers [ ] used the surface plasmon resonance (spr) detection with pretreatment of the bacteria (heat or ethanol treated or detergent-lysed cells) and the antibodies goat anti-e.coli o :h polyclonal antibody (pab) and mouse anti-e. coli o :h monoclonal antibody (mab) for signal amplification to obtain a limit of detection (lod) of cfu/ml. antibody-based indirect sensor (optical biosensor) for escherichia coli o :h with fluorescent labeling was reported by ho et al., [ ] in to reach a lod of cells/ml. detection and monitoring of pathogens in water continues to be a field with constant improvement and development of new tools that will permit relative low cost assays and rapid identification of multiple pathogens with limits of detection that meet regulatory goals. nevertheless, efforts to standardize techniques in the field have to include sample collection, sample concentration, sample purification, sample processing, analysis, and data collection are still remaining [ ] . sample collection has to be standardizing mainly if the target is on biofilms. validation of new emerging techniques has also to be completed. besides microarrays and biosensors, the integration of molecular technologies is promising. "lab-on-chip" (loc) represents an integration of sensors and microfluidic systems in a miniaturized tool that could archive real-time monitoring of samples [ ] . by this way, loc integrates several laboratory functions on a single chip and reduced sample and reagent consumption is automatized and has fast detection times and low limits of detection. however, there are still challenges related to developing practical loc components, assay procedures and validation of the on-chip detection approaches, and of course, the different scenarios that represent a water sample [ ] . since outbreaks continue to occur despite water quality monitoring, a new approach has been applied to solve the need for a conceptualization of risk and to provide guidance and legislations [ ] . quantitative microbial risk assessment (qmra) is a systematic quantitative assessment process to estimate the health risks or illness rates of human exposure to particular pathogens. the approach combines dose response information for the infectious agent with information on the distribution of exposures routes and is able to demonstrate if the health targets are met in a drinking water distribution system [ ] , which are suggested to be a health risk less than one infection per , individuals per year, or, a disease burden of − disability adjusted life year (daly) per person per year [ ] . this tool helps to predict the burden of waterborne diseases, set tolerable limits of waterborne disease, identify the means to reduce the risk to the consumers and determine measures to protect water safety [ ] . risk assessments involve five steps ( figure ) [ , [ ] [ ] [ ] [ ] . (i) hazard identification: consists in the identification and quality evaluation of the microbial hazard. this step takes into account the potential outcomes (health effects, disease outbreaks), pathogen properties (virulence, adaptation, resistance, and mutation) and hazard pathways; (ii) exposure assessment: is the estimation of the duration of human exposures to pathogens by specific routes, the determination of the size and nature of the population exposed and the barrier reduction and recontamination on water; (iii) dose-response assessment is the characterization of the relationship between dose and incidence of adverse effect in populations exposed to microbial pathogens. it comprises data of illness, secondary transmission and immunity in population. typically, the dose-response is derived from studies of exposure of human volunteers to different doses of the pathogen or is based on previous outbreaks [ ] [ ] [ ] . factors influencing the dose-response are the exposure route, exposure medium pathogen strain host endpoint and the data source. two commonly models are the beta-poisson model and the exponential model, described elsewhere [ , , ] ; (iv) risk characterization is the integration of information from hazard identification, dose-response assessment, and exposure assessment to estimate the magnitude of health effects; (v) risk management and communication is a decision-making process based in all previous steps in risk assessment [ , [ ] [ ] [ ] [ ] . in qmra, the different variants contribute to measuring the health risk but also introduce variability corresponding to dose-response sensitivity, detection methods, temporal and spatial heterogeneities in pathogen densities and uncertainty (i.e., unrepresentative population, modeling pathogen densities and knowledge of dose-response relationships) [ , [ ] [ ] [ ] . the advantage of probabilistic modeling is to distribute the uncertainties through the model [ ] . the qmra approach presents potential advantages and limitations of risk management options that could be evaluate by numerical stimulations to investigate their possible efficacy, and that risk below epidemiologically detectable levels may be estimated under specific circumstances [ ] . however, the greatest limitation on qmra is the lack of available data to carry out this assessment [ ] . qmra also depends on the method used for monitoring pathogens in water. actually, qmra approach is based exclusively on data obtained by cultivation methods, which are not allowing the complete characterization of risk, since as mentioned above, many waterborne microorganisms are not culturable or are inefficiently cultured, thus leading to underestimation of pathogens in water. therefore, selecting the optimal tool for pathogen monitoring in source water that gives the necessary information on occurrence, prevalence, virulence, and even viability of the pathogens influences the results of the qmra approach. polymerase chain reaction methods provide rapid detection of microorganisms but are complicated by the presence of inhibitory compounds in the sample and the detection of viable but non-culturable as well as non-viable pathogens. integrating the culture and pcr methods may help to overcome the weakness of each individual method [ , , ] . as mentioned before, current risk characterization approaches rely on detection of microbial indicators or "index pathogens", which does not accurately predict the occurrence of all pathogens. novel markers that better predict the presence of all pathogens of interest would improve risk assessment and management actions [ , ] . alternative indicators proposed are bacteroidales and lachnospiraceae, which are rich in host-specific bacterial organisms [ , ] , faecal bacteriophages, rotavirus, ascaris [ ] , firmicutes [ ] , bifidobacteria spp. [ ] , and methanobrevibacter smithii [ ] . commonly s rrna gene has been used as a marker to track host-specific organisms [ ] , but other authors such as villemur et al. [ ] and caldwell et al. [ ] suggest the use of dna (mtdna) to target the identification of human and animal origins. qmra has been used to estimate the health risk associated with bathers after surfing and swimming in dry weather and post-storm conditions near beaches [ ] [ ] [ ] , water distribution networks [ ] , recreational waters impacted by agricultural contamination [ , ] , and assessment of the efficiency of the applied treatment processes in drinking water [ ] . such investigations reinforce the need for preventive measures, such as those designed to prevent and take measures to reduce water contamination. a computational tool for quantitative microbial risk assessment, called qmraspot, was developed for rapid and automatic undertaking of qmra for drinking water. qmraspot uses four index pathogens: campylobacter, enterovirus, cryptosporidium, and giardia [ ] . also, a software infrastructure name frames (framework for risk analysis in multimedia environmental systems) have been created to assess the water health risk which allows describing the problem statement, integrating models and data-bases, and provides the infrastructure for performing sensitivity, variability and uncertain analyses [ ] . it is clear that qmra and management of water quality are important tools for governmental regulation and scientific analysis. nevertheless, it is also clear that these tools still are not adopted widely [ ] . efforts to encourage the use of the qmra approach have to be made to support implementation of water safety plans, improve understanding of vulnerabilities of drinking water distribution systems, assess risks associated with extreme events and establish the best management option for the given risk in order to ensure water safety [ ] . waterborne pathogens are a global concern for worldwide public health. since pathogens in water are still a major cause of severe illness and mortality, the control, monitoring and application of regulations for water quality are in urgent need and 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bifidobacterial diversity and the development of new microbial source tracking indicators a real-time qpcr assay for the detection of the nifh gene of methanobrevibacter smithii, a potential indicator of sewage pollution an environmental survey of surface waters using mitochondrial dna from human, bovine and porcine origin as fecal source tracking markers mitochondrial multiplex real-time pcr as a source tracking method in fecal-contaminated effluents qmraspot: a tool for quantitative microbial risk assessment from surface water to potable water comparison of recreational health risks associated with surfing and swimming in dry weather and post-storm conditions at southern california beaches using quantitative microbial risk assessment (qmra) microbial risks associated with exposure to pathogens in contaminated urban flood water qmra in drinking water distribution system estimated human health risks from exposure to recreational waters impacted by human and non-human sources of faecal contamination the authors would like to thank pathogens editors and the reviewers for their helpful comments. we are gratefully acknowledge the fie grate support of the natural sciences and engineering research council of canada (to j.h, rgpin- ) and from-fonds de la recherche du québec en nature et technologies (centre de recherche en infectiologie porcine et avicole, cripa-regroupements stratégiques rs- ) and from the dfait canada's emerging leaders in the americas program. we thanks to the conacyt mixed scholarship ( / ) of f.y.r.c., and to a.l.g.b., f.j.a.g. and the universidad autónoma de aguascalientes for their funds. josée harel had the original idea for the manuscript and, with all co-authors, carried out the design. flor yazmín ramírez-castillo drafted the manuscript, which was revised by all authors. abraham loera muro and mario jacques included the fluorescence in situ hybridization detection of actinobacillus pleuropneumoniae isolated from water sources. philippe garneau critically revised the manuscript and verified the detection methods. josée harel, mario jacques, alma lilián guerrero-barrera and francisco javier avelar-gonzález revised all the manuscript and profit it. all authors read and approved the final manuscript. the authors declare no conflict of interest. key: cord- -cb ntxs authors: nogales, aitor; l. dediego, marta title: host single nucleotide polymorphisms modulating influenza a virus disease in humans date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: cb ntxs a large number of human genes associated with viral infections contain single nucleotide polymorphisms (snps), which represent a genetic variation caused by the change of a single nucleotide in the dna sequence. snps are located in coding or non-coding genomic regions and can affect gene expression or protein function by different mechanisms. furthermore, they have been linked to multiple human diseases, highlighting their medical relevance. therefore, the identification and analysis of this kind of polymorphisms in the human genome has gained high importance in the research community, and an increasing number of studies have been published during the last years. as a consequence of this exhaustive exploration, an association between the presence of some specific snps and the susceptibility or severity of many infectious diseases in some risk population groups has been found. in this review, we discuss the relevance of snps that are important to understand the pathology derived from influenza a virus (iav) infections in humans and the susceptibility of some individuals to suffer more severe symptoms. we also discuss the importance of snps for iav vaccine effectiveness. influenza a viruses (iav) belong to the orthomyxoviridae family, and they contain a single-stranded (ss) negative-sense viral (v)rna genome formed by eight segments that are encapsidated into particles with an envelope ( figure a) . each of the vrna segments contains a long central coding region flanked at and termini by non-coding regions (ncrs), which work as promoters to initiate viral rna synthesis (transcription and replication). moreover, the packaging signals playing a role in the efficient encapsidation of the viral segments into nascent virions, are located at the and end of the coding regions ( figure b ) [ ] . structurally, vrnas form viral ribonucleoprotein complexes (vrnps), where vrnas are coated with multiple subunits of the viral nucleoprotein (np) and are associated with the heterotrimeric polymerase, which contains the polymerase basic and (pb and pb , respectively) and acidic (pa) proteins ( figure a ) [ ] [ ] [ ] . each vrnp acts as an independent transcription-replication unit using an uncommon mechanism among negative-sense rna viruses, given that viral rna synthesis occurs in the infected-cells nucleus. vrnas are used as templates by the viral polymerase to synthesize two positive-sense rna molecules, the complementary rnas (crnas), from which the same viral polymerase synthesizes more copies of genomic vrna, and the mrnas for viral protein synthesis [ ] [ ] [ ] [ ] [ ] [ ] . the small iav genome encodes for up to viral proteins through the viral envelope is decorated with the two viral glycoproteins hemagglutinin (ha) and neuraminidase (na) at a ratio of approximately four to one, respectively [ , ] . ha envelope protein mediates virus entry by binding to sialic acid-containing cell receptors, and then fusing endosomal and viral membranes during endocytosis [ , ] , while na is required for viral release from infected host cells, and it acts as a receptor destroying enzyme, cleaving terminal sialic acid residues from glycoproteins present at the cell surface [ ] [ ] [ ] . the matrix (m ) protein is also found in the viral membrane, although in much lower abundance than ha or na glycoproteins. m is a small transmembrane protein that forms a proton-selective ion channel in the viral envelope. m promotes uncoating of the vrnps after membrane fusion and the protein has also an essential role in viral assembly and release [ ] . under the viral envelop, there is an inner shell composed of the matrix (m ) protein, which interacts in the virion with the vrnp and the ha and na proteins. m apart from being a membrane-associated scaffold factor of the virion, acts as a crucial factor for different viral processes during infection, including virion assembly and budding [ ] [ ] [ ] . the nonstructural (ns) gene or segment of iav encodes an mrna transcript that is alternatively spliced to express two viral proteins, the nonstructural protein (ns ), produced from a continuous primary transcript, and the nuclear export protein (nep), which is produced by an alternatively processed transcript, using a weak splice site. nep is also located in the virion and may interact with m in the viral particle [ ] [ ] [ ] ( figure a) . during the infection, nep is responsible for the nuclear export of synthetized vrnp, ensuring that the vrnps are available for packaging [ ] . moreover, nep has also other functions during iav infection, contributing to viral budding and to regulate viral rna synthesis. ns is a multifunctional protein and a key viral factor that counteracts the host antiviral responses. ns has been shown to inhibit the production of interferon (ifn), the activity and expression of multiple interferon-induced genes (isg) and the processing and nuclear transport of host mrnas causing cellular shut-off [ , ] . segment of iav also encodes two proteins, the polymerase component pa and pa-x. pa is translated directly from the pa mrna, whereas pa-x is translated using a + frameshift mechanism from the same open reading frame (orf) [ ] . synergistically with ns , pa-x is also able to block the cellular antiviral responses by inhibiting host protein expression. moreover, the pa-x protein has been shown to modulate host inflammation, immune responses, apoptosis, and virus pathogenesis [ ] [ ] [ ] [ ] [ ] [ ] . human iav infections cause contagious respiratory diseases associated with mild to severe respiratory illness or even death, and they are considered as an important public health threat worldwide, which also results in significant economic losses [ ] [ ] [ ] . iav are divided into multiple subtypes, based on the ha and na glycoproteins. currently, there are ha (h to h ) and na (n to n ), but the growing iav surveillance programs and sequencing technologies could increase the number of subtypes in the following years. iav can infect a wide range of avian and mammalian species, although the natural reservoirs of iav are shorebirds and wild waterfowls [ ] [ ] [ ] [ ] . among all the ha and na subtypes, only h n and h n iav subtypes are circulating in human beings and they are responsible for annual recurrent epidemics that affect the entire world [ , ] . seasonal influenza infections are prevented and controlled through annual vaccination campaigns to decrease iav infections and viral transmission as well as to reduce their negative impact in the global economy. however, although vaccination remains the most effective approach to protect the population from seasonal infections, the effectiveness of current vaccination approaches is suboptimal [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . thus, the production of improved prophylactic approaches, including universal vaccines, are highly desired. concerns associated with iav are further aggravated by the adaptive capacity of the viruses to infect new hosts or escape to the immune system, as well as their ability to transmit efficiently in the population and the limited therapeutic options to treat viral infections [ , , , ] . because of the ability of iav to modify their genome using two main evolutionary mechanisms, antigenic drift and shift, viruses encoding novel antigenic proteins to which the population has limited or no preexisting immunity can be generated [ , , , ] . for that reason, seasonal vaccines have to be reformulated yearly to guarantee that the viral glycoproteins (ha and na) in the vaccine match seasonal viruses circulating worldwide [ , , ] . in addition, iav variability can lead to the generation of new virus strains with pandemic potential. for example, the first iav pandemic of this century occurred in and it is estimated that in approximately one year, the pandemic h n (ph n ) iav infected more than , human beings, causing near , deaths in over countries [ , ] . in addition, although only h n and h n are circulating in humans, the avian h , h , and h subtypes eventually cross the species barrier to infect humans, representing a new and serious public health problem [ , , [ ] [ ] [ ] . the cellular defense mechanisms provided by the innate immune system are a formidable barrier to inhibit virus infections [ ] and involve the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs). this recognition leads to the activation of signaling pathways and the production and secretion of ifns of type i (ifnα and ifnβ) and iii (ifnλ or il- a, ifnλ or il- b, and ifnλ or il- ) , and chemokines and cytokines involved in inflammatory processes [ ] . iav rnas are mainly recognized by the endosomal, membrane-associated prr toll-like receptors (tlrs) (double-stranded rnas, dsrnas) or / (ssrnas), respectively [ , ] , by the cytoplasmic prr retinoic acid-inducible gene i (rig-i), which detects dsrna and -triphosphates of the negative ssrna viral genome [ , ] , generated during replication of multiple viruses, by the nod-like receptor family member nod-, lrr-and pyrin domain-containing (nlrp ), which recognizes various stimuli (see below) [ ] and by the absent in melanoma (aim ) protein, recognizing not well-characterized influenza stimuli [ ] . the result of prr detection of viral pamps is the activation of multiple transcription factors, such as the nuclear factor kappa β (nf-κb), the activator protein (ap- ), and ifn regulatory factors (irf)- and irf- , which are responsible for the transcription of ifns [ , , ] and pro-inflammatory cytokines [ ] . secreted type i and iii ifns signal through different receptors in a paracrine or autocrine way to induce the transcription of ifn-stimulated genes (isgs), several of which counteract viral replication [ , , ] . just as an example mentioned below, ifitm is an isg playing antiviral roles against influenza virus infection and other viruses [ ] . type i and iii ifns signaling pathways lead to the post-translational phosphorylation of the signal transducer and activator of transcription (stat) and transcription factors [ ] , being the tyrosine kinase (tyk ) and janus protein tyrosine kinase (jak ) critical for the phosphorylation [ ] . moreover, stat is phosphorylated by ikkε during ifn signaling and this step is important for the ifn-inducible innate immune response [ , ] . upon phosphorylation, stat and stat associate with irf- forming the heterotrimeric isg factor (isgf ) complex [ ] . this heterotrimeric complex then translocates to the nucleus, and binds to ifn-stimulated response elements (isres) located in the promoters of isgs, up-regulating their expression [ , ] . inflammatory cytokines, such as interleukins (il)- a il- b and tumor necrosis factor (tnf)-α contribute to the proliferation and migration of different immune cells, such as monocytes, macrophages, neutrophils, and natural killer (nk) cells, to the infected tissue. nk cells have the ability to kill virus-infected cells, are important for the activation of a protective cytotoxic t lymphocyte (ctl) response [ ] , and nk-cell ifn-γ production is augmented by t-cell il- production in recall responses [ ] . neutrophils and resident alveolar macrophages are also important for virus clearance, due to their ability to destroy infected cells [ ] . in addition, cytokine signaling improves dendritic cells (dc) maturation, increasing the induction of adaptive immune responses by antigen presentation and co-stimulation [ , ] . these adaptive immune responses initiated upon innate immune activation are required for protection and viral clearance [ ] . nlrp is expressed by myeloid cells such as macrophages, monocytes, neutrophils, and dendritic cells [ ] or by human bronchial epithelial cells [ ] . upon stimulation, nlrp activates the inflammasome system, activating caspase- and leading to pro-inflammatory processes through the processing and activation of proil- b, proil- , and proil- [ ] . nlrp senses iav dsrna [ ] , and pb -f protein [ ] . furthermore, protein flux through the viral m ion channel activity in the trans-golgi network activates nlrp , leading to inflammasome activation [ ] . in addition to nlrp activation, iav activates the inflammasomes through aim , increasing iav-induced lung injury and mortality [ ] . the complement system is an important branch of innate immunity that plays an essential role in the clearance of pathogens. the complement system is triggered by three main pathways, the classical, the lectin, and the alternative pathways [ ] . the first two pathways are activated with the help of pattern recognition molecules, whereas the alternative pathway is activated spontaneously. interestingly, it is known that viruses are recognized by the three pathways. in the classical pathway, the c complex recognizes antigen-antibody complexes, which are formed on the pathogen surface. c qbp (complement c q binding protein) can bind to the globular heads of c q molecules, activating the classical pathway [ ] . on the other hand, in the lectin pathway, the mannan-binding lectin (mbl)/ficolin/mannan-binding lectin serin protease (map) complex recognizes specific carbohydrates on the pathogen surface. complexes activated after the classical and lectin pathways, cleave c and c , resulting in the generation of c bc a (c convertase). in the alternative pathway, spontaneous hydrolysis of native c results in the formation of c b-like c that binds factor b and after cleavage by factor d forms the initial c convertase [ ] . the three pathways converge at the cleavage of c into c a and c b by c convertases (c b, a and c b, bb). then, the c b molecules formed bind covalently to the c -convertases forming the c -convertases that cleave c into c a and c b. cd blocks c and c activation by preventing the formation of new c and c convertases [ ] . c b starts the formation of c b- or the membrane attack complex (mac). next, c binds to the membrane attached trimer and begins binding and polymerization of c that is inserted into the membrane, inducing virolysis [ ] . unregulated complement activation could play a central role in the acute lung injury (ali) pathology induced by highly pathogenic viruses, including severe acute respiratory syndrome (sars) coronavirus and avian iav h n , and h n [ ] . in virus-induced acute lung diseases, high levels of chemotactic, and anaphylatoxic c a can be generated as a result of excessive complement triggering and causing a "cytokine storm". accordingly, the blockade of c a signaling has been involved in treating the ali induced by highly pathogenic viruses [ ] . currently, particular attention is being paid to single nucleotide polymorphisms (snps) that are loci within the genome of an organism in which two or more alleles can exist. snps affect a single nucleotide or base pair and they are one of the most frequent types of genetic variations in the genome [ ] [ ] [ ] . snps need to be presented into the population with a frequency equal to or greater than % to be considered as polymorphisms. there are multiple types of snps, depending on their location that can be in different regions of the genes such as promoters, exons, introns or utrs ( figure ). snps in coding regions are classified as synonymous, when a nucleotide substitution does not change the amino acid sequence of the encoded protein, although other effects, such as changes in mrna structure or folding may account for variation in protein expression. on the other hand, non-synonymous snps are divided in missense or nonsense. in the first case, nucleotide substitution results in the change of one amino acid for another, affecting the protein sequence coded by a gene and therefore may lead to its dysfunction. in contrast, nonsense mutations are produced when instead of substituting one amino acid for another, the altered gene contains an early stop codon in the orf or a stop codon is abrogated, producing an elongated protein. this type of mutations results in shortened or elongated proteins leading typically to nonfunctional proteins. the functional role of snps in coding areas of the genome can be easily analyzed by studying the gene products. however, most snps fall within non-coding genome regions, therefore, predicting their effects is challenging. for example, snps in the promoter regions could affect their activity and regulation producing changes in gene expression levels. snps in utrs or intron regions have been related with an effect in protein translation or the production of splice variants of transcripts, leading to longer or shorter protein sequences, respectively. in summary, snps may influence gene regulation, the structure and stability of rna, the expression of rnas or proteins, the conformation and function of proteins, etc. thus, the identification of snps in genes and the analysis of their effects may lead us to better understand gene function or their impact on human health [ ] . in fact, snps that are or could be important for multiple human pathologies, such as cancer, diabetes, heart disease, schizophrenia, blood-pressure homeostasis, and autoimmune or metabolic diseases, have been identified [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, some described snps increase the human susceptibility to getting infected by viruses, bacteria or other pathogens [ , , [ ] [ ] [ ] [ ] [ ] [ ] . advanced sequencing and bioinformatics technologies have allowed the identification of a large number of human snps whose information is accessible in the databases. nevertheless, the biological significance and function for most of the snps found in the human genome remain unknown. currently, the scientific community recognizes the importance of this kind of genome variations that can act as biological markers and assist researchers in multiple aspects, such as: ( ) locate genes associated with multiple diseases, ( ) anticipate an individual's response to a specific infection, ( ) predict population responses to several treatments such as drugs or vaccines, ( ) design individualized therapies, ( ) identify markers for medical testing, ( ) perform pharmacogenetic studies, etc. this review focuses on the role of known snps on iav infection, as well as their impact on the effectiveness of vaccines against iav. instead of substituting one amino acid for another, the altered gene contains an early stop codon in the orf or a stop codon is abrogated, producing an elongated protein. this type of mutations results in shortened or elongated proteins leading typically to nonfunctional proteins. the functional role of snps in coding areas of the genome can be easily analyzed by studying the gene products. however, most snps fall within non-coding genome regions, therefore, predicting their effects is challenging. for example, snps in the promoter regions could affect their activity and regulation producing changes in gene expression levels. snps in utrs or intron regions have been related with an effect in protein translation or the production of splice variants of transcripts, leading to longer or shorter protein sequences, respectively. an snp is a variation on a single nucleotide which may occur at some specific point in the genome and that causes variations in dna sequences between members of the same species. (b) types of snps: dna variation can be located in non-coding or coding regions. snps within a coding sequence can be synonymous if they do not produce an amino acid change (silent mutation), or non-synonymous if they affect the protein sequence. nonsynonymous changes can be divided into missense (producing an amino acid change in the protein) or nonsense (producing a truncated or longer protein). an snp is a variation on a single nucleotide which may occur at some specific point in the genome and that causes variations in dna sequences between members of the same species. (b) types of snps: dna variation can be located in non-coding or coding regions. snps within a coding sequence can be synonymous if they do not produce an amino acid change (silent mutation), or non-synonymous if they affect the protein sequence. non-synonymous changes can be divided into missense (producing an amino acid change in the protein) or nonsense (producing a truncated or longer protein). risk factors, including underlying co-morbidities, age, and pregnancy, affect iav susceptibility, but do not explain all the conditions under which serious iav-associated disease can occur, making likely that snps in viral and host genes affect iav susceptibility and the outcome of the disease. in fact, there are some examples of the presence of snps in host genes affecting influenza severity (table ) , which will be discussed in this review. snps affecting iav disease have been found in genes recognizing viral components, in transcription factors important for ifn production and signaling, in isgs with antiviral activities, and in genes involved in inflammation. tlr recognizes dsrna, one of the iav replication intermediate products, and in turn activates ifn production, leading to an antiviral response. a missense mutation (f s) of the tlr gene was found in one out of three patients developing iav-associated encephalopathy (iae), a neurological consequence of severe viral infection [ ] . assays in tissue culture cells showed that a tlr receptor encoding the missense f s mutation was impaired in activating the transcription factor nf-κb, and in triggering downstream signaling via the ifnβ receptor, indicating that this genetic polymorphism could lead to increased iav replication [ ] . in a study of italian children diagnosed with iav h n infection, an additional tlr snp (rs , genotype c/t) was identified [ ] . this tlr snp was found in all the children developing iav-associated pneumonia ( cases). however, the snp was found in significantly less proportion in children with milder disease, suggesting a link between tlr and iav pathogenicity. furthermore, in a multicenter study involving adult cases of avian h n and ph n iav, in mainland china and hong kong, the tlr cc rs snp was associated with fatal cases [ ] . in addition to iav, there are other examples of snps in tlr or tlr signaling genes affecting viral infections. for instance, susceptibility to chikungunya virus (chikv) infection is highly increased in human and mouse cells with defective tlr molecules [ ] . furthermore, tlr snps, rs , and rs , leading to unknown functional consequences, were associated with an increased risk of chikv disease occurrence [ ] . patients with impaired tlr -mediated responses show an elevated susceptibility to herpes simplex- virus (hsv- )-mediated encephalitis by encoding tlr -deficient alleles [ , ] , or by encoding deficient traf , tbk and trif molecules, leading to impaired tlr- signaling [ ] [ ] [ ] . in a saudi arabian population, the tlr rs snp was strongly associated with hepatitis b (hbv) and hepatitis c (hcv) virus infections when compared to that in healthy control subjects [ , ] . the tlr rs c allele was also associated with hcv-related liver disease progression (cirrhosis and hepatocellular carcinoma) [ ] . however, the functional effects of these snps seem to be unknown. rig-i detects dsrna and -triphosphates of the negative ssrna iav genome, leading to innate immune responses activation [ ] . a caucasian male patient with severe iav h n infection during the swine flu pandemic showed two heterozygous variants (one in each chromosome): p.r h (snp rs ) and p.p s (snp rs ), located, respectively, in the caspase activation and recruitment domain (card) and rna binding domains of rig-i [ ] . these variants significantly decreased the recognition function of rig-i, and therefore, patient cells proved impaired antiviral responses to rig-i ligands and elevated proinflammatory responses to iav, providing evidence for dysregulation of the innate immune response and increased immunopathology [ ] . these results suggest that these rig-i polymorphisms may have contributed to severe iav outcome in this patient and reinforce that rig-i variants should be evaluated in future studies of host factors affecting ssrna virus infections. irf- is a transcription factor that increases interferon (ifn) production in response to viruses [ ] [ ] [ ] . a patient suffering from an unusual life-threatening disease after ph n infection encodes homozygous null mutations in the irf- factor. both irf- alleles from this patient encode mutations c. t>g/t (f v) and c. c>t/c (q x), which are mutations decreasing the ability of irf- to induce the transcription of ifn genes after iav infections [ ] . these findings suggest that irf- -dependent production of type i and iii ifns is required for controlling iav infections in humans. the rare allele a of two irf- snps, rs and rs , both located at exon/intron boundaries, were significantly associated with impaired levels of ifnα production by human plasmacytoid dendritic cells (pdcs) in response to human immunodeficiency virus (hiv- ) infection [ ] . therefore, these polymorphisms may affect the ability of human subjects to control hiv- infections, reinforcing the role of irf- in controlling viral infections. however, the effect of these snps should be further studied. irf- is a transcription factor essential for ifn signaling and the transcriptional induction of isgs [ ] . stat and stat , when phosphorylated, associate with irf- to form a heterotrimeric isg factor (isgf ) complex [ ] , which translocates to the nucleus, and binds isres present in the promoters of isgs, up-regulating their transcription [ , ] . a homozygous, loss-of-function mutation in irf- was described in a child born to first-cousin algerian parents and living in france affected by a severe pulmonary influenza infection [ ] . in particular, the homozygous mutation (c. g>a) occurred in the final nucleotide of exon and disrupted the essential splice site at the boundary of exon and intron , leading to deleted irf- proteins. the consequence of this mutation was an impaired activation of irf- , and therefore, an impaired transcription of isgs, many of which show antiviral activities [ ] . similarly, a family in which several members showed a surprising susceptibility to infection by different viruses, including iav, also showed to be irf deficient [ ] . the index patient, a boy with years born at term from healthy consanguineous parents (first cousins of portuguese origin and residing in venezuela) encoded a homozygous splicing mutation in the irf gene. the mutation, c. + g>t, was located in the donor splice site of introns and , leading to transcripts lacking exon . irf protein expression was undetectable in cells transfected with the c. + g>t irf construct, suggesting that either the protein was quickly degraded or the mrna was not translated. again, irf -deficient cells showed a profound defect in inducing the expression of multiple isgs [ ] . collectively, these findings show that human irf -and isgf -dependent type i and iii ifn responsive pathways are essential for controlling viral infections, including iav. the antiviral protein ifitm is an isg which abrogates the release of iav content from late endosomes into the cytoplasm [ ] . in addition, ifitm promotes the survival of mouse lung-resident cd + t cells following iav challenge, which may help clear the infection [ ] . furthermore, mice in which the expression of ifitm is abolished, showed severe disease after iav infection, compared to parental mice [ ] . one of the clearest associations of snps in genes affecting influenza severity is located in the isg ifitm . the human ifitm gene is encoded by two exons and is predicted to encode two splice variants that differ in the first amino-terminal amino acids. different studies have described the effect of ifitm snps in influenza disease severity. northern european patients infected with iav ph n virus requiring hospitalization showed over-representation of the snp rs in the ifitm gene, in which the majority t allele is replaced for a minority c allele [ ] . this leads to an alteration of the first splice acceptor site, originating an ifitm protein lacking the first amino acids (n∆ ) due to the protein starting from an alternative start codon. according to these results suggesting that this snp could affect influenza disease, the minority (cc) variant rendered homozygous cells more susceptible to iav infection, and this susceptibility correlated with decreased levels of ifitm protein expression in comparison to the majority (tt) variant cells [ ] . furthermore, cells expressing the n∆ protein showed an impaired ability to restrict viral replication when compared to wild-type ifitm cells [ ] . this data is consistent with previous results which show that the amino-terminal amino acids of ifitm are relevant for attenuating vesicular stomatitis virus (vsv) replication in vitro [ ] . moreover, the cc genotype was found in % of chinese patients showing mild disease after ph n virus infection compared to % in patients developing a severe ph n virus infection. in addition, the cc genotype was estimated to confer a six-fold increased risk for severe infection than the ct and tt genotypes [ ] , reinforcing the idea that ifitm is a factor affecting human iav disease [ ] . in another study, over-representation of the ifitm cc genotype was detected among fatal cases of chinese patients infected with iav ph n and h n viruses [ ] , and in a more general study, including twelve studies published before february with more than , subjects, revealed increased risk of severe influenza in both the east asian and white populations in the subjects encoding the ifitm cc genotype [ ] . another important snp (rs ) associated with risk of severe influenza in humans from the united states (us) infected with seasonal iavs is located in the -utr of the ifitm gene [ , ] . this snp affected ifitm expression being the risk allele associated with lower mrna expression. the mechanism for this lower mrna expression involves the decreased irf- binding and increased binding of the transcriptional repressor ccctc-binding factor (ctcf) in promoter-binding assays for the risk allele [ ] . moreover, the risk allele disrupted a cpg site that becomes differentially methylated in cd + t cell subsets, leading to less cd + t cells in the airways during natural influenza infection in the carriers of the risk allele, and suggesting that a critical role for ifitm may be to promote immune cell persistence at mucosal sites [ ] . interleukins a and b (il- a and il- b, respectively) are inflammatory cytokines that play critical roles in recruiting immune and inflammatory cells and developing adaptive immune responses. furthermore, accumulating evidence suggests that both cytokines play central roles in innate immunity against viral infections [ ] . the frequencies of snp (allele c) located base pairs upstream from the transcription start site (rs ), on the il- b promoter were associated with increased risk of influenza disease in chinese subjects [ ] . this nucleotide change is localized in a tata-box motif of il- b and modulates the transcription activity of il- b by binding to multiple transcription factors [ ] . the allele t of rs enhanced il- b protein expression, as indicated by several reports [ ] . people carrying allele t showed a higher il- b expression, which could lead to increased ifnγ production, which promotes virus clearance [ ] . in contrast, expression of il- b may be decreased in individuals who carry allele c, leading to a weaker immune response during viral infection. in addition, a t allele in il- a gene (snp rs ) increased the risk of iav ph n susceptibility, as observed in chinese subjects [ ] . the snp rs introduces a nonsynonymous mutation (a s) in il- a protein, suggesting that this genetic variant may lead to a functional variation in host susceptibility to ph n . nevertheless, the molecular mechanism needs to be evaluated and the real risk of these alleles should be analyzed in larger populations. tnf-α is a pro-inflammatory cytokine which orchestrates the host´s defense. a minor allele (a) at position - of tnf (snp rs ) was more frequent in greek patients infected with ph n virus compared to control subjects [ ] , and developing pneumonia was more uncommon in greek and mexican subjects with no copies of the minor allele compared to subjects with at least one copy of the minor allele [ , ] , leading to the hypothesis that this snp allele could be linked with an elevated susceptibility to infection with the ph n virus [ , ] . decreased tnf-α expression was observed in subjects encoding the minor allele at position - [ ] . this may explain how snps leading to lower production of tnf-α may predispose to more severe clinical symptoms following iav infections. however, the tnf-α rs minor a allele, associated with higher levels of tnf-α expression, was associated with susceptibility to japanese encephalitis virus infection in an indian population [ ] . the tnf-α rs minor a allele was a risk factor to develop liver cirrhosis and hepatocellular carcinoma following hbv infection in a han chinese population [ ] , suggesting that the protective or deleterious roles of tnf-α expression may vary depending on the infecting virus. chemokine receptor (ccr ) is expressed mainly on macrophages, t cells, and dendritic cells. ccr mediates leukocyte chemotaxis in response to its ligands, including mip- a, mip- b, and rantes. it can help direct multiple immune cell subsets, including regulatory t cells or th cells to sites of infection, supporting the antiviral immune response. evidence in humans support that homozygosity for the ccr -∆ allele, a naturally occurring polymorphism of ccr encoding a -bp deletion, prevents its expression on the cell surface, and is linked with an elevated susceptibility to west nile virus (wnv) [ ] and with increased severity of illness among patients infected with ph n [ ] , although this evidence is modest due to the limited number of subjects analyzed. in contrast, homozygous carriers of the ∆ mutation are resistant to hiv- infection because this molecule, absent in the cell surface in subjects encoding the deletion, is a molecule normally used by hiv- to enter cd + t cells [ ] . cd is an important complement regulatory protein which blocks c and c activation by preventing the formation of new c and c convertases, two proteases involved in inflammation and complement activation. consequently, cd protects cells from complement attack and decreases amplification of the complement cascade [ ] . the cd snp (rs , genotype t/t) was significantly associated with severe iav infection in chinese patients infected with ph n virus [ ] and was associated with increased death risk in greek patients [ ] . the rs snp of cd is located in the minimal promoter region [ ] and individuals with this genotype showed significantly lower levels of cd expression in comparison to those with the more frequent allele [ ] . therefore, patients who carry the t/t genotype may have more robust complement activation during iav infection, resulting in enhanced inflammation and disease severity [ , ] . according to these results, the polymorphism rs in gene cd was linked to disease severity in adult chinese cases of avian (h n ) and human ph n iav in another study [ ] . however, these findings need to be confirmed in bigger cohorts. c qbp can bind to the globular heads of c q molecules, activating the classical pathway of complement [ ] . an increased risk of severe disease after iav infection was found in patients homozygous for the minor allele of the snp rs in european and mexican populations [ , ] . however, the effect of this snps on gene expression and function is undescribed. soluble pattern-recognition molecules, forming part of the innate immune system, can neutralize iav infection. particularly, the serum mannose-binding lectin (mbl), several secreted human c-type lectins of the collectin family, collectin , and the pulmonary surfactant proteins (sp) -a , -a , and -d (sftpa , sftpa , and sftpd, respectively), may neutralize iav infectivity in vitro [ ] . mice lacking sp-a or sp-d were more susceptible to iav infection, indicating that sps exert relevant roles against iav infection [ ] [ ] [ ] . two frequent sp-a (sftpa ) missense alleles (rs -c, leading to the mutation q k and rs -a, leading to the mutation t n) were associated with acute respiratory failure, mechanical ventilation, and acute respiratory distress syndrome after infection with ph n virus in a spanish population [ ] . in addition to c-type lectins, s-type lectins have been described, such as galectins, which recognize galactose-containing oligosaccharides present in the cellular plasma membranes and in viruses, such as iav. importantly, intranasal treatment of galectin- enhanced survival of mice infected with iav by reducing viral load, apoptosis, and inflammation in the lung [ ] . moreover, galectin- knockout mice showed increased susceptibility to influenza virus infection than wild-type mice [ ] . to study human genetic susceptibility to avian iav h n infection, a genome-wide association study involving heavily-exposed healthy poultry chinese workers and iav h n patients was performed [ ] . functional variants of galectin- gene, including rs and rs , causing increased expression levels of galectin- expression, may confer more protection from iav h n infection to the carriers of these variants [ ] . the cleavage of the iav ha by host proteases is critical for viral infectivity. tmprss is a type ii transmembrane serine protease family member, which was shown to activate ha proteins of multiple human iavs in tissue culture cells. furthermore, deletion of tmprss in mice impairs the spread of h n influenza viruses, including the ph n swine iav [ ] . in addition, bodyweight loss and survival after h n iav infection were less severe in tmprss mutant mice compared to wild type mice [ ] . the genetic predisposition to severe ph n influenza virus was evaluated in chinese human subjects, finding that the gg genotype of rs , leading to increased expression of tmprss , was a risk variant to severe ph n influenza [ ] . furthermore, rs and rs , both of them associated with increased gene expression, were significantly associated with the susceptibility to iav h n [ ] . table . single nucleotide polymorphisms associated with susceptibility and severity of influenza infections. recognizes dsrna, triggering ifn production. rs not annotated; f s (nonsyn). rs (ncr). [ ] [ , ] rig-i detects dsrna and -triphosphates of the negative ssrna iav genome, leading to innate immune responses activation. rs ; r h (nonsyn). rs ; p s (nonsyn). [ ] irf- transcription factor that increases ifn production in response to viruses. rs ; f v (nonsyn) rs ; q x (nonsyn) [ ] irf- transcription factor essential for ifn signaling and the transcriptional induction of isgs. c. g>a occurred in the final nucleotide of exon and disrupted the essential splice site at the boundary of exon and intron (nonsyn). c. + g>t, was localized in the donor splice site of introns and and led to transcripts lacking exon (nonsyn). [ ] [ ] ifitm isg which abrogates the release of iav content from late endosomes into the cytoplasm. ifitm increases the survival of mouse lung-resident cd + t cells after iav infection, which can help clear the infection. rs , leading to an alteration of the first splice acceptor site, leading to an ifitm protein lacking the first amino acids (nonsyn). rs , is located in the -utr and affects ifitm expression with the risk allele showing lower mrna expression (ncr). [ , , , ] [ ] il- b inflammatory cytokine involved in the development of adaptive immune responses. furthermore, accumulating data has suggested that il- a and il- b have critical roles in innate immunity against viral infections. rs , located base pairs upstream from the transcription start site, on the il- b promoter. this nucleotide change is located in a tata-box motif of il- b, affecting the transcription activity of il- b (ncr). [ , ] galectin- recognizes galactose-containing oligosaccharides present in the cellular plasma membranes and in viruses, such as iav. -rs (ncr). -rs (ncr). [ ] tmprss type ii transmembrane serine protease family member which activates ha proteins of diverse human iav in tissue culture cells. deletion of tmprss in mice impairs the spread of h n influenza viruses, including the ph n . moreover, body weight loss and survival were less severe in tmprss mutant mice compared to wild type mice after infection with h n iav. -rs , localized in an intron (ncr). -rs , localized in an intron (ncr). [ ] syn-synonymous, nonsyn-nonsynonymous, ncr-non-coding region (intron, regulatory regions, promoter or utr). currently, iav vaccines are the main strategy to prevent iav infection, though their effectiveness is suboptimal in many cases. notably, the efficacy of vaccines against iav infections can fluctuate and there is a significant immune response variability across the population. factors such as previous exposure to iav infections or vaccines, age, and the closeness of the match between the vaccine and circulating strains are important to explain differences in vaccine effectiveness between seasons and group populations [ , , [ ] [ ] [ ] . however, multiple reports have demonstrated that the host genetic background and polymorphisms on key immune response genes modulate the immune response to infection or vaccination [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . therefore, new insights into iav-host interaction and immune response modulating factors could allow us to design better vaccination strategies. snps may modify the humoral immune response after iav vaccination. therefore, their impact on the immune responses induced after iav vaccination are being analyzed [ ] [ ] [ ] [ ] . the major histocompatibility complex (mhc) is localized in chromosome of the human genome, it includes multiple genes and exhibits considerable diversity between populations. moreover, in this genomic region, there is a higher presence of snps than in other sections of the genome. mhc class i and class ii molecules have an essential role in the adaptive immune system in response to infections. both classes of proteins bind peptide fragments derived from pathogens to be presented on the cell surface for recognition by appropriate t cells [ , , ] . in those genes, the human leukocyte antigens (hla) class i and ii are important because of their role in the immune system. gelder et al. studied whether hla class ii polymorphisms modulate anti-iav antibody responses to vaccination in a united kingdom population [ ] . for that, a cohort of hla-typed donors at risk was investigated, and hemagglutination-inhibition (hai) titers were evaluated before and days after the administration of seasonal trivalent influenza vaccine. a correlation between hla class ii alleles and iav hai titers in the influenza risk group was found. moreover, a positive association between non-responsiveness to influenza vaccine and hla-drb * and a negative association with hla-drb * and hla-dqb * - / [ ] was reported, suggesting that polymorphisms in hla class ii molecules affect antibody responses to iav vaccination. these findings are important because they could potentially identify individuals who may not be protected by current vaccination approaches. in another study, poland et al. analyzed the immunogenetic relationships between hla, cytokine and cytokine receptor gene polymorphisms in the induction of antibodies in response to inactivated seasonal vaccines [ ] . authors did not find statistically significant associations between hla class ii alleles and iav hai titers. however, they established a positive association of some hla class i alleles and iav h n hai titers, including hla-a* , a* , b* , b* , and c* . in contrast, they did not find associations between the hla-a, b or c alleles and hai antibody titers for iav h n . in addition, when authors evaluated a panel of cytokine and cytokine receptor snps, they identified several significant associations between snps, in regulatory or coding regions of cytokine (il- , il- b) or cytokine receptor (il- r, il- rb, tnfrsf a) genes and variations in hai antibody titers for iav h n [ ] (table ) . notably, snps from three genes, il- (rs ), il- b (rs ) and il- r (rs ) revealed links with iav h n -induced antibody responses in an allele dose-related way. the presence of snp allele c or g in the il- b or il- r genes, respectively resulted in reduced hai titers. however, high hai titers in the presence of minor snp allele g in the il- gene were observed [ ] . snps associations between cytokine or cytokine receptor genes and iav h n hai titers were also identified ( table ) . for example, a variant ga for non-synonymous snps within the il- receptor gene (rs ; d g) and tnf receptor gene (rs ; k e) displayed associations with lower hai titers, while a minor allele t variant (rs ) located in the region of the il- receptor gene was related with high antibody titers ( table ). these data suggest that host snps affect responses to influenza vaccine. mannose-binding lectin (mbl- ) is a protein that binds n-acetylglucosamine, mannose, and fucose on different microorganisms and activates the lectin complement pathway [ , ] . tang et al. studied the presence of snps in subjects who received an inactivated influenza vaccine. for that, authors classified the vaccine recipients in poor, normal or adverse responders. they observed that the g to a snp in the codon allele (rs ) in mbl- was associated with a decreased risk for the development of adverse or poor responses (table ) [ ] . in addition, they did not find a significant association between responses and either tnf-α or il- promoter snps among the response groups [ ] . cytokine expressed as a response to infections or tissue injuries. it plays an important role in host defense through the stimulation of acute-phase responses. -rs (ncr). -rs (ncr). iiv [ ] il- b cytokine that serves as a crucial inducer of th cell development. rs , located in ´utr (ncr). iiv [ ] ifn-b cytokine released as part of the innate immune response against infection by viruses or other pathogens. rs (ncr). iiv [ ] tnfrsf a cytokine receptor, its interaction with tnf-α control cell survival, apoptosis, and inflammation. rs (ncr). iiv [ ] il- r cytokine receptor involved in inflammatory and immune responses. rs , located in ´utr (ncr). iiv [ ] il- rb cytokine receptor that mediates the activation of the jak/stat signaling pathway leading to the expression of isg. rs , located in ´utr (ncr). iiv [ ] il- ra this cytokine receptor is important for the signaling pathway leading to immune cell differentiation and function. -rs (syn). -rs (ncr). iiv [ ] il- ra cytokine receptor that is involved in the inhibition of the synthesis of several proinflammatory cytokines. -rs (syn) -rs (ncr). iiv [ ] il- rb cytokine receptor that plays a role in th cell differentiation. rs ; d g (nonsyn). iiv [ ] il- rn cytokine receptor which modulates a variety of immune and inflammatory responses related with il- . -rs (syn). -rs located in ´utr (ncr). iiv [ ] tnfrsf b cytokine receptor involved in the recruitment of anti-apoptotic proteins. rs ; k e (nonsyn) iiv [ ] mbl- this calcium-dependent protein that plays an important role in innate immunity, and activates the lectin complement pathway. rs ; g d (nonsyn) iiv [ ] il- b (ifnl ) type iii ifn molecule, with brad functions in antiviral responses rs (ncr). iiv [ ] syn-synonymous, nonsyn-nonsynonymous, ncr-non-coding region (intron, regulatory regions, promoter or utr). iiv: inactivated seasonal vaccine. il- , interleukin . il- b, interleukin . ifn-b , interferon beta (ifnβ). tnfrsf a, tnf receptor superfamily member a. il- r , interleukin receptor type . il- rb, interleukin receptor subunit beta. il- ra, interleukin receptor subunit alpha. il- ra, interleukin receptor subunit alpha. il- rb , interleukin receptor subunit beta . il- rn, interleukin receptor antagonist. tnfrsf b, tnf receptor superfamily member b. mbl- , mannose binding lectin . il- b or ifnl , interferon lambda . other snps that are not related with immune responses have been also linked to vaccine effectiveness. egli et al. revealed that the presence of the t/g or g/g genotype (rs , minor-allele) in il- b (ifnλ ), a type iii ifn, was linked with increased seroconversion in recipients of an inactivated influenza vaccine (table ) [ ] . moreover, iav-stimulated b-and t-cells from the minor-allele carriers exhibited increased hla-dr and il- expression, respectively. in addition, the expression of il- b, but not il- a or il- , mrnas was significantly reduced in the rs , minor-allele carriers. authors also reported that the il- b rs polymorphism affected humoral responses to the iav vaccine, and had a strong outcome on cellular immune responses by modulating the th /th cytokine response [ ] . these findings are important because they will help to predict which individuals could not be protected by present vaccines and they can also be used to design personalized vaccine strategies to optimize the immune reaction. the sequencing of the human genome together with the development of novel bioinformatic tools have made possible the identification of multiple snps. more information is available for the scientific community in the databases. in addition, the identification and study of the human genome variability has opened the opportunity to investigate their association with the risk of developing multiple human diseases 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complex a journey through the lectin pathway of complement-mbl and beyond the role of mannose-binding lectin in health and disease host single-nucleotide polymorphisms and altered responses to inactivated influenza vaccine we apologize for those publications we could not refer due to space limitations. the authors declare no conflict of interest. key: cord- -ytr ds i authors: lutz, mirjam; steiner, aline r.; cattori, valentino; hofmann-lehmann, regina; lutz, hans; kipar, anja; meli, marina l. title: fcov viral sequences of systemically infected healthy cats lack gene mutations previously linked to the development of fip date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: ytr ds i feline infectious peritonitis (fip)—the deadliest infectious disease of young cats in shelters or catteries—is induced by highly virulent feline coronaviruses (fcovs) emerging in infected hosts after mutations of less virulent fcovs. previous studies have shown that some mutations in the open reading frames (orf) c and b and the spike (s) gene have implications for the development of fip, but mainly indirectly, likely also due to their association with systemic spread. the aim of the present study was to determine whether fcov detected in organs of experimentally fcov infected healthy cats carry some of these mutations. viral rna isolated from different tissues of seven asymptomatic cats infected with the field strains fcov zu or fcov zu was sequenced. deletions in the c gene and mutations in the b and s genes that have been shown to have implications for the development of fip were not detected, suggesting that these are not essential for systemic viral dissemination. however, deletions and single nucleotide polymorphisms leading to truncations were detected in all nonstructural proteins. these were found across all analyzed orfs, but with significantly higher frequency in orf b than orf a. additionally, a previously unknown homologous recombination site was detected in fcov zu . feline coronaviruses (fcovs) are endemic in the domestic cat population and are found worldwide with high seroprevalences. fcovs are positive-stranded enveloped rna viruses with a non-segmented genome of~ kilobases (kb) encoding for a polymerase (replicase complex a and b), four structural proteins (spike (s), membrane/matrix (m), envelope (e), and nucleocapsid (n)), and five nonstructural/accessory proteins (nsp) abc and ab [ ] . due to infidelity of the rna polymerase, fcovs show high mutation rates upon replication. this leads to the formation of quasispecies, a cloud-like appearance of multiple genetic virus variants that are linked by mutations [ , ] . virus variants that acquire high virulence can lead to feline infectious peritonitis (fip), an immune-mediated disease that is currently the most frequent fatal infectious disease in young pedigree cats and cats in shelters [ , ] . however, the pathogenesis of fip is still not fully understood [ ] . fcovs occur as two pathotypes, the low virulence so-called feline enteric coronaviruses (fecv) and the virulent fip viruses (fipv). the former dominates since fcov infection is via the fecal-oral route and primarily targets the intestine; it does, if at all, only induce mild enteritis. however, - % of adult cats and - % of kittens develop fip at some point after infection [ , ] as a consequence of de novo occurrence of highly virulent fcovs that arise from low virulent fcovs by mutations in the individual infected cat [ ] ; these are generally not horizontally transmitted. it was initially thought that the essential process for the development of fip would be a broadening of the viral target cell spectrum from enterocytes to also include monocytes/macrophages, which would be mediated by specific viral mutations and would allow systemic infection, leading to fip [ ] . in vitro studies subsequently revealed that it is effective and sustainable replication in macrophages rather than the capacity to enter the cells that confers virulence of fcovs [ , ] . also, it was soon shown that healthy carrier cats become systemically infected and carry low amounts of virus in different organs [ , ] . irrespective of the pathotype, fcovs can be classified into two serotypes based on their neutralization reactivity with s protein-specific monoclonal antibodies [ , ] . serotype ii fcovs have arisen from a double rna recombination event between canine coronavirus (ccov) and serotype i fcovs [ ] . only type ii fcov can be efficiently propagated in cell cultures [ ] . however, viruses of both serotypes can cause fip. previous studies found deletions in open reading frame (orf) c and mutations in orf b in fcovs from organs of cats that succumbed to fip [ , ] . these were not found in fecal fcov isolates from healthy carrier cats and were hence considered to be linked to the fip pathotype and, consequently, to the development of fip. in fact, an intact orf c was shown to be critical for replication in enterocytes and thus, shedding of the low virulent pathotype fcovs [ , ] . the fact that the deletions and mutations were unique to each cat supported the de novo mutation theory [ ] . fcovs found in fip often exhibit mutations in orf c that lead to truncation of the protein; however, a significant proportion (up to %) of fipv still have an intact orf c [ , , , , ] . hence, a virulence switch cannot be explained with orf c deletions alone [ , ] . nevertheless, mutations that affect the expression of the accessory proteins might contribute to increased viral replication in monocytes/macrophages [ , ] and, thereby, to the development of fip. conflicting evidence exists regarding the role of orf a and b. while some studies found deletions in orf to be associated with reduced in vivo virulence or in vitro replication [ ] [ ] [ ] [ ] , others did not detect such links [ ] [ ] [ ] . a high n protein diversity was identified as evidence of quasispecies formation, but not as a marker of pathotypes [ ] . mutations in the e or m genes were not found to be critical, neither in vivo [ ] nor for in vitro replication [ ] . the fcov s glycoprotein mediates viral cell entry through receptor binding and is thus responsible for cell tropism [ ] . early in vitro studies found that macrophage tropism can be promoted by introducing the s protein of a highly virulent type ii fcov strain into its low virulent counterpart, suggesting that this protein plays a role in virulence. however, the study did not associate a distinct mutation with the new features of the recombinant strain [ ] . specific s gene mutations were only identified in a more recent full genome sequencing approach, analyzing fcovs of both pathotypes. the majority of fipv pathotype fcovs were found to carry a mutation in a nucleotide position that was associated with an amino acid change in the putative fusion peptide of the s protein [ ] . the same mutation plus a substitution leading to an amino acid change in the heptad repeat (hr ) region, possibly leading to an altered fusogenic activity of the s protein and thus an altered cellular tropism, was found in another study [ ] . however, it was subsequently shown that the amino acid change described by chang and coworkers [ ] correlates with systemic spread of the virus and not directly with fip [ ] . another study examined a furin cleavage site between the receptor binding and fusion domain of the s gene. the site was found to be conserved in less virulent fcovs, whereas fip pathotype fcovs exhibited a higher variability in the core motif and surrounding residues. these mutations alter cleavability of the s protein by furin. depending on the exact position and the exchanged nucleotide, cleavage was either enhanced or suppressed [ ] . again, the study design did not consider systemically infected healthy carrier cats in this context. reverse genetics approaches with recombinant chimeric viruses in which genes of an avirulent (fcov black) strain were successively substituted by genes from a highly virulent serotype ii fipv strain ( - ) led to seroconversion and systemic infection, but did pathogens , , of not induce fip [ , ] . despite the fact that many comparative studies have described mutations only present in fip pathotype fcovs, their relevance for the development of fip remains unclear. based on the hypothesis that certain mutations are essential for the capacity of fcovs to spread systemically, the present study investigated a cohort of systemically infected healthy carrier cats at different time points post experimental infection for the presence of a range of mutations in the genes encoding for the s protein, nsp abc, and nsp b, which have been shown to have implications for the development of fip. it also assessed the changes in the swarm composition of the challenge stock virus present in the organs to determine if its complexity would increase or rather decrease with time. to track viral sequence mutations in organs of healthy fcov carrier cats, we investigated fcov sequences detected in the colon, liver, and thymus, as well as feces of seven experimentally fcov infected cats. in two animals, the thymus was found to be negative by fcov reverse transcription quantitative real-time pcr (rt-qpcr) and lung and tonsil were included as alternatives (table ) . fcov rt-qpcr was performed from undiluted and -fold diluted rna samples to detect any potential pcr inhibition and the rna dilutions with the lower cycle threshold (ct) value, corresponding to the higher viral load, were selected for analysis by one-step rt-pcr (table ) for further cloning and sequencing. due to low tissue viral loads and/or lack of primer hybridization due to possible sequence mismatches, in five of the samples, the analysis of mutations in either orfs abc or b was not possible (cat b: amplicon nsp ab from liver and tonsil; cat d : amplicon nsp ab from the liver; cats y and b: amplicons s-e and s-m from the thymus, respectively). in total, sequences were obtained. the plan was to sequence three colonies from each amplicon. due to the often low concentrations of eluted viral cdna, this was not possible for all samples. however, sequences for of the expected amplification reactions were obtained. we determined the sequence of the putative fusion region of the s protein from at least one amplicon from each organ of five cats ( b, b, b, d , y ). the one-step rt-pcr for the s gene already yielded an amplicon of the expected length ( bp) from the colon of five cats ( b, b, b, d , y ) and the liver of cat b, and after the nested pcr, amplicons with the expected length ( bp) were obtained from the liver of four cats ( b, b, y , b), the thymus of four cats ( b, b, b, y ), and the tonsil of cat b. from one cat (y ), no amplicon was obtained. to determine how a virus swarm changes in its composition and viral sequences during in vivo infection and with systemic spread, the genomic sequences of fcovs in the tissue and fecal samples were compared to those of challenge stock virus. the number of sequences identical to the consensus was always higher in the latter than in the tissue/fecal counterparts of the same strain and orf (table ). the number of single nucleotide polymorphisms (snps) per nucleotides analyzed did not differ significantly between challenge stock virus and tissue/fecal viruses. however, a tendency towards higher mutation frequencies was observed for orf b in the tissue/fecal viruses compared to the challenge stock virus (table , figure ). interestingly, zu orf showed higher mutation frequencies in the stock virus than in the tissue/fecal viruses. to determine whether there was a higher selective pressure on a certain gene after in vivo infection, we compared the mutation frequencies and resulting amino acid changes of genes a, b, c, and b ( table ). the variability per gene of the tissue/fecal fcovs of each cat and in the challenge to determine whether there was a higher selective pressure on a certain gene after in vivo infection, we compared the mutation frequencies and resulting amino acid changes of genes a, b, c, and b ( table ). the variability per gene of the tissue/fecal fcovs of each cat and in the challenge stock virus was small and ranged from . to . %. while the a, b, and c genes seemed to have equal variability, the b gene exhibited a significantly higher mutation frequency than the a gene ( figure ). to determine whether there was a higher selective pressure on a certain gene after in vivo infection, we compared the mutation frequencies and resulting amino acid changes of genes a, b, c, and b ( table ). the variability per gene of the tissue/fecal fcovs of each cat and in the challenge stock virus was small and ranged from . to . %. while the a, b, and c genes seemed to have equal variability, the b gene exhibited a significantly higher mutation frequency than the a gene ( figure ). we wanted to determine whether the length of infection had an effect on mutation frequencies. the overall comparison of fcov gene sequences from the different cats euthanized at different time points after infection did not reveal any significant differences (table ) . nevertheless, when the cats previous studies have provided evidence of a link between truncated c proteins and the development of fip [ , ] . therefore, we investigated the effect of the mutations and deletions found in the present study on the encoded proteins. table shows all deletions and snps detected in viral sequences from tissue and fecal samples and the challenge stock virus that led to a major alteration, i.e., leading to more than just one amino acid change, of the encoded protein. deletions were detected in five of the viral gene tissue/fecal sequences. most mutations of viral sequences led to truncated b proteins. two deletions and one snp led to truncated a and c proteins. two snps and one deletion caused the start codon or the stop codon to disappear. the challenge virus only contained one truncated b protein. to determine the serotype of the strains fcov zu and fcov zu and their tissue/fecal derivate strains, we constructed bootstrap phylogenetic trees with fcov and ccov sequences from the ncbi database (genbank). consensus sequences of virus sequences from the same cat and origin and the previous studies have provided evidence of a link between truncated c proteins and the development of fip [ , ] . therefore, we investigated the effect of the mutations and deletions found in the present study on the encoded proteins. table shows all deletions and snps detected in viral sequences from tissue and fecal samples and the challenge stock virus that led to a major alteration, i.e., leading to more than just one amino acid change, of the encoded protein. deletions were detected in five of the viral gene tissue/fecal sequences. most mutations of viral sequences led to truncated b proteins. two deletions and one snp led to truncated a and c proteins. two snps and one deletion caused the start codon or the stop codon to disappear. the challenge virus only contained one truncated b protein. to determine the serotype of the strains fcov zu and fcov zu and their tissue/fecal derivate strains, we constructed bootstrap phylogenetic trees with fcov and ccov sequences from the ncbi database (genbank). consensus sequences of virus sequences from the same cat and origin and the consensus sequence of the challenge stock virus were aligned to the reference sequences from the genbank for genes a, b, c, and b (figure a-d, respectively) . fcov zu and consensus sequences obtained from cat b inoculated with fcov zu clustered with type i fcovs in all genes, whereas in tissue and fecal samples of cats inoculated with the fcov zu strain, only viral consensus sequences encoding the a gene clustered with type i fcovs. sequences related to fcov zu encoding genes c and b clearly clustered with type ii fcovs. while sequences of the c and b genes could undoubtedly be assigned to serotype ii, because they clustered with the sequences of the corresponding serotype, this was not the case for the a and b gene. here, sequences clustered together but could not be unquestionably assigned to a given serotype. therefore, we speculate that the a and/or b gene of the challenge virus fcov zu harbors a new recombination site. pathogens , , x of consensus sequence of the challenge stock virus were aligned to the reference sequences from the genbank for genes a, b, c, and b (figure a-d, respectively) . fcov zu and consensus sequences obtained from cat b inoculated with fcov zu clustered with type i fcovs in all genes, whereas in tissue and fecal samples of cats inoculated with the fcov zu strain, only viral consensus sequences encoding the a gene clustered with type i fcovs. sequences related to fcov zu encoding genes c and b clearly clustered with type ii fcovs. while sequences of the c and b genes could undoubtedly be assigned to serotype ii, because they clustered with the sequences of the corresponding serotype, this was not the case for the a and b gene. here, sequences clustered together but could not be unquestionably assigned to a given serotype. therefore, we speculate that the a and/or b gene of the challenge virus fcov zu harbors a new recombination site. based on sequence alignments with defined serotype i (fecv-ucd (fj )) and serotype ii (fipv - (dq )) sequences, the recombination site could be tentatively mapped to the overlapping region between orf a and orf b ( figure s ). to evaluate the sequence evolution patterns of the fcov zu and zu challenge stock viruses for both the abc and b genes in each cat (i.e., at different time points post infection), bootstrap phylogenetic trees were constructed, with each available sequence obtained from different colonies in relation to the respective challenge stock virus consensus sequence (supplementary figures s -s ) . in cats b, b, b ( figures s and s ) , and d ( figures s and s ), the variability between the different sequences was very small and no subtree resulted from the analysis of either gene. the virus variation seemed to be evenly distributed across tissue and fecal samples independent of the time interval between infection and euthanasia ( , , or days) . in cat y , the gene abc based analysis revealed an equal distribution of virus variability in all examined tissue and fecal samples ( figure s ). however, for the b gene, the analysis revealed a higher variability, with viral sequences from different origin grouping to a subtree. the evolutionary pressure on the b gene seemed to be similar in all samples of this cat ( figure s ). phylogenetic analysis of cat y sequences based on genes abc and b showed subtrees in both genes. the sequence variability in the abc genes was smaller than in the b gene ( figures s and s ). analysis based on the b gene led to three different subtrees where viral sequences of the same tissue and feces grouped together and were clearly distinct from each other. also, in cat b, the variability in the b gene was slightly higher than in the abc genes ( figures s and s ). trees constructed with both genes formed subtrees. two colonies sequenced from the colon of this cat grouped together when the analysis was based on the abc genes. analysis of the b gene yielded two subtrees, one consisting of two sequences from the feces and one of each feces and a colon sequence. the genetic difference of the viral sequences identified in the colon of cat b to fcov zu (figures s and s ) was the highest of all sequences analyzed, potentially reflecting the longer time span after infection ( days) in this animal. to assess if the fcov zu strain evolved divergently in different sites (tissues and/or feces) of individual cats, bootstrap phylogenetic trees were constructed with all sequences from feces and tissues. figures and show the analysis based on abc and b genes, with all viral sequences of the same origin. also, these trees revealed a higher variability in the viral b genes ( figure ) compared to the abc genes ( figure ). with the exception of the tree based on the analysis of the abc genes from thymus, all trees generated subtrees (figure : colon; feces; liver). subtrees consisted of sequences originating from the same cat, except for one subtree for liver sequences where two sequences of cat y and y grouped together ( figure : liver). sequences that diverged in the analysis based on genes abc did not originate from the same animals and/or colonies as those diverging in the analysis based on gene b. sequences with mutations that led to truncated proteins did not cluster together but were rather dispersed and the majority were not represented in the subtrees ( figures and : red dots) . when the analysis was based on genes abc of the colon, feces, and liver of cat b, i.e., the cat that was sacrificed after the shortest time post infection ( days), the sequences always formed a subtree that was clearly distinct from the challenge virus fcov zu and sequences found in other cats infected with the same virus (figure : colon; feces; liver). this was not seen in the b gene analysis (figure : colon; feces; liver). the trees based on the b gene indicated that in cats y , y , d , and b, the b genes were generally more distant from parent fcov zu than from those of the other cats. orf b colon orf b feces orf b liver orf b thymus figure . phylogenetic analysis based on the sequences encoding for the nonstructural protein b of all sequences of colon, feces, liver, or thymus, respectively (red dot, sequence carries a deletion that leads to a premature stop codon ( b c , y c , d c ); blue dot, snp causes the disappearance of a start codon (y c , y c ); bar, mean number of differences per sites). unfortunately, none of the nested pcr products could be sequenced, neither by direct sequencing nor after cloning. however, sequences were obtained for five of the six samples that were positive after the first rt-pcr step. four of these sequences derived from the colon and one from the liver. all sequences had an atg codon at position , resulting in a methionine residue (figure ) . the fipv c je strain exhibits a ttg codon at this position, resulting in a leucine residue [ ] . at position , all sequences had a tct codon. none of our samples exhibited the serine to alanine mutation described in a minority of fipvs at this position [ ] . other snps detected in the immediate surrounding of position and position in our samples did not alter the amino acid figure . phylogenetic analysis based on the sequences encoding for the nonstructural protein b of all sequences of colon, feces, liver, or thymus, respectively (red dot, sequence carries a deletion that leads to a premature stop codon ( b c , y c , d c ); blue dot, snp causes the disappearance of a start codon (y c , y c ); bar, mean number of differences per sites). unfortunately, none of the nested pcr products could be sequenced, neither by direct sequencing nor after cloning. however, sequences were obtained for five of the six samples that were positive after the first rt-pcr step. four of these sequences derived from the colon and one from the liver. all sequences had an atg codon at position , resulting in a methionine residue (figure ) . the fipv c je strain exhibits a ttg codon at this position, resulting in a leucine residue [ ] . at position , all sequences had a tct codon. none of our samples exhibited the serine to alanine mutation described in a minority of fipvs at this position [ ] . other snps detected in the immediate surrounding of position and position in our samples did not alter the amino acid composition. overall, none of the mutations identified by chang and coauthors in the putative fusion region of the s protein were present [ ] . composition. overall, none of the mutations identified by chang and coauthors in the putative fusion region of the s protein were present [ ] . during the last years, many studies have been performed with the aim to identify mutations responsible for the virulence switch in fcovs towards the fipv pathotype. there has been evidence that mutations in accessory genes and the s gene of fcovs are associated with fip development. so far, most of these studies compared less virulent fcovs from healthy cats with fcovs in animals suffering from fip [ , , , [ ] [ ] [ ] [ ] . in the present study, we investigated the orfs abc and b and the s gene mutations of viral sequences identified in different organs and feces of healthy cats that had been experimentally infected with different fcov field strains and had developed a systemic fcov infection [ ] . in the original study, the challenge virus used for experimental infection had been isolated from the feces of naturally infected cats. since fcovs show high mutation rates, due to the infidelity of their rna-dependent rna polymerase and homologous recombination events [ ] , various fcov variants are transmitted in natural fcov infections [ ] ; therefore, due to our experimental setup, we hypothesized a similar scenario. such virus swarms are called quasispecies, defined by a dominant nucleotide sequence and its mutant spectrum. evolutionary selection acts on the entire quasispecies rather than on single viral mutants [ ] . in our study, we compared the swarm composition of the challenge stock viruses used for infection to the viruses detected in organs and feces at different time points after infection, focusing on the sequences of accessory genes. in parallel, we investigated the s genes for the presence of mutations described to have an impact on the development of fip [ ] . the analysis of the accessory genes of more than colonies of the challenge stock viruses confirmed the existence of one dominant sequence and several mutated derivates in each orf and strain (data not shown); this observation aligns with the quasispecies theory. in the viral genomes detected in the tissue and fecal samples of the infected cats, the extent of sequences identical to the consensus sequence was lower than in the challenge stock virus. additionally, viral sequences identified in cats that were euthanized at a later time point after infection overall showed significantly more variation than those from cats that were euthanized earlier, although due to the small number of cats analyzed, this significance could subsequently not be assigned to a specific accessory gene. a limitation for the interpretation of the results is the fact that only few cats (n = ) were included in this study, of which just one was infected with the fcov zu strain. furthermore, two of these animals had been subcutaneously vaccinated with the inactivated fcov zu strain before challenge with the homologous strain. these cats were included in the study to add a further time point to monitor mutation frequencies over time. as the vaccine was inactivated and unable to replicate, we did not expect to find it in the tissues and feces. the animals seroconverted and started to shed virus in the feces only upon challenge and at the same time, with no differences in titers between vaccinated during the last years, many studies have been performed with the aim to identify mutations responsible for the virulence switch in fcovs towards the fipv pathotype. there has been evidence that mutations in accessory genes and the s gene of fcovs are associated with fip development. so far, most of these studies compared less virulent fcovs from healthy cats with fcovs in animals suffering from fip [ , , , [ ] [ ] [ ] [ ] . in the present study, we investigated the orfs abc and b and the s gene mutations of viral sequences identified in different organs and feces of healthy cats that had been experimentally infected with different fcov field strains and had developed a systemic fcov infection [ ] . in the original study, the challenge virus used for experimental infection had been isolated from the feces of naturally infected cats. since fcovs show high mutation rates, due to the infidelity of their rna-dependent rna polymerase and homologous recombination events [ ] , various fcov variants are transmitted in natural fcov infections [ ] ; therefore, due to our experimental setup, we hypothesized a similar scenario. such virus swarms are called quasispecies, defined by a dominant nucleotide sequence and its mutant spectrum. evolutionary selection acts on the entire quasispecies rather than on single viral mutants [ ] . in our study, we compared the swarm composition of the challenge stock viruses used for infection to the viruses detected in organs and feces at different time points after infection, focusing on the sequences of accessory genes. in parallel, we investigated the s genes for the presence of mutations described to have an impact on the development of fip [ ] . the analysis of the accessory genes of more than colonies of the challenge stock viruses confirmed the existence of one dominant sequence and several mutated derivates in each orf and strain (data not shown); this observation aligns with the quasispecies theory. in the viral genomes detected in the tissue and fecal samples of the infected cats, the extent of sequences identical to the consensus sequence was lower than in the challenge stock virus. additionally, viral sequences identified in cats that were euthanized at a later time point after infection overall showed significantly more variation than those from cats that were euthanized earlier, although due to the small number of cats analyzed, this significance could subsequently not be assigned to a specific accessory gene. a limitation for the interpretation of the results is the fact that only few cats (n = ) were included in this study, of which just one was infected with the fcov zu strain. furthermore, two of these animals had been subcutaneously vaccinated with the inactivated fcov zu strain before challenge with the homologous strain. these cats were included in the study to add a further time point to monitor mutation frequencies over time. as the vaccine was inactivated and unable to replicate, we did not expect to find it in the tissues and feces. the animals seroconverted and started to shed virus in the feces only upon challenge and at the same time, with no differences in titers between vaccinated and non-vaccinated animals. for these reasons, we did not expect to see a difference in mutation frequencies due to vaccination, but we also cannot completely exclude it. only three colonies per amplicon of each tissue/fecal sample were sequenced. this sequencing approach was most likely picking the most represented sequences, which might not be enough to appropriately characterize the quasispecies swarm. a targeted high throughput sequencing study would allow for much greater sensitivity for detecting sequence variations in the virus population. nevertheless, taken together, the results point towards an expansion of the viral swarm with increasing infection time. yet, stabilization of the virus swarm over a longer period might subsequently occur. this has been shown in a previous study, which also provided evidence that persistently infected cats are likely no source of novel virus variants, since they carry highly conserved fcov swarms [ ] . single nucleotide polymorphisms (snp) were found randomly scattered across all accessory genes without defined patterns (hot spots) both in the viral rna from tissues/feces and the challenge viruses. this suggests that they are a consequence of the infidelity of the viral rna-dependent rna polymerase. the present study found a generally higher mutation rate in the b gene than in the abc genes; the difference was significant for orf a. this confirms previous findings suggesting that the amino acid sequence of the nsp b is less conserved than that of other nsps [ , ] . a bias could have been introduced since a non-proofreading taq polymerase was used for pcr amplification to increase the efficiency of the one-step rt-pcr because of the low viral loads in the tissues. but as sequencing errors are supposed to be equally randomly distributed across the genes, this would not impact on the difference of the mutation frequencies. interestingly, we found deleterious mutations and snps that led to truncated nsp b proteins in four viral sequences from the feces and colon of different cats infected with the fcov zu strain. truncated b proteins were previously shown to correlate with attenuated virulence: four well-characterized fcov strains (fecv - , fipv tn -hp, fipv ucd , and fipv df ) were found to be avirulent after they had acquired orf b gene deletions during in vitro passage in cell culture. in contrast, field fcov isolates consistently carried an intact b gene regardless of their pathotype [ ] . thus, it was postulated that an intact b gene confers a selective advantage in natural infection but is not necessary for in vitro growth [ ] . our study detected truncated b proteins solely in either feces or the colon, i.e., the main site of replication and persistence of less virulent fcovs, possibly representing virus variants that were indeed of low virulence and potentially even unable to infect monocytes/macrophages and to spread systemically. this would support the hypothesis that orf b mutations are not involved in the development of fip [ ] . we also identified each two sequences each with truncated a, b, and c proteins, respectively. these results differ from those of a previous study which sequenced the structural and accessory genes of fcov from feces and organs of fip cats and mainly found truncated c proteins in the diseased tissue of fip cats [ ] . the same study also claimed that orfs a, b, and a show the least variability among the other accessory and structural genes. the present study does not support this assumption, but rather indicates that the variability of the orf genes is equally distributed at least in systemic infections with fcovs of low virulence. at the same time, the absence of orf c deletions and orf b mutations that have previously been linked to the development of fip in fcovs identified in the organs of our cats provides indirect support for the previous hypothesis that these might indeed be a feature of fipvs [ ] . however, since healthy carrier cats also harbor virus in different organs, they are likely not a prerequisite of systemic spread in an infected animal. the orfs abc and b based phylogenetic analysis showed clustering of fcov zu with serotype i fcovs, whereas fcov zu sequences clustered with serotype ii fcovs. this was remarkable since both viruses were identified as type i fcovs after parts of the s gene had been sequenced in (dq and dq ) [ ] . serotype definition is commonly based on the reactivity of antibodies to the s protein, hence it is generally accepted that fcov type i and ii can be distinguished based on the s gene sequences [ , ] . the rna-dependent rna polymerase of covs is well known to be error-prone and to incorporate one mutation per kb [ ] ; this allows for three mutations per replication cycle of the kb fcov genome and can lead to deleterious mutations that yield non-viable viruses. to overcome this problem, the viruses might rely on homologous recombination. the latter mostly occurs at specific hotspots in the genome, where secondary rna structures are formed that cause the polymerase to pause. four such hotspots, leading to double recombination events that result in type ii fcovs, have previously been identified; upstream ones in the polymerase sequence and downstream ones in genes e and m, respectively [ ] . according to this configuration, after a double recombination event, the spike and nsp genes should belong to type ii, whereas the nsp should belong to type i. however, the exact locations of these recombination sites vary in the different strains, indicating that serotype ii fcovs continuously arise through independent recombination events [ , , ] . therefore, homologous recombination can also generate new variants as long as they do not have evolutionary disadvantages [ ] . this might also hold true for fcov zu where a recombination event in the a/ b gene resulted in a genome with type i fcov s gene, whereas the adjacent c gene as well as the b gene are those of a type ii fcov. controversial evidence exists concerning a set of defined s gene mutations first described in [ ] in fipvs for which a role in the development of fip was suggested. two years later, two studies investigated these mutations in more detail. one study compared fecal samples of healthy fcov carriers with fecal and/or ascites samples of natural fip cases by sequencing the accessory and the s genes. this revealed a conserved methionine residue at amino acid position , in / fcovs from healthy carriers, whereas the m l mutation was found at this position in / ascites samples of fip cats whose feces generally displayed both genotypes. the authors also investigated the animals for orf c mutations/truncations and concluded that these together with mutations at amino acid position , , account for the pathotype switch [ ] . another study, published in the same year, concluded that the spike mutations in question are relevant for systemic spread and are not associated with fip. the authors found fcovs that carried the m l mutations in the majority of extra-intestinal tissue samples obtained from both cats with fip and healthy cats, whereas the majority of fcovs derived from fecal samples had a methionine residue. percentages of mutated versus conserved sequences in the different types of samples did not significantly differ between fip cats and controls [ ] . we were also interested in the potential presence of these mutations in fcovs in our cohort of healthy carrier cats. sequence information covering the region of interest was obtained from four cats and two different organs: colon (four sequences, one per cat) and liver (one sequence). interestingly, none of these samples exhibited one or both mutations in question (m l and s a) [ ] . these results could indicate that the above-mentioned mutations are not even markers of systemic spread per se [ ] , but rather of another relevant aspect in the development of fip that, like systemic spread, is mediated by and/or occurs in monocytes/macrophages. however, our results are based on a single successfully sequenced extra-intestinal sample and are therefore of very limited value. mutations in the s gene are presumed to be associated with infection of monocytes and macrophages [ ] , however unfortunately only few samples ( out of ) showed a monocyte-associated viremia [ ] and due to the rna degradation, could not be further analyzed. at present, the relevance of the above-mentioned viral mutations for fip is still not clear. complementary transparent studies using clearly defined animal populations to obtain sufficient data for a definitive conclusion are clearly lacking and before these are available, no given mutation should be propagated as a diagnostic marker. a real-time rt-pcr has been marketed since august as a confirmatory diagnostic test for fip (fip virus realpcr™ test; idexx reference laboratories). a detailed description of the investigations on which the test validation was based seems to be lacking; test sensitivity ( . %) and specificity ( %) were generated in a population of cats "who were either healthy or had confirmed fip based on biopsy". it is questionable whether healthy cats are adequate controls for fip cases. after all, porter and coworkers found fcovs with the mutation described by chang and coworkers [ ] not only in % of the tissue samples from cats with fip, but also in % of the tissue samples from fcov positive cats without fip [ ] . therefore, with the current knowledge, we recommend considering the detection of the s gene mutations in question solely as further support of a diagnosis of fip, but not as definite proof. the exact role of the fcov spike protein in the pathogenesis of fip is still unknown. it is most likely the key determinant of cell tropism and is crucial for viral host cell entry [ ] . substitution by recombination of the s region of fipv - into fecv - was found to be sufficient to enable the hybrid virus to effectively infect macrophages in vitro [ ] . thus, it is likely not the receptor binding (via s ) but the fusion process that is critical in this context. also, for type i fcovs, which, unlike type ii fcovs, hardly grow in cell culture, the receptor is not yet known. interestingly, a cell culture adapted type i fcov strain (ucd ) was found to carry a mutation in the furin cleavage site that renders the s protein susceptible to cleavage by heparan sulfate [ ] , leading to the speculation that the amino acid composition of the furin cleavage site is critical for cell culture adaption and thus cell tropism. indeed, a strong correlation was later found between conservation of the furin cleavage site and fcov pathotype [ ] . unfortunately, attempts to target the furin cleavage site at the boundary of s and s [ ] did not yield conclusive results in our study (data not shown). fip pathogenesis includes several key processes, i.e., the establishment of systemic fcov infection; effective and sustainable viral replication in monocytes/macrophages; and activation of infected monocytes [ ] . efficient entry into monocytes/macrophages is an essential prerequisite for all these, but there are most likely additional s protein-unrelated factors that allow the increase in viral replication in monocytes/macrophages and their subsequent activation. therefore, s gene mutations likely only represent contributing factors among multiple events, ultimately resulting in fip. in conclusion, despite some limitations mentioned above, using known field viruses and a controlled experimental setting, we were able to establish that fcovs in the tissue and fecal samples of healthy fcov infected cats do not carry the orf abc, b, and s gene mutations that have previously been linked to the development of fip. these findings support the hypothesis that these alterations are linked to fipv pathotype viruses. however, like all other studies so far, they do not answer the question whether their occurrence does directly cause fip. interestingly, the viruses detected in the tissues also lacked mutations seen in association with systemic spread of fcovs [ ] , which indicates that they are not essential for this key prerequisite of fip. furthermore, we detected a homologous recombination site in the strain fcov zu that has not been described before. further comparative studies, and in particular, those using a ngs approach, are required to ultimately assess whether the development of fip can be attributed to virus characteristics alone or whether it represents an interplay between fipv pathotype viruses and a host prone to a specific ultimately detrimental immune response. the tissue and fecal samples used in this study originated from a study performed between and [ ] . the study was approved by the swiss ethics committee (tvb - ). specific pathogen-free (spf) cats aged between and weeks were orally infected with different fcov type i strains (fcov zu [dq . ] or fcov zu [dq . ]) isolated from feces of healthy field cats or from the intestines of cats that had been experimentally infected with the same virus strains. cats d , y , and y originated from an unpublished study where the cats had been subcutaneously vaccinated with the inactivated homologous fcov strain , , and weeks before challenge. cats d and y had been vaccinated with the inactivated strain mixed : with the adjuvant diluvac forte (intervet ltd., uk). for cat y , a cpg motif, as described in a previous vaccine study [ ] , had been added together with the adjuvant. cat y had been mock vaccinated. the challenge virus ( ml) was administered twice, as previously described [ ] . all infected cats had remained clinically healthy until euthanasia at , , , or days after infection (table ) . a full postmortem examination was carried out. the gross and histological examination did not reveal any pathological changes apart from lymphatic hyperplasia [ ] . challenge stock virus samples of the fcov zu and zu strains originated from the same material originally prepared and aliquoted for the experimental challenge. all samples used in the current study had been stored at − • c since . feces, colon, liver, and thymus or, when the thymus was not fcov positive by rt-qpcr, tonsils or lung, were analyzed. viral rna was isolated with the rneasy mini kit (qiagen, hombrechtikon, switzerland). approximately mg of tissue was taken from the frozen samples and placed in a ml eppendorf tube containing µl of qiagen lysis buffer rlt, . µl β-mercaptoethanol, and a mm steel bead (schieritz & hauenstein ag, arlesheim, switzerland). samples were homogenized and lysed by mixing at hz for min in a mixer mill device (qiagen). after mixing, samples were centrifuged shortly to reduce the foam that had formed during the homogenization step. the homogenate was loaded on a qiashredder spin-column (qiagen) and centrifuged at , rounds per min (rpm) for min; µl of % ethanol were added to the flow-through and mixed by pipetting. samples were transferred to rneasy spin columns placed in ml collection tubes. downstream operations were performed according to the manufacturer's instructions and rna was eluted with µl nuclease free h o and centrifugation at , rpm for min. fcov loads were determined by a rt-qpcr assay that detects a bp long amplicon of the b gene, modified from previously described protocols [ ] . briefly, µl reactions contained . µl × reaction buffer (one step rt qpcr mastermix plus low rox, rt-qprt- xlr, eurogentec, seraing, belgium), . µl each of forward and reverse primers ( µm) (microsynth, balgach, switzerland), . µl probe (microsynth), . µl euroscript reverse transcriptase & rnase inhibitor (eurogentec), µl template rna, and rnase free water to µl. the reaction underwent reverse transcription (rt) at • c for min, denaturation for min at • c, and cycles of • c for s and • c for min. to test for any inhibition, each rna sample was tested neat and after -fold dilution in rnase free water. all qpcr assays were performed using an abi fast sequence detection system (applied biosystems, thermofisher scientific, reinach, switzerland). positive and negative controls were included in each rt-qpcr run. synthesis of cdna and pcr amplification were performed with the superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, basel, switzerland) using specific primers. viral rna isolated from a cell culture supernatant infected with the fcov wellcome strain or from the fcov zu and fcov zu gut homogenates used for infection of the cats was always included as positive controls. also, two negative controls were included, of which one was kept open throughout the manipulation to control for airborne contaminations. pcr reactions ( µl) contained . µl × reaction mix (provided in kit), µl superscript iii rt/platinum taq mix, . µl each of forward and reverse primers ( µm) (microsynth), µl template rna, and autoclaved distilled water to µl. three novel assays were developed. one amplified parts of the s gene, the abc genes, the e gene, and parts of the m gene of the strain fcov zu . primers used for this amplicon (s-m, bp) were fcov_se_f ( -tgc tgt tta act act ggt tgt tgt gga- ) and fcov_sm_r ( -gca ccc gct ata cta agg ccg-a ). the second assay amplified parts of the s gene, the abc genes, and parts of the e gene of the strain fcov zu . primers used for this amplicon (s-e, bp) were fcov_snsp b.f ( -ctt ggt atg tgt ggc tac taa ttg g- ) and fcov_se_r ( -atc aac agg agc cag aag aag aca ct- ). the third assay amplified parts of the a and the whole b gene of both strains. for this amplicon (nsp ab, bp), already described primers were used, namely a-f ( -ctg cga gtg atc ttt cta g- ) [ ] and fcov r ( -aac aat cac tag atc cag acg tta gct- ) [ ] . all assays were run on a biometra tpersonal thermocycler (labgene, chatel-st-denis, switzerland). cycling conditions were • c for min, • c for min; cycles of • c for s, • c for s, and • c for min; min at • c and then cooling to • c for the first assay (s-m); and • c for min, • c for min; cycles of • c for s, • c for s, and • c for min; min at • c and then cooling to • c for the second and third assay (s-e and nsp ab). for the amplification of the s gene targeting the mutations m l and s a, a modified previously published nested rt-pcr assay was used [ ] . the fcov-ucd -s. ( -caa tat tac aat ggc ata atg g- ) forward and fcov-ucd -s. ( -ccc tcg agt ccc gca gaa acc phylogenetic and molecular evolutionary analyses were conducted using geneious prime (biomatters ltd.). nucleotide sequences were edited, assembled, and aligned. alignments were manually adjusted when necessary. bootstrap phylogenetic trees were constructed using the neighbor-joining algorithm and the tamura-nei genetic distance model [ ] . four bootstrap phylogenetic trees based on the a, b, c, or b genes were constructed with the consensus sequence of all viral rna sequences from the same tissue or feces of each cat and different fipv, fcov, and ccov strains retrieved from the genbank. additional neighbour-joining trees were constructed with either all abc or b sequences of the same cat or with all sequences from the same sample. the consensus sequences of fcov zu and fcov zu served as outgroups. variable sites and deletions of genes a, b, c, and b were analyzed with graph pad prism version . . (san diego, ca, usa). data were tested for normal distribution with the kolmogorov-smirnov test. statistical significance was determined using one-way anova analysis of variance with bonferroni correction to compare the mutational frequency of the different genes. mean mutation frequencies in challenge stock viruses and viral sequences from tissues/feces for orf abc and orf b of fcov zu and zu were evaluated using the wilcoxon signed-rank test. the time-dependent mutation frequency between genes was analyzed using kruskal-wallis with dunn's multiple comparison posttest. data were expressed as median with boxes and whiskers showing the maximum and minimum values within a group or as means in a bar chart. a p-value < . was considered statistically significant in all cases. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : nucleotide alignment of the orf abc region between fcov zu and defined serotype fecv-ucd (fj )) and serotype ii (fipv - (dq )) viral sequences. sequences were aligned using the geneious prime software (www.geneious.com, biomatters ltd.) and the muscle method. dots depict identical nucleotides. the grey bars indicating the different orf abc locations are based on the sequence of fcov zu . figure s : phylogenetic analysis based on the sequences encoding for nonstructural proteins abc for cats b, b, and b. (*, day p.i. of euthanasia; yellow dot, sequence carries snp that leads to a premature stop codon; bar, mean number of differences per sites), figure s : phylogenetic analysis based on the sequences encoding for nonstructural protein b for cats b, b, and b. (*, day p.i. of euthanasia; red dot, sequence carries a deletion that leads to a premature stop codon; bar, mean number of differences per sites). figure s : phylogenetic analysis based on the sequences encoding for nonstructural proteins abc for cats d , y , y , and b. (*, day p.i. of euthanasia; red dot, sequence carries a deletion that leads to a premature stop codon (y ) or a shortened protein without frameshift (y ); green dot, snp causes disappearance of a stop codon; bar, mean number of differences per sites). figure s : phylogenetic analysis based on the sequences encoding for nonstructural protein b for cats d , y , y , and b. (*, day p.i. of euthanasia; red dot, sequence carries a deletion that leads to a premature stop codon; blue dot, snp causes disappearance of a start codon; bar, mean number of differences per sites). cycling conditions were • c for min, • c for min; cycles of • c for s, • c for s, and • c for min; min at • c and then cooling to • c. for the second nested pcr step, if needed, two newly designed primer pairs amplifying the region of interest (amplicon bp), fcov-ucd -s. f ( -aat ggt gct tcc tgg ggt tg- ) and fcov-ucd -s. r ( -gca cct gca tag caa aag gc- ), and the phusion hot start ii high-fidelity dna polymerase (thermo scientific scientific) were used. then, µl of one-step rt-pcr cycling conditions were • c for min; cycles of • c for s, • c for s, and • c for min or alternatively, a gene ruler dna ladder mix (thermo scientific) or a -kilobase-pair dna ladder (eurogentec), was used for molecular size comparisons. appropriate bands were cut out with sterile razor blades and weighed cloning and sequencing the pcr amplicons were cloned into the pcr plasmid with the topo ta cloning kit for from each cloned rt-pcr amplicon, three colonies for each tissue/fecal sample as well as and colonies of the fcov zu and fcov zu challenge stock virus strains (for better characterization of the virus pool used for infection), respectively, were picked and cultured overnight at • c in luria-bertani-liquid-medium containing ampicillin. cultures were centrifuged at rpm for min and the pellets were used for further manipulation fcov zu tissue/fecal virus orf abc fcov zu tissue/fecal virus orf abc fcov zu tissue/fecal virus orf b fcov zu tissue/fecal virus orf b fcov zu & zu tissue virus partial s-gene: mt -mt ) the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses quasispecies theory and the behavior of rna viruses an overview of feline enteric coronavirus and infectious peritonitis virus infections a review of feline 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and mutation analysis of the spike protein in feline enteric coronavirus and feline infectious peritonitis detected in household and shelter cats in western canada pathogens mutation of the s and c genes in genomes of feline coronaviruses detection of feline coronavirus mutations in paraffin-embedded tissues in cats with feline infectious peritonitis and controls persistence, and transmission of natural type i feline coronavirus infection unfinished stories on viral quasispecies and darwinian views of evolution quasispecies composition and phylogenetic analysis of feline coronaviruses (fcovs) in naturally infected cats a comparison of the genomes of fecvs and fipvs and what they tell us about the relationships between feline coronaviruses and their evolution direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral rna full genome analysis of a novel type ii feline coronavirus ntu emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses recombination in large rna viruses differential susceptibility of macrophages to serotype ii feline coronaviruses correlates with differences in the viral spike protein cleavage of group coronavirus spike proteins: how furin cleavage is traded off against heparan sulfate binding upon cell culture adaptation immunization of cats against feline immunodeficiency virus (fiv) infection by using minimalistic immunogenic defined gene expression vector vaccines expressing fiv gp alone or with feline interleukin- (il- ), il- , or a cpg motif one-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses the neighbor-joining method: a new method for reconstructing phylogenetic trees this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to acknowledge enikö gönczi and andrea spiri for their excellent technical support. the laboratory work was performed using the logistics of the center for clinical studies at the vetsuisse faculty of the university of zurich. this work was mainly performed by mirjam lutz and aline r. steiner as partial fulfillment of their masters theses. the authors declare no conflict of interest.pathogens , , key: cord- -wsagoy authors: stranieri, angelica; scavone, donatella; paltrinieri, saverio; giordano, alessia; bonsembiante, federico; ferro, silvia; gelain, maria elena; meazzi, sara; lauzi, stefania title: concordance between histology, immunohistochemistry, and rt-pcr in the diagnosis of feline infectious peritonitis date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: wsagoy histology, immunohistochemistry (ihc), and reverse transcription polymerase chain reaction (rt-pcr) have been used to diagnose feline infectious peritonitis (fip), but no information regarding the comparison of their diagnostic performances on the same organ is available. the aims of this study were to determine the concordance among these tests and to evaluate which combination of tests and organs can be used in vivo. histology, ihc, and nested rt-pcr (rt-npcr) for feline coronavirus (fcov) were performed on spleen, liver, mesenteric lymph node, kidney, large and small intestine, and lung from fip and non-fip cats. sensitivity, specificity, predictive values, likelihood ratios, and concordance were calculated. ihc and rt-npcr had the highest concordance in lung and liver, histology and ihc in the other organs. the sensitivity of histology, ihc, and rt-npcr on the different organs ranged from . to . %, . to . %, and . to . %, respectively, and their specificity ranged from . to . %, % and . to . %. therefore, ihc is recommended when histology is consistent with fip. if rt-npcr is performed as the first diagnostic approach, results should always be confirmed with ihc. lung or liver provide accurate information regardless of the method, while ihc is preferred to rt-npcr to confirm fip in the kidney or intestine. feline coronavirus (fcov) is a widespread, highly contagious virus belonging to the species alphacoronavirus of the subgenus tegacovirus (genus alphacoronavirus, subfamily orthocoronavirinae, family coronaviridae, and order nidovirales) [ ] . fcov may induce a transient or persistent enteric infection, characterized by mild or absent clinical signs, or an invariably lethal systemic disease called feline infectious peritonitis (fip) [ ] [ ] [ ] . according to their pathogenicity, fcovs can be divided into two different biotypes: feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv), respectively [ ] . it has been supposed that mutations of the viral genome could be responsible for the pathotypic switch, but they are more likely associated with a change in tropism from enterocytes to regarding the fip group, five cats showed non-effusive forms of the disease, with primarily neurological (n • , , ) and ocular signs (n • , ), along with fever, lethargy, and anorexia. the remaining nine cats were affected by effusive fip, with abdominal (n • , , , , , , ) and/or thoracic (n • , , ) effusion. when present, the effusion displayed features typical of fip, such as yellowish color, viscous and sticky consistency, often associated with the presence of fibrin clots [ ] . the exceptions were represented by cats n • and , whose effusions, collected in vivo, were initially consistent with fip, while at necropsy had a purulent appearance, confirmed by cytologic evaluation, which allowed the categorization of the effusion as bacterial purulent effusion. regarding the non fip group, in four cases, fip was considered as a possible differential diagnosis because of the presence of hyphemia (n • ), body cavity effusion and icterus (n • ), anisocoria and tremors (n • ), neurological signs and thoracic effusion (n • ). in eight cats, fip was not clinically suspected, and death occurred for severe injuries (n • , , and ) or rodenticide poisoning (n • ) confirmed by necropsy, which revealed fractures and/or bleeding. the remaining cats were affected by inflammatory (n • ), infectious (n • ), and cardiac (n • , ) diseases. see supplementary table s for additional information regarding histologic lesions. histological examination was performed on tissue samples obtained from fip cats and obtained from non fip cats. supplementary tables s and s summarize histological findings recorded in each organ and results regarding macroscopic alterations; histology, ihc, and pcr recorded in all the tissues examined in this study, respectively. as reported in table s , histological lesions were also present in sections taken from organs not exhibiting evident macroscopic alterations. overall, / tissues ( . %) from fip cats exhibited pathogens , , of histological features consistent with the disease. different types of lesions consistent with fip were often simultaneously detected in the same animal and even within the same organ. most of the cats ( / , . %) in the fip group, in fact, showed lesions consistent with fip in more than one organ, with different distribution based on the organ. fibrinous serositis was consistently found, particularly on the spleen, liver, and lungs, while mesenteric lymph nodes were prevalently affected by granulomatous lesions, often with vasculitis. on the other hand, kidneys were often affected by lymphoplasmacytic infiltrates, in association either with granulomatous/pyogranulomatous lesions or vasculitis. small and large intestine showed the various distribution of the above-mentioned lesions. in cat n • , which presented neurological signs, histological lesions consistent with fip were found only in the central nervous system (cns) with the diagnosis of severe, chronic, multifocal to coalescent lymphoplasmacytic meningoencephalitis, and ependymitis. not infrequently ( / tissues, . %), the examined histological sections did not show either relevant lesions or lesions consistent with fip. in the fip group, the tissues that most often showed typical fip histological lesions (table ) were the lung, kidney, and mesenteric lymph node, followed by the liver and spleen, while the small and large intestine were the organs less frequently affected by lesions imputable to fip. as regards the non fip group, the histological examination did not show lesions consistent with fip in / samples ( . %), and, therefore, lesions potentially consistent with fip were found only in / tissues ( . %), collected from / cats ( . %). in this group, the organs that most frequently showed lesions possibly consistent with fip were the kidney, followed by the lymph node, spleen, and liver. lesions consistent with fip were never detected in the small and large intestine ( table ) . in a few cases, histological results were compatible but not highly suggestive for fip and were categorized as negative or positive depending on findings in other tissues. in particular, / tissues from fip cats showed lesions suspected for fip, and three were categorized as negative (small intestine and lung of cat n • and large intestine of cat n • ) and two as positive (lymph node and kidney of cat n • and , respectively). in only / samples from non fip cats, histology was non-conclusive, and the sample was categorized as positive considering findings in the other tissues (lung of cat n • ). all the cats ( . %) assigned to the non fip group tested negative at ihc for fcov antigen in all the examined organs, with the exception of columnar enterocytes of cat n • . this result was anyway considered negative because viral antigen was not found in the context of a histological lesion. on the other hand, / cats ( . %) assigned to the fip group tested positive in at least one of the examined organs, for a total of / immunohistochemically positive tissues ( . %). the only cat in the fip group with negative ihc in all the organs systematically sampled for this study was the cat n • , in which lesions morphologically consistent with fip and immunohistochemically positive were instead restricted on the cns only. fcov immunodetection was more frequent in lesions located in the pathogens , , of lung and lymph node, followed by the spleen, liver, and kidney, while the small and large intestine were instead less often found to be positive (table ). as regards the fip group, all the cats showed a positive result in at least one of the examined tissues, and / samples ( . %) gave a positive result, while the fcov genome was not found in / tissues ( . %). the organs which more frequently tested positive were the kidney and mesenteric lymph node, followed by spleen, lung, and large intestine, while the small intestine and liver were the least frequently positive organs. considering the non fip group, / cats ( . %) exhibited a positive result in at least one tissue, and overall, / samples ( . %) were positive, while fcov rt-npcr was negative in / samples ( . %). the organs displaying the most frequent detection of viral rna were the mesenteric lymph node and spleen. the kidney and small intestine were positive in only one case, while the large intestine, liver, and lung never tested positive ( table ). the percentage of cases for which both compared methods classified the same sample as negative or positive, together with the corresponding cohen's kappa coefficient are reported in table . the highest rate of concordance was found between histology and ihc either on the whole set of tissues or each specific tissue. exceptions were the lung, where the highest rate of concordance was found between ihc and rt-npcr, and the liver, on which ihc and rt-npcr had the same concordance of histology and ihc. the tissue with the highest rate of concordance (close to or higher than . %, k > . ) was the small intestine, followed by the liver, large intestine, and lymph node, while the kidney, spleen, and lung had the lowest concordance ( - %, k coefficients between . and . , i.e., strong). the highest rate of concordance was found in tissues with a high prevalence of double negative results. with regard to discordant results, the analysis of individual data (table s ) revealed that the occurrence of negative ihc in tissues with lesions consistent with fip was more frequent than positive ihc in tissues without lesions consistent with fip. more specifically, ihc was negative in all the six samples from the non fip group and / samples from the fip group that were histologically consistent with fip. conversely, in the fip group, ihc was positive in only / samples on which no lesions histologically consistent with fip were found. in particular, the spleen of cat n • showed a moderate increase in the plasmacytic component and perivascular macrophages, not organized in a typical fip lesion but immunohistochemically positive. as stated above, with the exception of the liver, that had an almost absolute concordance and a cohen's k coefficient that could be classified as "almost perfect", in all the other organs, the percentage of concordance varied between and % and the level of concordance measured by the k coefficient was strong ( . - . ) for the kidney and large and intestine, but moderate ( . - . ) for the lymph node, small intestine, and lung. with regard to discordant results, the analysis of individual data (table s ) revealed that positive rt-npcr in tissues without lesions potentially consistent with fip occurred more frequently than negative rt-npcr in tissues with lesions potentially consistent with fip. in particular, rt-npcr was negative in all the six samples from the non fip group and in / samples from the fip group that had lesions histologically consistent with fip. curiously, this latter sample was anyway positive in ihc. conversely, rt-npcr was positive in tissues from cats with fip and tissues from the non fip group in which no lesions consistent with fip were found at histology. more specifically, / rt-npcr-positive tissues from the non fip group did not have histological lesions at all (spleen of cat n • and n • , lymph node of cat n • , kidney of cat n • ), while in / rt-npcr-positive cases lymphoid hyperplasia was found (lymph node of cat n • , small intestine of cat n • ). in the fip group, no lesions at all were detected in six rt-npcr-positive cases (spleen of cat n • , kidney of cat n • , small intestine of cats n • and , large intestine of cats n • and ), while eight rt-npcr-positive cases had lesions not consistent with fip, such as lymphocytic hyperplasia (spleen of cat n • , liver of cat n • , lymph nodes of cats n • , and , large intestine of cat n • ), interstitial nephritis (cat n • ) and pulmonary emphysema (cat n • ). the percentage of concordance was higher than %, and the level of concordance measured by the k coefficient was almost perfect only for the liver and lung. in all the other organs, the percentage of concordance varied between and %, and the k coefficient was classified as strong ( . - . ). overall, double negative results (negative results in both rt-npcr and ihc) were more common than double positive results (positive result in both rt-npcr and ihc). as regards discordant results, the analysis of individual data (table s ) revealed that, with one single exception (lung of cat n • ) of positive ihc and negative rt-npcr, in all the other discordant cases, rt-npcr was positive in tissues with negative ihc. in particular, this occurred in the same cases from the non fip group and in / fip tissues in which histology was classified as negative and rt-npcr was positive (spleen of cats n • and , liver of cat n • , lymph nodes of cats n • , , and , kidney of cats n • , , and , small intestine of cats n • and , large intestine of cats n • , , and and lung of cat n • ), whose histological findings have been described above. additionally, ihc was negative, but rt-npcr was positive in the kidney of cat n • and the large intestine of cat n • , which, however, had histological lesions consistent with fip. the diagnostic performance of histology, ihc, and rt-npcr for the diagnosis of fip on the different tissues analyzed in this study are reported in table . histology showed absolute specificity and positive predictive value (ppv) only in the small and large intestine, while in all other organs except the kidney ( . %), specificity was between . and . %. the organs that less frequently showed lesions imputable to fip, leading to very low sensitivity, were the small and large intestine with a sensitivity of . and . %, respectively. the other organs also showed low sensitivity, except lung ( . %), which also showed the likelihood ratios (lr)-result closest to . (i.e., the lr− that indicates a good probability to exclude the disease), followed by the kidney and mesenteric lymph node. immunohistochemistry showed absolute specificity in all tissues and, consequently, a high ppv, while its sensitivity, as well as the negative predictive value (npv), were low, and the lr− was not close to . . conversely, specificity and ppv of rt-npcr were absolute only for the liver, large intestine, and lung, although, for the kidney and small intestine, both the specificity and the ppv and especially the lr+ were high. however, sensitivity and the npv of rt-npcr, although not absolute and probably not relevant for diagnostic purposes, were higher than for ihc. in addition, in this case, however, the lr− was always distant from the optimal value of . . this study was designed to achieve information about the concordance of different tests (histology, ihc, rt-npcr) performed on the same organs of cats with fip, aiming to find which one could be sampled in vivo to achieve an early diagnosis. cats with both the non-effusive and effusive form were included for a fair comparison between methods applied to the same tissues. in fact, despite the clinical distinction, mixed or transition forms are common [ ] , and effusion and granulomatous lesions can be present to a greater or lesser degree and coexist [ ] . of course, in the effusive form, analysis of the effusion is very useful for diagnosis, even though the sensitivity of immunostaining is not absolute, and rt-npcr lacks both sensitivity and specificity [ ] . therefore, tests on tissues might be necessary for diagnosis confirmation, even when effusion is present. the organ panel was selected with the major aim of including organs that can be sampled in vivo when trying to reach an early diagnosis in clinical practice. organs reportedly affected by fip lesions and, at the same time, accessible from a surgical point of view, were selected, aiming to guide the clinician to perform targeted surgical biopsies in cats suspected to have fip. the analysis of every single test demonstrated, as expected that none of the tests have both absolute specificity and sensitivity. histological lesions most frequently detected in the fip group comply with the ones described in the literature as strongly consistent with fip [ , , ] . in some organs from fip cats, histological lesions consistent with fip were not detected. however, cats with fip had histological lesions in more than one organ, except for two cats in which lesions were restricted to the kidney, which has been reported as one of the most frequently affected organs [ , ] , and cns. these findings suggest that the collection of biopsies from more than one organ might increase the probability of diagnosing fip. however, histological lesions consistent with fip, such as granulomatous inflammation and lymphoplasmacytic infiltrates [ ] , can also be detected in tissues from cats affected by diseases other than fip. these lesions, however, in the absence of granulomatous lesions, cannot be considered absolutely diagnostic for fip [ , ] . differently from histology, ihc was positive only in cats with fip. in non fip cats, ihc positive signal was rarely found on villous superficial columnar epithelial cells of the large intestine. this may occur in non-symptomatic carriers since colonic enterocytes are thought to be the main site of viral persistence in the gut [ , ] . the few immunohistochemical positive results in the small and large intestine are quite surprising, given the tendency of fip-related lesions to follow the course of the cranial mesenteric artery and their frequent involvement of the serosa of these organs [ ] . however, ihc may also be negative in thoracic or abdominal organs when lesions are localized only in the cns, as occurred in one cat of our caseload. it is noteworthy that revising the spleen of cat n • with a negative histological examination and positive ihc, only a mild increase in the perivascular number of macrophages was noticed multifocally, but was not considered as a typical lesion (figure a ). in light of the positivity to ihc (figure b) , these lesions could be considered as early lesions. this confirms that the presence of lesions potentially consistent but not absolutely diagnostic for fip should induce the pathologist to perform ihc since the combination of tests may be more informative than histology alone. pathogens , , x for peer review of one organ, except for two cats in which lesions were restricted to the kidney, which has been reported as one of the most frequently affected organs [ , ] , and cns. these findings suggest that the collection of biopsies from more than one organ might increase the probability of diagnosing fip. however, histological lesions consistent with fip, such as granulomatous inflammation and lymphoplasmacytic infiltrates [ ] , can also be detected in tissues from cats affected by diseases other than fip. these lesions, however, in the absence of granulomatous lesions, cannot be considered absolutely diagnostic for fip [ , ] . differently from histology, ihc was positive only in cats with fip. in non fip cats, ihc positive signal was rarely found on villous superficial columnar epithelial cells of the large intestine. this may occur in non-symptomatic carriers since colonic enterocytes are thought to be the main site of viral persistence in the gut [ , ] . the few immunohistochemical positive results in the small and large intestine are quite surprising, given the tendency of fip-related lesions to follow the course of the cranial mesenteric artery and their frequent involvement of the serosa of these organs [ ] . however, ihc may also be negative in thoracic or abdominal organs when lesions are localized only in the cns, as occurred in one cat of our caseload. it is noteworthy that revising the spleen of cat n° with a negative histological examination and positive ihc, only a mild increase in the perivascular number of macrophages was noticed multifocally, but was not considered as a typical lesion (figure a ). in light of the positivity to ihc (figure b) , these lesions could be considered as early lesions. this confirms that the presence of lesions potentially consistent but not absolutely diagnostic for fip should induce the pathologist to perform ihc since the combination of tests may be more informative than histology alone. our results demonstrated that the number of rt-npcr positivities exceeded those recorded in histology and ihc, due to the higher analytical sensitivity of this method [ ] and to the possible occurrence of viremic episodes. moreover, the viral burden in fcov infected cats is reported to be very variable [ , ] . therefore, some cats may have enough circulating viruses to be detected by molecular methods even when not affected by fip. the best concordance was found between histology and ihc, except for the lung, which showed the best concordance between ihc and rt-npcr. in non fip cats, the discordance between positive histology and negative ihc confirmed that lesions were due to causes other than fip, as hypothesized above. in fip cats, it may rather depend on the already reported variable distribution of lesions in different organs and viral antigens within lesions [ ] . in these cases, however, other tissues from the same cat were positive at both histology and ihc, confirming that multiple tissues should be sampled to improve the probability to detect fip lesions and/or positive ihc since ihc might not be confirmatory if only one section is analyzed [ ] . a substantial limitation of this study is that the sampling of organs post-mortem does not exactly represent what can be sampled in a clinical setting, our results demonstrated that the number of rt-npcr positivities exceeded those recorded in histology and ihc, due to the higher analytical sensitivity of this method [ ] and to the possible occurrence of viremic episodes. moreover, the viral burden in fcov infected cats is reported to be very variable [ , ] . therefore, some cats may have enough circulating viruses to be detected by molecular methods even when not affected by fip. the best concordance was found between histology and ihc, except for the lung, which showed the best concordance between ihc and rt-npcr. in non fip cats, the discordance between positive histology and negative ihc confirmed that lesions were due to causes other than fip, as hypothesized above. in fip cats, it may rather depend on the already reported variable distribution of lesions in different organs and viral antigens within lesions [ ] . in these cases, however, other tissues from the same cat were positive at both histology and ihc, confirming that multiple tissues should be sampled to improve the probability to detect fip lesions and/or positive ihc since ihc might not be confirmatory if only one section is analyzed [ ] . a substantial limitation of this study is that the sampling of organs post-mortem does not exactly represent what can be sampled in a clinical setting, and our results cannot be directly transferred to bioptic samples collected in vivo. moreover, tissues collected in vivo by bioptical means are reduced in size, and the possibilities of obtaining multiple sections from these kinds of samples, especially when they are sent to commercial laboratories, are low. unfortunately, the same approach adopted in this study would not have been possible in live pathogens , , of animals. the ultrasound-guided collection of bioptic samples immediately after euthanasia would be an alternative future approach to reproduce the clinical setting. however, results of histology, immunohistochemistry, and/or rt-npcr may provide positive results even when evident gross lesions are not detected. therefore, knowing which tissues might be preferable to collect, even if not affected by macroscopic or sufficiently large lesions to be detected by imaging means, could possibly be useful. the low concordance between rt-npcr and histology or ihc was generally due to positive rt-npcr in tissues that were negative for histology and/or ihc. this is likely due to the above mentioned high analytical sensitivity of rt-npcr, coupled with the possible systemic spread of fcov in infected cats not affected by fip [ , , ] . the single exception was represented by one lung sample from an fip cat, which had positive ihc but negative rt-npcr. different hypotheses could explain this discrepancy. first, lesions could be restricted to sections processed for ihc and not in those intended for rt-npcr, despite collected tissue sections always being adjacent to each other. second, the amount of viral rna in the sample intended for rt-npcr might not have been adequate to produce enough amplification products. third, the fcov genome within the lesion could have undergone mutation(s) that did not allow the binding of primers to target sequences. this latter hypothesis is unlikely, either because the -utr's target of the rt-npcr is well conserved in fcovs [ ] or because the kidney from the same cat was rt-npcr positive, although simultaneous infection by different mutated strains cannot be excluded. it is noteworthy that in non fip cats, positive results of rt-npcr may be misleading. positive results were found, especially in the mesenteric lymph node and spleen. these latter organs are thought to harbor high viral loads because of the migration of fecv infected monocytes through the vascular network [ ] . conversely, the absence of rt-npcr positive results in the liver and lung of non fip cats is partly surprising and in disagreement with those studies which identified these organs as probable sites of fcov persistence in healthy cats, as the results recorded in the large intestine. again, this finding could be imputable to the low sample size of non fip cats [ ] . however, in both fip and non fip cats, the rate of concordance between rt-npcr and ihc was very variable in the different tissues. again, this indicates that the probability of diagnosing fip, independent of the method used, increases when several organs are sampled. alternatively, it may be advisable to sample the liver, lung, and small intestine since these organs had the highest rate of positive results and may provide the same information independent of the diagnostic method used. indeed, the choice of tissue and test that more likely might provide diagnostic information should also be based on information about diagnostic accuracy. from this perspective, our results confirmed that, regardless of the examined organ, positive ihc is always diagnostic for fip since the specificity and positive predictive value reached %. on the contrary, negative ihc does not allow the exclusion of fip, based on the low sensitivity and negative predictive value and on the high negative lr. these results, in fact, indicate that, in the case of negative ihc, the chance that the cat is affected by fip is not null [ ] . nevertheless, it should again be considered that performing ihc on a wide range of organs and lesions increases the probabilities to diagnose fip. additionally, considering results from cns tissues from cat n • , which were not included in the calculation of diagnostic performance of ihc, the diagnostic accuracy of ihc would have reached %. this additional information confirms that ihc performed on multiple tissues collected at necropsy, including cns, may be absolutely diagnostic for fip. however, this information would also deviate from the aims of this study, which is intended to recommend organs to sample in clinical practice on live animals, based on the frequency of positive results. from this standpoint, the higher specificity of ihc compared with histology, coupled with the low sensitivity of histology, suggest that a negative histological result alone cannot rule out fip and that in case of strong clinical suspicion, it is advisable to obtain serial sections of the same tissue to perform ihc, to increase the probability of finding the fcov antigen within histological lesions, which may not have been included in the first slide. contrary to ihc, rt-npcr showed an overall higher sensitivity but lower specificity, as already reported [ ] , with rare exceptions. according to our results, positive rt-npcr cannot be considered diagnostic for fip, except for the liver, large intestine, and lung, in which this method exhibited absolute specificity. conversely, diagnostic accuracy on the kidney proved to be the highest, as demonstrated by the high positive likelihood ratio, that unlike predictive values, was not affected by the prevalence of the disease. in some cases, the sensitivity of rt-npcr was low, especially in the liver and large intestine. results regarding rt-npcr on the large intestine were quite surprising since samples from this latter organ are more likely expected to give false positive rt-npcr results due to the presence of fcov in the gastrointestinal tract of shedder cats. a possible explanation for this finding could be the action of fecal rnase on nucleic acids and their degradation. moreover, the high rate of negative rt-npcr results recorded in this study in the small and large intestine could depend on a decreased viral fecal excretion, which has been demonstrated to occur in fip affected cats, hypothetically due to the impaired tropism of mutated fcov for intestinal epithelial cells [ , ] . on the other hand, non fip cats are less likely to shed the virus in the feces compared with fip cats [ ] . nevertheless, the small sample size of the non fip group could also have affected the results, and also performing the rt-npcr on feces would have been useful to confirm if the cats were not shedding fcov at the moment of sampling. specificity was notably low in the mesenteric lymph node and spleen, suggesting that an rt-npcr positivity in these organs, which are frequently sampled in clinical routine for diagnostic purposes, cannot be considered as diagnostic for fip. this seems to be in contrast with the high sensitivity and specificity of quantitative rt-npcr on the mesenteric lymph node that has been recently reported [ ] . overall, the low specificity of rt-npcr makes it mandatory to perform histology and ihc independently of the rt-npcr results, except in the liver and lung, where a positive result is absolutely specific for fip. tissue samples were collected post-mortem at the veterinary teaching hospital of milan from cats affected by fip or other systemic diseases or serious injuries, deceased or euthanized, and subjected to necropsy for diagnostic purposes. all the above methods were performed within routine diagnostic procedures and with the owner's written informed consent about the use of tissues and samples for research. therefore, according to the ethical committee of the university of milan (decision n • , ), residual aliquots of tissues were used for research purposes without any additional formal request of authorization to the ethical committee. the inclusion criteria were: • the possibility to collect and process tissues within six hours after death; • the availability of signalment, history, and clinical information (physical examination, clinical pathology, diagnostic imaging, depending on the clinical presentation). this information, along with necropsy findings and histology results, was used to classify the cats in the fip or non fip group [ ] . samples were collected from the spleen, liver, mesenteric lymph node, kidney, large intestine, small intestine, and lung of the necropsied cats, paying particular attention to sample tissues with gross lesions, whenever present, and to always include the organs serosa (see table for a detailed list of sampled organs). when possible, data regarding evident macroscopic alterations frequently seen in fip, namely: thickening of the serosa, focal or diffuse nodular lesions, organs increase in size, were recorded (see supplementary table s ). samples were then processed as described below. for diagnostic purposes, tissues from other organs showing macroscopic alterations, as well as organs not visibly affected but plausibly involved based on the patient's history (e.g., brain and cerebellum from cats with neurological signs), were also sampled during a necropsy to perform routine histology and immunohistochemistry. since rt-npcr was not performed on these tissues, the corresponding results were not included in comparison with rt-npcr but were used to achieve accurate classification of cases in the fip or non fip group as detailed below. from each organ, a sample was taken and sectioned with a sterile scalpel in two immediately adjacent halves of approximately one-centimeter diameter each. if evident nodular lesions were present, the two halves were obtained, sectioning the sample in the center of the lesion. one half was placed in plain tubes and immediately frozen at − • c for molecular biology, while the other half was collected into % neutral-buffered formalin to perform histology and immunohistochemistry. histopathology was performed by an experienced veterinary pathologist, blinded to both gross lesions as well as to the patient's signalment and history. formalin-fixed samples were sent to the department of comparative biomedicine and food science of the university of padua. sections ( µm) obtained from formalin-fixed paraffin-embedded samples were stained with hematoxylin-eosin for histology with an automated stainer (autostainer xl, leica biosystems, wetzlar, germany). for immunohistochemistry (ihc), µm paraffin sections were placed on surface-coated slides (superfrost plus, thermofisher scientific, milan, italy). immunostaining was performed with an automatic immunostainer (ventana benchmark xt, ventana medical system, tucson, az, usa). antigen retrieval was performed with standard cc (heat induced epitope retrieval, hier, in tris-edta buffer ph at • c for min). as the primary antibody, a mouse monoclonal antibody against the feline coronavirus was used (dilution : , clone fipv - serotec, oxford, uk). after incubation at • c for min, a kit with a secondary antibody with horseradish peroxidase (hrp)-conjugated polymer that binds to mouse and rabbit primary antibodies (ultraviews universal dab, ventana medical system, tucson, az, usa) was used. all reagents were dispensed automatically except for the primary antibody, which was manually dispensed. fip positive tissues were used as internal positive controls. for negative controls, the antibody diluent (ventana medical systems) was applied instead of the primary monoclonal antibodies. from frozen-thawed samples, rna was obtained using a nucleospin rna kit (macherey-nagel, bethlehem, pa, usa). twenty milligrams of tissue were thinly shredded on sterile plates using sterile scalpels and vigorously vortexed in ra lysis buffer until completely dissolved. all the further steps were performed according to the manufacturer's instructions. rna samples were then frozen at − • c or immediately used for rt-npcr. rt-npcr targeting a bp product of the highly conserved untranslated region ( utr) of the genome of both type i and type ii fcov was used [ ] . rna pre-analytical quality control targeting vertebrate s rrna locus [ ] was performed on randomly selected samples (results not shown). fcov rt-npcr positive rna was used as positive control and a fip-negative sample as negative control. rt-npcr products were visualized under uv transilluminator on a . % agarose gel stained with ethidium bromide. cats included in this study were divided into two groups based on the following criteria. fip group: cats suspected to have fip based on history and/or clinical, hematological, biochemical, and serum protein electrophoretic alterations [ ] on which diseases other than fip were excluded, showing gross and histological lesions consistent with fip in at least one tissue, as reported below, including tissues sampled for diagnostic purposes and not included in this study (e.g., brain or cerebellum). • non fip group: cats with or without a clinical presentation potentially consistent with fip, on which diseases other than fip were already diagnosed on a clinical basis (e.g., based on results of diagnostic imaging or cytology) and/or without any histological lesion consistent with fip. for each organ, lesions were considered consistent with fip if showing one or more of the following typical fip patterns ( figure ) [ , ] : pyogranulomas on one or more serosal surfaces; • granulomas with or without necrotic areas; • lymphocytic and plasmacytic infiltrates in specific sites (e.g., band-like infiltrate in serosal surfaces, perivascular infiltrate in meninges and cns); • granulomatous to necrotizing vasculitis and fibrinous serositis. pathogens , , x for peer review of • non fip group: cats with or without a clinical presentation potentially consistent with fip, on which diseases other than fip were already diagnosed on a clinical basis (e.g., based on results of diagnostic imaging or cytology) and/or without any histological lesion consistent with fip. for each organ, lesions were considered consistent with fip if showing one or more of the following typical fip patterns ( figure ) [ , ]: conversely, histological lesions were considered non-consistent with fip if changes consistent only with diseases other than fip or no lesions at all were present. histological lesions not diagnostic for fip as a standalone, but consistent with fip if associated with signalment, history, gross lesions, and other histological lesions (e.g., pyogranulomas) suggestive of fip, were considered potentially consistent with fip. these lesions (e.g., multifocal perivascular increase in macrophages) were categorized as positive or negative depending on the lesions recorded in other organs, as it would happen in a clinical setting. as previously described by pedersen and kipar, immunohistochemistry was considered positive if fcov antigen was detected within typical histological lesions above described [ , ] . on the contrary, according to kipar and colleagues, the detection of fcov antigen in few scattered tissue macrophages (e.g., kupffer cells in the liver, pulmonary interstitial macrophages in the lungs as well as individual sinus macrophages in the lymph nodes) or epithelial cells (e.g., intestinal columnar cells, likely infected by fecv, figure ) only, was not considered as a positive result [ ] . • pyogranulomas on one or more serosal surfaces; • granulomas with or without necrotic areas; • lymphocytic and plasmacytic infiltrates in specific sites (e.g., band-like infiltrate in serosal surfaces, perivascular infiltrate in meninges and cns); • granulomatous to necrotizing vasculitis and fibrinous serositis. conversely, histological lesions were considered non-consistent with fip if changes consistent only with diseases other than fip or no lesions at all were present. histological lesions not diagnostic for fip as a standalone, but consistent with fip if associated with signalment, history, gross lesions, and other histological lesions (e.g., pyogranulomas) suggestive of fip, were considered potentially consistent with fip. these lesions (e.g., multifocal perivascular increase in macrophages) were categorized as positive or negative depending on the lesions recorded in other organs, as it would happen in a clinical setting. as previously described by pedersen and kipar, immunohistochemistry was considered positive if fcov antigen was detected within typical histological lesions above described [ , ] . on the contrary, according to kipar and colleagues, the detection of fcov antigen in few scattered tissue macrophages (e.g., kupffer cells in the liver, pulmonary interstitial macrophages in the lungs as well as individual sinus macrophages in the lymph nodes) or epithelial cells (e.g., intestinal columnar cells, likely infected by fecv, figure ) only, was not considered as a positive result [ ] . rt-npcr was considered positive if it showed a bp band on agarose gel electrophoresis [ ] . for histology, ihc and rt-npcr, true-positive (results consistent with fip in cats with fip) and false-positive results (results consistent with fip in cats without fip), as well as true-negative (results not consistent with fip in cats without fip) and false-negative results (results not consistent with fip in cats with fip) were recorded. sensitivity, specificity, accuracy, positive and negative predictive values, as well as positive and negative likelihood ratios (lr+ and lr−, respectively) were then calculated for histology, ihc, and rt-npcr. the concordance between histology, ihc and rt-npcr was assessed using cohen's k coefficient. concordance was classified as absent (k < ); minimal ( < k < . ); weak ( . < k < . ); moderate ( . < k < . ); strong ( . < k < . ), and almost perfect ( . < k < ) [ ] . rt-npcr was considered positive if it showed a bp band on agarose gel electrophoresis [ ] . for histology, ihc and rt-npcr, true-positive (results consistent with fip in cats with fip) and false-positive results (results consistent with fip in cats without fip), as well as true-negative (results not consistent with fip in cats without fip) and false-negative results (results not consistent with fip in cats with fip) were recorded. sensitivity, specificity, accuracy, positive and negative predictive values, as well as positive and negative likelihood ratios (lr+ and lr−, respectively) were then calculated for histology, ihc, and rt-npcr. the concordance between histology, ihc and rt-npcr was assessed using cohen's k coefficient. concordance was classified as absent (k < ); minimal ( < k < . ); weak ( . < k < . ); moderate ( . < k < . ); strong ( . < k < . ), and almost perfect ( . < k < ) [ ] . in conclusion, results from this study prove that the frequency of positive results obtained with different methods (histology, ihc, and rt-npcr) on fip cats varies depending on the analyzed tissue. even if with a low frequency, some histological results consistent with fip were also recorded in non fip cats. conversely, ihc had a % specificity, and, therefore, it may be advisable to use ihc to confirm and exclude the disease in the presence of histological lesions consistent with fip. on the other hand, rt-npcr is not recommended as a first diagnostic approach, i.e., on fine-needle aspiration biopsy (fnab), due to its lower specificity and sensitivity. therefore, it is always advisable to confirm possible positive or negative rt-npcr results with ihc. however, data concerning concordance suggest that, when both tests cannot be performed, it would be preferable to examine either the lung or liver, on which the probability to obtain accurate information, independent of the analytical method, is good. instead, when the kidney or intestine are sampled, ihc should be preferred to rt-npcr as a confirmatory test since the concordance of the two methods is not sufficiently high, and the specificity is higher for ihc than for rt-npcr. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / / s , supplementary table s . details on histological lesions recorded for each sample included in the study. abbreviations. eo, eosinophilic, f, fibrinous; g, granulomatous; lp, lymphoplasmacytic; n, neutrophilic; m, macrophagic; pv, perivascular. lesions highly suggestive and classified as consistent with fip are reported in italic. a lesions compatible with fip, classified as negative (not consistent with fip). b lesions compatible with fip, later on, classified as positive (consistent with fip). supplementary table s . results of macroscopic and histological examination, immunohistochemistry, and rt-npcr obtained on each collected sample. negative (not consistent with fip) and positive (highly suggestive, therefore compatible with fip) results are listed. abbreviations: n, negative; n/a, not applicable (sample not collected); n/d, not done; p: positive. a tale of two viruses: the distinct spike glycoproteins of feline coronaviruses feline coronavirus in multicat environments an update on feline infectious peritonitis. companion anim feline coronaviruses: pathogenesis of feline infectious peritonitis replication of feline coronaviruses in peripheral blood monocytes amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis persistence and evolution of feline coronavirus in a closed cat-breeding colony the molecular dynamics of feline coronaviruses feline infectious peritonitis: still an enigma? clinical and laboratory features of cats with feline infectious peritonitis-a retrospective study of confirmed cases ( - ) comparison of the performance of laboratory tests in the diagnosis of feline infectious peritonitis diagnosis of feline infectious peritonitis: update on evidence supporting available tests an update on feline infectious peritonitis: diagnostics and therapeutics performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases diagnosis of non-effusive feline infectious peritonitis by reverse transcriptase quantitative pcr from mesenteric lymph node fine-needle aspirates sensitivity of tru-cut and fine-needle aspiration biopsies of liver and kidney for diagnosis of feline infectious peritonitis limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis a review of feline infectious peritonitis virus infection: - feline infectious peritonitis diagnosis of feline infectious peritonitis: a review of the current literature cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis some aspects of humoral and cellular immunity in naturally occuring feline infectious peritonitis chronic kidney disease in aged cats cellular composition and interferon-γ expression of the local inflammatory response in feline infectious peritonitis (fip) sites of feline coronavirus persistence in healthy cats natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats high viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (fcov) type i and in naturally fcov-infected cats detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr on the use and computation of likelihood ratios in clinical chemistry feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral c gene spike protein fusion peptide and feline coronavirus virulence feline coronavirus infections two universal primer sets for species identification among vertebrates the measurement of observer agreement for categorical data funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -h kot og authors: schäfer, alexandra; baric, ralph s. title: epigenetic landscape during coronavirus infection date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: h kot og coronaviruses (cov) comprise a large group of emerging human and animal pathogens, including the highly pathogenic severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) strains. the molecular mechanisms regulating emerging coronavirus pathogenesis are complex and include virus–host interactions associated with entry, replication, egress and innate immune control. epigenetics research investigates the genetic and non-genetic factors that regulate phenotypic variation, usually caused by external and environmental factors that alter host expression patterns and performance without any change in the underlying genotype. epigenetic modifications, such as histone modifications, dna methylation, chromatin remodeling, and non-coding rnas, function as important regulators that remodel host chromatin, altering host expression patterns and networks in a highly flexible manner. for most of the past two and a half decades, research has focused on the molecular mechanisms by which rna viruses antagonize the signaling and sensing components that regulate induction of the host innate immune and antiviral defense programs upon infection. more recently, a growing body of evidence supports the hypothesis that viruses, even lytic rna viruses that replicate in the cytoplasm, have developed intricate, highly evolved, and well-coordinated processes that are designed to regulate the host epigenome, and control host innate immune antiviral defense processes, thereby promoting robust virus replication and pathogenesis. in this article, we discuss the strategies that are used to evaluate the mechanisms by which viruses regulate the host epigenome, especially focusing on highly pathogenic respiratory rna virus infections as a model. by combining measures of epigenome reorganization with rna and proteomic datasets, we articulate a spatial-temporal data integration approach to identify regulatory genomic clusters and regions that play a crucial role in the host’s innate immune response, thereby defining a new viral antagonism mechanism following emerging coronavirus infection. the severe acute respiratory syndrome coronavirus (sars-cov) emerged in / , most likely in the guangdong province, china. from the initial outbreak, sars-cov rapidly spread across the globe causing infections and~ deaths in countries; mortality rates approached % in aged individuals [ ] [ ] [ ] . from its animal reservoir in chinese horseshoe bats (genus rhinolophus), the sars-cov was thought to have adapted to palm civets and raccoon dogs in open markets, before finally colonizing human populations [ ] . more recent studies have shown that sars-cov, as well as a large reservoir of sars-like bat cov (sl-cov) have the ability to efficiently utilize the human angiotensin-converting enzyme (ace ) receptor for docking and entry and replicate efficiently in primary human airway epithelial cells. these data document the presence of a large animal reservoir (orfs) . the ′ end is capped and contains a leader sequence (l). sars-cov encodes for orfs, including orf , which is processed into nsp to nsp , structural orfs (s, e, m, and n) in grey, and luxury downstream orfs ( a, b, , a, b, a, b and b). nsp: nonstructural protein; s: spike; e: envelope; m: matrix; and n: nucleoprotein. innate immunity is one of the earliest barriers to coronavirus infection. following infection, pathogen recognition receptors (prrs) such as retinoic acid-inducible gene i (rig-i), melanoma the end is capped and contains a leader sequence (l). sars-cov encodes for orfs, including orf , which is processed into nsp to nsp , structural orfs (s, e, m, and n) in grey, and luxury downstream orfs ( a, b, , a, b, a, b and b). nsp: nonstructural protein; s: spike; e: envelope; m: matrix; and n: nucleoprotein. innate immunity is one of the earliest barriers to coronavirus infection. following infection, pathogen recognition receptors (prrs) such as retinoic acid-inducible gene i (rig-i), melanoma differentiation-associated gene (mda ), toll-like receptors (e.g., tlr , and ) and other sensing molecules recognize pathogen-associated molecular patterns (pamps) in viral components, such as viral structure proteins or viral nucleic acid. successful recognition initiates a signaling cascade that activates an antiviral state in the host. several main players of innate immunity, such as signal transducer and activator of transcription (stat ), myeloid differentiation primary response gene (myd ), tlr , tlr and tlr /tir-domain-containing adapter-inducing interferon-β (trif), function to dampen infection severity during coronavirus infection in vivo [ ] [ ] [ ] [ ] [ ] . similarly, interferons (ifn−alpha (α) and ifn-beta (β), ifn-gamma (γ)) also play critical roles in controlling sars-cov in vivo and in vitro [ ] [ ] [ ] [ ] [ ] , ] . data from the / outbreak have demonstrated that differential ifn and interferon-stimulated gene (isg) expression levels in patients correlated with sars disease outcomes. several mouse models for sars-cov pathogenesis confirmed protective roles of myd , tlrs and select isgs [ , , , ] . like other viral pathogens, coronaviruses such as sars-cov and mers-cov have evolved genetic functions that delay and/or antagonize pathogen recognition as well as isg effector functions. sars-cov encodes several proteins that modulate innate immune signaling through the antagonism of the induction of interferon. as mentioned above, several nsps that are encoded by orf (orf a/b), like nsp , nsp papain-like protease, nsp and nsp antagonize various sensing or signaling programs and nfκβ, or function to cap viral messenger rnas (mrnas) and evade interferon-induced protein with tetratricopeptide repeats (ifit) - isgs [ , [ ] [ ] [ ] [ ] [ ] (figure ). these nsps show high homology to proteins of other human coronaviruses and are critical for efficient viral replication. several downstream open reading frames like orf a, orf b and orf also antagonize sensing or signaling pathways, or block karyopherin nuclear import [ , ] (figure ). mers-cov also encodes several luxury functions with interferon antagonism activities, including orf a, orf b and perhaps orf , noting that orf b antagonizes phosphodiesterase activity and rnase l activation [ ] [ ] [ ] [ ] [ ] . however, how the exact underlying mechanisms allow these antagonistic molecules to interfere with the effector molecules that establish an antiviral state, assist in wound repair, or prime and enhance an adaptive immune response, which is critical for clearance, is still under study. recent studies have suggested that rna viruses like coronaviruses and influenza viruses are able to manipulate the host's epigenome, potentially heralding entirely new mechanisms of viral antagonism and new targets for therapeutic intervention and control [ , ] . the purpose of this review is to summarize the available methodology to study the epigenetic mechanisms that allow study of the epigenome during infection with coronavirus and other rna viruses. epigenetic regulation bridges genotype and phenotype by changing the function of the gene locus without changing the sequence of the underlying dna. over the last decade, research efforts have revealed a dynamic range of epigenetic factors that shape and regulate chromatin status, leading to changes in host gene expression patterns, and therefore to alterations in phenotypes. epigenetic modifications are significant in regulating cellular mechanisms and pathways during embryonic development, in memory function, in immunity and in disease [ , ] . while mutations directly affect the genetic material by changing the genetic code, epigenetic modifications change the chromatin structure or modify the nucleic acid without altering the genetic code. this makes epigenetic modifications reversible, flexible, and quickly responsive to changes in the environment and other exposures. based on this ability, the study of epigenetic modifications is an important interface between the environment and the genome [ ] . over the last decade, epigenetics research has made rapid progress in understanding developmental biology, memory, and inheritability functions. more recently, it has become increasingly important in studies of oncology, adaptive and innate immunity, and infectious diseases [ , ] . it is becoming well established that many dna viruses, and to some lesser extent rna viruses, have evolved functions that antagonize the regulatory machine of the host epigenome, leading to regulated changes in host gene expression that lead to a favorable environment for virus replication and spread [ ] . over the last years, the development of many biochemical and in particular high-throughput approaches have revolutionized our understanding of chromatin biology and function. chromatin biology is now at the point where studies can be performed that use its tools to discover and validate new players and pathways in epigenetics and their role in a variety of biological disciplines, including developmental biology, oncology and infectious diseases, like bacterial and viral infections. the human genome project (hgp) was officially completed in , providing the research community with a detailed map of the genetic organization and structure of the human genome as well as the epigenome [ ] . another benefit of the human genome project was the development of next-generation sequencing (ngs) technology. epigenetics research adopted the ngs techniques early on by refining methods like chip-seq, rna-seq, and medip-seq [ , ] . today, these are routine methods for the investigation of genome-wide changes in dna methylation, histone modification, and dna-protein interactions. similarly, in the field of infectious disease research, high-throughput dna analyses have enabled the genome-wide examination of epigenetic modifications and dna methylation, providing systematic, large-scale association testing with disease phenotypes. it is likely that many common diseases, cancers, and infectious disease outcomes in humans are mediated by genetic and environmental factors. likewise, epigenome-wide association studies (ewas) provide a systematic identification of genome-wide epigenetic variants associated with disease outcomes [ ] . ewas can collect information about variation of epigenetic markers, global epigenetic patterns, and genome-wide distribution of epigenetic markers which can provide functional correlation with genotypes and phenotypes associated with particular pathological or non-pathological outcomes, defining new disease-associated marks [ , ] . in particular, the encyclopedia of dna elements (encode) project has advanced our understanding of the principles of genome, epigenome and chromatin organization, discovering and identifying formerly unknown histone modifications, nucleosome positions, and chromosome-wide maps of regulatory chromatin structures [ , , ] . gencode, a part of encode, now contains an extensive catalogued transcript, and pseudogene and long noncoding rna (lncrna) resources, helping to develop and to identify histone modifications and variants from several combinatorial patterns that define active promoters/tsss (transcription start sites), transcribed gene bodies, inactive regions, and enhancers [ , ] . several techniques described below are now commonly used in studies that integrate different data types including transcriptomics, proteomics, and epigenomics [ ] . these allow us to validate and discover new molecular pathways that could lead to new discoveries in developmental biology, memory, and disease. the genetic information in eukaryotic cells is encoded in the chromosomes and mitochondrial dna. chromosomes exist in deoxyribonucleoprotein complexes called chromatin. chromatin is found in two variations: the euchromatin and the heterochromatin, which were originally distinguished cytogenetically by giemsa staining procedures. darker staining heterochromatin indicates tightly packaged protein and nucleic acid complexes found at centromers and telomers. these contain mostly inactive satellite dna as opposed to the lighter-stained loosely-packed euchromatin, which defines genome regions under active transcription and gene expression [ ] . chromatin organization is complex and composed of a specialized set of proteins-the histones (h)-that organize the dna into the nucleosome. the nucleosome is composed of a tightly-packed histone octamer consisting of the core histones h , h , h a, and h b with roughly base pairs of dna wrapped around it, much like beads on a string. this structure maintains stability and most importantly, protein and transcription factor accessibility to the dna genome, allowing the chromatin to guarantee tight packaging of the genomic dna, accurate replication, and distribution into the daughter cells during cell division, as well as transcriptional regulation of gene expression [ ] . the histone's n-termini, the so-called histone tails, extend from the globular protein unit and as such are targets for post-translational modifications. at this time, several chemical modifications have been identified and characterized: lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation. these modifications are found on h a, h b, h , and h histone subunits [ , ] . in , jenuwein and colleagues described the histone code, hypothesizing that a coding mechanism within the chromatin structure is regulated by chemical modifications to the histone tail, a concept which is now well supported by the literature [ ] . we now know that distinct modifications of the histone tails interact with different sets of chromatin-associated proteins ( table ) . as a result, modifications on the same or different histone tails may be interdependent and generate various combinations on any one nucleosome, thereby supporting the modification-induced recruitment of chromatin-associated proteins. consequently, the specificity of the downstream information is guaranteed and a specific crosstalk between histone modifications is possible. the last decade has shown that this regulatory instance of nucleosomes and chromatin structure on the genome has emerged as a critically important determinant of cellular transcription, replication, and differentiation state. the histone tails of the nucleosome are subject to post-translational modulations. these modulations are covalently attached to the tails and include methylation of arginine residues, and methylation, acetylation, ubiquitination, phosphorylation, and sumoylation of serines (s) and threonines (t) (table , figure a ). over the last decade, many modifications have been associated with active or non-active transcription. modifications like the acetylation (ac) of h and h , as well as the di-or tri-methylation (me) of h on lysine (h k ), are associated with an active transcription state. on the other hand, methylation of h k and h k are now associated with transcriptional repression of the particular gene [ ] . based on their function within gene expression the particular histone marks can be found in distinct localizations within a gene region [ ] . likewise, many modifications are almost uniquely associated with gene organization components, like promoters, tsss, enhancers or gene bodies, slicing sites, and transcriptional end sites (tes). these histone marks help to organize the chromatin by modulating accessibility, thereby defining regulatory regions and elements, like promoters, enhancers and insulators, within the genome [ ] . the histone tails of the nucleosome are subject to post-translational modulations. these modulations are covalently attached to the tails and include methylation of arginine residues, and methylation, acetylation, ubiquitination, phosphorylation, and sumoylation of serines (s) and threonines (t) (table , figure a ). some of the histone mark distributions are uniquely associated with the transcription rates of particular genes. as mentioned above, main histone marks for regulating the tss are h k me and h k me ; both modifications are exclusively found at the tss and in the appropriate promoter region of the particular gene [ ] . h k me is the main modification for an active promoter region and therefore actively transcribed chromatin, while h k me on the other hand, is the main modification found at repressed promoters [ ] . despite these differences, both modifications perform a crucial function in bivalent or 'poised' promoters [ ] . a promoter occupied with both h k me and h k me can be rapidly activated or inactivated for transcription, making both histone modifications signature configurations for poising bivalent promoters for alternate fates: active and repressed gene transcription [ , , ] . other histone marks are preferentially located in enhancer regions or within the gene body. for example, h k ac and h k me are enriched at active enhancer sequences, active promoters are flanked by h k ac and h k me , and gene bodies show enrichment of h k me . the state of the chromatin is modulated by a large number of proteins which can be seen as 'writers', 'readers', and 'erasers' [ ] . 'writers' are responsible for encrypting the information capacity of nucleosomes by adding distinct post-translational modifications to the histone tails. generally, 'writers' are acetylases, methylases, and phosphorylases that specifically add the appropriate modification to the histone tails. 'erasers' antagonize the function of the 'writers', and remove the histone modifications. these enzymes include deacetylases, demethylases, and phosphatases. 'writers' and 'erasers' modulate the assembly, placement, recognition, and modification. the recognition of histone modifications is mediated by 'readers'; proteins which are tightly regulated by phosphorylation and dephosphorylation through signaling pathways, recruitment and binding of co-factors, like transcription factors and adaptor proteins [ ] . overall, histone modification patterns are dynamic and reflect the activation state of a gene, the elongation state and the splicing patterns of the pre-mrna transcript. to match the complexity of the modification patterns, 'readers' are often organized in protein complexes, containing a bromodomain, chromodomain, and tudor domains and harboring several putative modification-dependent binding sites. many of the histone modifying enzymes belong to complex protein superfamilies that show stringent substrate, catalytic, and tissue specificity. this way, these proteins regulate dna accessibility together with atp-dependent chromatin remodeling complexes, which are mediating remodeling of nucleosomes, like moving, ejecting or restructuring nucleosomes. this mediates pioneer transcription factors, that are involved in recruiting transcription machinery complexes, like the rna polymerase ii (pol ii) complex, or insulator proteins, to bind at nucleosome-free dna regions and initiate gene expression. this guarantees a specific modification and response to environmental stimuli [ ] . based on the theory of the histone code, the same histone mark can have very different physiological outcomes depending on the location in the chromatin, the neighboring modifications, and the combination of modifications [ ] . in other words, to understand the function of a single histone mark, the combination and the co-occurrence with other marks needs be considered. this complexity determines that a combination of multiple histone modifications can have a cascading effect with a variety of different outcomes ranging from transcription repression to transcription activation to transcription termination. these varying outcomes regulate combinatory and sequentially downstream functions, generating distinct signatures for every individual gene [ , ] . this circumstance influences strongly the nature of the modification-binding proteins, the so-called readers and the following down-stream processes. this way, a rather small set of separate histone modifications results in a broad range of different outcomes for the cell. overall, it is not surprising that these chromatin-modifying enzymes play an important role in maintaining chromatin structure and dynamics. it is important to note, however, that chromatin marks can be easily reversed. as a consequence, they can rapidly respond to external stimuli, thereby regulating the accessibility of the underlying dna to the transcriptional machinery and ensuring the correct association of expressed genes in the appropriate situation [ , ] . in all mammalian cells, dna methylation takes place post dna replication. it occurs at the position of the cytosine ring within cpg nucleotides by adding a methyl group to create -methylcytosine ( mc). the modification is mediated by a family of enzymes, the dna methyltransferases (dnmts) ( figure b ). dnmt a and dnmt b have been described as de novo methyltransferases, preferentially targeting unmethylated cpg islands (cgis) in the genome to initiate dna methylation [ ] . studies have shown that dnmt functions as maintenance methyltrasferase, ensuring that the methylation status is maintained during dna replication and following cell division [ ] . however, dna methylation as an epigenetic marker is highly dynamic, and therefore crucial in gene silencing and gene regulation, the establishment of heterochromatin, and in regulating the stability of the chromosome [ ] . hypermethylation of repetitive dna sequences in combination with certain histone marks results in the condensation of chromatin and therefore in the establishment of heterochromatin [ ] . recently, dna hydroxymethylation ( hmc) has been identified as another form of dna methylation. several studies have shown that enzymes of the ten-eleven translocation (tet)-family catalyze the modification and that it has a major role in embryonic neuronal development. [ , ] . the presence of mc cgis plays a critical role in regulation of gene expression. more than % of coding genes contain cgis in promoter-associated regions. these cgis are generally unmethylated, and therefore easily accessible to transcription factors and other chromatin-associated proteins for the expression of most housekeeping genes and other regulated genes [ ] . however, de novo methylation of those promoter-associated cgis will repress and silence promoter activity. transcriptional inactivity at a methylated promoter region can be reversed by methyltransferases, rendering the dna sequence into active chromatin, therefore demonstrating another instance of gene regulation [ , , , ] . starting in the early s, the field of non-coding rnas (ncrnas) evolved from its historic origins as "junk rna" and quickly expanded into its own field of research. based on their function and their genetic origin ncrnas can be divided into long non-coding rnas (lncrnas) and small non-coding rnas (sncrnas), based on whether each rna is greater than or less than bp in length [ ] . for many years, lncrnas were considered to be unimportant junk byproducts of evolution and were ignored by most of the research community. however, this group of rnas is now recognized as a critical regulator in chromatin remodeling, transcriptional regulation, and post-transcriptional processing [ ] . epigenetics and micrornas (mirnas) regulate whole gene expression patterns transcriptionally and post-transcriptionally, respectively ( figure c ) [ ] . at the same time, epigenetics and mirnas control each other to form a regulatory circuit and to maintain normal physiological functions [ ] . several mirnas have been identified that target genes that control epigenetic pathways, like dnmts and histone methyltransferases (hmts), thus controlling chromatin structure by regulating by regulating histone modifier molecules. the expression of mirnas on the other hand is regulated by histone modification and dna methylation, forming an epigenetics-mirna regulatory circuit [ ] . a number of high throughput technologies have been developed to study the epigenetic landscape and epigenetic modifications genome-wide and on sequence-specific levels ( figure ). control epigenetic pathways, like dnmts and histone methyltransferases (hmts), thus controlling chromatin structure by regulating by regulating histone modifier molecules. the expression of mirnas on the other hand is regulated by histone modification and dna methylation, forming an epigenetics-mirna regulatory circuit [ ] . a number of high throughput technologies have been developed to study the epigenetic landscape and epigenetic modifications genome-wide and on sequence-specific levels ( figure ). ) that are associated with functional gene components (dashed arrow ), like promoters, transcriptional start sites, enhancers, gene bodies, slicing sites, and transcriptional end sites [ ] . tss: transcription start site; tf: transcription factor; tfbs: transcription factor binding site; faire: formaldehyde-assisted isolation of regulatory elements; chip: chromatin immuno-precipitation; medip: methylated dna immunoprecipitation. as a broad strategy to identify modifications of histones and the localization of histone marks across the genome, faire was developed and has been applied to understand the chromatin status of target cells and of dna viruses under different conditions of infection [ ] . faire is a method to isolate regulatory elements from eukaryote chromatin, thereby taking advantage of the fact that dna segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes. the faire method involves crosslinking the chromatin by adding formaldehyde, which preferentially targets heavily-condensed, transcriptionally-repressed chromatin over transcriptionally-active chromatin. the crosslinked chromatin is then sheared by sonication; phenol- control epigenetic pathways, like dnmts and histone methyltransferases (hmts), thus controlling chromatin structure by regulating by regulating histone modifier molecules. the expression of mirnas on the other hand is regulated by histone modification and dna methylation, forming an epigenetics-mirna regulatory circuit [ ] . a number of high throughput technologies have been developed to study the epigenetic landscape and epigenetic modifications genome-wide and on sequence-specific levels ( figure ). ) that are associated with functional gene components (dashed arrow ), like promoters, transcriptional start sites, enhancers, gene bodies, slicing sites, and transcriptional end sites [ ] . tss: transcription start site; tf: transcription factor; tfbs: transcription factor binding site; faire: formaldehyde-assisted isolation of regulatory elements; chip: chromatin immuno-precipitation; medip: methylated dna immunoprecipitation. as a broad strategy to identify modifications of histones and the localization of histone marks across the genome, faire was developed and has been applied to understand the chromatin status of target cells and of dna viruses under different conditions of infection [ ] . faire is a method to isolate regulatory elements from eukaryote chromatin, thereby taking advantage of the fact that dna segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes. the faire method involves crosslinking the chromatin by adding formaldehyde, which preferentially targets heavily-condensed, transcriptionally-repressed chromatin over transcriptionally-active chromatin. the crosslinked chromatin is then sheared by sonication; phenol- control epigenetic pathways, like dnmts and histone methyltransferases (hmts), thus controlling chromatin structure by regulating by regulating histone modifier molecules. the expression of mirnas on the other hand is regulated by histone modification and dna methylation, forming an epigenetics-mirna regulatory circuit [ ] . a number of high throughput technologies have been developed to study the epigenetic landscape and epigenetic modifications genome-wide and on sequence-specific levels ( figure ) . ) that are associated with functional gene components (dashed arrow ), like promoters, transcriptional start sites, enhancers, gene bodies, slicing sites, and transcriptional end sites [ ] . tss: transcription start site; tf: transcription factor; tfbs: transcription factor binding site; faire: formaldehyde-assisted isolation of regulatory elements; chip: chromatin immuno-precipitation; medip: methylated dna immunoprecipitation. as a broad strategy to identify modifications of histones and the localization of histone marks across the genome, faire was developed and has been applied to understand the chromatin status of target cells and of dna viruses under different conditions of infection [ ] . faire is a method to isolate regulatory elements from eukaryote chromatin, thereby taking advantage of the fact that dna segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes. the faire method involves crosslinking the chromatin by adding formaldehyde, which preferentially targets heavily-condensed, transcriptionally-repressed chromatin over transcriptionally-active chromatin. the crosslinked chromatin is then sheared by sonication; phenol- ), like promoters, transcriptional start sites, enhancers, gene bodies, slicing sites, and transcriptional end sites [ ] . tss: transcription start site; tf: transcription factor; tfbs: transcription factor binding site; faire: formaldehyde-assisted isolation of regulatory elements; chip: chromatin immuno-precipitation; medip: methylated dna immunoprecipitation. as a broad strategy to identify modifications of histones and the localization of histone marks across the genome, faire was developed and has been applied to understand the chromatin status of target cells and of dna viruses under different conditions of infection [ ] . faire is a method to isolate regulatory elements from eukaryote chromatin, thereby taking advantage of the fact that dna segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes. the faire method involves crosslinking the chromatin by adding formaldehyde, which preferentially targets heavily-condensed, transcriptionally-repressed chromatin over transcriptionally-active chromatin. the crosslinked chromatin is then sheared by sonication; phenol-chloroform is added to separate protein (nucleosome-depleted) dna fragments from nucleosome-covered dna. downstream detection methods include microarrays, ngs, or quantitative pcr. the regions isolated and detected by faire are largely coincident with the location of open chromatin, such as dnase hypersensitive sites, tss, enhancers, and actively-transcribed promoters [ , [ ] [ ] [ ] . this technique is used to determine whether a given protein binds to, or is localized to, a specific dna sequence in vivo. cross-linked chromatin is sheared and the dna-binding protein of interest is precipitated by using a protein-specific antibody. the bound dna is then isolated by reverting the cross-linking and can be analyzed by utilizing microarrays (chip-on-chip), next-generation sequencing (chip-seq), and quantitative pcr (chip-pcr) [ , ] . the method is strictly dependent on the availability of high quality antibodies to the target protein. the availability of antibodies and the quality of the antibody used in the chip determines the quality of the data generated by the study. in general, only antibodies with high sensitivity and specificity should be considered for use, because this will allow the detection of enrichment peaks without substantial background noise [ ] . the combination of the chip technology with next-generation sequencing allows and improves the characterization of binding sites for transcription factors and other dna-binding proteins and the identification and characterization of dna sequence motifs across the entire genome. the advancement in high resolution is crucial in profiling nucleosome positioning, the systematic cataloguing of histone modification patterns, and the establishment of precise histone modification maps throughout the entire genome [ , , ] . to quantify the global distribution of active and inactive states of chromatin across the genome, several methods and technologies have been developed to measure the methylation status across the genomic dna [ ] . recently, the methylated dna immunoprecipitation (medip) technique was developed and has proven to be a versatile, unbiased approach to study the methylation status of either the whole genome or specific regions of interest. in brief, genomic dna is sheared and precipitated with a monoclonal antibody that recognizes -methylcytidine. another approach based on immune precipitation is the methyl-dna binding protein chip using the methyl-cpg-binding domain protein (mbd ), a member of the mbd protein family. the resulting enrichment of methylated dna can be determined by pcr to assess the methylation state of cpg islands in individual promoters or gene regions of interest. alternatively, precipitated methylated dna can be combined with large-scale analysis using microarrays or next-generation-sequencing [ , ] . there are other complementary approaches to study the genome-wide methylation status of chromatin based on methylation arrays and methylation-sensitive and methylation-insensitive restriction enzymes. the methylation array technology is based on the infinium methylationepic technology (formerly the infinium human methylation array) which allows low sample input and fast read-out but has the disadvantage of not covering all annotated genes and shows bias which is based on the array technology. another approach uses restriction enzymes, like hpaii/mspi, which are blocked or not blocked by cpg methylation. after treatment of the total dna with the enzymes the distribution and extent of dna methylation can be analyzed by quantitative pcr targeting regions of interest. however, the enzymatic approach is prone to bias based on the sequence specificity of the utilized restriction enzymes. this limits the analysis to certain sequence motifs, which can be unevenly distributed across the whole genome [ ] . based on functional and spatial patterns, the immune system is broadly divided into two broad arms: the innate immune system and the adaptive immune system. both systems include a wide range of cell types that communicate via direct cell-cell interactions or by the secretion of mediators such as interleukins, cytokines, and chemokines. the innate immune system not only regulates cell intrinsic defense programs in response to microbial attack but also has a critical role in activating and shaping the adaptive immune response. the innate immune system accomplishes this by being able to generate and drive a transcriptional response that is both cell-and stimulus-specific. based on these mechanisms, the signal-specific induced response guarantees initiation of the appropriate innate and adaptive immune responses that have the greatest potential to successfully control a particular pathogen [ ] [ ] [ ] . much of the innate immune response is regulated by membrane-bound and intracellular pamps, like tlrs, rig-i, mda- and cyclic gmp-amp synthase (cgas)-stimulator of interferon genes (sting) and other sensors that detect invading pathogens [ , ] . these pamps use unique and overlapping signaling cascades to activate effector transcriptional programs that regulate antimicrobial defense pathways. most of the research has focused on elucidating the exact signaling programs that regulate antimicrobial defense to different pathogens and the microbial countermeasures that inactive specific pathways [ , ] . more recently, a growing body of evidence has determined that chromatin modifications and epigenetic regulation play a crucial role in shaping the activated host response to a microbial invasion [ , , ] . advances in sequencing technologies have significantly increased our ability to sensitively and specifically measure the transcriptional state at a single-cell level. systems biology approaches have revealed the more complex gene interaction networks that become activated or repressed. these mechanisms have been essential in understanding the functional specialization of cells as individual units of the innate immune system, the flexibility in mounting innate and inflammatory immune response, and in deciphering the mechanism of communication and interactions within specific cell populations. a basic feature of innate immune cells is the ability to start a transcriptional response program that is specific to the stimulus, and then mounting a signal with a high degree of cell type and stimulus specificity [ ] [ ] [ ] . recent studies have involved epigenetic factors in every aspect of activation and shaping innate and adaptive immune responses. major contributions are the: → recruitment of transcription factors/machinery; → prevention of unwanted expression of potent mediators; and → repression or activation of secondary gene programs [ , ] . the main players of the innate immune system are primary response genes like ifn and tumor necrosis factor (tnf), which are rapidly induced and whose promoters show the characteristics of a poised promoter. often, the promoters of these genes also contain cpg islands which are resistant to epigenetic modifications like dna methylation and histone tail modification. these common modifications can be found at promoters of highly active transcribed genes, which also show high levels of rna pol ii occupancy [ ] . to the contrary, isgs usually display low levels of activating histone marks like h k me , h ac, and low level rna pol ii occupancy [ ] . these genes often require additional transcription factors and chromatin remodelers, like recruitment of the atp-dependent chromatin remodeling complex switch/sucrose non-fermentable (swi/snf) to initiate transcription [ , ] . two cell types of the innate immune system, dendritic cells and macrophages, are the primary sensors of 'danger' signals. once these cells are activated, it is especially important that their signals are both cell-specific and stimulus-specific to ensure the initiation of a temporal and spatial response. these cell-specific signals can be mediated through cell-cell contact or by secretion of ifn and tnf. thus, the ability of their epigenome to change within minutes after a stimulus is not just essential for initiating a rapid antiviral host response but is also essential to ensure a persistent and specific defense response. this way, epigenetic mechanisms are responsible for the priming and the memory of these responses and for guaranteeing a functional and highly regulated host response beyond the initial activation wave. besides the histone marks for poised promoters h k me and h k me , an important role as a major player in regulating the activation of ifn has been described for h k me . fang et al. correlated the levels of h k me modification with the level of interferon expression in vitro. h k me is a repressive histone mark that contributes to dna methylation and heterochromatin formation and thereby prohibits histone tail acetylation by recruiting the transcriptional repressor of the heterochromatin protein family [ ] . however, in the above study, fang et al. were able to demonstrate that the overall levels of h k me mark in the promoter region of the type i interferon and the expression of isgs inversely correlates in dendritic cells, defining this histone modification as an important regulator of the ifn response [ , ] . on the other hand, h k me is a histone modification exclusively found at active promoters and is therefore often enriched in promoter regions regulating tlrs. a recent study has shown that min after lipopolysaccharide (lps) stimulation of macrophages and dendritic cells, the overall histone acetylation and the binding of polymerase ii (pol ii) at the specific promoters was tremendously increased, demonstrating an efficient and specific induction of the innate immune response by epigenetic control mechanisms [ ] . interferons are important mediators of an antiviral state and initiators of pathogen-driven immune response by the inactivation of isgs [ , ] . therefore, it is likely that many viruses have evolved antagonistic mechanisms to overcome specific isg effectors [ ] . as discussed earlier, ifn and innate immune responses are subject to extensive epigenetic regulation, mediated by specific epigenetic marks, and the manipulation of histone modification enzymes, dna methylases, and chromatin remodeling complexes. viruses have evolved mechanisms to disturb and antagonize these epigenetic regulatory programs by ( ) interfering with the host's histone modification enzymes [ ] ; ( ) interfering with the host's chromatin remodeling machinery [ ] ; and ( ) encoding for viral proteins that interact directly with the host's modified histones [ , ] . marazzi et al. have demonstrated that the highly pathogenic h n influenza a virus inhibits the initiation of the host innate immune response in part by interfering with the epigenetic control of gene expression. using histone mimicry, it has been proposed that the carboxyterminus of the h n nonstructural protein ns shares homologue sequences with the aminoterminus of the histone h tail [ ] . essentially, the viral ns protein mimics the histone tail of the h histone and thereby interacts with the transcription complex, which usually docks to the h k mark to initiate transcription [ , ] . previously, we compared isg profiles of pathogenic influenza viruses and coronaviruses in calu cells, a human airway epithelial cell line, by using a transcriptomics and proteomics dataset [ ] . the infection of calu cells with the tested respiratory viruses resulted in diverse virus-specific isg expression signatures. the highly pathogenic h n avian influenza a (hpia) virus showed a rapid manipulation of isg with strong down-and up-regulation of specific isg sets at h post infection. in contrast, the pandemic h n strain showed no isg modulation, and the infected cells mounted a robust ifn-induced antiviral state starting at h post infection. sars-cov infection of calu cells also showed a strong induction of isg effectors, but the response was significantly delayed with peak expression at to h post infection. in , the newly emerged mers-cov showed delayed isg production with effects visible at -h post infection (hpi), with significant inhibition of expression of specific isg subsets. overall, the viral manipulation of the host antiviral ifn response results in successful virus infection and viral replication, defining a viral antagonistic approach, which may be a mechanism to interfere with the host's innate immune response [ ] . in our laboratory, by using chip-pcr approaches, we could determine differential occupancy of histone marks at the promoters of isg genes. we showed that the promoter regions of isg genes contained more histones with active marks of h k me than the repressive h k me mark, therefore favoring open chromatin and promoting active transcription and isg expression during h n - and sars-cov infection. in contrast, infection of calu with the highly pathogenic hpai and mers-cov resulted in increased levels of h k me and decreased levels of h k me occupancy at the promoter regions of subsets of specific isgs, which were not induced, demonstrating that these viruses have developed antagonistic mechanisms to specifically target the ifn arm of the innate immunity ( figure ). we have expanded this dataset by using a genome-wide chip-seq approach. this allows us to choose any set of cellular effector molecules and to study their histone modification profile during infection. figure shows as an example the expression profile of isgs during the early phase mers-cov infection. as already described by menachery et al., expression of isgs effectors occurred rather late after infection at hpi to hpi. however, mers-cov infection in calu induces the upregulation and down-regulation of isgs. we then applied chip-seq data to the expression data. when we looked at the expression level of isg transcripts at hpi, we could see how the histone modification promoter profile at hpi corresponded with the transcriptomic data. isgs that were downregulated during infection showed increased occupancy of h k m modification in their promoter region (indicated in yellow) and isgs that were upregulated during infection showed increased occupancy of h k me within their promoter region (blue). while histone mimicry has been identified for h n virus, the responsible binding motif is not contained in ns protein encoded by h n -vn , although the ns protein may contribute to this phenotype [ ] . this suggests that different ns proteins may mediate the downregulation of subsets of isgs by different mechanisms depending on influenza type a virus (iav) strain. for h n -vn , ns may inhibit isg expression by mimicking different histones, targeting histonemodifying enzymes, or disrupting a histone adaptor protein complex [ ] . several ifn antagonists have been identified; however, the nature of the accessory protein mediating isg downregulation by interfering with the host's epigenome remains to be identified [ , ] . we have expanded this dataset by using a genome-wide chip-seq approach. this allows us to choose any set of cellular effector molecules and to study their histone modification profile during infection. figure shows as an example the expression profile of isgs during the early phase mers-cov infection. as already described by menachery et al., expression of isgs effectors occurred rather late after infection at hpi to hpi. however, mers-cov infection in calu induces the up-regulation and down-regulation of isgs. we then applied chip-seq data to the expression data. when we looked at the expression level of isg transcripts at hpi, we could see how the histone modification promoter profile at hpi corresponded with the transcriptomic data. isgs that were downregulated during infection showed increased occupancy of h k m modification in their promoter region (indicated in yellow) and isgs that were upregulated during infection showed increased occupancy of h k me within their promoter region (blue). calu cells are a continuous human airway epithelial cell line that can be differentiated in ciliated cells and are commonly used to study respiratory cell function under different physiological stresses and conditions [ ] . by utilizing calu cells, we have developed a robust human model platform to study innate immune regulatory control and epigenetics following emerging coronavirus and influenza virus infections as well as other highly pathogenic viruses ( figure ). the first step is to define expression changes following treatment with defined perturbations, like various cytokines, to identify smaller subsets of effector gene expression patterns (rna and protein) that are downstream of a specific signaling pathway. in parallel, the calu cells are then infected with different highly pathogenic emerging or contemporary respiratory viruses and global proteomic and transcriptomic expression patterns characterized at different times post infection ( figure ). by filtering expression changes to specific subsets of cytokine specific gene sets after infection, novel patterns of virus-induced regulatory control are revealed while identifying novel gene sets for downstream epigenetic and virus studies. for example, the same set of isgs are either globally induced rapidly or differentially induced following h n and h n infection. in contrast, isg expression patterns are significantly delayed but either globally or differentially induced following sars-cov and mers-cov infection, respectively [ ] . expression pattern differences were independent of any specific transcription factor function, but rather were regulated primarily by epigenetic control mechanisms. importantly, these studies seed future studies using other viruses while histone mimicry has been identified for h n virus, the responsible binding motif is not contained in ns protein encoded by h n -vn , although the ns protein may contribute to this phenotype [ ] . this suggests that different ns proteins may mediate the downregulation of subsets of isgs by different mechanisms depending on influenza type a virus (iav) strain. for h n -vn , ns may inhibit isg expression by mimicking different histones, targeting histone-modifying enzymes, or disrupting a histone adaptor protein complex [ ] . several ifn antagonists have been identified; however, the nature of the accessory protein mediating isg downregulation by interfering with the host's epigenome remains to be identified [ , ] . calu cells are a continuous human airway epithelial cell line that can be differentiated in ciliated cells and are commonly used to study respiratory cell function under different physiological stresses and conditions [ ] . by utilizing calu cells, we have developed a robust human model platform to study innate immune regulatory control and epigenetics following emerging coronavirus and influenza virus infections as well as other highly pathogenic viruses ( figure ). the first step is to define expression changes following treatment with defined perturbations, like various cytokines, to identify smaller subsets of effector gene expression patterns (rna and protein) that are downstream of a specific signaling pathway. in parallel, the calu cells are then infected with different highly pathogenic emerging or contemporary respiratory viruses and global proteomic and transcriptomic expression patterns characterized at different times post infection ( figure ). by filtering expression changes to specific subsets of cytokine specific gene sets after infection, novel patterns of virus-induced regulatory control are revealed while identifying novel gene sets for downstream epigenetic and virus studies. for example, the same set of isgs are either globally induced rapidly or differentially induced following h n and h n infection. in contrast, isg expression patterns are significantly delayed but either globally or differentially induced following sars-cov and mers-cov infection, respectively [ ] . expression pattern differences were independent of any specific transcription factor function, but rather were regulated primarily by epigenetic control mechanisms. importantly, these studies seed future studies using other viruses and/or segwaying into primary human airway epithelial cells (hae), primary type ii pneumocytes (at ), lung fibroblast (lf), pulmonary endothelial cells (mev) and resident immune cell populations in the lung [ , ] . utilizing these model systems, we aim to study genome-wide histone modifications, dna methylation patterns, and the chromatin landscape after virus infection across different cell types in the lung, revealing cell type-specific regulatory features that function to regulate infection outcomes. the goals of recently performed studies in our laboratory sets the stage to determine if host epigenetic processes play a crucial role in controlling transcriptional regulatory networks that antagonize or promote mers-cov and h n infection and pathogenesis. while the molecular mechanisms regulating epigenetic control remain elusive following emerging cov infection, a growing body of evidence suggests that viruses, even rna viruses that replicate in the cytoplasm, interfere with the host's epigenome. and/or segwaying into primary human airway epithelial cells (hae), primary type ii pneumocytes (at ), lung fibroblast (lf), pulmonary endothelial cells (mev) and resident immune cell populations in the lung [ , ] . utilizing these model systems, we aim to study genome-wide histone modifications, dna methylation patterns, and the chromatin landscape after virus infection across different cell types in the lung, revealing cell type-specific regulatory features that function to regulate infection outcomes. the goals of recently performed studies in our laboratory sets the stage to determine if host epigenetic processes play a crucial role in controlling transcriptional regulatory networks that antagonize or promote mers-cov and h n infection and pathogenesis. while the molecular mechanisms regulating epigenetic control remain elusive following emerging cov infection, a growing body of evidence suggests that viruses, even rna viruses that replicate in the cytoplasm, interfere with the host's epigenome. host-pathogen relationships are plastic and dynamic. in particular, selective pressure put on the pathogen by the host increases the degree of plasticity and adaptability of the pathogen. on the other hand, the host phenotype is altered by the pathogen and the pathogen's virulence, which leads to a co-adaptation that influences pathogen and host equally. for a successful viral life cycle, viruses have co-evolved with the host, which often means that the virus has to adjust to the host's immune system, resulting in distinct mechanisms to repress or evade the host's immune response. coevolution with the host may well have expanded the capabilities of epigenetic regulation, adding additional mechanisms and sources of dynamic reversible phenotype variation [ ] . the evolution of viral antagonistic mechanisms which interfere with the host's gene expression ability by modifying histone marks and therefore chromatin dynamics, enabling viruses to attack entire immune gene host-pathogen relationships are plastic and dynamic. in particular, selective pressure put on the pathogen by the host increases the degree of plasticity and adaptability of the pathogen. on the other hand, the host phenotype is altered by the pathogen and the pathogen's virulence, which leads to a co-adaptation that influences pathogen and host equally. for a successful viral life cycle, viruses have co-evolved with the host, which often means that the virus has to adjust to the host's immune system, resulting in distinct mechanisms to repress or evade the host's immune response. co-evolution with the host may well have expanded the capabilities of epigenetic regulation, adding additional mechanisms and sources of dynamic reversible phenotype variation [ ] . the evolution of viral antagonistic mechanisms which interfere with the host's gene expression ability by modifying histone marks and therefore chromatin dynamics, enabling viruses to attack entire immune gene clusters, has not been heavily investigated and may represent a productive arena for future study. mechanisms by which viruses could modulate the host's chromatin include sequestration and displacement of chromatin-associated proteins, interference with the chromatin remodeling machinery, dna-binding transcription factors, histone modifying enzymes, and direct alteration of methylation and acetylation state of histones and histone mimicry [ , , , ] . several complementary approaches can be combined to study, define, and map the epigenetic landscape during coronavirus infection and other highly pathogenic human viruses ( figure ). combining systems biology approaches like epigenomic, transcriptomic and proteomic datasets provides a data integration approach to identify regulatory genomic clusters and regions that play a crucial role in the host's antiviral response (figure ) [ , ] . moreover, systems biology approaches allow the development of models to make comparisons of data across pathogens to better predict complex biological systems. for example, the integration across systems-based data types will allow us to predict gene expression and to infer new gene regulatory networks. using comparatively generated data from multiple levels of biological systems will allow the association between phenotypic outcome and variation, and the prediction of gene expression using only a few epigenetic features ( figure ) [ , ] . these genome-wide based predictions are essential to define and interpret gene regulatory networks (grns). by linking epigenetic features to gene activation, gene expression levels are integrated into enrichment profiles which can be interpreted as clusters of differential enrichment patterns and then used to map interactions of virus and host to infer the identity of genes that are implicated in disease and pathogenesis associated with the virus [ ] . mechanisms by which viruses could modulate the host's chromatin include sequestration and displacement of chromatin-associated proteins, interference with the chromatin remodeling machinery, dna-binding transcription factors, histone modifying enzymes, and direct alteration of methylation and acetylation state of histones and histone mimicry [ , , , ] . several complementary approaches can be combined to study, define, and map the epigenetic landscape during coronavirus infection and other highly pathogenic human viruses ( figure ). combining systems biology approaches like epigenomic, transcriptomic and proteomic datasets provides a data integration approach to identify regulatory genomic clusters and regions that play a crucial role in the host's antiviral response (figure ) [ , ] . moreover, systems biology approaches allow the development of models to make comparisons of data across pathogens to better predict complex biological systems. for example, the integration across systems-based data types will allow us to predict gene expression and to infer new gene regulatory networks. using comparatively generated data from multiple levels of biological systems will allow the association between phenotypic outcome and variation, and the prediction of gene expression using only a few epigenetic features (figure ) [ , ] . these genome-wide based predictions are essential to define and interpret gene regulatory networks (grns). by linking epigenetic features to gene activation, gene expression levels are integrated into enrichment profiles which can be interpreted as clusters of differential enrichment patterns and then used to map interactions of virus and host to infer the identity of genes that are implicated in disease and pathogenesis associated with the virus [ ] . integration across data types. epigenetic approaches combined with transcriptomics and proteomics datasets provide a spatial-temporal data integration approach to identify regulatory genomic clusters and regions playing a role in viral infection. integration across data types. epigenetic approaches combined with transcriptomics and proteomics datasets provide a spatial-temporal data integration approach to identify regulatory genomic clusters and regions playing a role in viral infection. recent advancements through basic research in the understanding of the mechanisms involved in viral chromatin modification in lytic viruses have opened a new window into previously unknown mechanisms of viral antagonism and host-virus interactions, including genetic factors that influence both protective or pathogenic host responses. the subsequent identification and improved understanding of these mechanisms open new avenues for the development of antiviral drugs by illuminating new targets for specific inhibitors. established model systems for latent and persistent viruses such as human immunodeficiency virus (hiv- ) and herpesviruses have already demonstrated modulation of the viral infection by chromatin [ , ] . as described here, a growing number of studies also show modulation of viral infection by chromatin in lytic virus infections. the emerging parallels between the existing knowledge of chromatin's effect on and interaction with latent and persistent viruses and the emerging understanding of its comparable interaction with lytic viruses suggest that a greater focus on chromatin-based therapies for a variety of virus families could reveal fundamental new landscapes of virus-host interaction that play critical roles in disease severity. management and prevention of sars in china severe acute respiratory syndrome the chronology of the - sars mini pandemic severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats sars-like wiv -cov poised for human emergence a sars-like cluster of circulating bat coronaviruses shows potential for human emergence a decade after sars: strategies for controlling emerging coronaviruses middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread. microbes infect recent insights into emerging coronaviruses severe acute respiratory syndrome: identification of the etiological agent a 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conducting airways of the lungs interference of viral effector proteins with chromatin, transcription, and the epigenome pathogens hijack the epigenome: a new twist on host-pathogen interactions bugs in the system understanding gene regulatory mechanisms by integrating chip-seq and rna-seq data: statistical solutions to biological problems methods of integrating data to uncover genotype-phenotype interactions transcriptional activation and chromatin remodeling of the hiv- promoter in response to histone acetylation inhibition of the histone demethylase lsd blocks alpha-herpesvirus lytic replication and reactivation from latency key: cord- -fzba wn authors: chauhan, ravendra p.; gordon, michelle l. title: a systematic review analyzing the prevalence and circulation of influenza viruses in swine population worldwide date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: fzba wn the global anxiety and a significant threat to public health due to the current covid- pandemic reiterate the need for active surveillance for the zoonotic virus diseases of pandemic potential. influenza virus due to its wide host range and zoonotic potential poses such a significant threat to public health. swine serve as a “mixing vessel” for influenza virus reassortment and evolution which as a result may facilitate the emergence of new strains or subtypes of zoonotic potential. in this context, the currently available scientific data hold a high significance to unravel influenza virus epidemiology and evolution. with this objective, the current systematic review summarizes the original research articles and case reports of all the four types of influenza viruses reported in swine populations worldwide. a total of articles were found eligible through screening of pubmed and google scholar databases and hence were included in this systematic review. the highest number of research articles (n = ) were reported from asia, followed by americas (n = ), europe (n = ), africa (n = ), and australia (n = ). the h n , h n , h n , and a(h n )pdm viruses were the most common influenza a virus subtypes reported in swine in most countries across the globe, however, few strains of influenza b, c, and d viruses were also reported in certain countries. multiple reports of the avian influenza virus strains documented in the last two decades in swine in china, the united states, canada, south korea, nigeria, and egypt provided the evidence of interspecies transmission of influenza viruses from birds to swine. inter-species transmission of equine influenza virus h n from horse to swine in china expanded the genetic diversity of swine influenza viruses. additionally, numerous reports of the double and triple-reassortant strains which emerged due to reassortments among avian, human, and swine strains within swine further increased the genetic diversity of swine influenza viruses. these findings are alarming hence active surveillance should be in place to prevent future influenza pandemics. influenza viruses are the members of orthomyxoviridae family and have a wide host range [ ] [ ] [ ] [ ] [ ] [ ] . due to unique physiology, swine are considered the "mixing vessel" for influenza viruses [ ] . four types of influenza viruses have been reported in swine i.e., influenza a virus (iav), influenza b virus (ibv), influenza c virus (icv), and influenza d virus (idv). the genomes of iav and ibv have eight gene segments of single-stranded negative sense rna while the genomes of icv and idv have seven gene segments [ ] . among the eight gene segments of iav and ibv, the hemagglutinin (ha) and neuraminidase (na) are most significant and crucial for the pathogenicity of these viruses which neuraminidase (na) are most significant and crucial for the pathogenicity of these viruses which determine the antigenic properties. the ha gene regulates the attachment of virus particles to the host receptor while na gene regulates the release of progeny virus into the host cell. co-infection of swine with two or more iav strains may trigger the reassortment [ ] which in turn, could facilitate the emergence of new influenza virus strains [ ] [ ] [ ] . point mutations which occur due to an errorprone rna polymerase that lacks the ability of proof-reading and corrections during replication may also complement the genetic diversity of the influenza viruses [ ] . the mechanisms of reassortment and point mutations may give rise to "antigenic shift" and "antigenic drift" within ha and na genes, respectively, facilitating the emergence of new subtypes and lineages of influenza viruses. as a result, total ha and na subtypes of iav [ ] [ ] [ ] and two lineages (victoria/b and yamagata/b) of ibv have been reported so far in different hosts [ , ] . the host range of iav and ibv is determined by their specificity to sialic acid receptors. the ha proteins of iav can bind to α- , and α- , sialic acid receptors present in avian and human trachea, respectively [ ] [ ] [ ] . interestingly, swine trachea has both, α- , as well as α- , sialic acid receptors, due to which swine can become infected with avian and human strains of influenza viruses [ ] . the genomes of icv and idv have a gene segment termed as "hemagglutinin-esterase-fusion" (hef) which carries out the functions similar to that of ha and na genes of iav and ibv. the hef is responsible for attachment and release of icv and idv virus particles into the host cell [ ] [ ] [ ] . the particles of both virus types icv and idv bind to -o-acetylated sialic acid receptors of the host [ ] . several studies have shown that human and avian origin influenza viruses can be transmitted to swine in natural settings and thus may evolve into new strains of reassorted influenza viruses [ , ] . historically, the first flu pandemic (spanish flu) hit the human population in [ ] and killed approximately million people globally [ ] . the influenza pandemic emerged as a result of reassortment in which human h virus acquired avian (poultry) n neuraminidase along with internal protein genes and evolved into what is now termed as "classical h n " virus [ ] ( figure ). the second flu pandemic occurred in (asian flu) and was traced to the h n virus which killed approximately two million people [ ] . the third flu pandemic hit the human population in (hong kong flu) with an h n outbreak and killed approximately two million people [ , ] . the second flu pandemic occurred in (asian flu) and was traced to the h n virus which killed approximately two million people [ ] . the third flu pandemic hit the human population in (hong kong flu) with an h n outbreak and killed approximately two million people [ , ] . the most recent flu pandemic (swine flu) originated in swine in mexico during march-may [ ] databases. this systematic review is part of a research project which has already obtained the relevant ethical approvals from the animal research ethics committee (arec), university of kwazulu-natal, durban, south africa; arec reference: arec/ / d. additionally, the authors have the required permission to do research in terms of section of the animal diseases act, (act no. of ) from the department of agriculture, forestry and fisheries (daff), government of the republic of south africa; daff reference: / / / / ( ). the original research articles and case reports on the serological and virological prevalence of all the four genera of influenza viruses i.e., iav, ibv, icv and idv were downloade [ ] [ ] [ ] nigeria h n , h n , a(h n )pdm , h n none [ , [ ] [ ] [ ] [ ] [ ] [ ] ghana h n none [ ] egypt h n , h n , h n , a(h n )pdm none [ , ] kenya h n , h n , a(h n )pdm none [ ] [ ] [ ] benin, cote d'ivoire none none [ ] reunion island a(h n )pdm none [ ] togo a(h n )pdm none [ ] uganda iav none [ ] [ ] [ ] [ ] nigeria h n , h n , a(h n )pdm , h n none [ , [ ] [ ] [ ] [ ] [ ] [ ] ghana h n none [ ] egypt h n , h n , h n , a(h n )pdm none [ , ] kenya h n , h n , a(h n )pdm none [ ] [ ] [ ] benin, cote d'ivoire none none [ ] reunion island a(h n )pdm none [ ] togo a(h n )pdm none [ ] uganda iav none [ ] asia china h n , h n , h n , a(h n )pdm , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n icv, idv bhutan h n , a(h n )pdm none [ ] cambodia h n , h n , a(h n )pdm none [ , ] japan h n , h n , h n , a(h n )pdm icv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] south korea h n , h n , h n , a(h n )pdm , h n , h n , h n none [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] thailand h n , h n , h n , h n , a(h n )pdm none [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] viet nam h n , h n , h n , a(h n )pdm , h n none [ ] [ ] [ ] [ ] india h n , h n , h n , a(h n )pdm none [ , ] lebanon h n none [ ] malaysia h n , h n none [ ] laos h n none [ ] russia h n none [ ] taiwan iav ibv [ , ] indonesia h n none [ ] sri lanka h n , a(h n )pdm none [ ] kazakhstan h n , h n none [ ] australia australia h n , h n , h n , a(h n )pdm none [ ] [ ] [ ] [ ] [ ] [ ] [ ] denmark h n , h n , h n none [ ] [ ] [ ] united kingdom h n , h n , h n , a(h n )pdm , h n ibv, icv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] finland h n , h n , h n , a(h n )pdm none [ , ] france h n , h n , h n , a(h n )pdm none [ ] [ ] [ ] [ ] [ ] germany h n , h n , h n , a(h n )pdm none [ ] [ ] [ ] [ ] greece h n , h n , h n , a(h n )pdm none [ ] italy h n , h n , h n , a(h n )pdm idv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] spain h n , h n , h n none [ ] [ ] [ ] [ ] netherlands h n , h n , h n none [ ] norway a(h n )pdm none [ ] [ ] [ ] [ ] poland h n , h n , h n , a(h n )pdm none [ ] [ ] [ ] czechoslovakia h n none [ ] hungary h n none [ ] czech republic h n , h n , h n none [ ] republic of ireland h n , h n , h n none [ ] luxembourg none idv [ ] multiple european nations h n , h n , h n none [ ] north america canada h n , h n , h n , a(h n )pdm , h n , h n none usa h n , h n , h n , h n , a(h n )pdm , h n , h n ibv, idv mexico h n , h n , h n , a(h n )pdm , h n none [ ] [ ] [ ] [ ] [ ] [ ] guatemala h n , a(h n )pdm none [ ] cuba h n , a(h n )pdm none [ , ] trinidad & tobago h n , a(h n )pdm none [ ] south america argentina h n , h n , h n , a(h n )pdm none [ ] [ ] [ ] [ ] [ ] brazil h n , h n , h n , a(h n )pdm none [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] colombia a(h n )pdm none [ ] peru a(h n )pdm none [ ] chile iav, h n none [ ] [ ] [ ] pathogens , , of the first report of iav in cameroonian swine appeared when a(h n )pdm virus was documented during - . the youngest infected swine was four-month old which suggested that a(h n )pdm virus can infect the young piglets [ ] . nine more swine herds in cameroon were found infected with a(h n )pdm and h n viruses during may-june [ ] . a multiple-site study including free-roaming and penned swine along with domestic poultry and columbiformes birds between december and august identified one iav positive swine at each of the two study sites. the inter-species transmission of iav was ruled out as all the birds were negative for the iav [ ] . the first evidence of the past iav infection in nigerian swine appeared in when h n and h n virus antibodies were detected in swine sampled at three different locations [ ] . shortly after that, in , the first report of a(h n )pdm virus appeared in the nigerian swine when one swine herd was found seropositive for a(h n )pdm virus. interestingly, eight other swine herds were found seropositive for the h n and four herds were found positive for human-like h n viruses. the seroprevalence of iav further increased as swine herds were detected positive for a(h n )pdm virus and herds were found seropositive for h n virus in [ ] . the active infection (viral rna) of a(h n )pdm virus was first reported in the nigerian swine between july -june when a(h n )pdm virus isolates were retrieved from the swine in lagos. the zoonotic transmission of a(h n )pdm virus to the exposed human workers was ruled out as all the human samples were negative for the a(h n )pdm virus [ ] . nineteen more a(h n )pdm and five human-origin h n viruses were identified in the nigerian swine during - [ ] . later one more report of the human strain of h n virus appeared in swine during january-february [ ] . a high seroprevalence of iav in a commercial piggery was reported in lagos. total human and swine sera samples were screened which determined that % human and % swine sera had antibodies for iav depicting the past exposure [ ] but the active infection was absent given that all nasal swabs were negative for iav infection [ ] . lately, highly pathogenic avian influenza virus (hpaiv) strain h n was detected in swine samples between december and february during an ongoing h n disease outbreak in nigerian poultry [ ] which indicated the inter-species transmission of h n virus from poultry to swine [ ] . a molecular study reported the negative prevalence of avian-like h n and h n viruses in egyptian swine in may [ ] but the serological investigation identified h n virus antibodies in seven and h n virus antibodies in four swine sera samples [ ] which suggested a past exposure of these swine to the viruses. the active h n infection in egyptian swine was again ruled out in october as the viral rna could not be detected in swine samples but interestingly, the antibodies against avian-like h n , h n , and a(h n )pdm viruses were detected in swine sera samples which suggested a past exposure [ ] . interestingly, of the swine nasal swab samples collected during and were found positive for iav active infection using rt-pcr. as a result, ha subtyping identified avian-origin h n , seven h n and a(h n )pdm viruses [ ] . the first report of iav in swine in kenya appeared in may when a(h n )pdm virus were detected in eight swine samples collected from the asembo and kibera counties and at a nairobi based abattoir. the extended serological study further identified h n and h n viruses in swine during august to december [ ] . the active iav infection was reported in four household members pathogens , , of having acute respiratory illness while the backyard swine were negative for the iav and ibv in kiambu county during september -august . on the contrary, the serology identified the iav antibodies in swine sera samples suggesting a past exposure of iav [ ] . the a(h n )pdm virus was again reported in five swine samples collected from a slaughterhouse in kenya during september -september . interestingly, all the human subjects including the slaughterhouse workers or the traders and farmers who had visited the slaughterhouse were negative for iav, hence ruled out the zoonotic transmission [ [ ] and in togo during october -january [ ] . the human strain of h n virus was detected in swine in ghana during january-february [ ] . one more report documented iav in swine in two districts of uganda in [ ] . overall, influenza viruses have been reported in swine from eight african countries including cameroon, nigeria, egypt, kenya, reunion island, togo, ghana, and uganda ( figure a ). the a(h n )pdm virus, which originated in mexican swine in , has been reported in all except ghana and uganda. interestingly, the hpaiv strain of h n has been reported in swine in nigeria and egypt while hpaiv strain h n and low pathogenic avian influenza virus (lpaiv) strain h n have also been reported in the egyptian swine (table ) . china is considered the epicenter of influenza viruses [ ] . the first seroprevalence of iav in chinese swine was documented during - when antibodies for h n , h n , h n , h n , and seven h n viruses was detected in swine sera obtained from apparently healthy swine [ ] . the first ever report of icv in swine was documented from the apparently healthy swine in beijing when icv isolates were retrieved during january-december [ ] . three isolates of reassortant h n virus were identified after an influenza-like illness triggered abortions and mortalities in sows on a swine farm in november [ ] . the same year, lpaiv strain h n was isolated from the sick or dead swine in china which was the first ever isolate of h n virus retrieved from a swine [ ] . first human-origin h n and four human-origin h n virus isolates in chinese swine were retrieved during - [ ] . further, two isolates of swine h n viruses, four isolates of avian-origin hpaiv strain h n and two isolates of h n viruses were detected in swine nasal swab and lung tissue samples collected from swine in central provinces of china during - [ ] . surprisingly, two isolates of equine influenza virus h n were also detected in swine during december and january [ ] . another report of avian-origin h n virus in chinese swine was documented during - when four h n virus isolates with closely related nucleotide sequences were retrieved from swine [ ] . each of the two different investigations reported h n , one h n and nine h n virus isolates from chinese swine during - [ , ] ; the h n virus and all nine isolates of h n viruses were either double or triple-reassortant viruses [ ] . the first report of hpaiv strain h n in swine was documented during october -may when two h n virus isolates were retrieved from apparently healthy swine [ ] . the third report of avian-origin h n virus in chinese swine appeared when apparently healthy swine across four provinces viz., yunnan, guangdong, fujian and zhejiang were found h n positive over a four-year period during march -march . the frequent interactions of birds to the swine at the study sites was reported which was suspected to be the most likely source of infection [ ] . further, a novel strain of avian-origin h n virus was isolated from a chinese swine in [ ] . several classical and avian-like h n , eurasian avian-like h n , triple-reassortant h n , h n , h n and a(h n )pdm viruses were reported in chinese swine between and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a triple-reassortant h n virus having the internal genes of avian, human, and swine lineages of influenza viruses was reported from a two-month old piglet on a guangdong based swine farm in january [ ] . three reassortant h n virus isolates having internal genes of a(h n )pdm virus were reported in swine between november and june [ ] . a three-year old boy was diagnosed with european origin avian-like h n virus on a family swine farm in a rural area of the jiangsu province in december which speculated a zoonotic transmission from swine to the boy [ ] . the first report of h n avian-origin influenza virus in a domestic swine in hubei province further extended the diversity of swine influenza viruses and provided another evidence of interspecies transmission of avian influenza virus to the swine under natural conditions [ ] . several other avian-origin h n , h n , h n , h n , h n , and h n virus antibodies were detected in swine in china during april -june [ , [ ] [ ] [ ] . another interspecies transmission of avian-like h n virus in southern china was observed when swine and swine farm workers were identified to be infected with avian-like h n swine influenza virus between march and march [ ] . further a zoonotic transmission of h n virus was identified at a shandong based swine farm during may -april when h n virus antibodies were detected in swine and four farm workers. the wild birds visiting swine feeding sites at the swine farm were speculated to serve as the carrier for h n virus [ ] . zoonotic transmission of h n virus was reported on a swine farm in shandong province between march and february among the swine exposed human workers having influenza-like illness. as a result, five of the ( . %) nasal swab samples were found iav positive; a married couple exposed to swine were found infected with h n virus [ ] . the iav infection was also documented in wild boars in jilin province of china between april and february [ ] . the first report of the idv prevalence in chinese swine documented idv positive swine in the guangdong province in [ ] . the swine idv sequences shared high similarity ( - %) with idv sequences reported earlier from the bovine species in china [ ] which indicated the transmission of idv from bovine to swine in china. hong kong is a special administrative region while tibet is an autonomous administrative region under the control of people's republic of china. the h n and h n virus isolates were successfully retrieved from apparently healthy swine in hong kong during july -june [ ] . further, classical swine h n , h n and avian-like h n viruses were identified in hong kong based swine between march -june ; two independent introductions of the avian-like h n viruses were ascertained from avian species to the swine [ , , ] . the first information of iav seroprevalence in tibetan swine appeared during april-december when antibodies against h n and h n viruses were detected in swine sera collected from tibet [ ] . the first report of h n seroprevalence in swine in bhutan appeared when h n virus was detected in backyard as well as breeding swine during october and february [ ] . the h n virus was reported in swine over a five year-period between - while the a(h n )pdm and h n viruses were identified only in [ ] . later three triple-assortant h n viruses were isolated and sequenced from the backyard swine between may and july [ ] . the antibodies against a/hong kong(h n ) virus termed as "a/swine/wadayama/ / " were first detected in japanese swine in [ , ] . the h n virus seroprevalence in japanese swine was further documented in sendai city during to [ ] ; the transmission between human and swine was also suggested [ ] . the first active iav infection was reported when two reassortant h n virus isolates were retrieved from the japanese swine having influenza-like disease in . the isolated h n virus was believed to be a recombinant of h n and h n viruses [ ] . further swine were diagnosed with h n antibodies in toyama prefecture between - . a lower seroprevalence was observed during the summer months while the seroprevalence was relatively higher during the winter season [ ] . again, one more h n virus was isolated and characterized from the sows in ehime prefecture in september [ ] . intriguingly, h n , h n and h n viruses were detected in swine imported from the united states, however, all the imported swine from the europe were negative for the iav infection. this was the first report of the iav infection in the imported swine [ ] . the icv seroprevalence ( %) in japanese swine was first reported in the hyogo prefecture during july -june [ ] but swine in yamagata prefecture were found seronegative for the icv between august and march which suggested a localized transmission of icv in swine within hyogo prefecture [ ] . several other reassortant h n virus isolates were reported in japanese swine after [ ] . one novel reassortant h n virus appeared to have emerged from the a(h n )pdm virus was reported in swine in gunma prefecture while two other h n viruses appeared to have emerged from the japanese h n viruses with internal genes from a(h n )pdm virus. one more h n virus was detected in swine which was closely related to the japanese h n virus [ ] . the immunohistochemistry identified lesions in the lungs of the sick swine infected with reassortant h n virus [ ] . additionally, several h n and h n viruses have also been reported in japanese swine between and [ , ] . interestingly, six h n virus isolates were identified with reassorted genes from a(h n )pdm virus while one h n isolate appeared to have h gene from japanese swine influenza virus with internal genes of a(h n )pdm virus. further, one h n virus isolate was determined to have genes of japanese swine influenza and a(h n )pdm viruses [ ] . these results reflected the occurrence of the reassortment events between japanese swine influenza and a(h n )pdm viruses. iav seroprevalence has lately been reported in wild boars (sus scrofa leucomystax) in japan. three wild boars in the yamaguchi prefecture were found seropositive for a(h n )pdm virus while nine wild boars in tochigi prefecture were seropositive for the swine h n virus. but, the active iav infection could not be identified in these wild boars as all the nasal swab samples were negative for iav and ibv [ ] . in a more recent investigation, fifteen wild boars were found seropositive for a(h n )pdm virus in kagoshima prefecture between november -december while two of these fifteen wild boars had antibodies against h n and h n viruses as well [ ] . this reflected a past exposure of the japanese wild boars to the iav strains. the first active iav infection in the korean swine was identified in december when three h n virus isolates were recovered from the swine experiencing an acute influenza-like respiratory disease. the close relatedness of these korean swine h n isolates with human-origin h n viruses reported from korea between - suggested the events of reverse zoonosis [ ] . one unique h n virus isolate was detected in swine which had seven gene segments originated from hong kong avian-origin h n virus isolated in and the ns gene originated from hong kong h n virus isolated in . additionally, four typical swine influenza h n viruses were identified in swine [ ] . several h n , h n , and h n viruses were detected in symptomatic south korean swine after [ ] [ ] [ ] [ ] [ ] [ ] . the iav localization in the swine lung tissues was confirmed by immunohistochemistry [ ] . total avian-origin h n viruses of eurasian lineage were identified in swine in different south korean provinces during - which suggested cross-species transmission of h n virus [ ] . three h n virus isolates closely related to us isolates of h n were obtained from -day-old piglets in korea in january . the other swine farms in the proximity of this index farm were negative for the h n virus [ ] . further, one h n , two h n , and one h n subtypes of iav identical to the american strains based on their ha and na gene sequences were obtained from swine nasal swab, lung, and thoracic fluid samples during - which suggested that there was no probability of arising of these iav strains in korea through recombination [ ] . two novel isolates of swine h n virus with high genomic similarity to each other were retrieved from two different swine farms in korea during march-april which would be due to a common origin of these isolates. these viruses had human-like h gene while other gene segments originated from swine influenza viruses within korea. high reactivity of the swine sera samples to h n virus antibodies suggested a previous exposure and probability of the swine to swine transmission of h n virus [ ] . the human to swine transmission of a(h n )pdm virus was reported in chungbuk province where a(h n )pdm virus isolates were recovered from swine lung tissues [ ] . the reassortment between a(h n )pdm and swine h n viruses emerged into a novel reassortant h n virus in swine [ ] . a triple-reassortant h n virus was identified in swine during december -may which indicated the iav reassortment was taking place in korean swine [ ] . a swine fever eradication campaign identified nine a(h n )pdm , two classical h n and one h n viruses in wild boars which were hunted and killed in south korea during [ ] . more recently, a complete genome sequence of h n virus was reported from a domestic swine in korea in [ ] . the occurrence of iav in thai swine was first reported during november-december . active h n infection was detected in one swine while several other swine had h n antibodies [ ] . two h n virus isolates from thai swine were first recovered in january [ ] . several studies reported h n , a(h n )pdm , h n , and h n viruses in swine exhibiting respiratory disease symptoms between to . intriguingly, one swine sample was found co-infected with four iav subtypes including h n , h n , h n , and h n viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the first evidence of h n seroprevalence in thai swine was documented in when eight h n positive swine sera samples were identified [ ] . later ten h n and two h n virus isolates were retrieved from piglets aged between to weeks during - [ ] . interestingly, most of the virus isolates retrieved in this study were obtained from to week-old piglets which was in agreement of a previous report stating that swine influenza viruses can be successfully retrieved from piglets less than ten weeks of age [ ] . a zoonotic transmission of iav was reported at a thai swine farm where all the swine were found positive for either h n or h n virus. interestingly, two farm owners, swine handlers, four veterinarians, five farm cleaners and two farm office workers also reported iav seroprevalence. this study claimed that there was transmission of swine influenza viruses from swine to human however the possibility of human to swine transmission was ruled out [ ] . after a respiratory disease outbreak in nursery piglets, nasal swabs were found positive for a(h n )pdm virus between december and march . fifteen sera samples of the farm workers along with three sera from dogs and one serum obtained from a cat were negative for iav, hence the interspecies transmission of iav was ruled out [ ] . the first report of active infection with reassortant h n virus in thai swine appeared in february but the follow up screenings conducted after two and three months, respectively confirmed the cessation of the active infection as the viral rna was not detected anymore [ ] . the reshuffling and reassortment of iav internal genes were reported in thai swine in february . the ha and na genes of h n virus isolates clustered with the eurasian swine-like iav lineage while the h n viruses diverged and formed a separate group. all the internal genes of h n and h n virus isolates appeared to be derived from a(h n )pdm viruses which confirmed the events of reassortments [ ] . the events of reverse zoonoses were suggested after the detection of a(h n )pdm virus seroprevalence in vietnamese swine during october -march [ ] . one more evidence of reverse zoonosis was identified during february-march after six triple-reassortant h n viruses having a novel cluster of the triple reassortant internal gene (trig) cassette were isolated. the ha and na genes of these reassortant h n isolates originated from human h n viruses reported between - while the other six internal genes had a high similarity with the korean and american isolates [ ] . two more studies reported the h n , a(h n )pdm , hin , and h n virus isolates during february -december from clinically healthy swine with no influenza disease symptoms [ , ] . additionally, the antibodies for a(h n )pdm and h n viruses were detected in swine which suggested a past exposure of swine to these viruses [ ] . a high seroprevalence of h n , h n and h n viruses was detected in human and swine sera in calcutta, india during - [ ] . the first active infection of iav in indian swine appeared in when a(h n )pdm virus isolates were reported from a swine farm located in uttar pradesh. interestingly, the retrieved a(h n )pdm virus sequences were similar to the north american and korean viruses which might be either because of trade or long-distance transmission [ ] . after an influenza outbreak on lebanese poultry farms in the farmers fed the carcasses of the dead flocks to the swine. intriguingly, a following investigation found that three swine were seropositive for the h n virus while approximately one-third of the poultry farm workers were seropositive either for h or h viruses [ ] . these results revealed the interspecies transmission of iav among poultry, farm workers and swine. the seroprevalence of h n and h n viruses in four to six-month-old malaysian swine at swine farms was reported during may-august . co-infections of h n and h n were detected in swine samples [ ] . the seroprevalence of h n virus in swine samples obtained from the slaughterhouses in laos was reported between may to january [ ] . a full-length genome sequence of a reassortant h n virus was reported from a russian swine in . the ha and na genes of this virus isolate shared % identity with the h n viruses that were reported from humans in the usa in the s [ ] . the human to swine transmission of iav was speculated after iav antibodies were detected in taiwanese swine during june -may . the results were further confirmed with virus isolation which retrieved iav isolates [ ] . more recently, ibv of victoria/b lineage was detected in swine nasal swab samples collected from apparently healthy swine at three swine farms in [ ] . an active iav infection in swine within four provinces in indonesia was identified during - . interestingly, h n virus isolates were successfully retrieved and sequenced [ ] . the first report of influenza in sri lankan swine was documented during - after one human-like h n virus was identified. later, a(h n )pdm virus isolates were identified in swine during - . a spillover of these viruses from human to swine was speculated [ ] . one recent investigation in kazakhstan during - identified nine h n and eight h n viruses in human while seven h n and four h n viruses were identified in swine. interestingly, of the human samples were also positive for ibv infection while the swine samples were negative for ibv [ ] . in summary, the influenza viruses have been reported in swine in asian countries including china, japan, thailand, south korea, viet nam, cambodia, taiwan, india, bhutan, russia, laos, malaysia, lebanon, indonesia, kazakhstan, and sri lanka ( figure b ). apart from the most common iav strains of h n , h n , h n , and a(h n )pdm viruses, several avian-origin h n , h n , h n , h n , h n , h n , h n , h n , and h n influenza viruses were also reported in chinese swine. horse to swine transmission of equine influenza virus h n was reported in china. additionally, avian-origin h n , h n viruses were identified in south korean swine while h n was reported in indonesian swine. interestingly, after the swine were fed upon dead poultry carcasses in lebanon the h n virus was detected in lebanese swine. the ibv was reported in asian swine only in taiwan while strains of icv were reported in swine in china and japan while idv was recently reported in chinese swine (table ) . swine influenza was first reported in australian swine only in after a swine farm owner reported coughing symptoms among swine. simultaneously, some of the human workers on the farm also developed influenza like symptoms and hence stayed out of the farm until recovery. later, the farm owner also developed similar symptoms following which he was tested for a(h n )pdm virus which resulted positive. as a result, a representative number of swine showing coughing symptoms and loss in appetite were sampled for molecular diagnostics and serology which confirmed that swine were positive for h n virus [ ] . second report of iav in australian swine appeared on a queensland farm in august when a veterinarian observed elevated temperature, coughing and loss of appetite in swine. simultaneously, two of the staff members on the farm exhibited influenza-like symptoms and hence were sampled for diagnostic testing using nasal swabs. interestingly, both the staff members and four of the swine were found positive for the a(h n )pdm virus. sequencing identified that the ha gene of a(h n )pdm virus retrieved from a staff member was identical to the virus retrieved from the swine which suggested transmission of a(h n )pdm virus between swine and human [ ] . third report of iav in australian swine appeared when a respiratory disease outbreak in swine and the farm workers occurred in perth, western australia during which identified iav positive swine. sanger sequencing of ha and na genes identified six novel hin , three novel h n , one a(h n )pdm and two seasonal h n viruses in swine. on the contrary, only one out of eight human workers were found positive for seasonal h n virus. this study could not conclude the event of zoonotic transmission of iav between swine and human workers at the farm [ ] . the fourth report of iav was documented when iav positive swine were identified at a commercial swine farm in western australia during july-september and later during september-november . additionally, swine were determined to be iav positive in southern queensland. the complete genomes of iav isolates retrieved in western australia and queensland were successfully sequenced which identified seven h n , two human-like h n and one h n virus [ ] . overall, four reports of iav outbreaks in swine in new south wales, queensland and western australia were available ( figure c ). the h n , h n , h n and a(h n )pdm subtypes have been reported from australian swine with relatively low prevalence. the h n virus was identified in swine lung tissues or trachea of two of the deceased sows after an influenza-like disease erupted at two swine farms in january . interestingly, it was also reported that the identical virus was detected in wild ducks in germany [ ] . since it was already established that h n from wild ducks can successfully infect swine if inoculated via intranasal route [ ] hence this observation suggested the transmission of h n from wild ducks to the swine [ ] . a second investigation isolated three avian-like h n , two h n and twelve human-like h n viruses from eight commercial swine farms in march [ ] . denmark has been running a passive surveillance program for iav detection in swine since . the h n virus having the h gene which evolved from h n avian-like viruses and n gene which evolved from human h n viruses was reported in swine during - [ ] . this was an example of how iav can evolve through reassortment and may emerge into a new iav strain. the other investigation included swine sampling at different time intervals to assess the persistence of iav shedding in danish swine which detected one avian-like h n and reassortant h n viruses. this study observed that most of the swine were shedding iav right before achieving six weeks of age. surprisingly, a piglet as young as just three days was found infected with iav [ ] . two h n isolates having h genes from seasonal human influenza along with internal genes that originated from a(h n )pdm virus and na genes from contemporary n swine influenza viruses that have been in circulation in denmark were retrieved from young piglets at two locations during - [ ] . h n virus was also detected from piglets having respiratory illness and from sows with reproductive problems in commercial piggeries in [ ] . the h n virus antibodies were first detected in english swine in which revealed the past exposure of swine to h n virus [ ] . later, the antibodies for h n and h n viruses were detected in swine at a slaughterhouse in england during - [ ] . interestingly, this serological investigation also reported the antibodies for ibv in eight and for icv in swine [ ] . a molecular investigation identified a novel h n virus in swine in england which had six of its rna segments closely related to those of human viruses while two rna segments were identical to those of equine viruses which concluded that the h n strain may have evolved due to reassortment between human h and equine h n viruses [ , ] . the first report of a(h n )pdm virus in english swine appeared in september when histology and immunofluorescence assays followed by molecular diagnostics and sequencing confirmed four a(h n )pdm virus infected swine in the northern ireland [ ] . after this, more a(h n )pdm virus isolates were reported in swine in england during september -october which revealed that a(h n )pdm virus was in circulation in english swine during the flu pandemic [ ] . the same year, four h n virus isolates were reported in english swine which had six internal genes of a(h n )pdm virus along with ha and na genes of h n virus hence were identified as the novel reassortant h n strains [ ] . in a more recent study, two more iav positive swine were identified in the united kingdom in [ ] . however the first report of seroprevalence of h n virus in finnish swine appeared in during an investigation which detected h n virus antibodies in swine at seven swine farms which further increased to swine farms in [ ] but the first isolate of avian-like swine h n virus (indicative of active infection) was detected from the lung tissues of a swine in february . later, the first a(h n )pdm virus in finnish swine was detected in november [ ] . three more swine were identified with iav antibodies during may -january which was due to a past exposure to iav [ ] . the h n viruses in turkey and swine were identified after the swine influenza outbreak hit the turkey population in brittany, france in february which suggested that iav transmission happened from swine to turkey [ ] . later two strains of h n virus were isolated from six swine exhibiting influenza-like illness in brittany during - [ ] . another investigation reported h n , h n , and h n viruses in swine herds experiencing respiratory disease in brittany region [ ] . a negative prevalence of iav was reported in wild boars in camargue during september -november given that all the nasal swabs obtained from either hunted or trapped wild boars along with all the sera samples were negative for iav [ ] . a more recent investigation reported the zoonotic transmission of a(h n )pdm virus from swine to a farmer in january . this farmer along with a veterinarian collected nasal swab samples from three pregnant sows exhibiting influenza-like illness on the swine farm and submitted to a local diagnostic laboratory for analysis which, as a result, were found iav positive. few days later, the farmer and the veterinarian both developed the influenza-like symptoms. the farmer was later diagnosed with a(h n )pdm virus [ ] . sixty-five iav positive wild boars were identified across five german states during - . cloning and sequencing identified h n and h n viruses in these wild boars [ ] . later thirteen h n , three reassorted a(h n )pdm and four h n viruses were detected in swine during - . interestingly, the a(h n )pdm virus isolates had high similarity with the a(h n )pdm viruses reported earlier in humans within germany which suggested a reverse zoonotic transmission of the a(h n )pdm virus [ ] . a nationwide sero-surveillance identified , swine with h n , , swine with human-like h n , , swine with human-like h n and , swine with a(h n )pdm virus antibodies during june -december which reflected a high seroprevalence of influenza viruses in german swine population [ ] . later iav positive swine exhibiting influenza-like illness were detected between january -december . subtyping successfully distinguished of samples into h n , h n , h n and a(h n )pdm viruses. the h n virus was the most widely occurring in german swine while a(h n )pdm virus had the lowest prevalence [ ] . the h n , h n , h n , and a(h n )pdm viruses were detected in swine sera samples collected from apparently healthy swine at swine farms during - and from swine farms during - [ ] . the seropositivity of italian swine to h n virus was first reported during december -november when swine were detected with h n antibodies [ ] . the first report of h n active infection in italian swine appeared during an influenza disease outbreak between to which identified h n viruses [ ] . further, four h n viruses were detected in swine nasal swabs originated from three swine farms and an abattoir during - [ ] . later h n and h n viruses were detected in swine during - . interestingly, four human sera samples were also positive for h n and samples were positive for h n viruses which might be due to the transmission between human and swine [ ] . further iav seroprevalence was detected in the age group of three-month to four-year old swine during - [ ] . the first report of a(h n )pdm virus in italian swine appeared after a respiratory disease outbreak in piggeries in lombardia region of northern italy in november . piglets experienced diarrhea and weight loss while the sows experienced reduction in reproduction rate [ ] . two more a(h n )pdm virus isolates were reported in female swine in sicily in december [ ] while five isolates of a(h n )pdm virus were identified in swine at three different locations during - [ ] . a novel strain of reassorted h n virus having - % identity through six gene segments with a(h n )pdm virus along with ha and na genes similar to h n virus was reported in swine in mantua province [ ] . reassorted h n viruses were again detected in piglets during - [ ] . seroprevalence of italian wild boars with one h n , ten h n , and one h n viruses at two different locations was reported during . on the contrary, active infection was found only in three wild boars whose nasal swabs were positive for the iav [ ] . one more investigation reported active infection of iav in wild boars while wild boars had iav antibodies during july-december [ ] . further molecular and serological investigations detected avian-like h n viruses in italian wild boars [ ] . the first complete genome sequence of idv in italian swine was retrieved from a symptomatic sow in which was identified to be closely related to the idv sequence reported in oklahoma swine in [ ] . further idv prevalence in italian swine was reported when , three and four swine were found positive for idv antibodies in veneto, emilia romagna and lombardia regions, respectively during june -may . as a result, swine clinical samples collected during - were investigated retrospectively for idv prevalence but were reported negative. an extended serological investigation detected idv antibodies in swine sera samples collected during . these findings suggested that idv was in circulation in italian swine population only after [ ] . isolation and characterization of h n , nine h n and one h n viruses reported the prevalence of influenza viruses for the first time in spanish swine herds experiencing the respiratory illness and pneumonia during november -april [ ] . more strains of h n , h n and h n viruses were isolated, sequenced and characterized in spanish swine during - [ ] [ ] [ ] . interestingly, five h n , three h n , and four h n virus isolates retrieved between january and august had significant similarities with other european isolates which was an evidence of continent-wide transmission of these swine influenza viruses [ ] . a molecular investigation reported a negative prevalence of idv in swine in luxembourg during but later successfully detected three idv positive swine during - . further, the serological investigation confirmed that swine in luxembourg were free from idv during but interestingly, idv antibodies were detected in swine samples collected during - . these observations suggested that idv was not in circulation in swine in luxembourg during - but became prevalent at a low frequency later during - [ ] which was almost the same time idv was reported in italian swine populations [ ] . a serological investigation of swine in the netherlands identified h n , h n , and h n virus antibodies in swine herds during january-may [ ] with no further evidence of iav in swine in the country after that. after the swine which were experiencing influenza-like illness were found infected with a(h n )pdm virus on a norwegian swine farm in october the surveillance was expanded to the nearby swine farms which determined that of these farms were positive for the a(h n )pdm virus. intriguingly, one human subject at the index farm who had influenza-like symptoms was also found positive for a(h n )pdm virus. this study suggested that the symptoms first appeared in the human subject at the index farm and later the disease got transmitted to the swine. hence the findings of this study suggested the reverse zoonosis of the influenza virus from human to pig [ ] . further molecular and serological investigations identified more swine herds that were positive for iav during september -october [ ] . a more comprehensive nation-wide surveillance in norwegian swine identified a(h n )pdm virus positive swine herds during which later increased to swine herds in [ ] . later more swine were found infected with a(h n )pdm virus in norway between april and july and reported that the iav infected swine took longer to weigh kg body mass [ ] . the first active iav infection in swine in poland was reported in when oral fluid samples collected from three swine farms detected iav [ ] . soon after, five avian-like h n viruses were reported from the swine lung tissues during - [ ] . later a serological surveillance identified h n , h n , h n , and a(h n )pdm virus antibodies in swine during march -february [ ] . surprisingly, of these swine had antibodies against all four iav subtypes i.e., h n , h n , h n , and a(h n )pdm viruses [ ] suggesting the past co-infections. the human-like h n virus was isolated from a swine in czechoslovakia during - [ ] ; however, no other reports ever appeared from the country in later years. complete genome of an h n virus was reported from a hungarian swine having fever and conjunctivitis in may [ ] . this was the only report of h n virus in the swine in hungary. a large-scale investigation across seven european countries reported a high seroprevalence (> %) of iav antibodies in swine populations of belgium, germany, spain, italy while a relatively lower (< . %) seroprevalence was observed in swine populations of czech republic, poland and ireland during - . antibodies against h n , h n , and h n viruses were reported in swine from the european countries under surveillance except poland where swine had antibodies against only h n virus [ ] . a virological surveillance across five european countries including belgium, united kingdom, italy, france and spain reported iav positive swine during - . the h n , h n , and h n viruses were detected in swine from belgium, italy, and spain while the samples from united kingdom and france were found infected with h n and h n viruses [ ] . briefly, the virological and/or serological prevalence of influenza viruses in european countries ( figure d ) identified the strains of h n , h n , h n , and a(h n )pdm viruses in swine populations of the united kingdom, ireland, italy, germany, france, norway, finland, denmark, belgium, spain, poland, greece, hungary, netherlands, czech republic, and czechoslovakia while the swine in luxembourg and italy were found infected with idv. shortly after a respiratory disease outbreak in swine in manitoba, an autopsy was done on a dead swine on march , . the histopathology confirmed the bronchitis in the deceased swine and a strain of iav designated as "s/manitoba/ / " was characterized using iav antisera [ ] . the first report of h n virus in canadian swine appeared in quebec during s- s when five genotypes of h n virus were identified [ ] . since then several studies have reported h n , h n and h n viruses in canadian swine [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . another study reported nine isolates of swine influenza viruses with an antigenic variant from the sick swine having proliferative pneumonia in quebec, canada during - [ ] . in a retrospective diagnosis, only one formalin-fixed paraffin embedded swine lung tissue collected during was found iav positive with immunohistochemistry. this investigation suggested that immunohistochemistry can be useful in retrospective diagnosis of the swine influenza virus [ ] . the broncho-intestinal pneumonia in lung tissues of dead swine was reported on a swine farm which exhibited disease symptoms including coughing, weight loss, and labored breathing. interestingly, before the onset of the disease symptoms, this farm conducted a routine serological surveillance of influenza virus which identified h n virus in only one of the twelve swine samples [ ] . following this surveillance, a three-month old swine from the same farm was found positive for avian influenza virus h n . the complete genome of this h n virus was reported in . this was the first ever report of an avian-origin h n virus in swine. the proximity of the swine farm to a natural lake where several wild bird species including waterfowls which were reported to visit frequently might be the reason behind the introduction of this avian influenza virus strain to the swine [ ] . later three avian-origin h n influenza virus isolates were recovered from swine in eastern ontario exhibiting weight loss and coughing during october . on a nearby farm located approximately kms away, another h n virus isolate was recovered from the swine. there was no recorded movement of the swine between these two farms. since these were avian-origin h n viruses hence the role of birds in transmission cannot be ruled out. later, on a third farm, where an influenza like disease had been affecting mainly the nursery piglets, an h n virus was recovered in may [ ] . reassortant h n and h n viruses were detected in swine nasal swab or lung tissue samples obtained from three-week old piglets and sows exhibiting typical influenza-like symptoms in ontario during - [ ] . first triple-reassortant (avian/classical swine/human triple-reassortant) h n viruses from four swine and one human nasal samples were identified in ontario during . the phylogenetic analysis determined that all the virus sequences were % identical to each other which apparently emerged from triple-reassortant h n viruses reported in us based swine in [ ] . one more report of triple-reassortant h n (trh n ) viruses appeared on the swine farms located in saint-hyacinthe, assomption and saint-foy during early . the trh n viruses identified in this study were determined to be closely related to north american/canadian trh n viruses reported earlier [ ] . later a(h n )pdm and h n viruses having internal genes of triple reassortant h n virus were reported in swine in four provinces including manitoba, alberta, saskatchewan and quebec during [ ] . the first evidence of a(h n )pdm virus in canadian swine appeared in after the human workers at a swine farm developed influenza-like illness. the investigation identified that two farm workers along with swine were positive for the a(h n )pdm virus. transmission of a(h n )pdm virus from human to swine was suggested [ ] . the same year more swine were detected with a(h n )pdm virus after a respiratory disease outbreak hit the alberta swine farms [ ] . a reverse zoonotic transmission of a(h n )pdm virus to swine from a human subject who visited mexico and returned to the swine farm was reported in april . as a result, ten swine having severe disease were sacrificed for necropsy which identified lesions in the bronchioles corresponding to the influenza virus disease. virus isolation and sequencing identified the a(h n )pdm virus. additionally, a(h n )pdm virus was identified in two more human subjects who were exposed to the swine hence indicated the occurrence of zoonoses on the swine farm [ ] . later during summer , ten more a(h n )pdm viruses from five swine herds in manitoba were reported. virus shedding was observed up to days post-infection after the appearance of the clinical symptoms in swine [ ] . this observation was in agreement of a previous report which documented the experimental infection of swine in the laboratory and determined that virus shedding occurs until th day after appearance of the clinical symptoms [ ] . another investigation reported nine a(h n )pdm and four h n viruses after an influenza-like disease outbreak on a quebec based swine farm in december [ ] . the effect of microclimatic conditions on the transmission dynamics of swine iav in the barns was studied which observed that high relative humidity in the environment during summer months suppresses the aerosol transmission of the droplets which in turn decreases the transmission of iav [ ] . the high relative humidity in the environment would facilitate the generation of larger droplets which do not tend to shrink easily and hence are less likely to be aerosol transmitted to a longer distance as they fall on the ground quickly after their formation [ , ] . as a result, a lower transmission of iav is observed usually during the summer months. on the contrary, the iav transmission increases during winter months when relative humidity is relatively lower [ ] . the iav was first isolated from the nasal discharge of a swine in the united states in [ ] and from the human in [ ] . the first report of human-origin iav in swine appeared in the united states on may after an unexpected result was observed when the serum sample of a sick swine obtained from a state prison farm located in new jersey neutralized the antibodies of human influenza virus. a series of investigations made a strikingly new observation that swine had suffered from a human strain of influenza virus [ ] . serological investigations conducted during s suggested that the weight loss and mortalities among swine were due to swine influenza viruses [ , ] . swine influenza viruses were isolated from febrile swine at nine occasions during - in wisconsin and nebraska [ ] . additionally, swine influenza antibodies were also detected in swine sera samples collected from six farms [ ] . a virological surveillance conducted in memphis, tennessee and madison, wisconsin during may to june successfully isolated influenza viruses from swine nasal swabs collected at abattoirs; approximately of which were characterized to be swine h n viruses. additionally, the serological surveillance identified that % of the swine sera samples had swine h n virus antibodies [ ] . a small percentage ( . %) of swine sera samples were found positive for the swine h n viruses which was further confirmed by virus isolation [ ] . interestingly, this study identified inter-species transmission of swine influenza viruses between human and swine [ ] . a novel swine-origin h n virus termed as "a/new jersey/ (hsw n )" was detected at fort dix army training camp in new jersey in january . the outbreak was localized and was limited to fort dix only. as a result, soldiers were found infected with this novel virus; of which had severe respiratory disease with one death due to viral pneumonia [ ] [ ] [ ] . since this novel swine-origin h n virus quickly disappeared from fort dix hence the epidemiology and the origin of the disease could not be ascertained [ ] . the h n and h n virus antibodies were detected in swine sera collected from an abattoir in north-west united states. interestingly, a higher iav seroprevalence was observed during the fall and early winter months. virus isolation and sequencing identified that the h n viruses were closely related to the classical h swine influenza virus [ ] . classical swine-like h n and triple-reassortant h n viruses were identified in swine samples collected across states in the usa during - [ ] . the minnesota veterinary diagnostic laboratory (mvdl) detected large number of h n , h n and h n subtypes of iav in swine samples during - and again during - . interestingly, some of the samples were co-infected with h n and h n viruses [ ] [ ] [ ] . a second-generation reassortant h n virus having genes from a reassortant h n and classical h swine influenza viruses was obtained from the lung tissue samples of a dead sow at an indiana swine farm in november [ ] . a novel subtype of h n virus termed as "a/swine/minnesota/ / (h n )" was identified during a severe respiratory disease outbreak on a swine farm in minnesota in october . sequencing observed that the ha gene of this strain was closely related to swine influenza h n virus while the na gene was related to classical h n virus which suggested that the novel h n virus emerged due to reassortment between h n and h n viruses in the midwest united states [ ] . further an h n subtype of iav which may have emerged as a result of a reassortment between avian and swine influenza viruses was identified on a commercial swine farm in minnesota in april and again in september [ ] . the first evidence of a(h n )pdm virus infection in us swine appeared when four a(h n )pdm and one triple-reassortant h n viruses were identified and characterized in the exhibition swine in the states of minnesota and south dakota in [ ] . during last ten years, a large number of h n , h n , h n , a(h n )pdm along with reassortant iav subtypes have been reported in the us swine populations [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the united states has a large feral swine population which is considered a reservoir of h n and h n viruses [ ] . the swine-like h n , avian-like h n , swine-like h n , swine-like h n , human-like h n , a(h n )pdm along with avian-like h n and h n viruses were identified in feral swine samples collected across states in the usa between october -september [ ] . histological examination of the lung tissues obtained from two backyard piglets suffering from pneumonia and weight loss in colorado in november suggested that the piglets were infected with swine influenza virus which were later confirmed to be infected with iav subtype a(h n )pdm virus. since the piglets were raised at the house of a pharmacist hence a possible human to swine transmission was speculated given the possibility of an occupational exposure of the pharmacist to the a(h n )pdm virus at the pharmacy [ ] . the first report of ibv infection in swine appeared when swine in the midwest united states were found infected with ibv lineages of yagamata/b and victoria/b [ ] . this was a new finding because initially ibv was thought to have a host range limited to human, pheasants, horses and seal [ ] [ ] [ ] [ ] . a novel strain of swine influenza virus was detected in oklahoma swine exhibiting influenza-like symptoms in april . the nasal swab samples taken from the swine were negative for the iav infection. hence the virus isolation was attempted in swine testicle cells; the cells in culture showed influenza-like cytopathic effects by third day. electron microscopic observations revealed particles typical of a virus of orthomyxoviridae family, but the rt-pcr was negative for the ibv and icv. after ultracentrifugation was used for virus isolation, the genome of the virus was sequenced using ion torrent sequencing. the genome sequence analysis along with genetic and biochemical investigations revealed that the isolated virus was a novel orthomyxovirus having % overall identity at amino acid level with human influenza c virus [ ] . since this novel virus was genetically and antigenically distinct from icv therefore, later was proposed to be categorized as a new genus of orthomyxoviridae family which was later accepted as influenza d virus (idv) [ ] . later, two feral swine which were shot dead in a cotton field in texas in june were found infected with a(h n )pdm virus. the significant identity of a(h n )pdm virus isolated from these two feral swine with human a(h n )pdm virus suggested a possible transmission between human and the feral swine [ ] . another study reported seroprevalence of h n virus in one feral swine from mississippi and in five feral swine from the state of california in but a negative seroprevalence was reported in the feral swine samples obtained from the states of florida, oklahoma and missouri. additionally, the seroprevalence of iav was reported in feral swine from texas where a total of out of feral swine sera were found positive for h n and h n viruses [ ] . another investigation detected h n virus rna in only one feral swine from a pool of samples collected across states in the usa during - which indicated a negligible active influenza infection in us feral swine population. on the contrary, elisa identified iav antibodies in feral swine samples while the serological subtyping identified h n virus antibodies in feral swine samples collected from states which indicated a significant past exposure of us feral swine to the h n virus [ ] . further, seroprevalence of idv was reported in feral swine samples collected from oklahoma, texas, hawaii and north carolina during october -september which provided the first evidence of past idv infections in us feral swine [ ] . a study investigating virus shedding in nursery piglets found that all piglets under investigation were shedding h n virus starting seventh day of arrival into the barns until th day. shedding was still observed in some piglets until th day [ ] . interestingly, of these nursery piglets were also identified shedding h n virus starting at the third day of arrival into the barns until st day over a -day observation period [ ] . this was the new information which identified that young nursery piglets could get infected with iav. the oral fluid samples collected from neonatal piglets at four oklahoma based swine farms during may-august [ ] were found infected with different iav subtypes including h , n , h , and n . this study supported the use of swine oral fluid samples in iav diagnostics [ ] . the swine oral fluid samples were also collected in north and south carolina during june to august using the cotton rope hanging method [ ] . in this method of sampling, swine are encouraged to chew the rope, as a result, saliva accumulates on the rope which is later squeezed to collect the sample aseptically. one of the benefits of this method of sampling is that each sample does not represent an individual swine but rather represents multiple swine that chewed the rope while hanging inside the pen [ ] . another benefit of this sampling method is that swine oral samples may contain contaminants like feed and feces but this method minimizes the chances of such contaminations in the sample [ ] . another investigation carried out metagenomic sequencing of swine nasal and rectal swabs obtained from apparently healthy swine which identified iav positive swine at three abattoirs and a buying station in usa in august [ ] . in a striking observation, an avian-lineage h n virus was isolated and sequenced from - -month-old gilts on a missouri based swine farm in december [ ] . the investigators collected more samples at different time points for next few months at the same farm to assess the transmission of h n virus among swine. no other samples were found positive for the h n virus which suggested that the h n virus did not transmit from swine-to-swine and therefore disappeared from the index farm. interestingly, this extended study identified three h n viruses infecting swine [ ] . one large-scale study identified that percent ( / , ) of the swine samples were positive for the iav in mid-west united states between july -march , however, sequencing could identify only h and h subtypes among positive samples [ ] . a human to swine transmission of iav was suggested when two human-like h n virus isolates were identified from an oklahoma based swine farm in which had high similarity with the human-like h n viruses reported earlier from baltimore [ ] . maya people represent ethnolinguistic groups in south and central america. the practice of household swine keeping put the maya people at high risk of contracting the swine influenza viruses. thirty-one sera samples collected from the maya people in mexico were identified having antibodies against h n and h n viruses while other sera had antibodies against the h subtype of iav, representing a past exposure to these viruses [ ] . however, this study did not include swine samples for investigation but since swine were household animals in their backyard hence the iav seroprevalence of the maya people could be because of a past transmission of these viruses from the backyard swine [ ] . a retrospective study identified antibodies against swine-like h n , a(h n )pdm , h n , and human-like h n viruses in backyard swine in mexico between to . this investigation retrospectively determined that the classical-swine h n virus was most widely present in mexican swine before the influenza pandemic [ ] . further, a significant number of swine experiencing respiratory illness had h n or h n virus antibodies in commercial piggeries in sonora province of mexico during october -march . the molecular diagnostics and subtyping determined four h and two h viruses while other iav positive samples could not be subtyped given the low viral load [ ] . during the influenza virus pandemic in mexico in , a(h n )pdm virus was first identified in a single swine nasal swab. additionally, h n , a(h n )pdm and ibv viruses were detected in four symptomatic humans [ ] . the a(h n )pdm virus isolate retrieved from the swine was believed to be the first from the sister lineage of the pandemic influenza virus isolates reported in mexico [ ] . further iav isolates were retrieved from mexican swine having respiratory illness during - . intriguingly, this study identified reassorted genotypes of iav in mexican swine [ ] . this investigation also reported that iav introduction into mexican swine may have occurred through three different routes; human to swine transmission; reassortment between human-like h n and a(h n )pdm virus; and through the long-distance movement of the swine from usa and europe. a periodic introduction of iav in mexican swine occurred with the import of american and european swine to mexico over two decades in s and s before the influenza pandemic [ ] . fifty-eight iav whole genome sequences were retrieved from mexican swine during - . genome sequence analysis identified classical h n , h n , and a(h n )pdm viruses. interestingly, the data obtained in this study suggested independent evolution of iav in the mexican swine population in different regions of the country. phylogeny determined that mexico city was the source of the influenza pandemic which erupted during march-may [ ] . later a reassortant h n virus which had the genes from human and swine influenza viruses was isolated and sequenced from a swine in november [ ] . the molecular diagnostics identified a total of iav positive commercial and backyard swine in guatemala during - which resulted into three a(h n )pdm and one h n virus isolates [ ] . the first report of a(h n )pdm virus in commercial piggeries in cuba appeared in november when swine were found positive for a(h n )pdm virus across five swine farms [ ] . further, five more iav positive swine were detected in pinar del rio province of western cuba having respiratory illness and interstitial pneumonia. however only one iav positive sample could be successfully subtyped as a(h n )pdm virus having reassorted internal genes, all except the na gene [ ] . in a more recent investigation, a high seroprevalence of iav ( / ) was detected in swine in trinidad and tobago which later identified h n and a(h n )pdm viruses in swine [ ] . in summary, the h n , h n , h n , and a(h n )pdm viruses were reported in north american swine population. interestingly, the avian influenza virus strain h n was detected in us based swine while h n and h n were identified in the canadian swine and h n was reported in the mexican swine in ( figure e ). mexico city was identified to be the origin of influenza pandemic. it was also ascertained that a(h n )pdm virus was present in mexican swine well before pandemic erupted. after influenza virus outbreak hit a swine farm in buenos aires in november , one of the five dead swine were diagnosed with viral pneumonia through immunohistochemistry. a full genome of h n virus sharing - % nucleotide sequence identity with h n viruses reported in north america during - was recovered from the swine [ ] . an h n virus was reported from a swine after a swine farm manager along with his spouse experienced influenza-like symptoms few days before the outbreak erupted in the swine at a buenos aires based farm in june . the influenza disease symptoms lasted for about a week in nursery piglets. immunohistochemistry identified necrotizing bronchiolitis in four of the swine post-mortem samples while one sample had severe inflammation in the bronchiolar epithelia. the serological investigation detected iav antibodies in most of the sera samples collected after days of onset of clinical symptoms however the active infection was reduced to only six swine [ ] . the third investigation carried out histopathology which identified lung lesions compatible to the influenza virus infection in nine swine necropsy samples at a buenos aires based swine farm in october and later in eight swine necropsy samples originated from a santa fe based farm in may . the swine at buenos aires farm were found infected with h n virus while the swine at the santa fe farm retrieved one h n and three human-like reassortant a(h n )pdm virus isolates which had triple reassortant internal genes. this was the first report of human-like reassortant a(h n )pdm virus in swine in argentina [ ] . later two more investigations using histopathology, immunohistochemistry, serology, and molecular analyses reported h n , h n , and reassortant h n viruses with a(h n )pdm internal genes in swine in argentina during - [ , ] . several h n , h n , h n , human-like h n , and a(h n )pdm viruses have been identified in brazilian swine from the minas gerais, parana, rio grande do sul and sao paulo provinces in brazil during and after [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a technician who visited a minas gerais swine farm experiencing influenza outbreak developed similar respiratory disease symptoms. the nasal swab sample was obtained from the technician, as a result, one a(h n )pdm virus was isolated which was closely related to the a(h n )pdm viruses reported from the swine herd in the minas gerais which was recently visited by the technician. hence it was concluded that a zoonotic transmission from swine to the technician occurred at the minas gerais swine farm [ ] . an immunohistochemical investigation demonstrated microscopic lesions suggesting broncho-interstitial pneumonia in the lung tissues of four severely sick piglets at a swine farm located in parana province in february . the a(h n )pdm viruses were isolated from two piglets. additionally, a novel reassortant h n virus was also recovered [ ] . one more investigation identified that a(h n )pdm virus was the most prevalent iav subtype in sows. the co-infections of sows with a(h n )pdm , h n , or h n subtypes were also documented in rio grande do sul province. these findings were noteworthy because the coinfections may trigger reassortments and thus may facilitate emergence of novel strains of iav [ ] . later two more h n viruses were isolated and characterized from swine in rio grande do sul province during . the sequences of both the isolates had high nucleotide similarity to each other in different genome segments in the range of . % to % which suggested a common source of origin of both isolates [ ] . the backyard productive systems (bps) for raising swine, cattle, and poultry are popular in chile. a molecular investigation reported a negative active iav infection across bps units within ten counties in chile during - but the serological investigation detected iav antibodies in swine at two bps units which suggested a past exposure of swine to the iav [ ] . interestingly, the ha gene sequence of an h virus was obtained from a domestic muscovy duck at one of the bps which appeared to have originated from a wild bird. this suggested a spillover of the iav from wild reservoir to the domestic poultry [ ] . another study reported the prevalence of h n virus in swine reared at different bps having poultry and swine in el yali wetland during - [ ] . one more study identified four swine sera samples ( / ; . %) that were found positive for iav antibodies collected from different bps in central chile. one pool of swine nasal swab samples ( / ; . %) was also detected iav positive with real-time rt-pcr. interestingly, . % chicken, . % ducks and . % geese samples collected from bps in central chile also had active iav infections. the breeding practice of poultry and swine in the bps was determined to be a major risk factor for iav transmission [ ] . briefly, the iav strains of h n , h n , h n , and a(h n )pdm viruses have been reported from the swine in argentina and brazil while a(h n )pdm virus was reported in swine in colombia and peru. swine in chile were found infected with h n virus ( figure f ). in summary, total research articles were identified which reported several influenza viruses in swine populations globally. the highest number of studies were reported from asia (n = ), followed by north america (n = ), europe (n = ), south america (n = ), africa (n = ) and australia (n = ). the highest number of reports per country were documented in united states (n = ) followed by china (n = ) and canada (n = ). until february , influenza viruses have been reported from countries worldwide. four subtypes of iav including h n , h n , h n , and a(h n )pdm viruses were most frequently detected in swine populations ( table ) . most of the large-scale studies used serological investigations including elisa, hemagglutinin inhibition (hi), neuraminidase inhibition (ni), virus neutralization (vn), or microneutralization (mn) assays for the determination of the seroprevalence and subtyping of the influenza viruses in swine. several investigations used virus isolation for the confirmation and subtyping of iav. most of the virological investigations used one-step real-time rt-pcr and/or reverse-transcription pcr for influenza virus detection and subtyping. sanger sequencing or next-generation sequencing using miseq or ion torrent sequencing successfully generated the influenza virus sequences from the swine samples for epidemiological interpretations. histological examinations including immunohistochemistry or immunofluorescence were used to examine the swine lung or other internal organ tissue samples for the influenza virus diagnostics (table ) . as of february , influenza viruses have been identified and reported in swine from countries worldwide (table ; figure ). the influenza viruses have been detected in different sample types including swine sera, nasal, tracheal, oropharyngeal, nasopharyngeal swabs as well as oral fluids collected from the live swine. nasal and snout wipes, lung homogenates and fecal slurry samples were also used. additionally, the lung as well as other internal organ tissues ( table ) obtained from either dead or sacrificed swine have also been used for the detection of iav symptoms i.e., lesions in lungs, pneumonia, bronchitis, bronchiolitis etc. various methods have been used for the detection of influenza viruses in swine samples depending on the sample type, sample numbers and objective of the study. virus isolation methods, using either mdck, caco- , hrt , or swine testicle cells or the pathogen free embryonated chicken eggs, although considered the gold standard [ ] [ ] [ ] have largely been taken over lately by the sequencing approaches which tend to provide a considerably faster identification of the iav subtypes. the additional benefit of sequencing over virus isolation is that the sequences would be useful for analyzing the influenza virus outbreak clusters [ ] , virus evolution or reassortment [ ] using phylogenetic analyses in different gene segments. a recent study reported that next-generation sequencing can be useful in the influenza virus diagnosis and for the identification of the novel virulence markers and drug resistance [ ] . most of the studies have used real-time rt-pcr with matrix-gene specific oligonucleotide primers and taqman probe for iav detection [ , , ] . the conserved sequences of the matrix-gene specific primers can detect any iav subtype in the swine samples [ ] . most of the studies used subtype-specific real-time rt-pcr for the iav subtyping, however, few studies opted for the conventional approach of reverse-transcription pcr followed by sanger sequencing for amplification of the ha and/or na genes for retrieving the sequences for phylogenetic analyses to identify the subtypes. although the real-time rt-pcr is a powerful and rapid tool for the subtyping of iav strains, it is more expensive than reverse-transcription pcr. a few studies have reported reverse-transcription pcr based amplification of all the eight gene segments of iav to generate the whole genome sequences [ , , , ] but in most cases, the whole genome sequences were generated using miseq next-generation sequencing approach [ , , , ] . a great advantage of this sequencing approach is that it can identify novel influenza viruses in the swine samples [ , , ] . most of the serological investigations used one or more methods for influenza virus detection and subtyping in the swine samples e.g., elisa, hi, ni, mn, or vn assays. the serological methods are useful in large-scale surveillances for screening large number of samples in a limited time. however, the molecular detection assays are more reliable than the serological methods given the higher sensitivity, but the serological assays are rapid and affordable hence are preferred for large-scale surveillance studies. the molecular and serological investigations report either active infections (viral rna) or past exposures (antibodies) in swine samples, respectively. the molecular detection approaches followed by sequencing are largely used for the research focusing on the influenza virus epidemiology [ , , , , , , , ] . [ , , , , , , , , , , , , , , , , , [ ] [ ] [ ] , , , , , , , , , , , ] . [ , , , , , , , , , , , , , , , , , ] . lung/liver/internal organ tissues rna extraction, real-time rt-pcr, reverse transcription-pcr, ligation, hi assay, virus isolation (mdck cells/spf chicken eggs), sanger and next-generation sequencing, hematoxylin-eosin staining, immunohistochemistry, immunofluorescence h n , h n , reassortant h n , h n , h n , a(h n )pdm , h n , idv [ , , , , , , , , , , , , , , ] . lung homogenate rna extraction, real-time rt-pcr, multiplex rt-pcr, single step rt-pcr, virus isolation (mdck cells/caco- cells/spf chicken eggs), sanger sequencing, membrane enzyme immunoassay, hi assay h n , h n , h n , reassortant h n , a(h n )pdm [ , , , ] . fecal slurry rna extraction, qrt-pcr iav [ ] . rectal swab nucleic acid extraction, reverse transcription, metagenomic sequencing iav [ ] several studies used histological examinations e.g., immunohistochemistry or immunofluorescence to identify the iav symptoms in lung or other internal organs of either dead or severely sick swine sacrificed for the investigations [ , , , ] . immunohistochemistry provides a rapid and affordable diagnosis of the influenza virus disease using swine tissue samples [ ] . one major benefit of immunohistochemistry is that it can be used in the retrospective analysis of the archived tissue samples [ ] . a large number of investigations have reported sub-clinical influenza virus infections in asymptomatic (apparently healthy) swine [ , , , , , , ] , indicating that influenza infections can go undetected while the swine may be shedding virus and hence may infect other swine and farm workers in contact [ ] . intriguingly, most of the swine samples processed in australia, europe, and north america were obtained from the symptomatic swine while most of the chinese swine samples were collected from asymptomatic swine ( figure ). symptomatic swine may exhibit mild or severe influenza like symptoms [ , , ] , including fever, coughing, sneezing, pneumonia, bronchitis, reduced appetite, diarrhea, nasal and/or ocular discharge, conjunctivitis, weakness, anorexia, prostration, weight loss, abortion in sows, and mortality in some cases [ , , , , , , ] . most studies where the swine were severely infected reported reduced appetite and weight loss [ , , , ] . due to iav infection, the swine takes longer to weigh kg body mass [ ] , hence the iav disease burden affects the swine farmers economically. varying rates of mortality of swine due to iav infections were reported from around the world ranging from . % to % [ , , , , , , , ] . this wide difference in mortality rate could be due to novel virus strains emerged through reassortments within the swine [ , , ] or inter-species transmission, e.g., avian to swine transmission, resulting into severe disease outbreaks [ , , , ] . one example of the emergence of a novel influenza virus strain is the emergence of a(h n )pdm strain due to reassortment between avian and swine iav strains in swine which resulted into influenza pandemic [ ] . additionally, the emergence of iav subtype h n is another classic example of influenza virus reassortment which resulted into severe disease outbreaks in japanese and korean swine populations during s and s [ , , ] . strains of iav can infect the swine of any age group; piglets as young as one week may become infected with iav naturally. interestingly, a study in denmark observed a piglet as young as just three days was infected with iav despite having maternally derived iav antibodies [ ] , suggesting that the infection might have occurred from the infected sow which was shedding the virus [ ] . however, the symptoms of the influenza-like illness in swine may last only for one week but the virus shedding may still persist until days after appearance of the influenza-like symptoms [ , ] . this phenomenon may have serious implications in influenza virus spill over to the non-infected swine as well as to the exposed farm workers due to prolonged virus shedding. three other studies observed virus shedding in swine and reported that the virus shedding may persist until the day [ ] , day [ ] , or day [ ] after onset of the clinical symptoms in swine. this variation in the duration of the virus shedding might be strain dependent, which needs to be further investigated. a higher rate of virus shedding and iav prevalence was reported during the fall and early winter months than summer season because the high relative humidity present in the environment during summer decreases the transmission of influenza virus [ ] . the high relative humidity in summer facilitates the generation of larger droplets which are less likely to be aerosol transmitted to a longer distance as they tend to fall on the ground quickly after their formation [ , ] . several cases of inter-species transmission were identified which documented transmission of iav between human and swine or between birds and swine. the occurrence of the avian influenza virus strains in swine in china (h n , h n , h n , h n , h n , h n , h n , h n , h n ), united states (h n , h n , h n ), canada (h n , h n ), south korea (h n , h n ), nigeria (h n ), and egypt (h n , h n , h n ) serve as the evidence of interspecies transmission of avian influenza viruses to swine. the first evidence of avian influenza virus active infection in swine appeared in in canada when h n virus was isolated from a swine. later several other avian-origin iav strains were detected and sequenced in swine in china, canada, and south korea ( figure ) . a large number of investigations have reported sub-clinical influenza virus infections in asymptomatic (apparently healthy) swine [ , , , , , , ] , indicating that influenza infections can go undetected while the swine may be shedding virus and hence may infect other swine and farm workers in contact [ ] . intriguingly, most of the swine samples processed in australia, europe, and north america were obtained from the symptomatic swine whi egypt is recognized as a "hot spot" for the influenza virus reassortment due to its geographical location [ ] . the role of migratory wild birds in the introduction of avian influenza in egypt has been already established [ , ] , and the highly pathogenic strains of the iav have previously been detected in migratory birds in egypt [ ] . since migratory wild birds were reported to harbor in the vicinity of cairo [ ] therefore, the probability of the migratory bird-swine interaction in the regions remain high which very well explains the occurrence of highly and low pathogenic strains of avianorigin iav in swine in cairo, egypt. given the "mixing vessel" nature of the swine, the occurrence of avian-origin iav strains in swine is alarming in terms of iav reassortment and evolution which may trigger the emergence of novel iav strains of pandemic potential in the future. further, the multiple reports of double or triple reassortant iav strains in swine are evidence that iav co-infections may facilitate the antigenic diversity of the influenza viruses; and as a result, new ha and na subtypes of iav may be continually added to the existing ha and na subtypes in the future. intriguingly, the frequency of the occurrence of double or triple-reassortant iav strains in swine has dramatically increased in the recent decades [ , , , , , , , ] . one unique example of the reassortment and evolution of the pandemic strain of iav in swine was the emergence of a(h n )pdm virus in swine in mexico which evolved due to the reassortment between avian and swine iav strains [ ] . while an overwhelming majority of investigations reported iav in the swine across the world ( figure a) , there were only a few reports which documented either active infections or past exposures of the swine to the influenza virus types ibv [ , , , ] (figure b ), icv [ , , ] ( figure c ) or idv [ , [ ] [ ] [ ] [ ] ] (figure d) . a low prevalence of ibv was observed in swine given that only one study reported the ibv antibodies in swine samples in england during - [ ] with no evidence of further spill over to other european countries. the active infection of ibv was later reported in us swine when two ibv isolates were obtained in . a recent study from taiwan reported three strains of the victoria/b lineage of ibv in naturally infected swine in [ ] , again various studies have spotted wild birds visiting the swine farms or in the vicinity which suggested that wild birds may have served as the carriers for the introduction of the different avian-origin iav subtypes to the swine populations [ , , , , ] . the highest number of avian-origin iav strains were reported in chinese swine which shows frequent avian-swine interaction in china, a country that has historically been an epicenter for influenza virus disease [ ] . egypt is recognized as a "hot spot" for the influenza virus reassortment due to its geographical location [ ] . the role of migratory wild birds in the introduction of avian influenza in egypt has been already established [ , ] , and the highly pathogenic strains of the iav have previously been detected in migratory birds in egypt [ ] . since migratory wild birds were reported to harbor in the vicinity of cairo [ ] therefore, the probability of the migratory bird-swine interaction in the regions remain high which very well explains the occurrence of highly and low pathogenic strains of avian-origin iav in swine in cairo, egypt. given the "mixing vessel" nature of the swine, the occurrence of avian-origin iav strains in swine is alarming in terms of iav reassortment and evolution which may trigger the emergence of novel iav strains of pandemic potential in the future. further, the multiple reports of double or triple reassortant iav strains in swine are evidence that iav co-infections may facilitate the antigenic diversity of the influenza viruses; and as a result, new ha and na subtypes of iav may be continually added to the existing ha and na subtypes in the future. intriguingly, the frequency of the occurrence of double or triple-reassortant iav strains in swine has dramatically increased in the recent decades [ , , , , , , , ] . one unique example of the reassortment and evolution of the pandemic strain of iav in swine was the emergence of a(h n )pdm virus in swine in mexico which evolved due to the reassortment between avian and swine iav strains [ ] . while an overwhelming majority of investigations reported iav in the swine across the world ( figure a) , there were only a few reports which documented either active infections or past exposures of the swine to the influenza virus types ibv [ , , , ] (figure b ), icv [ , , ] (figure c ) or idv [ , [ ] [ ] [ ] [ ] ] (figure d) . a low prevalence of ibv was observed in swine given that only one study reported the ibv antibodies in swine samples in england during - [ ] with no evidence of further spill over to other european countries. the active infection of ibv was later reported in us swine when two ibv isolates were obtained in . a recent study from taiwan reported three strains of the victoria/b lineage of ibv in naturally infected swine in [ ] , again there was no further report of dissemination to nearby asian countries. the ibv infected swine were apparently healthy with no signs of influenza disease. pathogens , , x of there was no further report of dissemination to nearby asian countries. the ibv infected swine were apparently healthy with no signs of influenza disease. the first report of icv appeared in chinese swine after the virus was isolated from apparently healthy swine in in a routine diagnostic procedure at an abattoir in beijing [ ] . later icv seroprevalence was reported in english and japanese swine during s- s [ , ] with no further evidence of circulation anymore thereafter. the idv was first detected and characterized in in oklahoma based swine in the united states which appeared to have made a species jump from cattle to swine [ , ] . interestingly, a complete idv genome was retrieved from a symptomatic sow in italy in which was found closely related to the idv genome reported in from oklahoma, usa [ ] . this might have happened due to the trade of the cattle or swine between italy and the united states. a recent study from china identified idv sequences which shared a high similarity ( %- %) with the idv sequences reported earlier from the cattle in china [ ] which was another evidence of bovine to swine transmission of idv. the idv has been in circulation in swine in the current decade with reports emerging from swine in italy, luxembourg, china and the united states. in summary, iav was first isolated from a swine in usa in [ , ] and later antibodies for the human influenza viruses were reported in swine at the state prison of new jersey in [ ] . more iav outbreaks and cases in swine in north america were reported during - ; the frequency has now dramatically fallen in the last two decades (figure ). this might be due to the first report of icv appeared in chinese swine after the virus was isolated from apparently healthy swine in in a routine diagnostic procedure at an abattoir in beijing [ ] . later icv seroprevalence was reported in english and japanese swine during s- s [ , ] with no further evidence of circulation anymore thereafter. the idv was first detected and characterized in in oklahoma based swine in the united states which appeared to have made a species jump from cattle to swine [ , ] . interestingly, a complete idv genome was retrieved from a symptomatic sow in italy in which was found closely related to the idv genome reported in from oklahoma, usa [ ] . this might have happened due to the trade of the cattle or swine between italy and the united states. a recent study from china identified idv sequences which shared a high similarity ( %- %) with the idv sequences reported earlier from the cattle in china [ ] which was another evidence of bovine to swine transmission of idv. the idv has been in circulation in swine in the current decade with reports emerging from swine in italy, luxembourg, china and the united states. in summary, iav was first isolated from a swine in usa in [ , ] and later antibodies for the human influenza viruses were reported in swine at the state prison of new jersey in [ ] . more iav outbreaks and cases in swine in north america were reported during - ; the frequency has now dramatically fallen in the last two decades (figure ). this might be due to improved swine influenza surveillance and vaccination in north america in recent decades. the h n , h n , h n , and a(h n )pdm viruses were reported from the commercial, backyard, exhibition, feral swine and wild boars in the united states. as of february , the highest number of reports of influenza virus infections in swine in a country were documented in the united states (n = ) followed by china (n = ) and canada (n = ). the highest number of iav positive swine samples were reported in the united states ( / ) followed by china ( / ). one of the factors behind the higher number of iav cases in swine in the united states compared to china would be related to the disease symptoms. a majority of the north american swine samples that were screened for the iav infections had either mild or severe symptoms of influenza-like illness which would have made it visually easier to identify iav infected swine in the united states. on the contrary, a smaller number of chinese swine exhibited influenza-like disease symptoms while a large proportion of the chinese swine population appeared to have sub-clinical infections with no symptoms. this would have made it difficult for identifying the influenza virus infected swine during surveillances in china. the first report of idv in in oklahoma swine reflected the antigenic diversity and evolution of influenza viruses in the us swine. however, the recent influenza virus disease prevalence in north american swine appeared to have declined, nevertheless, given the large swine population of the continent, the surveillance should continue to track the influenza virus diversity and evolution. the first serological evidence of iav in european swine was documented from the czechoslovakia during - , but the first h n virus in european swine was isolated in belgium in which was apparently transmitted from wild ducks in germany to the swine in belgium. since then several h n , h n , h n , and a(h n )pdm viruses have been detected in commercial and backyard swine as well as in wild boars within europe. the incidence of iav in european swine has increased several folds in the past two decades with a relatively high number of iav positive swine samples ( / ). most of the iav positive european swine were reported having influenza-like symptoms at the time of sampling. germany reported the highest number of iav positive swine in europe where the pork industry is considered the third largest globally after china and the united states. importantly, the idv was more recently identified in the european swine, first in italy in , and later a retrospective study identified idv infection in swine samples collected in luxembourg during - which indicated that the circulation of idv in european swine took place only after . the evidence has suggested the bovine to swine transmission of idv. this observation is interesting because until recently more emphasis has been given to the avian-swine interaction and the bovine-swine interactions have been neglected from the influenza virus spill over perspective. the first occurrence of iav in asian swine can be traced back to , but the iav prevalence has increased multi-fold in the recent two decades. the iav subtypes h n , h n , h n , and a(h n )pdm have become endemic in swine in several asian countries. the highly pathogenic avian-origin iav strains of h n and h n have been reported from chinese swine while h n has been documented in swine in viet nam and indonesia. the highly pathogenic strains of h n have been reported in south korean swine. several lpaiv strains including h n , h n , h n , h n , h n , and h n have also been documented in chinese swine. the studies suggested a frequent interaction between wild birds and swine in china which appeared to have transmitted avian-origin iav strains in the chinese swine. occurrence of equine influenza virus h n in chinese swine further expanded the genetic diversity of swine influenza viruses. h n , h n , h n , and a(h n )pdm viruses were reported from the commercial, backyard, exhibition, feral swine and wild boars in the united states. as of february , the highest number of reports of influenza virus infections in swine in a country were documented in the united states (n = ) followed by china (n = ) and canada (n = ). the highest number of iav positive swine samples were reported in the united states despite an avian to human transmission of certain avian influenza virus strains including h n virus, only a limited human to human transmission of avian influenza viruses was established in the past [ , ] . with the passage of these avian-origin iav strains in a mammalian host like swine, a high probability remains of these avian influenza virus strains to adapt and gain the ability of the human to human transmission, if happens, the consequences would be devastating for the public health. australian swine were free from influenza virus until the year when a new south wales swine farm reported an influenza-like outbreak in the swine. the zoonotic transmission of the a(h n )pdm virus was reported to the farm workers and the farm owner. until now, there have only been four iav reports in australian swine which reflects a low prevalence of iav in australian swine. new zealand is yet to officially report the influenza virus prevalence in swine and remains free from the disease. a retrospective study identified that the a(h n )pdm virus was present in mexican swine as early as , well before the influenza pandemic occurred during march-may . a high genetic diversity of iav in mexican swine due to live swine imports from north america and europe during s laid the foundation of the emergence of zoonotic strain of a(h n )pdm virus in mexican swine [ ] . the highest number of iav positive swine in central america were reported from mexico followed by guatemala. interestingly, a report of the highly pathogenic h n virus in mexican swine in further triggers the alarm in the context of a potential novel iav reassortment. outbreaks of iav in the south american swine populations occurred during the last two decades, with the highest prevalence reported in the brazilian swine. a considerable proportion of cases showed sub-clinical infections with no symptoms which might have made it more difficult to detect the infected swine. the reports of iav active infections or the seroprevalence appeared in the african swine only during last two decades. until february , iav have been detected in swine in cameroon, nigeria, egypt, kenya, reunion island, uganda, togo, and ghana. however, south africa has a considerable swine population [ ] , but currently there is no published report on the prevalence of active iav or other influenza virus infections in the south african swine. this might be because of the lack of an active surveillance for the detection of the influenza virus disease in the swine in south africa. the reports of reassortant, double-reassortant and triple-reassortant influenza viruses in asian, north american and european swine strengthens the concept of swine being the "mixing vessel" in terms of influenza virus reassortment and evolution. the multiple reports of avian-origin iav strains including highly pathogenic h n , h n and h n in swine are alarming given the fact that the avian-origin strains may adapt in swine to facilitate the emergence of a reassortant pandemic strain. the highest number of influenza virus studies in swine population have been reported from the united states (n = ) followed by china (n = ). also, the united states reported the highest numbers of iav cases in swine. due to widespread active surveillance, the united states has significantly brought down the influenza virus disease in swine in the last two decades. conversely, the iav disease burden has increased multi-fold in chinese swine in the last two decades. additionally, the occurrence of several high-and low-pathogenic avian-origin iav strains in the chinese swine population may put the country at greater risk of an influenza pandemic for the future. given the "mixing vessel" nature of swine physiology, the occurrence of several avian-origin iav strains and multiple reports of double-reassortant and triple-reassortant iav subtypes in chinese swine are alarming because reassortments in swine may facilitate the emergence of a new iav strain of pandemic potential. in the background of the current corona virus pandemic (covid- ) which originated in china, the presence of 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pathogenic avian influenza virus (h n ) clade . . . infection in migratory birds the etiology of swine influenza detecting human-to-human transmission of avian influenza a (h n ) barrier to bird flu transmission found in humans pig farming in rural south africa: a case study of uthukela district in kwazulu-natal this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -b ujorz authors: koyama, takahiko; weeraratne, dilhan; snowdon, jane l.; parida, laxmi title: emergence of drift variants that may affect covid- vaccine development and antibody treatment date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: b ujorz new coronavirus (sars-cov- ) treatments and vaccines are under development to combat covid- . several approaches are being used by scientists for investigation, including ( ) various small molecule approaches targeting rna polymerase, c-like protease, and rna endonuclease; and ( ) exploration of antibodies obtained from convalescent plasma from patients who have recovered from covid- . the coronavirus genome is highly prone to mutations that lead to genetic drift and escape from immune recognition; thus, it is imperative that sub-strains with different mutations are also accounted for during vaccine development. as the disease has grown to become a pandemic, b-cell and t-cell epitopes predicted from sars coronavirus have been reported. using the epitope information along with variants of the virus, we have found several variants which might cause drifts. among such variants, a>g variant (p.d g) in spike protein b-cell epitope is observed frequently in european countries, such as the netherlands, switzerland, and france, but seldom observed in china. in late , a new coronavirus, sars-cov- , causing acute respiratory distress syndrome, was first reported in wuhan, china. despite a lockdown of the city, the number of patients increased exponentially, while in parallel the virus spread across the globe. the world health organization (who) declared a pandemic on march . currently, no treatments or vaccines are scientifically proven to be effective against the virus. safe and effective vaccines for sars-cov- are urgently needed to mitigate the pandemic. to that end, a clinical trial of mrna- with full spike protein as an antigen started on march [ ] . pharmaceutical companies are currently investigating repurposed compounds from other infections as potential treatments for covid- . for instance, lopinavir and ritonavir are both hiv protease inhibitors; however, the derived treatment benefit was dubious in a lopinavir-ritonavir clinical trial that was recently reported [ ] . remdesivir, an rna polymerase inhibitor originally intended to treat ebola virus, appears to have in vitro activity against sars-cov- [ ] and preliminary clinical activity [ ] . additionally, convalescent immunoglobulins derived from recovering patients are currently being investigated as a potential treatment for the disease [ ] . until a widely available, efficient vaccine exists, these treatments are the best hope to reduce mortality. typically, surface proteins outside of the viral virion are selected for antigens so that antibodies generated from a vaccine-trained b-cell can bind to the virus for neutralization. in addition to the b-cell epitope requirement, the antigens must generate antigenic peptides, which bind to the major histocompatibility complex (mhc) molecules to be presented. by presenting a peptide, a b-cell can become stimulated by a helper t-cell and become a plasma cell to generate antibodies. a fraction of stimulated b-cells are transferred to the germinal center, where they are further enhanced from random somatic mutagenesis induced by activation-induced deaminase (aid) allowing stronger binding to the antigen. therefore, the resulting antibodies have differences in binding epitope and protein sequences in variable antibody regions. the antigens introduced as vaccines need to account for current major sub-strains to prevent potential escape from immune recognition. genetic drift takes place when the occurrence of alleles or variant forms of a gene increase or decrease over time [ ] . genetic drift is measured by the changes in allele frequencies and continues until one of two possible events occurs: the involved allele is lost by a population or the involved allele is the only allele present in a population at a particular locus. genetic drift may cause a new population to be genetically distinct from the original population. this study's objective is to interrogate currently identified sub-strains of sars-cov- and identify genetic drifts and potential immune recognition escape sites that would be integral for the development of a successful vaccine. predicted b-cell and t-cell epitopes were obtained from results of assays performed for sars-cov and sequence alignments between sars-cov and sars-cov- from the recent work by grifoni et al. [ ] . the sequence identity and similarity of spike protein between the strains was . % and . %, respectively, after running needle pairwise alignment [ ] . as shown in figure , the spike protein sequences of sars-cov and sars-cov- have high similarity in the regions of interest, which are colored in blue. for instance, in the segment ranging - , out of ( %) residues are identical, out of ( %) residues are similar, and out of ( %) residues are dissimilar. therefore, we assume that epitopes predicted from sars-cov results are reliable. in total, variant data files in general feature format (gff ) were downloaded from china's national genomics data center (ngdc) (https://bigd.big.ac.cn/ncov/release_genome?lang=en) on march . they provide the variant information from the global initiative on sharing all influenza data (gisaid) [ ] , genbank, ngdc genome warehouse, and national microbiology data center (nmdc). sample information is provided in supplementary table s . samples with hyper mutations and large gaps were considered to be of low quality and were discarded from the analysis. the gff files were processed to extract sample information, including genome accession number, geographic location, sample collection date, coordinate information, base changes, genes, amino acid changes, and variant types, and were then organized into a database. we searched for variants located within each predicted epitope and then tabulated these, as shown in table . additionally, country-based statistics of the prevalence of a>g variant (p.d g) were generated, as shown in table . twelve distinct variants were found within b-cell epitopes of spike protein (s), nucleocapsid protein (n), and membrane protein (m), respectively, as listed in table . also, twenty-one distinct variants were identified in t-cell epitopes. among the twelve variants in the b-cell epitopes, a>g variant (p.d g) in one of the epitopes in spike protein between residue and stands out, with samples in total samples. the variant is located in the middle of that epitope and the amino acid change in the a>g variant (p.d g) involves a change of large acidic residue d (aspartic acid) into small hydrophobic residue g (glycine). such large differences in both size and hydrophobicity in the middle of the epitope would compromise the binding affinity to antibodies trained by vaccines with wild-type spike protein. most of the samples with the variant were collected in europe, in particular the netherlands ( out of ), switzerland ( out of ), and france ( out of ), as shown in table . in these countries, the majority of infected patients possess the variant; therefore, vaccine design and convalescent plasma antibody treatment might require further considerations to accommodate the drift. the immunogenicity of sars-cov- proteins can be extrapolated from very close sequence homology to sars-cov- . five regions of immunodominance, including residues from to in the spike protein, have been reported from sars-cov- and % homology is observed in that region with sars-cov- . notably, the d residue is conserved between the two sars strains. a spike glycoprotein peptide encompassing residues - derived from a convalescent sars-cov- patient was successfully able to elicit humoral immune response and prevent infection in non-human primates, underscoring the immunogenic importance of this region [ ] . in addition to the netherlands, switzerland, and france, our data indicate that the d g sub-strain is frequently detected in brazil, finland, and belgium. however, given the small sample size, it is hard to ascertain whether d g is the dominant strain in these countries. a recent report corroborated our findings of high prevalence of d g in europe [ ] . within the analyzed patient cohort, the variant was first observed in epi_isl_ , collected on january , , in a sample from germany. subsequently, the variant was detected in epi_isl_ , collected on february , , in a sample from wuhan, china. notably, these two samples do not share common variants besides p.d g. it is unclear whether the variant emerged in china and disseminated to europe or this variant emerged independently in china and europe. intriguingly, in our data the d g variant was detected only in out of chinese patients analyzed. the reports of reinfection and relapse of covid- disease suggest that eliciting an effective and lasting host immune response to facilitate viral clearance can be a challenge, at least in some patients. as viruses mutate during replication, host antibodies generated in the earlier phase of the infection may not be as effective later on [ ] . while the precise effects of glycine in lieu of aspartic acid in residue on immunogenicity and virus neutralization potential are currently unknown, it may confer conformational changes, which may affect binding. although a single amino acid change may not affect binding to antibodies, a new variant in the same epitope may emerge as the viral genome evolves. in fact, we observed p.v l in our data set, suggesting that a second or a third hit could occur in the same epitope while a vaccine is being developed. therefore, it is imperative that currently known variants of covid- , as well as new variants that may occur as the viral genome mutates, are carefully considered in the design of a vaccine. the highly prevalent a>g (p.d g) variant in the european population may cause antigenic drift, resulting in vaccine mismatches that offer little protection to that group of patients. innovative vaccine design methods, including using highly conserved internal epitopes, recombinant proteins spanning epitopes, or pooling multiple vaccines, will be required to combat the inherent antigenic drift. consideration of drift variants in sars-cov- will offer cross-protection across different sub-strains and obviate the need for reformulation of the vaccine for each distinct sub-strain. additionally, consideration of drift variants in convalescent immunoglobulin treatment strategies will also result in better patient outcome. in conclusion, consideration of antigenic drift in the different sub-strains of the virus is imperative in the design of a "one size fits all" universal vaccine to offer protection against the deadliest outbreak in this century. immunogenicity study of -ncov vaccine (mrna- ) to prevent sars-cov- infection a trial of lopinavir-ritonavir in adults hospitalized with severe covid- remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro compassionate use of remdesivir for patients with severe covid- convalescent plasma as a potential therapy for covid- genetic drift, selection and the evolution of the mutation rate a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- a general method applicable to the search for similarities in the amino acid sequence of two proteins global initiative on sharing all influenza data-from vision to reality immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates patient-derived mutations impact pathogenicity of sars-cov- . medrxiv covid- infection: the perspectives on immune responses key: cord- - cakgmsj authors: oxford, alexandra e.; halla, fabio; robertson, evan b.; morrison, brad e. title: endothelial cell contributions to covid- date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: cakgmsj understanding of the clinical, histological and molecular features of the novel coronavirus (severe acute respiratory syndrome coronavirus (sars-cov- )) has remained elusive. coronavirus disease (covid- ) caused by this virus has unusual clinical presentation with regard to other related coronaviruses. recent reports suggest that sars-cov- , unlike other related viruses, infects and replicates within endothelial cells, which may explain a significant portion of the observed clinical pathology. likewise, mounting evidence associates vascular and endothelial cell dysfunction with increased mortality. this review focuses on understanding how endothelial cell pathology is caused by sars-cov- at the molecular and cellular levels and how these events relate to covid- . a detailed examination of current knowledge regarding canonical inflammatory reaction pathways as well as alteration of endothelial cell-derived exosomes and transdifferentiation by sars-cov- is included in this assessment. additionally, given an understanding of endothelial contributions to covid- , potential therapeutic aims are discussed, particularly as would affect endothelial function and pathology. the new strain of coronavirus (severe acute respiratory syndrome coronavirus (sars-cov- )) has created a global health crisis that is unlike any pandemic witnessed since the spanish flu in . it is now reported that % of coronavirus disease- (covid- )-related deaths come from cardiovascular complications, with most of the remaining % attributed to respiratory failure separate from myocardial injury and unknown causes [ , ] . many cases of sars-cov- infection are asymptomatic, causing subclinical or mild symptoms [ ] . however, immunologic overdrive has been strikingly common in those who progressed to a later stage of coronavirus disease , presenting some similar clinical findings as those associated with macrophage activation syndrome (mas) [ ] . the associated increase in serum cytokines (termed a "cytokine storm") progresses to cause acute respiratory distress syndrome (ards). in addition, endothelial cell infection promotes the formation of thrombus associated with an elevated neutrophil count [ ] . it is important to note that though endothelial infection has been validated, the virus remains highly replicative in the throat and upper respiratory tract (as is symptomatically consistent with respiratory viruses of this type), and has been found to be inactive (un-replicative) within the blood [ ] . while the cardiovascular complications in patients with covid- are becoming more apparent, the underlying mechanisms leading to these issues are not fully understood. this review will focus on the concept of endothelial cell infection and dysfunction as an active driver of covid- , which begins as a respiratory illness, with vascular pathology contributing significantly to the most negative patient outcomes. to better explain the varying symptoms and severities that arise from sars-cov- infection, the stepwise progression of covid- is utilized. disease progression consists of three phases: the early infection phase, the pulmonary phase, and the hyperinflammation phase [ ] . initial infection by sars-cov- begins the incubation period (during which infectivity may peak), and continues until the onset of symptom presentation [ ] . beginning at the point of initial infection, and continuing through the incubation period and infectious course, sars-cov- binds to the angiotensin-converting enzyme (ace ) receptor on human lung epithelium. as described in the following, ace is found on a variety of other cell types, including the vascular endothelium and intestinal epithelium (see table ). the widespread expression patterns of this receptor may contribute to the varied, systemic symptoms observed. additionally, infection by sars-cov- may result in the disease state of covid- . the pulmonary phase (phase ii) of covid- is associated with an established pulmonary disease, caused by infection of local epithelium and viral multiplication. this stage is commonly associated with viral pneumonia, cough, and fever. stage ii may be further subdivided into stages iia (pulmonary disease without hypoxia) and iib (pulmonary disease with hypoxia) [ ] . the hyperinflammation phase is when additional systemic, inflammatory symptoms coincide with sars-cov- infection and antiviral type i and iii interferon production [ ] . this phase of the disease aligns with the idea that covid- is associated with severe endothelial cell infection and inflammation leading to an increase in cardiovascular complications. it is important to note that disease severity may be highly dependent on patient characteristics, including incidences of comorbidity (particularly of inflammatory conditions such as cancer and diabetes) [ ] . a "cytokine storm" is defined as an excessive immune response towards an external stimulus. cytokines play an essential role in the innate immune system against viral infections. however, an excessive first-line response to viruses can sometimes become more harmful than beneficial [ ] . cytokine storms progress rapidly in a very complex manner, resulting in a high mortality rate. huang et al. [ ] showed a history of increased proinflammatory cytokines in sars-cov- -related viruses (including sars and middle east respiratory syndrome-cov (mers-cov)), with these patients displaying increases in il- β, il- , il- , ifnγ, ip , and mcp similar to those found in mas. similarly, the study reported that covid- patients also had an increase in proinflammatory cytokines, leading to the activation of t helper cell responses. however, these cytokines led to a further increase in additional cytokines (gcsf, ip , mcp , mip a, and tnfα), which were associated with increased icu admission and disease severity. essentially, the release of copious amounts of pro-inflammatory cytokines induces and sustains the systemic inflammatory response in severe covid- . following excessive cytokine release, sars-cov- infections lead to the activation of additional immune cells that harm healthy cells and disturb normal physiological functions [ , ] . for example, the release of interferons i and iii, though antiviral in function, may cause significant damage to the lung epithelium following extended exposure, as may be observed in the cytokine storm. this tissue damage may worsen clinical outcomes [ ] . an increased neutrophil to lymphocyte ratio, serum levels of inflammatory cytokines and chemokines, and inflammatory markers in blood have been associated with increased disease severity and death. however, the observed neutrophil insurgence is likely not an effective defense against viral pathogens and could be suppressing t cell-mediated antiviral activities [ ] . in addition, increased inflammatory markers in blood of covid- patients (including c-reactive protein, ferritin, and d-dimers) have been reported, suggesting that there is hyperactivity of the coagulation system [ , , ] . an elevated neutrophil count in covid- patients suggests the pathogenic role of neutrophil extracellular traps (nets), which have been found to contribute to thrombosis in other similar pandemic viruses h n , sars-cov, and mers-cov [ ] . nets function to disarm pathogens through the use of molecular complexes targeting dna and promoting its destruction. common proteins such as neutrophil elastase, cathepsin g, and specific histones are associated with nets [ ] . nets are commonly understood as the immune system's first line of defense towards infection, either by the secretion of antimicrobial substances or the engulfment of the pathogen. however, it has shown that an overactive netosis state can lead to more harm than help to the host. moreover, nets are responsible for initiating thrombotic events in arteries, veins, and microvasculature by activating the contact pathway of coagulation via electrostatic interactions [ ] and the intrinsic pathway by presenting tissue factor [ , ] . serum samples in patients with covid- display large amounts of net remnants including mpo-dna complex, citrullinated histone h , and cell-free dna, promoting venous thrombosis and inflammation of the vessel wall [ ] . the serum of covid- patients seems to have contents which stimulate netosis; the exact contents are yet to be understood and encourages more research concerning nets in covid- infected individuals. when added to control neutrophils, covid- sera are potent stimulators of netosis [ ] . the presence of net remnants, and the activation of netosis via covid- sera, may suggest that patients suffer from a pro-netosis stage. this stage may contribute to virally damaged epithelial cells, activated platelets, activated endothelial cells, and excessive release of inflammatory cytokines. consequently, inhibitors of nets are of interest as a potential treatment to reduce the severity of sars-cov- infection [ ] . table . cell type and tissue distribution of ace and transmembrane serine protease (tmprss ) protein expression. detection of ace or tmprss is indicated by a plus (+) and unsuccessful detection is also shown (-). infection of blood vessel endothelium leading to systemic circulation of the virus is virtually unheard of in previous viral outbreaks. neither taxonomic sister virus sars, nor additional pandemic virus h n (influenza a of the family orthomyxoviridae, the cause of both the influenza and the swine flu pandemics), showed such endothelial disruption [ , ] . nevertheless, sars-cov- exhibits an ace-mediated mechanism of endothelial infection. the coronavirus spike protein attaches to ace to enter the host cell [ ] . this receptor is commonly found on epithelial type ii cells, renal, intestinal, cardiac, and endothelial cells [ , ] . as analyzed by varga et al., the distribution of this receptor is largely consistent with the clinical findings for target organ failure (e.g., cardiac and renal) but does not fully correlate with the broad symptomatology [ ] . this study found the presence of viral bodies and host inflammatory cells in several organs, suggesting that sars-cov- leads to the induction of endotheliitis [ ] . endothelial cell infection that proceeds via ace shows how sars-cov- can replicate into a wide range of cells, which may explain some of the clinical symptoms found in covid- patients. however, how tissues with restricted serum, and thereby viral particle, access become infected is unknown. following the entry of sars-cov- into the host cell, host ribosomes bind to and facilitate the translation of the positive-sense viral rna genome. translation of the viral genome results in the production of polyproteins capable of forming the replication-transcription complex subsequent to processing by viral protease activity. the replication-transcription complex then acts to produce positive-sense genomic rna for future packaging, as well as mrna transcripts for viral structural and accessory proteins (nucleocapsid, envelope, membrane, and spike proteins). these viral mrna transcripts are then trafficked to, and translated by, the endoplasmic reticulum of the host cell. the newly produced viral proteins, as well as the genomic rna, are then packaged by the golgi apparatus and exocytosed as a fully functional viral particle [ , ] . sars-cov- has been demonstrated to use the ace receptor on human endothelial cells for cell entry. in fact, it has been shown that only coronaviruses which express the sars-cov spike protein can utilize the ace receptor [ ] . this was originally reported in a study conducted using the sars-cov permissive african green monkey kidney cell line vero e . vero e have been subsequently used extensively to characterize this family of viruses and the molecular features of sars-cov spike protein-ace receptor interaction [ ] [ ] [ ] . sars-cov and sars-cov- both utilize a conserved spike protein (also called the s protein). in sars-cov, the s protein has two distinct subunits-s and s . s was determined to have a high-affinity association with respective receptors in particular the ace receptor [ , ] . using a carboxy terminally tagged form of the s domain as bait, vero e cell lysate was incubated and s immunoprecipitated. after gel purification, interacting proteins were identified by mass spectrometry. three proteins were identified as putative s interactors with ace ( fragments which comprised % of human ace ) being the strongest cell surface receptor candidate. this finding piqued the interest of researchers studying sars as the tissue distribution and localization of ace make it an appropriate receptor for sars-cov [ ] . this work was then validated in a vero e cell line cloned from human-lung endothelial cells where ace -s binding was confirmed using blocking antibodies [ ] . in a separate study, transmembrane serine protease (tmprss ) was shown to activate the viral s protein which affects viral entry but not replication [ ] . tmprss accomplishes this by cleaving the glycoprotein hemaglutenin which is necessary for the fusion of viral and host cell membranes. this forces a conformational change after endocytosis and allows for viral invasion into the host cell cytoplasm [ ] . betacoronaviruses (such as sars-cov, sars-cov- , and mers) have only two distinct methods of entering endothelial cells. the first is through the endosomal pathway. it has been shown that mers, which binds to and enters cells in a similar method (though with the utilization of the dipeptidyl peptidase- receptor) as sars, can bind to cathepsin l in the endosome in the absence of tmprss and facilitate entry into the host cell cytoplasm [ ] . however, this method results in a greater than -fold reduction in infectivity compared with tmprss -mediated entry [ ] . notably, tmprss and ace are found in their highest concentrations in endothelial cells located in the kidneys, as well as the lung and heart, where sars and mers coronaviruses produce the most life-threatening damage [ , ] . ace contains two virus-binding locations which provide a tight bond between this protein and sars-cov- [ , ] . sars-cov- forms a larger binding surface with ace than with sars-cov, increasing the binding affinity. binding hotspots within ace are designated as hotspot and hotspot , which likely form weak salt bridges (electrostatic interaction between amino acid side chains at secondary and tertiary levels of organization) between amino acids in the native protein that are then broken upon binding of the virus spike protein [ ] . hotspot breaks and forms two hydrogen bonds with the virus, whereas hotspot breaks the weak salt bridge and forms a single hydrogen bond with the main chain of sars-cov- while maintaining one weak salt bridge. both hotspots exhibit stronger and more stable binding through the sars-cov- -ace interface than the native ace intramolecular interactions [ , ] . while ace and tmprss are the most well-characterized viral entry factors, it is important to note that these proteins are not the only ones that have been reported to facilitate this process [ ] . sars-cov- portrays an evolutionary strategy that distinguishes it from other zoonotic origin strains, illustrating the possibility of human epithelial sodium channel alpha subunit (enac-alpha) mimicry in covid- . enac is associated with the renin-angiotensin-aldosterone system (raas) and regulates sodium ion concentration and water homeostasis in distal lung airway tissue [ ] . contrary to other areas of the human body, enac sodium transport is not involved in direct salt balance, but rather in maintaining appropriate hydration of the inner surface epithelial layer [ ] . therefore, the key role of enac in alveolar tissue is to control the volume of airway-surface liquids. in a study performed by stoner et al., enac was evaluated in flat epithelia from toad urinary bladder and frog skin. the study portrayed the great specificity of enac for sodium; however, little was known about the exact mechanism of activation [ ] . importantly, enac needs to be proteolytically activated to function in controlling fluid reabsorption in the airways [ ] . it is now reported that furin cleaves enac between arginine and serine residues in the th and th position (rsar/sass) to become activated [ ] . when compared to sars-cov strains, a unique sequence insertion at the s /s site of the spike protein is found in sars-cov- [ ] . sars-cov- is coated with spike glycoprotein (s) that requires proteolytic cleavage for activation [ ] . after interaction of s with the ace receptor, sars-cov- entry into host cells occurs in two steps. once the s subunit of the s protein engages the ace receptor of the host cell, viral entry into the host cell is facilitated via s /s cleavage [ ] . the s site is then cleaved to allow the viral-host cell membranes to fuse [ ] . the ability of the virus to facilitate the use of host mechanisms to cleave at s /s furthers belief that sars-cov- has developed an evolutionarily beneficial insertion to achieve increased host infection. walls et al. show that the furin-like cleavage motifs at the s /s site are present in other viruses, but the exact mimicry of enac-alpha is unique to sars-cov- [ ] . as explained earlier, the ace receptors are present in a variety of different locations ranging from alveolar to intestinal cells [ ] . remarkably, enac-alpha are present in apical membranes of similar cells as those of ace [ , , ] leading to the hypothesis that sars-cov- leverages the protease network that is responsible for the cleavage of enac, which could subsequently lead to excessive sodium and liquid buildup in the lungs explaining clinical findings of ards. anand et al. found that the viral receptor ace , enac-alpha, and furin were coexpressed in the colon (immature enterocytes) and pancreas (ductal cells) of human tissue and tongue (keratinocytes) of mouse cells [ ] . moreover, pcsk and pcsk are expressed in the intestinal, pancreas, nasal cavity, tracheal epithelial cells, and podocytes of the kidney, similarly to that of the ace receptor [ ] . thus far, we have discussed the viral mechanisms of sars-cov- and resultant covid- sequelae as they relate to endotheliitis and endothelial cell infection mediated by viral spike protein-ace interaction. we now turn our focus towards potential therapies that can be derived from an understanding of infection and disease progression as fundamentally concerned with endothelial cells and their subsequent pathophysiology. when considering potential treatments for viral infections of this nature, targets may vary based upon treatment intention. the following discussion will concern both the prevention of cellular infection and symptomatic amelioration following infection. prevention of initial cellular entry and infection should be considered separately from ameliorative therapies, as it may negate the need for further symptomatic treatment. as discussed previously, sars-cov- finds its receptor in the highly expressed ace [ , ] . this transmembrane receptor functions primarily in the raas, as part of the body's innate homeostatic balance, acting to maintain fluid balance and blood pressure [ , ] . an understanding of this initial viral binding event allows for the proposition of recombinant ace as a competitor against viral binding to the endogenous receptor. if successful, recombinant soluble receptor domains serve to "distract" the virus from endogenous ace , thereby preventing or reducing cellular infection [ ] . this approach has seen initial success in an april study of endothelial organoids. using an anatomically and physiologically analogous endothelial tissue model, monteil et al. was able to show a significant decrease ( - -fold decrease) in viral rna upon treatment with human recombinant soluble ace (hrsace ) at doses of - µg/ml [ ] . the validity of this approach is bolstered by the recent successful completion of phase i and ii safety trials of clinical grade hrsace (gsk ) in the treatment of ards. it is important to note that the trial of gsk concerned itself with the ability of recombinant ace to attenuate lung injury through its innate physiological actions, and not as a competitor for viral binding [ , ] . however, the safety of the drug as demonstrated in phase ii trials, considered in conjunction with the proven ability to reduce viral load in an endothelial model, points to potential applications in this additional therapeutic role. the viral spike protein itself may also present a therapeutic target. published in , in response to the current pandemic, a study conducted by wang et al. has confirmed the first known human monoclonal antibody against sars-cov- . human monoclonal antibody d is a neutralizing antibody against the s subunit of the spike protein, and has been shown effective in the inhibition of cellular infection of veroe cells in vitro. additionally, as d shows cross-reactivity and neutralization of the s in both sars-cov and sars-cov- , wang et al. propose that this antibody may be effective in the treatment of future coronavirus strains. it is important to note that the specific mechanism of action of d remains unknown, and is thought to be unrelated to receptor-binding interference, which serves to further differentiate d from other similar potential therapeutics [ ] . in addition to prevention of initial ace binding, our previous discussion of sars-cov- activation allows for investigation into additional targets. of these targets, the most prescient appears to be the tmprss [ , ] . as of june , tmprss inhibitor camostat mesylate is undergoing phase ii trials [ ] . this is a placebo-controlled, double-blinded study, with an intervention group of mg oral dosing three times daily for seven days in patients over years of age [ ] . the primary outcome measure of this study is viral load as assessed by qpcr from days to . secondary measures include changes in viral load at different time points after treatment, changes in symptom severity as assessed by the covid- daily self-score tool, changes in infection status, body temperature, and symptom frequency [ ] . investigation into camostat mesylate for covid- is based upon previous studies showing the efficacy of the drug candidate in sars-cov- [ ] these studies were primarily interested in the epithelial cells of the lungs, and not the vasculature [ ] . however, as direct endothelial infection relies upon the action of this serine protease similarly to epithelial cell infection, it remains possible that camostat mesylate may be efficacious in the prevention of endothelial cell infection. however, additional research into the potential effects on the vasculature is needed. another successful strategy to inhibit sars-cov- has been through the use of remdesivir [ ] . this drug has an adenosine nucleoside triphosphate analog that interferes with viral rna polymerase and is classified as a delayed chain terminator [ ] . remdesivir has exhibited promise in the clinic in early studies resulting in a median recovery time of days versus days for placebo [ ] . in addition, mortality was reduced from . % to . % by remdesivir [ ] . this drug has shown promising results. however, the morbidity and mortality rate is still cause for concern. therapeutic approaches concerned with the abruption of viral replication processes (including remdesivir) may present relevant targets for future research not reliant on ace and tmprss . these targets may include those relating to transcription, metabolism, and biomolecule synthesis [ ] . endothelial pathology as a result of covid- progression can be roughly broken into three subcategories: coagulopathy, inflammation (including cytokine storm), and edema. as such, potential endothelial concerned treatments for covid- may be subdivided similarly. though both coagulative and edemic pathologies have their roots in inflammation, there are additional considerations that warrant the division as follows. a primary vascular concern of covid- appears to be that of rampantly dysregulated thrombotic events [ , ] . these clotting abnormalities have resulted in stroke and disseminated intravascular coagulation (dic) in severe covid- patients, and may represent a significant cause of mortality [ , ] . though blood thinners, such as heparin, have been shown to reduce mortality in severe covid- cases, this acts only after the development of the clotting pathology, and not to prevent the dysregulation of the clotting cascade [ , ] . to prevent aberrant thrombosis, it is helpful to investigate the endothelial pathology that allows for upregulation of the clotting cascade. in covid- , sars-cov- infection of endothelial cells and subsequent cell death, allows for pathological exposure of blood to the thrombogenic basement membrane of the blood vessel which contains collagen [ ] [ ] [ ] . collagen exposure activates the clotting cascade. as such, actors within the clotting cascade present potential therapeutic targets. of these targets, p-selectin may be appropriate, as antibodies to this cell adhesion molecule have been used to prevent thrombosis [ ] . additionally, pro-inflammatory cytokines interleukin beta (il- β) and tumor necrosis factor (tnf), are integral to the clotting cascade as they function cyclically to trigger platelet production and the upregulation of additional adhesion molecules [ , ] . as such, antibodies against these cytokines may help to temper the hypercoagulative state and prevent fatal dic. as this clotting pathology is often inflammatory in nature, many of the proposed therapies in the treatment of the cytokine storm may be efficacious in the treatment of the clotting abnormalities and will be discussed in more detail in the following section. a cytokine storm refers to a hyperinflammatory condition related to the uncontrolled release of cytokines [ ] . this condition is seen as controlled inflammation (pain, heat, redness, and swelling) that occurs as a healing response to injury and infection, becomes overactive and systemic as opposed to strictly regulated and locally active [ ] . in covid- , the cytokine storm is seen to worsen the inflammatory symptoms of the disease, including the aforementioned clotting pathologies and subsequent multi-system organ damage [ , ] . therapeutic candidates to treat this inflammatory condition include corticosteroids and monoclonal antibodies, among others [ , ] . an ongoing trial, currently in phase iii, tests the effects of cytokine-targeting medications in individual and combination therapy [ ] . this study tests il- antibodies tocilizumab (anti il- r) and siltuximab (anti il- ), as well as il- β receptor antagonist anakinra. intervention groups include individual treatment with each of the three pharmaceuticals, as well as combination therapy with anakinra and either tocilizumab or siltuximab. the primary outcome measure of this trial is time to clinical improvement as assessed by hospital discharge or improvement in clinical indicators. secondary outcome measures include, among others, incidences of adverse events, mean change in oxygenation, and mean change in clinical scores [ ] . the successful use of anti-interleukin drugs to treat the inflammatory symptoms seen in severe covid- would have marked effects on endothelial pathology as these cells are highly responsive to cytokine signaling [ ] . this ameliorative effect would be seen in the downregulation of the prothrombotic cascades as initiated by cytokine signaling, as well as decreased inflammatory leukocytic cascades. as mentioned, corticosteroids may be effective in the treatment of inflammatory pathology. a randomized controlled trial conducted by the recovery collaborative group tests the efficacy of dexamethasone in the treatment of hospitalized covid- patients. the primary outcome measure of this study is mortality at days. patients received oral or intravenous dosing at mg/day or continued care without corticosteroid intervention. this study found that dexamethasone was effective in lowering mortality at days when compared to control patients receiving mechanical ventilation or supplemental oxygen. however, the mortality rate among patients included in the intervention group was not found to be lower when compared to hospitalized patients not receiving ventilation or external oxygen [ ] . edema, or swelling, is a fundamental inflammatory response that occurs primarily due to vascular leakage. similar to other hallmark inflammatory responses of pain, redness, and heat; edema occurs primarily as a result of endothelial cell function or dysfunction [ ] . as reviewed by teuwin et al., in covid- , the overactive edemic response may occur due to a weakening of the endothelium as a result of cell death secondary to direct endothelial infection [ ] . additionally, the actions of cytokines il- β and tnf weaken intercellular tight junctions and result in the overproduction of hyaluronic acid (ha) in the extracellular matrix of endothelial cells [ ] . increases in vascular leakage due to cell death following direct endothelial infection may be lessened with the use of antiviral medications. these pharmaceuticals may prevent intracellular viral replication and resultant cell death [ , ] . a potential agent in this approach is ribavirin, which may inhibit viral rna polymerase and subsequent viral replication. ribavirin was tested in both sars and mers and found significant safety concerns limiting its efficacy, though as of now, suffers from a dearth of sars-cov- research [ ] [ ] [ ] . additionally, previous clinical trials showing toxicity related to necessary dosage were concerned mainly with the complete cessation of viral activity, and not with symptomatic amelioration. lower dosages that were shown to be ineffective when used as the sole antiviral treatment in sars, may be effective in combination therapy or in purely symptomatic aims [ ] [ ] [ ] . the weakening of the endothelium may also occur as a result of increased contractility and the loosening of intercellular tight junctions due to the inflammatory properties of cytokines [ , ] . again, edema may be treated similarly to cytokine storm pathology as the causative agents of inflammation are cytokines such as il- [ ] . as mentioned, edema may be inflammatory in origin. in covid- , the actions of cytokines il- β and tnf can be seen to upregulate ha-synthase- , resulting in increased ha concentrations in the extracellular matrix of endothelial cells. this leads to marked water retention and vascular leakage and may therefore contribute to severe respiratory pathology and ards [ , ] . potential therapeutics in this aim include anti-inflammatory drugs against il- β as discussed previously. through this review, we have discussed the mechanisms, pathologies, and proposed treatments of sars-cov- infection with a focus on the effects of direct endothelial infection and dysfunction. though the vasculature itself appears to play a large role in the pathogenesis of the disease, there is additional merit in a discussion of non-vascular infection and pathology that occurs as a function of initial endothelial infection. the following will discuss potential endothelial-derived mechanisms of extravascular infection and pathology. the healthy endothelium has roles beyond those of a vascular nature. in uninfected cells, these functions allow for and maintain the health of endothelial tissue, as well as the overall health of the organism [ , ] . however, upon endothelial infection, innate processes may become factors driving further pathogenesis. the transdifferentiative capacity of endothelial cells, as well as the innate exocytotic machinery of these cells, may be such infectious considerations [ ] [ ] [ ] . transdifferentiation describes the process whereby a mature cell undergoes transformation into another cell type [ , ] . endothelial cells retain this ability into maturity and have been found to assume multiple extravascular phenotypes, as shown by fluorescent fate mapping [ , ] . evidence suggests that descendent cell types may include cerebellar granule neurons, pancreatic acinar and β cells, duodenal and ileal crypt cells, ependymal cells, and skeletal myocytes among others [ ] . additionally, genetic fate mapping in rhabdomyosarcoma has given further credence to the transdifferentiative role of endothelial progenitors in disease pathogenesis [ ] . this propensity for transdifferentiation does not, on its own, imply a further infectious route for sars-cov- from endothelium to descendent cell. however, when considered in conjunction with the extravascular labeling that results from intravenous adeno-associated virus injection (a common labeling method in model systems), relevant conclusions may be drawn. notably, if the ability of intravenous adenovirus to label extravascular cells is due to endothelial transdifferentiation, then this labeling (adenovirus infection in descendant cells upon transdifferentiation) may allow for the inference of a similar route for sars-cov- infection [ ] [ ] [ ] . in other words, the transdifferentiation of endothelial cells infected with sars-cov- may result in the creation of extravascular descendent cells that are similarly infected (much in the same way that adenovirus injection results in extravascular labeling), thereby furthering systemic sars-cov- infection. this analysis should not be interpreted to imply significant similarity between sars-cov- and adenovirus beyond that as a model of endothelial infection and viral propagation subsequent to transdifferentiation. the final consideration of endothelial transdifferentiation is that of process and function. due to the limited conditions under which transdifferentiation successfully occurs, endothelial sars-cov- infection may alter or disrupt this process [ ] . alternatively, successful transdifferentiation of an infected cell may result in a descendent cell with pathologies independent from those occurring due to an active infection, though more research is needed. in addition to transdifferentiation, exocytosis is a possible route for endothelial-derived extravascular sars-cov- infection. similar to transdifferentiation, exocytosis can be considered as an innate function of healthy endothelial cells, which may become a driver of further infection and pathology following viral infection with sars-cov- [ , , ] . as reviewed by hromada et al., non-pathological endothelial extracellular vesicles have functions relating to immunity, cell-to-cell communication, and tissue regeneration [ ] . as discussed, sars-cov- is a membrane-bound virus that binds to ace [ , ] . viral binding and enzymatic activities support viral membrane fusion with the endothelial cell membrane, thereby allowing viral rnas, transcription factors, and additional viral proteins to enter the endothelial cell and begin the viral replicative processes that signify an active infection [ ] . exocytotic mechanisms may persist during a viral infection, and thus contribute to pathogenesis. in this case, a portion of the exocytosed cell membrane in any vesicle may be of viral origin, and thus may contain the spike proteins needed for viral binding [ , ] . additionally, during exocytosis of the extracellular vesicle, intracellular endothelial cell contents may be packaged within the vesicle. the inclusion of these components within an exocytosed vesicle is a non-pathogenic mechanism that allows for cell-to-cell communication and transport [ ] . however, in the case of an active sars-cov- infection, viral components within the endothelial cell may also be packaged within the vesicle [ ] . as a result, there is potential for the creation of an endothelial extracellular vesicle possessing membrane-bound spike proteins externally, as well as viral rna and transcription factors internally [ , ] . this may allow for an endothelial extracellular vesicle to effectively become a virus-like particle capable of transmitting sars-cov- to other vascular and extravascular cells. additionally, uninfected endothelial cells may play a role in increasing susceptibility to sars-cov- infection. this increased susceptibility may occur due to the exocytosis and subsequent fusion of endothelial vesicles possessing ace within their membrane [ , ] . conferred sars-cov- susceptibility may subject the recipient cells to the viral pathology upon infection. combating the covid- pandemic will require a comprehensive understanding of the sars-cov- pathology caused at the molecular and cellular levels. given the high vaccine failure rate for many common seasonal viruses, researchers should continue to explore both vaccine approaches, and additional antiviral strategies. in this review, we have focused our attention on endothelial cells as an emerging covid- contributor and potential therapeutic target. this review has covered the clinical covid- manifestations that could be linked to endothelial cell dysfunction, the molecular biology of sars-cov- entry into ace -expressing endothelial cells, immune reactions that 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emergence of novel coronavirus -ncov: need for rapid vaccine and biologics development date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: gu mt zp novel coronavirus ( -ncov) is an emerging pathogen that was first identified in wuhan, china in late december . this virus is responsible for the ongoing outbreak that causes severe respiratory illness and pneumonia-like infection in humans. due to the increasing number of cases in china and outside china, the who declared coronavirus as a global health emergency. nearly , cases were reported and at least other countries or territories have reported coronavirus cases as early on as february. inter-human transmission was reported in a few countries, including the united states. neither an effective anti-viral nor a vaccine is currently available to treat this infection. as the virus is a newly emerging pathogen, many questions remain unanswered regarding the virus’s reservoirs, pathogenesis, transmissibility, and much more is unknown. the collaborative efforts of researchers are needed to fill the knowledge gaps about this new virus, to develop the proper diagnostic tools, and effective treatment to combat this infection. recent advancements in plant biotechnology proved that plants have the ability to produce vaccines or biopharmaceuticals rapidly in a short time. in this review, the outbreak of -ncov in china, the need for rapid vaccine development, and the potential of a plant system for biopharmaceutical development are discussed. the global health system consists of a network of organizations, including many private and public health sectors operating at different regional or global levels that have developed a stringent system that can provide effective protection to humans against emerging and re-emerging diseases. though mortality associated with various infectious diseases have reduced in recent years and global life expectancy has increased in many parts of the world, infectious disease threats still remain one of the major global challenges and concerns even now [ ] . the global health system is often confronted by emerging pathogens responsible for expanding an array of infectious diseases such as zika, chikungunya, ebola, nipah, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and influenza. the emergence of the novel coronavirus ( -ncov) has recently added to the list of problematic emerging pathogens in the st century, which was suspected to originate from the persons exposed to a seafood or wet market in wuhan, hubei province, china, suggesting animal-to-human transmission [ , ] . this virus strain is previously unknown and was reported to infect humans for the first time. the virus continues to expand rapidly throughout the world. many confirmed and susceptible cases have been identified in wuhan, china, and exported cases have also been reported in neighboring countries including thailand, japan, korea, taiwan, and other countries including the united states, canada, and european countries, which proves that the virus has the potential for quick dissemination across borders. in response to the rapid spread of the virus, many countries have tightened their border security, investigating people showing symptoms, and have taken necessary emergency steps to control its spread. due to the increasing number of cases in china and other countries, the who has declared the -ncov outbreak a global health emergency of international concern on january [ ] . coronaviruses (covs) belongs to the family coronaviridae, subfamily coronavirinae, and the order nidovirales. they are classified into four genera such as alphacoronavirus and betacoronavirus, both of which infect mammals, whereas gammacoronavirus infect avian species, and deltacoronavirus infect both mammalian and avian species. it is a large enveloped virus with a positive sense, single-stranded rna genome of about to kb that is distributed broadly among birds, humans, and other mammals such as camels, bats, mice, dogs, and cats [ ] . the genome is surrounded by a helical capsid and an envelope; the spike protein forms large protrusions in the envelope in the shape of a crown, which gives the virus a coronal appearance. the word 'corona' in latin means crown [ , ] . human coronaviruses (hcovs) are a major group of coronaviruses that are known respiratory pathogens associated with respiratory and intestinal infections of varying severity, including the common cold, pneumonia, and bronchiolitis. human coronaviruses such as hcov- e, oc , nl , and hku , usually cause mild infection in humans. however, the onset of betacoronaviruses, severe acute respiratory syndrome coronavirus (sars-cov) outbreak in guangdong province, china in , and the middle east respiratory syndrome coronavirus (mers-cov) disease outbreak in in the middle east resulted in high pathogenicity in humans, which demonstrated the lethality of virus when they cross the species barrier from animals to humans. both viruses are believed to be originated from bats and subsequently transmitted to humans [ ] . hcovs has evolved rapidly in recent years due to mutation, high nucleotide substitution rates, its ability to establish infection in a new host, and cross-species transmission [ ] . in december , china detected many cases of viral pneumonia-like disease similar to sars that were confirmed to be caused by novel betacoronavirus, provisionally called novel coronavirus ( -ncov). since then, the novel coronavirus outbreak has raised attention throughout the world. although the potential cause of the disease is still unknown, initial reports predicted that the virus is possibly of zoonotic origin. -ncov is the causative agent for severe respiratory infection in humans termed as novel coronavirus-infected pneumonia (ncip) [ ] . ncov is the third known coronavirus that causes fatal respiratory diseases in humans after highly pathogenic viruses sars-cov and mers-cov. chinese researchers isolated the novel coronavirus from the infected patient in early . as the virus is closely related to other bat coronaviruses, it is suspected that the bats are the primary reservoir for the virus. however, it is still unclear that, if the virus transmitted to humans directly from the bats or whether through an intermediate host. detailed understanding of the enzootic patterns of the virus, its evolution, and surveillance are essential to control the disease and possibly to prevent the future epidemics of similar viruses. the explosive outbreak of -ncov in china has been spread rapidly in many countries, probably by human movement and travel. the geographical distribution of the virus has been increasing since the outbreak. in early february , thousands of cases were confirmed in china and more than cases were reported outside of china, and numbers of cases are escalating daily in all the provinces in china and other countries. the virus has transmitted rapidly and many cases were reported globally. as of february , , nearly , affected cases and fatalities were reported in china. most of the confirmed cases were from the wuhan city in hubei province. outside china, other cases were confirmed globally, and one death was reported in philippines. in singapore, cases were confirmed, in thailand, in japan, in korea, each in australia and malaysia, in united states, and some cases were reported in germany, france, vietnam, uae, canada, india, philippines, italy, uk, russia, nepal, sri lanka, cambodia, belgium, finland, sweden, and spain as on early february [ ] . chinese health authorities, who, and most of the countries have responded fast, and immediate actions were taken to contain the virus. all the countries have implemented stringent border control, screening travelers for possible infection, and travel restrictions in order to prevent its spread. the rapid spread of this infection is frightening and also it causes both mortality and financial loss, which present the global concern of this emerging disease. the transmission of -ncov is often spread from person to person through the respiratory droplets generated during coughs or sneezes from an infected person. human-to-human transmission is reported in countries such as germany, japan, vietnam, and the united states [ ] . the confirmed cases through inter-human transmission have increased the fear and panic accompanying the -ncov outbreak. it is still unknown whether the virus spreads only through human contact or if there is possible transmission through oral-fecal contact as well. the incubation time varies from - days after infection. the clinical presentation of this infection resembles sars-cov characterized with fever, dry cough, and shortness of breath in most of the cases, whereas non-respiratory symptoms such as headache, muscle ache, dyspnoea, rhinorrhoea, sneezing, sore throat, diarrhea, nausea, and vomiting are also reported in few patients. the affected persons also develop acute respiratory distress syndrome. cases with critical illness showed respiratory failure, septic shock, and organs failure, which require intensive care support [ , , ] . at this time, the knowledge about this virus is limited. new cases and mortalities are increasing daily. as a newly emerging viral infection, there is no vaccine or anti-viral therapeutics to treat human coronavirus infection till now. as of now, preventing infection is the current priority for disease control. the current protocol for infected patients is to quarantine and provide supportive management and palliative care. the best way to avoid the virus infection is to keep oneself away from infected people and the utmost personal hygienic care is essential. quarantine measures shall be taken to separate, restrict the movement of infected people, and also the normal population from the regions where there is an epidemic outbreak. the who recommended precautionary measures to the general public, such as frequently cleaning hands, wearing a face mask, avoiding close contact with the infected persons or farm animals, and avoiding consumption of raw or half-cooked meat/eggs and following good food safety practices [ ] . there is an urgent need to develop rapid diagnostic tools and vaccines or post-exposure prophylaxis to treat this infection. reliable, timely laboratory diagnosis and an effective vaccine are crucial for effective disease management and public health intervention. an effective vaccine should be affordable, and also the production platform should produce suitable vaccine candidates rapidly at low cost, especially during a disease outbreak. the advantages and disadvantages of the current expression systems for recombinant protein production are given in table . currently, plant expression system offers many advantages over other conventional systems that have the potential to tackle the production of vaccine candidates rapidly at affordable cost facilitating the global vaccination programs, especially in resource-poor nations where the vaccines are needed most [ ] . in recent years, plants are being focused more on the production of biopharmaceuticals and virus-like particles (vlp's). the technologies employed for the production of plant-made vaccines are stable nuclear expression, transient expression, and chloroplast expression. based on several vital factors like yield, quality, time, and cost, appropriate technology can be chosen for producing biopharmaceuticals. recent advancements in the plant expression strategies, especially the development of transient expression system or viral vectors, resulted in a huge increase in the protein yield that makes this plant host system, a promising system for the production of various biopharmaceutical proteins [ ] . several reports in the last two decades have enough evidence to prove that the plant produced biopharmaceuticals are as effective as the mammalian cell-based proteins and also elicit potent neutralizing antibodies, or shown therapeutic effects against the particular pathogen or infection [ ] [ ] [ ] . the use of plants for the production of recombinant proteins and biopharmaceuticals has been gaining importance since the plant produced biologic taliglucerase alfa has been commercialized in against gaucher's disease that proclaimed a new era for plant made biopharmaceutical and triggered the innovation in the field of biopharmaceuticals [ ] . furthermore, many plant-produced candidates are in the clinical pipeline close to commercialization. some of the examples of plant-produced recombinant antigens and monoclonal antibodies for infectious diseases are given in table . currently, countries, including thailand, india, japan, korea, and the european community, are majorly involved in developing plant biopharmaceuticals against several human diseases [ ] . many reports reviewed the importance of plant expression system for the rapid production of candidate vaccines and therapeutic antibodies against infectious diseases [ ] [ ] [ ] [ ] [ ] [ ] . west nile virus pe tobacco [ ] many biopharmaceutical companies have shifted their momentum towards the plant expression system by knowing its importance. various plants such as tobacco, duckweed, moss, alfalfa, and other plants have been used for the production of plant-made pharmaceuticals. in september , leaf bio, inc., the commercial partner of mapp biopharmaceutical, inc. (mapp), received fast track designation by the u.s fda for their plant-made biopharmaceutical called zmapp for the treatment of ebola virus disease. the authorization of plant-based biopharmaceutical zmapp for emergency use in the earlier ebv outbreak by fda is potentially a major breakthrough in the plant molecular farming field, as it serves as an endorsement of the technology to potential investors and grant funding agencies [ , , ] . many vaccine antigens expressed in plants are in clinical or advanced pre-clinical trials. major players in the global plant-based biologics market include plantform, ibio inc., mapp biopharmaceutical, inc., pfizer inc., ventria bioscience, medicago inc., greenovation biotech gmbh, kentucky bioprocessing, phycobiologics inc., synthon, fraunhofer ime, healthgen, planet biotechnology, and icon genetics gmbh. as plant-made biopharmaceuticals provide efficacious and cost-effective strategies to protect against emerging infectious diseases, plant expression systems can be employed for the development of vaccines against ncov. transient expression in plants can be adapted for biopharmaceutical protein production when it is necessary to produce 'rapid response vaccines' as it produces more protein in a short time. the plant-based biopharmaceutical production against -ncov will include the identification of potential epitopes and production of full-length viral surface proteins present in the envelope region or production of subunit vaccines expressing immunogenic region or chimeric proteins. the receptor-binding domain (rbd) in spike protein located on the outer membrane of coronavirus mediates receptor binding and plays a major role in virus entry into the host cell [ ] . this protein could be used as a potential vaccine candidate as it is the major target for neutralizing antibodies [ ] . hence, it could be considered to develop potential effective vaccines or therapeutics against coronavirus infection. as the virus uses angiotensin-converting enzyme (ace ), the host cell receptor for its cell entry similar like sars-cov [ ] [ ] [ ] , the monoclonal antibodies that are identified and tested to be effective against sars virus protein or specific to ace can be produced in plants and shall be tested for its efficacy against ncov. earlier reports showed several vaccines and monoclonal antibody candidates in response to sars-cov and mers-cov, which could be tested and used for passive immunotherapy for an immediate immune response [ ] [ ] [ ] . the possibility of producing a vlp based vaccine is also feasible by expressing all the structural viral proteins that can assemble into vlps in plants. the structural and functional studies of viral proteins in ncov might help in designing better vaccines and specific therapeutics. producing viral proteins in plants may further be helpful to evaluate their potential in developing diagnostic kits or the protection/therapeutic tools that can be manufactured fast in order to manage the highly infectious ncov. this will open an avenue to characterize recombinant immunogenic viral proteins and provide a proof of concept for using plants as a robust, rapid, and flexible platform for producing protein/research reagents, which are highly essential to face potential ncov outbreaks. the coronavirus outbreak has been declared a global health emergency and represents one of the greatest risks to global health, as the virus has a tendency to infect a large number of human populations, and the outbreak can cause severe medical complications with economic impact, particularly in middle-income countries where resources are limited for early diagnosis and preventive measures. human mobility, air travel, and international trade can likely increase the number of cases in other regions as well. continued surveillance along with the robust response of government agencies, medical practitioners, and researchers, is highly essential for the effective management of this emerging pathogen. public health officials need to identify the source and virus reservoir, transmission cycle, pathogenesis, inter-human transmission, and clinical manifestations, which might be helpful to develop animal models, diagnostic reagents, anti-viral therapies, and vaccines against this pathogen. as the virus emerged suddenly and became a serious global concern, there is a need for rapid vaccine development. although classical expression systems for biopharmaceutical proteins are still amenable, the development of transient expression in plants has deeply influenced the pharmaceutical sector to produce affordable vaccines and biologics rapidly at low cost. hence, the plant expression platform shall be employed for biopharmaceutical production to accelerate the fight against this deadly infectious disease. the collaborative efforts of researchers are highly desirable to use a plant expression platform for producing an efficient cost-effective vaccine to control this 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coronaviruses into the spotlight severe acute respiratory syndrome structure of sars coronavirus spike receptor-binding domain complexed with receptor angiotensin-converting enzyme is a functional receptor for the sars coronavirus receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars neutralizing human monoclonal antibodies to severe acute respiratory syndrome coronavirus: target, mechanism of action, and therapeutic potential antibodies and vaccines against middle east respiratory syndrome coronavirus recent advances in the vaccine development against middle east respiratory syndrome-coronavirus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -oyg oyx authors: li, shasha; yang, jinping; zhu, zixiang; zheng, haixue title: porcine epidemic diarrhea virus and the host innate immune response date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: oyg oyx porcine epidemic diarrhea virus (pedv), a swine enteropathogenic coronavirus (cov), is the causative agent of porcine epidemic diarrhea (ped). ped causes lethal watery diarrhea in piglets, which has led to substantial economic losses in many countries and is a great threat to the global swine industry. interferons (ifns) are major cytokines involved in host innate immune defense, which induce the expression of a broad range of antiviral effectors that help host to control and antagonize viral infections. pedv infection does not elicit a robust ifn response, and some of the mechanisms used by the virus to counteract the host innate immune response have been unraveled. pedv evades the host innate immune response by two main strategies including: ) encoding ifn antagonists to disrupt innate immune pathway, and ) hiding its viral rna to avoid the exposure of viral rna to immune sensors. this review highlights the immune evasion mechanisms employed by pedv, which provides insights for the better understanding of pedv-host interactions and developing effective vaccines and antivirals against covs. porcine epidemic diarrhea virus (pedv) is the etiological agent of porcine epidemic diarrhea (ped) that causes an acute and highly contagious enteric disease of swine characterized by vomiting, diarrhea, dehydration, and anorexia in pigs of all ages, especially resulting in severe diarrhea and high mortality rate in piglets. in serious cases, outbreak of ped even leads to a mortality rate of % in pigs [ ] [ ] [ ] . the causative agent of ped was first described in the s in england [ ] . in , an unrecognized enteric disease was reported in several european countries [ ] . the viral diarrhea was collectively designated "ped" in [ ] . endemic ped had been described in both developed and developing countries, but with a low impact on the swine industry until . in , outbreaks of ped caused by highly pathogenic variant pedv strains occurred in china, and this was immediately reported in other asian counties, causing up to % mortality in suckling piglets [ , [ ] [ ] [ ] . in april , pedv entered the united states (us) for the first time and the virulent strains spread rapidly across the us [ ]. apart from almost % mortality rate in suckling piglets, pedv infection also damaged the growth performance of finishing pigs [ ] . the highly pathogenic strains of pedv spread worldwide, resulting in serious problems to the swine industry and substantial economic losses [ , , , ] . vaccination used to be the main strategy to prevent and control the rate of pedv infection [ ] , however, the current available pedv vaccines cannot provide complete protection for the pigs affected by the highly pathogenic strains. the optimal vaccines should induce efficient maternal antibodies in sows that could be transmitted to the offspring and protect neonatal suckling piglets from pedv. the vaccination (s ) protein [ ] . apart from apn, pedv is able to bind to sialic acids [ ] . it remains unknown whether pedv uses sugar coreceptors during viral infection [ , ] . pedv infects multiple cell lines from different species including bat and primate (human and non-human) in vitro. the ability of pedv to infect the cells of different species indicates that the virus utilizes the evolutionarily conserved cell components as receptors, thereby enhancing the potential for cross-species and potentially, zoonotic transmission [ , ] . the highly pathogenic variant strains of pedv were identified in and caused a high morbidity of up to % in piglets, and since then, these strains become dominant, leading to most of the acute outbreaks of ped worldwide [ , , ] . the high virulence of these strains is critically associated with the immune evasion mechanisms employed by the virus. pedv has evolved different strategies to delicately manipulate and damage the host innate immune system for their multiplication. clarification of these mechanisms is critical for understanding the host range, tropisms, pathogenesis, and for developing effective vaccines and antiviral drugs to curb the spread of pedv in pigs. in this review, we provide an overview of different mechanisms used by pedv to evade host innate immune responses. pedv is an enveloped, positive-sense, single-stranded rna virus. the pedv genome constitution represents a standard cov arrangement. the viral genome is approximately kb in length, containing a terminal cap, a poly (a) tail, as well as seven open reading frames (orfs), including the orf a, orf b, s, orf , e, m, and n genes ( figure ) [ ] [ ] [ ] . the n terminal orf a and orf b encode two large replicase polyprotein precursors (pp a and pp ab), which are subsequently processed into nonstructural proteins (nsp to ). orf a encodes pp a which is cleaved by viral proteases into nonstructural proteins (nsp -nsp ). the orf b generates five additional nonstructural proteins (nsp - ) that are proteolytically cleaved by the viral proteases from pp ab [ ] . nsp contains two papain-like protease domains (plp and plp or pl pro ) that cleave the nsp - region of the replicase polyprotein at three sites. nsp , a chymotrypsin-like enzyme also known as c-like protease, cleaves the polyprotein at remaining cleavage sites [ ] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf [ ] [ ] [ ] . the nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [ ] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf [ ] [ ] [ ] . the nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [ ] . the viral proteins of pedv perform different biological functions during viral entry, replication cycle and propagation (table ) . pedv s protein, a type i membrane glycoprotein protein located on the envelope of the virus, consists of an n-terminal signal peptide, a large extracellular region, a single transmembrane domain, as well as a short cytoplasmic tail [ , ] . the ectodomain of s protein comprises s and s subunits. the n-terminal s region, containing n-and c-terminal domains (s -ntd and s -ctd), is mainly responsible for receptor binding [ ] . the c-terminal membrane-anchored s region is mainly involved in triggering the fusion of the viral envelope with host cell membranes [ , ] . the interaction of the cov s protein with its host cell surface receptor is a key determinant for host tropism. the s -ctd of most known members of α-cov genus, including pedv, interacts with aminopeptidase n (apn) to entry into the target cell [ , , [ ] [ ] [ ] [ ] . in addition, s protein contains the epitopes that are the major targets of the neutralizing antibody. n protein is the most abundant viral protein during the early phase of infection in cov-infected cells [ ] . similar to the n proteins of other covs, the pedv n protein has multiple functions, such as acting as a structural protein that forms nucleocapsid with viral genomic rna, playing important roles in viral replication, transcription, and assembly [ , ] . the expression of the n protein in intestinal epithelial cells extends the s-phase of cell cycle, causes endoplasmic reticulum (er) stress, and upregulates interleukin- expression [ ] . moreover, the n proteins of several α-covs and β-covs, including pedv, pdcov, sars-cov, and mouse hepatitis virus (mhv), have been identified as innate immunity antagonists [ ] [ ] [ ] [ ] . however, the involved antagonistic mechanisms are particularly different. pedv m protein participates in virion assembly and virus budding through collaboration with other viral proteins, and engages in the induction of neutralizing antibodies against pedv [ , ] . pedv m protein is distributed throughout the cytoplasm. it induces the cell growth retardation in intestinal epithelial cells (iec) and arrests the cells in s-phase [ ] . in addition, pedv m protein is identified as an interferon (ifn) antagonist with an unrecognized mechanism [ ] . pedv e protein is important for the virus packaging and budding [ ] . it is predominantly localized in the er, having no effect on cell growth, cell cycle and cyclin a expression in iec. however, it causes er stress and activates the nuclear factor-κb pathogens , , of (nf-κb) pathway which is responsible for the up-regulation of il- and the anti-apoptotic protein bcl- expression [ ] . orf has been predicted to possess multiple transmembrane domains [ ] , while it is predominantly distributed in the cytoplasm [ ] . orf also detains cells at s-phase, facilitating vesicle formation, and thus promoting pedv multiplication [ ] . a recent study suggests that orf interacts with s protein during pedv assembly and consequently benefits viral replication [ ] . cov nsps play multiple roles in the synthesis or processing of viral rna, or in virus-host interactions aiming to create an optimal environment for virus replication, such as facilitating viral entry, viral gene expression, rna synthesis, and virion release. nsp is a n-terminal cleavage product of orf a polyprotein [ ] , a -kda protein, that exists only in α-covs and β-covs [ ] . the nsp of α-covs is not very similar to β-covs nsp with regard to sequence homology and size [ , ] . based on the sequence alignment analysis of the genomes of different covs, the viral nsp can be regarded as a genus-specific marker [ ] . moreover, β-covs nsp has been widely reported to inhibit host protein expression. however, the biological functions of α-covs nsp remain largely unknown. despite the lack of overall sequence similarity, the nsp of different covs shares a similar function to interfere with host protein expression [ ] . these studies suggest the importance of nsp in the life cycle of different lineages of covs. it is shown that tgev nsp inhibits host gene expression and is critical for viral virulence [ ] . pedv nsp induces the degradation of cbp and nf-κb to abate ifn response [ ] , but the detailed mechanisms remain unclear. the sizes and amino acid sequence identity of nsp are variable among different covs [ ] . nsp of mhv and sars-cov are involved in viral rna synthesis [ ] . pedv nsp has unknown functions in replication, and may implicate the virus-host interactions and virulence. nsp is the largest nsp protein, containing two papain-like protease (plp and plp ) domains, of which pedv plp acts as a viral deubiquitinase (dub), to negatively regulate type i ifn signaling [ ] . covs plps domains exhibit multiple functions, serving as a viral protease, dub, as well as an ifn antagonist [ ] . covs, like other positive-stranded rna viruses, induce membranous rearrangements of varying morphologies that are essential for rtcs anchoring [ , ] . the cov-induced replicative structures consist of double-membrane vesicles (dmvs) and convoluted membranes (cms), which form a large reticulovesicular network that are critical for viral replication and transcription [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . among the cov nsps, nsp , nsp , and nsp include the hydrophobic transmembrane domains engaging in anchoring the viral rna synthesis components to the membranes [ ] . for mers-cov and sars-cov, co-expression of nsp and nsp is required to induce dmvs [ ] . sars-cov nsp has membrane proliferation ability as well, which also contributes to dmvs formation [ ] . the structure and functions of α-cov nsp are largely unknown [ ] . nsp is also a marker for cov-induced membrane structures; some results indicate that the nsp - of pp a act as a large complex through multidomain structure or scaffold during viral rna replication progress, before its cleavage into individual products [ ] . covs nsp encodes a c-like proteinase ( cl pro ). the polyproteins pp a and pp ab are processed into individual elements of replicase by c-like protease and plps [ ] . moreover, pedv nsp plays a crucial role in virus replication and also blocks host innate immune responses [ ] . crystallographic or nuclear magnetic resonance structures have shown that nsp , nsp , nsp , nsp , nsp , and nsp have the plprob and the adp-ribose -phosphatase (adrp) activity [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the crystal structure of sars-cov nsp suggests that nsp is dimeric and it is able to bind to single-stranded rna [ ] . the crystal structure of sars cov nsp protein suggests that nsp is a zinc-finger protein, which is existent exclusively in covs so far [ ] . moreover, nsp - have rna binding activity and nsp encodes a single rna-dependent rna polymerase (rdrp). the biochemical characterization and crystallization of sars cov nsp and nsp manifests that eight copies of nsp and eight copies of nsp form a supercomplex [ ] . the complex is supportive for nucleic acid binding and may be associated with the processivity of viral rdrp [ , ] . recently, structural studies have described that the sars-cov nsp polymerase binds to the nsp and nsp complex [ ] that may increase the polymerase activity of nsp rdrp [ ] . cov nsp , a ntpase/helicase, is also determined to play essential roles in viral replication [ ] . cov nsp is a multifunctional protein with - pathogens , , of exoribonuclease activity and n- -methyltransferase [mtase] activity [ , ] . nsp catalyzes the n -methylation of gppp-rna to form a cap- structure. cov nsp encodes an endoribonuclease (endou), performing functions through a hexamer in many covs [ ] [ ] [ ] . the endoribonuclease activity of nsp is not essential for cov replication [ , ] . for covs, the end of the viral genomic rna and subgenomic mrna (sgmrna) is supposed to have cap structures: an n- methylated guanosine nucleoside (m gpppn) (cap ) and a methyl group at the -o-ribose position (cap ) of the first nucleotide [ ] . these cap structures enhance the initiation of translation of viral proteins, protect viral mrnas against cellular - -exoribonuclease and limit the recognition of viral rna by host innate system [ , ] . nsp is proposed to catalyze the first step of the -capping reaction of viral rnas [ ] . the methylation of the two sites in the cap are catalyzed by three nsps; nsp (the n- -mtase), nsp (the -o-methyltransferase), and nsp [ , [ ] [ ] [ ] [ ] . in addition, the - exoribonuclease activity of nsp is involved in a replicative mismatch repair system during rna synthesis, which improves the replication fidelity of cov [ ] . although these nsps have been demonstrated to play essential roles in viral replication, transcription and/or post-translational polyprotein processing [ ] , the nsp - of pedv and other covs are poorly characterized to date, except for sars-cov. nsp is an ifn antagonist; nsp inhibits type iii ifn response; nsp is involved in nucleic acid binding; nsp enhances the inhibitory effect of nsp on ifn-β production. [ , [ ] [ ] [ ] nsp encoding a rdrp; viral replication [ ] nsp a ntpase/helicase that is essential for viral replication [ ] nsp n- -mtase; catalyzing n -methylation of gppp-rna to form a cap- structure; - exoribonuclease activity involves in a replicative mismatch repair system during rna synthesis; ifn antagonist [ , , , host cells generally defend against virus infection by mounting an innate antiviral immune response to prevent the spread of the infection and aid in initiating an adaptive immune response which eventually removes the viruses from host. therefore, the first barrier to restrain viral infections is the host innate immune system, which is related to multiple proteins and mechanisms, including ifns, inflammatory cytokine, apoptosis, autophagy, and so on. the activation of type i ifn responses is composed of three stages: ( ) recognition of pathogen-associated molecular pattern (pamp) by prrs; ( ) secretion of type i ifns through paracrine or autocrine pathways; and ( ) expression of numerous antiviral ifn-stimulated genes (isgs) which bring the host into the antiviral state [ ] . at least three important prrs have been identified in recognition of viral nucleic acids, including retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) (detection of viral rna in the cytoplasm) [ ] , the membrane-bound toll-like receptors (tlrs) (recognition of viral rna or dna in the endosome) [ ] , as well as a structurally unrelated group of viral dna sensors (e.g., cgas (cyclic gmp-amp synthase) and ifi ) localized in the host cytoplasm and/or nucleus [ ] . in the cytosol, the formation of specific secondary structure of viral rna is closely related to viral rna delivery and replication. these molecular signatures are detected by rlrs including rig-i (also known as ddx ), melanoma differentiation-associated gene (mda ), and laboratory of genetics and physiology- (lgp ) [ , ] . prrs recognize pamps, inducing an intracellular signaling cascade, thus leading to the activation of transcription factors such as irfs and nf-κb, which in turn induce the production of ifns [ ] . both rig-i and mda have two n-terminal card domains to interact with the card domains of the downstream adaptor proteins, and a dead/h-box rna helicase domain for rna binding. however, lgp does not have the n-terminalcard domains, and the involved functions remain unclear [ ] [ ] [ ] . dsrna, as a specific secondary structure of viral rna, can be sensed by rig-i/mda to induce ifn-α/β production through the cascade activation of the rlr pathway [ ] [ ] [ ] . activated rig-i/mda forms homo-oligomers and recruits the adaptor mitochondrial antiviral signaling (mavs) to induce the formation of signaling complex of mavs with other proteins on mitochondria. tnf receptor-associated factor (traf ) associates with tnfr -associated death domain protein (tradd), tripartite motif (trim ), and pyruvate carboxylase (pc), resulting in the activation of iκb kinases (ikks). ikks (ikkα, ikkβ and ikkγ) phosphorylate nf-κb inhibitor (iκb), leading to the ubiquitination of iκb and its subsequent degradation. nf-κb is then activated and translocated into the nucleus to turn on the expression of proinflammatory and type i ifn. mavs also recruits and activates tbk /ikkε by trafs that are pre-associated with tbk /ikkε, via direct interaction between the sdd domain of tbk /ikkε and the coiled-coil domain of trafs [ ] . activated tbk /ikkε phosphorylates ifn regulatory factors (irf /irf ), that further dimerize and import into the nucleus to promote type i ifn production. on the other hand, mavs interacts with mita (also known as sting), that is located in mitochondria and the endoplasmic reticulum (er) membrane. mita interacts with the trap complex, which may be involved in recruiting tbk and ikkε to phosphorylate irf . ubiquitination and deubiquitination are decisive in the regulation of rlr pathways activation [ ] . upon binding to viral dsrna, rig-i and mda undergo conformational changes and release the n-terminal tandem card domains [ ] [ ] [ ] . the card domains of rig-i are modified by lysine (k ) polyubiquitin chains through the ubiquitin ligases trim , rnf , and riplet. this modification is crucial for rig-i to recruit mavs [ , ] . in addition to the ubiquitination of rlrs, the polyubiquitylation of traf and traf also play an important role in the regulation of innate immune signaling by activation of tbk and ikks, respectively. the k polyubiquitin chains can be removed by dubs such as the tumor suppressor protein cyld, duba and a , providing a mechanism to downregulate immune responses [ ] . ubiquitination and deubiquitination are in a dynamic equilibrium to maintain immune homeostasis. type i ifns are secreted by secretory cells and peripheral cells through self-secretion or paracrine secretion manners. extracellular type i ifns bind to a heterodimeric complex composed of subunits of ifn-α receptors (ifnar ) and (ifnar ) located on the cell surface, which activates the tyrosine kinase (tyk )/janus kinase (jak ) signal transducer. tyk /jak subsequently induces the phosphorylation of transcription factors stat and stat , which dimerize and in turn recruit ifn-regulatory factor (irf ), to form stat -stat -irf trimerized complex (isgf ). this complex then translocates to the nucleus, where it binds to the ifn-stimulated response elements (isre motif; conserved sequence is tttcnntttc) [ ] . the binding of isgf to isre finally triggers expression of ifn-stimulated genes (isgs) that directly or indirectly exert antiviral effects in host cells [ ] . three types of ifns (types i, ii and iii) have been identified. type i and type ii ifns have been widely reported. type ii ifns only contain ifn-γ. ifn-γ is produced by natural killer (nk) cells and activated cd + and cd + t cells in response to the cytokines such as interleukin- (il- ) and il- [ ] . ifn-γ binds to the type ii ifn receptor composed of two subunits, ifngr and ifngr . ifngr and ifngr induce the formation of stat -stat homodimers. stat -stat homodimers translocate to the nucleus and bind to the promoter of the ifn-γ-activation site (gas) elements, to initiate the transcription of ifn-γ-regulated genes [ ] . type iii ifns have been explored in recent years, to unravel the underlying mechanisms that manipulate host innate immune responses. type iii ifns in humans contains ifn-λ (interleukin ), ifn-λ (il- a), ifn-λ (il- b), and ifn-λ [ ] [ ] [ ] . their expression profiles, signaling pathways, and gene expression programs resemble those of type i ifns. the production of type i and iii ifns are both initiated through the recognition of pamps or damage associated molecular patterns (damps) by prrs [ ] . despite the fact that the same transcriptional factors are required for the activation of promoters of type i and iii ifns, the nf-κb pathway is a pivotal regulator in ifn-λ production, whereas the irfs pathway dominates type i ifns expression. the promoter of ifn-λ includes more nf-κb binding sites compared with in the ifn-β promoter [ ] . the signal transduction of type iii ifns depends on the ifn-λ-specific receptor, il- ra chain and il- r chain [ , ] . synthetic ifn-λ binds to il- rα and induces a conformational change within the receptor subunits, that triggers the activation of the receptor-associated tyrosine kinases (tyk and jak ), which then phosphorylate stat and stat . stat and stat are heterodimerized and interact with irf to form the isgf transcription complex that binds to isre in the promoters of isgs, to induce the expression of hundreds of proteins with antiviral functions [ ] . induction of ifn-α/β is the most rapid and effective mechanism for hosts to initiate antiviral innate immune responses. sars-cov, mhv and many other covs are sensitive to ifns. a great number of viral dsrnas intermediates are generated during covs infection that contribute to ifn production, but these covs remain highly pathogenic. as a matter of fact, covs have developed a set of elaborate mechanisms to evade or inhibit the host antiviral innate immune response during virus evolution [ , ] . the evasive strategies utilized by pedv are classified into four major types: ( ) inhibition of rlrs-mediated ifn production pathways, ( ) inhibition of the activation of transcription factors responsible for ifn induction, ( ) disruption of the signal cascades induced by ifn, and ( ) hiding its viral rna to avoid the exposure of viral rna to immune sensors. in the past decade, accumulating evidence demonstrates that pedv n protein, nsp , plp , nsp , nsp , and nsp antagonize type i ifn or type iii ifns production [ , , , , , , , ] . this explains why only weak ifns' and cytokines' expression is detected in pedv-infected cells [ , ] . n protein, as an abundantly produced structural protein within cov-infected cells, has multiple functions, including virus replication, transcription, and assembly [ , ] . pedv n protein has been identified as an ifn antagonist that blocks the expression of ifn-β and isgs by suppression of the pathogens , , of irf and nf-κb activities [ ] . pedv n protein inhibits the activation of the ifn-β promoter induced by tbk and its upstream rig-i, mda- , visa, and traf , while not affecting the activation of the ifn-β promoter driven by irf . further experiments confirm that n directly interacts with tbk to obstruct the association between tbk and irf , which inhibits tbk -induced irf phosphorylation and ifn-β production [ ] . moreover, the effect of pedv n protein on type iii ifn production has also been evaluated [ ] . n protein inhibits polyinosinic-polycytidylic acid (poly(i:c))-induced ifn-λ production by blocking the nuclear translocation of nf-κb, but does not antagonize the type i or type ii ifn expression induced by poly(i:c) in ipec-j cells [ ] . recent studies show that sars-cov n protein inhibits type i ifn production through suppressing trim -mediated rig-i ubiquitination [ ] . the mers-cov n protein also blocks ifn production by interacting with trim [ ] . in addition, both mhv and sars-cov n proteins perturb the function of cellular protein activator of protein kinase r (pact), which can bind to rig-i and mda to activate ifn production, and thus antagonize type-i ifn signaling [ ] . these results indicate the important function of the covs n protein in modulating host innate immune response. whether pedv n protein targets trim or pact should be investigated. although several studies have been performed to understand the pathogenicity of pedv, there remains limited information about the interaction between viral proteins and host cell factors during viral infection. cov n protein is a vital viral protein involved in virus replication. current researches have indicated that n protein interacts with many host proteins, such as hcypa [ ] , proteasome subunit p [ ] , smad [ ] , hnrnp-a [ ] , and the chemokine cxcl [ ] . in the host cells, a large number of host proteins reveal various functions. however, for the virus, the genome only encodes several limited viral proteins. therefore, these viral proteins have to be multifunctional, which is pivotal to virus replication and existence. pedv nsp is the n-terminal cleavage product from polyproteins pp a and pp a/b processed by nsp and nsp [ ] and is about amino acids in length [ , ] . nsp of many α-cov and β-cov exhibits both functional conservation and mechanistic diversity in suppressing host gene expression and ifn signaling. for sars-cov, nsp triggers the decay and cleavage of host mrna and inhibits host protein translation, subsequently inhibiting type i ifn production [ , ] . sars-cov nsp also blocks the expression of ifn-inducible genes, by restraining the signal transduction during virus infection [ , ] . the tgev nsp considerably suppresses host protein expression during viral infection [ ] . structural studies show that the core structure of pedv nsp is highly similar to those of sars-cov nsp and tgev nsp [ ] . pedv nsp inhibits host gene expression and three motifs (amino acids to , to , and to ) form a stable functional region for inhibition of host protein synthesis, differing considerably from sars-cov nsp [ ] . pedv nsp has been identified as an ifn antagonist, which constrains poly (i:c)-induced ifn-β promoter activity [ ] . nsp significantly inhibits the activation of ifn-β promoter triggered by irf , whereas it does not inhibit irf phosphorylation and its nuclear translocation. nsp interrupts the association of irf with creb-binding protein (cbp), by promoting cbp degradation in the nucleus via the proteasome-dependent pathway. cbp/p , the transcription co-activator camp responsive element binding protein (creb), forms a complex with the activated irf in nucleus. the irf -cbp/p complex binds to the positive regulatory domain (prd) regions of the ifn-β promoter, to assemble the enhanceosome with nf-κb and other factors, which ultimately turn on the transcription of type i ifn genes [ ] [ ] [ ] . therefore, pedv nsp blocks type i ifn production in the nucleus. activated nf-κb induces the production of type i ifns and proinflammatory cytokines and is important for inhibiting viral infection. pedv nsp has been shown to interfere with the nf-κb activity [ ] and is the most potent suppressor of proinflammatory cytokines at early infection. it inhibits the phosphorylation and degradation of iκbα, and blocks p nuclear translocation, leading to the suppression of both ifn and the early production of pro-inflammatory cytokines [ ] . moreover, pedv inhibits type iii ifn production and nsp , nsp , nsp , nsp , nsp , nsp , nsp , orf , e, m, and n are identified as type iii ifn antagonists. among these antagonists, nsp is the most potent suppressor [ ] . pedv nsp blocks the nuclear translocation of irf and decreases the amounts of peroxisomes and then suppresses irf -mediated type iii ifns. the conserved residues of pedv nsp protein are crucial for ifn suppression [ ] . multiple effects of nsp on modulating innate immune response during pedv infection suggest the vital role of nsp in the pedv replication cycle. the antiviral innate immune signaling pathways are regulated by several posttranslational modifications (ptms), such as phosphorylation, ubiquitination, glycosylation, neddylation and sumoylation [ ] , of which ubiquitination is a critical modification to modulate the stability and activity of prrs and other components of innate immune signaling pathways. during viral infection, a reciprocatory action (occurrence of ubiquitination and deubiquitination) helps maintain the homeostasis of host immune responses. hence, deubiquitinases (dubs) are indispensable in the regulation of virus-induced type i ifn signaling [ ] . many host dubs have been reported engaging in the regulation of innate immune signaling pathways [ ] [ ] [ ] . in recent years, a variety of viral dubs have been discovered to target key components of type i ifn pathway during various rna virus infections. for example, foot-and-mouth disease virus leader proteinase (fmdv lb pro ) [ ] , and porcine reproductive and respiratory syndrome virus nsp (prrsv nsp ) possess ubiquitin-deconjugating activity to deubiquitinate key host components [ , ] . to counteract host antiviral response, covs likely take advantage of dub activity to break host innate immunity. indeed, the plps of mouse hepatitis virus a (mhv-a ) [ ] , sars [ ] , and human cov nl have dub activity and antagonize ifn induction [ ] . pedv plp has been reported as having a deubiquitinase activity as well, and it can be co-immunoprecipitated by rig-i and sting. as mentioned above, fmdv lb pro , mhv plp and sars plps all counteract host innate immune response through blocking the ubiquitination of the components of rlrs pathways. similarly, pedv plp removes the ubiquitinated conjugates from rig-i and sting by its dub activity, to negatively regulate type i ifn production. pedv plp probably interacts with rig-i and sting, which prevents the activation of rig-i and sting by hindering the recruitment of downstream signaling molecules. as expected, the interference with the ubiquitination of rig-i and sting by plp clearly benefits pedv replication [ ] . pedv nsp contains two core domains of plps (plpl and plp ). it is determined that pedv plp , but not plp , inhibits the ifn-β promoter activation in hek t cells. the dub activity of plp is highly dependent on its catalytic activity. three catalytically inactive mutants of pedv plp (c a, h a and d a) are defective in the deubiquitination of its targets and fail to impair virus-induced ifn-β production. sars-cov plp interacts with mdm (mouse double minute homolog) to deubiquitinate and stabilize mdm , approving the degradation of p and the suppression of ifn signaling [ ] . pedv infection degrades p by upregulation of mdm expression [ ] . pedv plp may be responsible for targeting the p pathway and inhibiting p -dependent apoptosis, leading to immune evasion. a recent study determined that tgev pl inhibits the ifn-β expression and interferes with the rig- and sting-mediated signaling pathway through a viral dub activity [ ] . it suggests that different viral proteins are involved in the deubiquitination of host proteins for different covs. however, these studies offer a probability to design a common therapeutic against different viral dubs to reduce the replication and pathogenesis of covs. therefore, further studies are required to understand more about the substrate specificity of these viral dubs and clarify the precise functions of cov protease/dub activity. notably, c pro is a critical ifn antagonistic protein identified in multiple different families of viruses. the c pro of picornaviruses, including fmdv [ ] , hepatitis a virus (hav) [ ] , enteroviruses (ev , ev-d ) and coxsackieviruses (cvb , cv-a , cv-a ) [ ] [ ] [ ] [ ] , antagonize innate immune signaling by targeting the critical components of the ifn pathways for proteolysis. a newly emerged picornavirus, seneca valley virus (svv), has also evolved an effective mechanism to escape host antiviral innate immune using its c pro . moreover, c pro cleaves the signaling components (mavs, trif, and tank) of type i ifn pathway and induces the degradation of the transcription factors irf and irf to constrain host antiviral response [ , ] . cov nsp is called c-like protease ( cl pro ), that resembles the c pro of other rna viruses. for cov, the polyprotein precursors (pp a and pp b) are mainly processed to generate mature nonstructural proteins by cl pro . to date, the cl pro of covs, including pedv and pdcov, have been confirmed to antagonize type i ifn production by the cleavage of nf-κb essential modulator (nemo) and stat [ , , ] . nemo is essential for rna virus-induced activation of nf-κb, irf , and irf [ ] . nemo is required for mavs-induced ikkα/β activation and is also crucial for the activation of tbk /ikkε [ ] . to establish successful infections, pedv targets nemo to subvert host innate immune responses. pedv nsp significantly inhibits sendai virus (sev)-induced ifn-β synthesis and the process depends on its protease activity [ ] . further experiments show that pedv nsp inhibits rig-i/mda signaling and targets the upstream of tbk . the cleavage of nemo by nsp is identified as responsible for this inhibitive effect. the pedv nsp -mediated cleavage of nemo efficiently blocks nemo-mediated downstream signaling. the cleavage site within nemo that is grasped by nsp has been determined. of these reported immune evasion strategies employed by covs, the cleavage of innate immune adaptors is a particularly effective manner to disrupt antiviral responses. nsp is essential for the life cycle of pedv and other covs [ , ] . it is a potential target for the development of anti-coronaviral therapeutics. although pedv nsp does not target stat mediated type i ifn signaling pathway, pedv nsp has been reported to inhibit the stat and stat induced activation of isre [ ] . nsp competes with karyopherin α (kpna ), which is an adaptor mediating nuclear translocation of isgf , in combination with stat , to block isgf nuclear transport. however, the expression and phosphorylation of stat and stat are not affected by pedv nsp . in fact, pedv infection degrades stat , leading to the inhibition of ifn signaling [ ] . therefore, other pedv encoded proteins likely target ifns mediated signaling. covs belong to rna viruses, which produce several rna species, such as dsrna intermediates and rna with a -triphosphate during replication. these rna intermediates are potent stimulators of prrs and are associated with the organelles of viral rna replication, dmvs [ , ] . dmvs formed from membranous rearrangements seem to sequester the replication intermediates using membrane-bound vesicles or invaginations to keep away from prrs. therefore, the form of dmvs may be a strategy for pedv to escape innate immune recognition in the cytosol (figure ). however, whether dmvs alone are sufficient to shield rna from prrs remains unknown. besides, the replication organelles, the endoribonuclease activity and viral end rna capping/protection mechanisms are also the critical ways of avoiding rna recognition or protecting it from degradation [ , ] . ( ) pedv nsp cov nsp has endou catalytic activity that was initially thought to play a vital role in virus replication. however, the catalytic-defective endou of mhv shows only a subtle defect in viral replication compared to wt virus in fibroblasts [ ] . similar results are found for the nsp mutants of sars-cov and hcov- e [ ] . these findings suggest that the endou activity of nsp is not required for rna synthesis. recently, nsp has been demonstrated to act as a new ifn antagonist of covs [ , , ] . recent reports indicate that covs' endou activity is essential for prevention of rna recognition by mda , protein kinase r (pkr), and oas/rnase l system [ ] . pkr and oas/rnase l recognize and destroy foreign rna in the cytosol to defend viral infections. to counteract the function of pkr and oas/rnase l, the virus hides or modifies its viral rna, to avoid the exposure of viral rna to these molecules. in all covs, the endou catalytic domain in nsp is highly conserved. pedv endou activity has been indicated as having an antagonistic effect on ifn signaling [ ] . the endou activity of pedv nsp not only inhibits the type i ifn response in porcine macrophages, but also antagonizes the type iii ifn response in porcine epithelial cells. the replication of endou-mutant pedv (icpedv-enumt) is considerably impaired in porcine epithelial cells compared to the wild type pedv (icpedv-wt). the icpedv-enumt clearly induces early and robust type i and type iii ifns production, as well as isgs' expression compared with that induced by icpedv-wt. the endou-deficient pedv infected animals also show reduced viral shedding and mortality. these results indicate that the endou activity of pedv nsp plays a vital role in evading host antiviral innate immunity (figure ) [ ] . ( ) pedv nsp cov nsp is a -o-methyltransferase ( -o-mtase). to evade recognition by the host immune sensors, many covs encode methyltransferases involved in the capping of viral rna. this modification makes the viral rna indistinguishable with host cell mrna, which is important to avoid the recognition of viral rna by mda ( figure ) [ , ] . methylation of the two sites in the cap is catalyzed by three nsps, including nsp (the n- -mtase), nsp (the -o-mtase), and nsp [ , [ ] [ ] [ ] [ ] . for example, sars-cov nsp acts as a -o-mtase to prevent innate immune recognition and promote viral proliferation [ , , ] . pedv nsp and nsp have been identified as the viral ifn antagonists [ , ] . the overexpression of nsp or nsp remarkably inhibits ifn-β production, but nsp appears to play a more important role in innate immunity regulation than nsp [ ] . nsp is a highly conserved methyltransferase which contains an invariant kdke motif within the methyltransferase core [ ] . this kdke motif is required to mediate its activity. notably, the mutation of any of the kdke active sit has been shown to abolish the -o-mtase activity [ ] . pedv nsp kdke motif plays a critical role in the inhibition of type i ifn production, suggesting the important role of the -o-mtase in pedv-mediated immune evasion. pedv nsp negatively regulates rlr-mediated signal pathway activation, and inhibits the expression of the ifn-stimulated ifit family members (ifit , ifit , ifit ), which in turn promotes pedv replication. taken together, these results demonstrate that pedv nsp negatively regulates cellular antiviral response to promote viral replication [ ] . screening inhibitors targeting the -o-mtase of nsp might be a prominent strategy to inhibit cov infections and develop antivirals for treatment of the diseases caused by covs. additionally, covs nsp also includes the -to- exoribonuclease (exon) activity [ ] . a mutation of tgev nsp exon generates lower levels of dsrna than wildtype tgev and thus triggers a reduced antiviral response [ ] . nsp exon activity is also critical for the resistance of host innate immune response in mhv-infected cells [ ] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. activity is also critical for the resistance of host innate immune response in mhv-infected cells [ ] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. heat shock protein (hsp ) belongs to the small heat shock proteins family, which has been identified as a multifunctional protein involved in cytoskeletal stability, proinflammatory processes, and the inhibition of apoptosis [ , ] . several hsps have been reported to be implicated in pedv infection in vitro and in vivo [ , ] . indeed, the infection of many viruses up-regulates hsp expression by different mechanisms to delay cellular apoptosis, then supplies sufficient time for viral replication [ , ] . however, pedv infection significantly induces the decreased expression of hsp in vero cells [ ] and marc- cells [ ] . hsp activates the phosphorylation of nf-κb, and thus promotes the mrna expression of ifn-β in marc- cells. as hsp is an upstream regulator of antiviral immune signaling, overexpression of hsp significantly inhibits the pedv replication. pedv has developed a strategy via mediating the suppression of hsp production to escape from host innate immune response [ ] . hsp , the most conserved hsp, is also important for the multiplication of several covs. the recruitment of hsp is thought to be a viral survival strategy for several viruses in their hosts [ ] . the relationship between hsp and pedv should be exploited further in future. viral infection triggers host immune response to induce ifns' and inflammatory cytokines' production. released ifns elicit the expression of numerous isgs which limit viral replication in the infected cells. however, the release of excessive amounts of ifns and inflammatory cytokines will lead to autoimmune and auto-inflammatory diseases. the concomitant uncontrolled apoptosis is also one outcome that is harmful to the host. to maintain the reaction in a proper balance, hosts have evolved a series of effective mechanisms to control the antiviral innate immune response [ ] . in contrast, viruses often break this balance, causing improper apoptosis reaction, which benefits viral replication. pedv infects various host cells including vero, pk- and marc- and cause obvious cytopathic effects. pedv-induced apoptosis of the infected cell has been demonstrated both in vitro and in vivo [ ] . apoptosis is induced through the activation of apoptotic caspases, including caspase- , - , - , - , - , - , and - [ ] . pedv infection results in obvious caspase- and caspase- activation, as well as the cleavage of apoptosis-inducing factor mitochondria-associated (aifm ) and poly adp-ribose polymerase (parp), which leads to apoptotic nuclear fragmentation. pedv spike protein s significantly elicits host cell apoptosis, while the nsp - and other structural proteins (m, n, e, s , and orf ) have none or few effects on cell apoptosis. therefore, s protein is probably the critical protein mediating the apoptosis induced by pedv [ ] . the multiple stages of cov replication cycle are closely associated with cellular membrane compartments, especially the endoplasmic reticulum (er). the shape and functions of er can be influenced by different physiological states and environmental conditions. when protein synthesis amounts surpass the folding capacity, the accumulation of a large amount of unfolded proteins in the er leads to er stress. consequently, cells manifest a corresponding biological reaction that is widely known as the unfolded protein response (upr) [ ] . once the upr is induced, it alleviates the problems by host protein translation inhibition (by the transducer pkr-like er protein kinase (perk)-induced phosphorylation of eif a), stimulating protein folding. if homeostasis cannot be re-established, apoptosis eventually is triggered. indeed, the activation of upr regulates a wide variety of signaling pathways, such as apoptosis, autophagy, mitogen-activated protein (map) kinase activation, and innate immune response [ ] . furthermore, α-cov and β-cov may induce er stress in the infected cells [ ] . pedv orf , as the only accessory protein encoded by pedv, is thought to be related to virus production and virulence of pedv [ ] . a series of studies suggest that orf plays multiple roles, in addition to acting as an ion channel during pedv replication. recent studies show that pedv orf consists of four transmembrane domains (tmds) and localizes in the cytoplasm in the aggregation manner [ ] . orf is a transmembrane protein, and the confocal microscopy analysis indicates that the aggregated orf localizes in the er to induce the er stress associated with either apoptosis or autophagy. however, pedv orf induces the autophagy via driving conversion of lc -i to lc -ii, but not influencing the apoptosis. orf -induced autophagy is dependent on er stress response. pedv orf triggers er stress response via the up-regulation of grp protein expression and the activation of the perk-eif α signaling pathway. moreover, orf protein is identified as an ifn antagonist to block ifn response by an unknown mechanism in pedv-infected cells [ ] . the functions of pedv orf should be further exploited. pedv has caused epidemic and endemic infections in pig populations in many countries and has become a major economic threat to the swine industry. previous studies have identified viral factors that target key signaling molecules in the rlrs' pathways, as well as viral factors that target the downstream signaling pathways responsible for isgs induction. of the pedv-encoded proteins, at least viral proteins have been identified as type i ifn antagonists [ , ] . the mechanisms utilized by pedv nsp , plp , nsp , and n protein to antagonize type i ifn production have been clarified (figure , [ , , , , ] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp , as well as nsp , has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. have been clarified (figure , [ , , , , ] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp , as well as nsp , has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. removes ubiquitinated conjugates from rig-i; pedv nsp induces cleavage of nemo; pedv n protein directly interacts with tbk to obstruct the association between tbk and irf ; pedv nsp causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p activation. pedv nsp inhibits type i ifn production and nsp enhances the inhibitory effect of nsp on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp blocks the nuclear translocation of irf and reduces the amounts of peroxisomes. pedv nsp inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp interacts with stat and stat to block nuclear translocation of isgf . removes ubiquitinated conjugates from rig-i; pedv nsp induces cleavage of nemo; pedv n protein directly interacts with tbk to obstruct the association between tbk and irf ; pedv nsp causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p activation. pedv nsp inhibits type i ifn production and nsp enhances the inhibitory effect of nsp on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp blocks the nuclear translocation of irf and reduces the amounts of peroxisomes. pedv nsp inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp interacts with stat and stat to block nuclear translocation of isgf . author contributions: the manuscript was prepared by s.l., j.y., z.z., and h.z., and reviewed by the co-authors. all authors have read and agreed to the published version of the manuscript. outbreak of porcine epidemic diarrhea in suckling piglets porcine epidemic diarrhea in china experimental infection of pigs with a new porcine enteric coronavirus, cv an apparently new syndrome of porcine epidemic diarrhoea virus-like particles associated with 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in the nuclear magnetic resonance structure of the replicase nonstructural protein from the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells sars coronavirus nsp protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp -induced rna cleavage severe acute respiratory syndrome coronavirus nsp protein suppresses host gene expression by promoting host mrna degradation severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp and rational design of an attenuated strain structural basis for the inhibition of host gene expression by porcine epidemic diarrhea virus nsp mechanisms of activation of interferon regulator factor : the role of c-terminal domain phosphorylation in irf- dimerization and dna binding virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation an atomic model of the interferon-β enhanceosome viral deubiquitinases: role in evasion of anti-viral innate immunity ubiquitin signaling in immune responses deubiquitinases in the regulation of nf-kappab signaling pathogens ubiquitin-specific protease b negatively regulates ifn-β production and antiviral activity by targeting tank-binding kinase the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production sars 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(pedv)-infected vero cells identification of differentially expressed proteins in porcine alveolar macrophages infected with virulent/attenuated strains of porcine reproductive and respiratory syndrome virus epstein-barr virus upregulates phosphorylated heat shock protein kda in carcinoma cells using the phosphoinositide -kinase/akt pathway down-regulating heat shock protein is involved in porcine epidemic diarrhea virus escaping from host antiviral mechanism recruitment of hsp chaperones: a crucial part of viral survival strategies caspases control antiviral innate immunity porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor caspase function in programmed cell death signal integration in the endoplasmic reticulum unfolded protein response coronavirus infection, er stress, apoptosis and innate immunity regulation of stress responses and translational control by coronavirus porcine epidemic diarrhea virus orf protein causes endoplasmic reticulum stress to facilitate autophagy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are thankful to everybody who participated in the studies. the authors declare no conflict of interest. key: cord- -tek u authors: sinderewicz, emilia; czelejewska, wioleta; jezierska-wozniak, katarzyna; staszkiewicz-chodor, joanna; maksymowicz, wojciech title: immune response to covid- : can we benefit from the sars-cov and mers-cov pandemic experience? date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: tek u the global range and high fatality rate of the newest human coronavirus (hcov) pandemic has made sars-cov- the focus of the scientific world. next-generation sequencing of the viral genome and a phylogenetic analysis have shown the high homology of sars-cov- to other hcovs that have led to local epidemics in the past. the experience acquired in sars and mers epidemics may prove useful in understanding the sars-cov- pathomechanism and lead to effective treatment and potential vaccine development. this study summarizes the immune response to sars-cov, mers-cov, and sars-cov- and focuses on t cell response, humoral immunity, and complement system activation in different stages of hcovs infections. the study also presents the quantity and frequency of t cell responses, particularly cd (+) and cd (+); the profile of cytokine production and secretion; and its relation to t cell type, disease severity, and utility in prognostics of the course of sars, mers, and covid- outbreaks. the role of interferons in the therapy of these infections is also discussed. moreover, the kinetics of specific antibody production, the correlation between humoral and cellular immune response and the immunogenicity of the structural hcovs proteins and their utility in the development of a vaccine against sars, mers, and covid- has been updated. coronaviruses (covs) were discovered in the s as zoonotic spherical pathogens causing mostly respiratory or enteric diseases [ , ] . coronaviruses vary in size and are enveloped with club-shaped spikes on their surface [ ] [ ] [ ] . a helically symmetrical nucleocapsid comprising positive-sense single-stranded rna is one of the largest virus genomes, ranging from to kilobases in length [ ] . although covs are distributed mainly among mammals and birds, over the last years some cov infections have resulted in lethal epidemics in humans. since , when the first human coronavirus (hcov) was identified, seven hcovs species have been described [ ] . four of them, hcov- e, hcov-oc , hcov-nl and hcov-hku , lead to mild diseases such as the common cold, while the sars-cov, mers-cov, and sars-cov- caused severe disorders, manifesting acute respiratory system failures and fatalities [ ] . the first identified hcov, sars-cov, originated from southern china in and induced an epidemic of severe acute respiratory syndrome (sars) with a mortality rate of - % [ ] [ ] [ ] . the first case of mers-cov, it was also found that the total number of t lymphocytes, cd + , cd + , and naive cd + t cells was still lower one year post-sars-cov infection compared to the control value [ ] . only cd + t cells returned to normal level in the recovery period, probably as the effect of recirculation between blood and organs [ ] . the ability of the mers-cov to infect t cells and the activation of extrinsic and intrinsic apoptosis pathways in t cells was also proven [ ] . similar to sars-cov infection, effective transmission of mers-cov resulted in downregulation of th and high frequencies of reactive cd + t cells in the first phase of the disease, but not in the convalescent phase [ , ] . furthermore, a correlation between th and th downregulation and the fatality rate of mers-cov and sars-cov infection was found [ , [ ] [ ] [ ] . the newest reports have also documented an assessment of the t cell numbers in covid- patients. similar to sars and mers, the number of total t cells, cd + , and cd + t cells was significantly diminished in covid- patients in comparison to healthy controls and positively correlated with the severity of the disease [ , ] . moreover, an age-dependent reduction in t cell numbers was observed in covid- , with the lowest t cells numbers detected in individuals older than years old, indicating an enhanced susceptibility to sars-cov- infection in elderly patients. furthermore, besides the decreasing number of t cells, the limited function of these cells has been described as a result of enhanced expression of immune-inhibitory factors, such as programmed death receptor (pd ) or hepatitis a virus cellular receptor (tim- ) [ ] . a flow cytometry analysis illustrated significantly greater expression of the pd and tim- on t cell surfaces isolated from covid- patients in comparison to healthy controls [ ] . growing pd and tim- levels were found as patients progressed from prodromal to overtly symptomatic stages, suggesting that the surviving t cells lost their functionality, particularly in patients requiring intensive care unit care. moreover, stimulation of peripheral blood mononuclear cells (pbmcs) from group of severe covid- patients with peptides covering all viral proteins activated both cd + and cd + sars-cov- -specific t cells [ ] . furthermore, a greater cd + :cd + ratio in covid- patients was observed in comparison with control individuals [ ] . similar to sars-cov, sars-cov- -specific cd + t cells were identified as central memory t cells, based on cd ra and ccr expression. a mixed phenotype of cd + t cells in covid- patients was also documented [ ] . a low content of cd + t cells in peripheral blood of covid- patients and a negative relationship between viral and cd + titers were also reported [ ] . moreover, sars-cov- -specific cd + and cd + t cells were detected in~ % and % of covid- convalescents, respectively [ ] . the response of cd + t cell to sars-cov- s protein was also correlated with the specific igg and iga titers in patients who recovered from covid- [ ] . li et al. [ ] established detailed maps of t cell immune responses to sars-cov using pbmcs from sars convalescents. the new t cell epitopes were identified which induced a response to eight of fourteen sars proteins: replicase, orf , orf , orf , spike (s), envelope (e), membrane (m), and nucleocapsid (n). almost % of the responses were focused on structural proteins (s, e, m, and n), principally on s protein ( %), whereas the most abundant in sars-cov proteome replicase was much less immunogenic [ , ] . immune-informatic tools were used to identify significant cytotoxic t lymphocyte (ctl) and b cell epitopes in the sars-cov- surface glycoprotein [ ] . ahmed et al. [ ] documented s and n sars-cov- protein-derived epitopes, which were comparable to the sars-cov map b cell and t cell epitopes. the surface glycoprotein of sars-cov- was found to have . % identity and . % similarity to sars-cov [ ] . moreover, five ctl epitopes, three sequential b cell epitopes, and five discontinuous b cell epitopes in the viral surface glycoprotein were detected and described [ ] . despite their high similarity to sars-cov, of identified sequential ctl and b cell epitopes were at least partially unique to sars-cov- compared with bt-cov, sars-cov, and mers-cov. a molecular dynamics stimulation showed that all ctl epitopes bind strongly to the peptide-binding groove of corresponding mhc class i molecules [ ] . these features of ctl epitopes suggested their potential in induction of immune responses and, thereby, utility in a vaccine against sars-cov- . the t cell responses against the s and n proteins were documented as the most long-term reaction in sars-cov-infection [ ] . similar results, showing strong specific t cell response against structural proteins, including s, n, m, and e proteins, were noted in mers-cov infections [ ] [ ] [ ] [ ] . specific sars-cov- s and n proteins were also documented as the most immunogenic and greatly expressed during covid- [ , , ] . although cd + t cell activation was reported against s, m, and n proteins, as well as against nsp , nsp , orf a, and orf , only s protein induced a robust response [ ] . activation of cd + t cells was detected against sars-cov- s and m proteins and at least eight orfs [ ] . similar results were presented by le bert et al. [ ] , who proved the reactivity of both cd + and cd + t cells to the n protein and non-structural (nsp and nsp of orf ) proteins of sars-cov- in covid- convalescents. it was also shown that sars convalescents responded to the n protein of sars-cov- [ ] . uninfected individuals also revealed sars-cov- -specific cd + , indicating possibility of cross-reactive t cell stimulation with the other hcovs [ , ] . interestingly, sars-cov- -specific t cells in controls expressed a different pattern of immunodominance in comparison to sars and covid- convalescents [ ] . patients who recovered from sars or covid- responded mainly to n protein, whereas the control group revealed dominant reactivity to both n protein and orf -encoded proteins [ ] . cytokines, produced mainly by immune cells like macrophages, b and t lymphocytes, and mast cells, modulate the balance between humoral and cell-based immune responses [ ] . their concentration in biological fluids may be an important marker of immune system activity and disease progress. cytokines include several protein groups which vary in function, cell secretion, and target action. the current study reviewed the role of interleukins (ils) with tumor necrosis factors (tnfs), chemokines and interferons (ifns) in the immune response to hcovs. a comparison of the content of proinflammatory th and th cytokines in the serum of sars patients with healthy controls documented a significantly greater concentration of tnf-α, il- , il- , il- , and il- in the early stage of the sars-cov infection [ , ] . decreasing levels of these cytokines were correlated with the course of recovery from sars-induced pneumonia. furthermore, significantly greater contents of il- , il- , and il- were reported in fatal sars cases [ ] . the enhanced secretion of il- α, il- β, il- , il- il- , and ifn-γ as an antiviral and inflammatory response to mers-cov was also documented [ , , , ] . moreover, il- and il- were produced in a greater amount in response to mers-cov than sars-cov [ ] . interestingly, in vitro studies showed that enhanced il- and il- levels in sars and mers patients were observed exclusively in the presence of s protein [ , ] . among the cytokines involved in the immune response against hcovs, several have been proposed as potential predictors of disease cause and progression. it has been documented that increased il- concentration in plasma of sars patients was significantly increased in severe cases, but not in convalescent or control subjects, suggesting a positive correlation between serum il- level and disease severity [ ] . inversely, il- and tgf-β concentrations were significantly reduced in sars patients with a severe course of the disease [ ] . tnf-α was considered a predictor of disease progression due to its greatest level in the early stage of recovery [ ] . moreover, a decreased content of il- and increased level of il- were only found in convalescent patients [ ] . it was also proven that the il- profile in patient's serum indicated the cause of pneumonia-a significantly lower il- concentration was detected in sars patients compared to others. a detailed analysis showed that in a group of sars patients with severe symptoms, cytokine secretion was varied among different t cell subpopulations. it was shown that ifn-γ and tnf-α were produced both by cd + and cd + t cells, whereas the production of il- was typical exclusively for cd + [ ] . moreover, in this group of patients the number of polyfunctional memory cd + t cells producing more than one cytokine was significantly higher compared to sars patients with a mild or moderate course of disease [ ] . a similar effect was not observed for cd + t cell responses, although intensified degranulation was observed in severe course of sars via cd a activation on cd + t cells surface [ ] . stimulation of pbmcs from recovered sars patients with peptides overlapping the entire e protein, a membrane component of sars-cov, resulted in cytokine production by both cd + and cd + t cells [ ] . similar observations have been reported in studies concerning covid- . the lack of expression of the receptor for sars-cov- on t cells, ace , suggested that the limited t cell number in covid- patients was likely caused by the influence of cytokine signaling and not by the direct infection of t cells [ , ] . the stimulation of pbmcs from covid- patients with s protein peptides resulted in production of effector or th cytokines (ifn-γ, tnf-α, and il- ) and, to a lesser extent, th (il- , il- , il- , and il- ) and th (il- a, il- f, and il- ) cytokines [ ] . however, among the numerous serum cytokines, only tnf-α, il- , il- , and il- levels were significantly increased in sars-cov- infected patients [ , , ] . these changes were characteristic of severe progression of the disease, supporting the hypothesis that covid- is driven by proinflammatory cytokines, which are responsible for histological changes and clinically full-blown cases of the disease. among detected cytokines, il- appears to be the most significantly involved in covid- progress. chen et al. [ ] detected an enhanced level of il- r in severe cases of covid- , although no significant differences among examined and control groups were detected in il- [ ] . the presence of il- , greatly expressed in fatal sars cases, was also not detected in the plasma of covid- patients [ ] . moreover, the concentration of tnf-α, il- , and il- was negatively correlated with amounts of total t cells, cd + t cells, and cd + t cells, respectively. furthermore, serum concentrations of il- , il- , and tnf-α were significantly lower in patients in the disease resolution in comparison to the disease period, whereas the total number of t cells, cd + t cells, and cd + t cells was restored during the decline period of covid- . these results suggested that in sars-cov- infections, a high serum concentration of tnf-α, il- , and il- negatively regulated t cell survival and/or proliferation [ ] . interestingly, the production of cytokines by cd + (mainly il- and ifn-γ and trace amounts of il- , il- , il- , or il- α) was also reported in covid- convalescents [ ] . thus, the functional response of cd + against sars-cov- was suggested in recovered patients. chemokines are essential in determining immune cell localization [ ] , and some of them act as factors involved in response to hcovs. enhanced contents of ip- /cxcl- , mcp- /ccl- , mip- α/ccl- , and rantes/ccl- were identified in the lungs and peripheral blood of sars and mers patients [ , , , ] . moreover, both the production and secretion of these molecules were greater in response to mers-cov in comparison to sars-cov [ ] . the upregulation of cxcl- at both transcriptional and translational levels was proven in murine epithelial cells, lung fibroblast cells, monocyte-derived macrophages, and dendritic cells as a result of the overexpression of mers-cov n protein [ ] . high secretion and a persistent increase of cxcl- in mers-cov patients were associated with disease severity [ ] . mers-cov infection also resulted in cxcl- chemokine production by th cells [ , ] . the presence of chemokines and their action has not been reported in covid- , although the number of studies concerning sars-cov- infection is still limited. among crucial elements of the immediate antiviral response, interferons (ifns) are pivotal for limiting viral replication and spread. therefore, ifns were extensively studied as potential therapeutic tools of sars-cov and mers-cov infections. the presence of an enhanced level of ifn-γ was documented in sera of sars-cov-and mers-cov-infected patients [ , ] . similar to the other cytokines, the ifn-γ profile was correlated with the cause of pneumonia. ifn-γ production was significantly greater in sars patients compared to others [ ] . on the other hand, further studies documented relatively low ifn-γ production in response to sars-cov infection. zhou et al. [ ] found greater levels of ifn-γ in sera of mers patients compared to sars-cov-infected patients. moreover, scagnolari et al. [ ] showed that ifn-γ production in response to sars-cov was significantly lower compared to well-established ifn-inducing viruses, such as vesicular stomatitis (vsv) and newcastle viruses (ndv), suggesting a limited role of ifns in early host defense against sars-cov infection. the lack of the antiviral ifn response to sars-cov with simultaneous enhanced secretion of several proinflammatory cyto-and chemokines suggested that the virus suppresses the induction of ifn production [ ] . the natural host defense based on ifn action may be restricted because of the documented inhibition of ifns type i and cytokines production in toll-like receptor (tlr) , tlr , and retinoic acid-inducible gene (rif-i) pathways in response to sars-cov infection. this limitation occurs via suppressing the activation of transcription factors, such as interferon regulatory factor (irf ), nuclear factor (nf)-κb, and adaptor related protein complex (ap ) and downregulation of tnf receptor associated factor (traf) and traf [ ] . it was also documented that mers-cov n and m proteins inhibited the gene expression of ifns type i and iii, resulting in the host antiviral response impairment [ , ] . similar results were documented for sars-cov- infection. chen et al. [ ] showed the production of ifn-γ by cd + t cells in response to sars-cov- tended to be lower in severe ( . %) than in moderate ( . %) cases of covid- . however, among the very few reports concerning the role of ifns in covid- disease was a study documenting a lack of ifn-γ in the serum of patients infected with sars-cov- [ ] . on the other hand, further studies documented the utility of ifns in the treatment of sars-cov infection. chen et al. [ ] confirmed virus susceptibility to exogenous type i ifn. it was also shown that early administration of ifns-i decreased immunopathological changes via downregulation of the expression of factors inducing apoptosis; upregulation of hypoxia/hyperoxia-related genes and the regulation of tlr, cytokine, and chemokine signaling; and expression of mhc-, lysosome-, and fibrosis-related genes [ , ] . however, high-level virus replication resulted in retardation of ifns-i signaling, which promoted the cumulation of pathogenic inflammatory monocyte macrophages and resulted in increased cytokine and chemokine levels in lungs, vascular leakage, and reduced virus-specific t cell responses, and thereby strong lung pathology. animal models showed that genetic ablation of ifn-α/β receptor (ifnar) depletion protected from lethal infection, without affecting viral load [ ] , suggesting that ifns therapy may be effective mainly in the early stage of infection. it was also shown that ifns-i and tlr agonists were the most effective in sars and mers therapy, which activates interferons [ ] . the best results were observed for ifn-β a, which reduced mortality by % in comparison to patients, who received ifn-α a. the efficacy of ifns was lower in older patients [ ] . the ability to induce ifns mrna accumulation by sars-cov in pbmcs from healthy donors was also investigated by castilletti et al. [ ] , who proved that combination of ifn-α and ifn-γ strongly inhibited virus replication, while single cytokines were much less effective. an analysis of molecular mechanisms of the immune response to hcovs showed that effective cytokine production correlated to the availability of functional hcovs receptors. the effective increase in il- level was similar to concentration observed for s protein binding to sars-cov functional receptor, ace , or to neutralizing monoclonal antibody. it was documented that il- production also depended on nf-κb activation and translocation and was suppressed by an nf-κb inhibitor [ ] . moreover, the latest studies suggested that protein s could activate pbmcs via the tlr ligand. it was demonstrated that a lack of functional tlr , tlr , and tlr adaptor molecule (tram) enhanced the possibility of sars-cov infection, reduced lung function and increased lung pathology and mortality. the suppression of tlr adaptor molecule (trif) in mice infected with sars-cov resulted in changes in inflammation and positively correlated with acute respiratory distress syndrome [ ] . on the other hand, infection of macrophages with mers-cov resulted in a reduced capacity to produce tnf-α and il- and enhanced the il- secretion [ ] . the role of mers-cov s protein in upregulation of the irak-m expression, which is a negative regulator of tlr signaling, as well as expression of the transcriptional repressor ppar-γ was documented. moreover, it was documented that the immunosuppressive effect was mediated by dipeptidyl peptidase (dpp ), which competitively inhibits mers-cov via binding to common for mers-cov and dpp functional receptor, dpp r [ , ] . in human dendritic cells (dc), the induction of c-c motif chemokine receptor (ccr) , ccr , and ccr in the presence of sars-cov was detected [ ] . the sars-cov infection induced also significant upregulation of tnf-related apoptosis-inducing ligand (trail) gene expression in dcs [ ] . it was demonstrated that, in mers-cov infection, c-type leptin receptor (clr) was also upregulated and a retinoic acid-inducible-i-like receptor (rlr) pathway was activated [ ] . the main aspects of t cell response in hcovs infections are shown in figure . the animal model of sars-cov infection documented activation of the complement cascade in the lungs and showed that absence of complement significantly reduced the pathological changes in the respiratory tract, even though the viral load is the same. in the lungs of the transgenic mice deficient in c (c −/− ), which is the central component of the complement system, significantly lower neutrophils and inflammatory monocytes were presented than in infected controls [ ] . moreover, diminished cytokine and chemokine contents in both the lungs and serum of c −/− mice were humoral immune response restrains the infection via neutralizing antibodies production and prevents reinfection in the future [ ] . in sars, the presence of igg, igm, and iga antibody responses was detected during both the infection and convalescent phases, although with variable dynamics [ ] [ ] [ ] [ ] . the presence of specific igg and igm antibodies was also documented in the first two weeks of the sars-cov infection ( . % and %, respectively) [ , , , ] . the levels of igg and igm increased during the next two weeks to % and %, respectively. the serum samples examined days after the onset of disease were positive only for sars-specific igg [ , ] . a study analyzing the kinetics of specific antibody contents in plasma of sars patients presented by mo et al. [ ] also showed a further significant increase in igg antibody levels. the highest concentration of igg was documented on day , remaining at the same level until day . then, the igg content gradually decreases until day . the igm antibody level peaked shortly after its detection and, in contrast to previous studies, declined until day when igm was undetectable [ ] . similar results were found by chen et al. [ ] , who suggested that sars-cov-specific igg antibody, persisting for a longer time than specific igm and iga antibodies, was the primary humoral immune response against sars. however, a significantly lower level of igg was detected in severe than in mild or recovering sars patients, which may be a result of some kind of immune system dysfunction in long-suffering acute patients. however, li et al. [ ] reported that strong t cell responses correlated significantly with a higher level of neutralizing antibody activity. in contrast to memory t cell responses, ensuring long-term protection, the antibody response was transient in convalescent sars patients [ ] . cao et al. [ ] documented the presence of specific antibodies within three years from the onset of sars symptoms in . % of examined samples. however, six years post-infection, sars-cov-specific igg and ag-specific memory b cells were undetectable in sars convalescents, whereas memory t cell responses to a pool of sars-cov s peptides were revealed in % of convalescents. the most recent study reported presence of long-lasting memory t cells responding to sars-cov n protein in sars convalescents years after the sars pandemic [ ] . moreover, memory t cell response was stronger in former patients, who revealed severe clinical manifestations during sars [ ] . similar to sars-cov infection, igm and igg levels increased during the first week after sars-cov- infection. the greatest concentration of igm was detected in the second week, after which its content was reduced to initial level in most patients, whereas the igg level remained at a high level for a long period [ ] . interestingly, the igm and igg antibody levels were not significantly different among mild, severe, and critical disease groups [ ] . however, the levels of igg, iga, and ige were greater in covid- fatalities in comparison to survivors [ ] . igm and igg against n and s proteins of sars-cov- were also detected in covid- convalescents [ ] . the igg titer remained high for at least days post-discharge, whereas igm was detected only in newly recovered patients [ ] . moreover, negative correlation between viral and igg titers [ ] and a significant positive correlation between the content of neutralizing igg and the number of n protein-specific t cells was observed, suggesting interdependence between humoral and cellular immunity in sars-cov- infection [ , ] . the kinetics of specific igg and igm antibodies were also analyzed in the serum of mers patients. robust serological responses were detected in most patients during the second or third week after symptom onset [ ] [ ] [ ] . specific igm antibodies were detectable at the same time or slightly later than igg [ ] . interestingly, the whole group of survivors, and only half of all fatalities, produced igg and neutralizing antibodies [ ] . although the mers-cov antibody response in the early phase of infection correlated with reduction of the disease severity [ ] , the presence of antibodies did not allow to the virus removal from the lower respiratory tract [ ] . mers-cov-specific igg was also detectable one year post-infection in all severe disease survivors [ ] . on the other hand, alhetheel et al. [ ] found a very low rate of mers-cov-igg positive patients and a lack of correlation between nucleic acid and serological analysis [ ] . the presence of specific iga in serum and respiratory tract secretions of mers patients was also confirmed. moreover, the correlation between iga and igg concentration in serum of mers-cov-infected individuals was proven [ , ] . however, as the majority of studies concerning humoral response in mers used a limited number of patients, using serological analysis is not recommended as a tool to determine disease severity or prognosis. animal models showed that the main antibody responses were induced by the most exposed s protein of sars-cov [ ] [ ] [ ] . mice immunization with a vector encoding a transmembrane domain of s protein resulted in neutralizing antibody production and action. in consequence, viral replication in the lungs of mice was significantly reduced and immune defense was provided by a humoral but not a t cell-dependent immune mechanism [ ] . however, deming et al. [ ] showed that the efficacy of the humoral response to sars-cov s protein depended on the homology of the virus strain. vaccines including a virus replicon expressing sars-cov s protein ensured complete shortand long-term protection against homologous strain challenge in young and senescent mice. on the contrary, the implementation of a construct encoding a synthetic s protein gene of the most genetically different human strain resulted in complete short-term protection in vaccinated young mice and limited protection in senescent animals [ ] . high tolerance for the vaccine encoding the sars-cov s protein and its high immunogenicity has also been documented in humans, with specific antibodies being detected in % of subjects [ , ] . moreover, sars-cov-specific cd + t cell responses were observed in all vaccinated patients and cd + t cell responses in % of individuals [ ] . neutralizing b cell antibody responses to the sars-cov s protein were also major in sars convalescents, suggesting that a spike-based vaccine can be sufficient for a preventive vaccine, as it was previously demonstrated in animal models [ ] . as was mentioned above, the strongest response against sars-cov s protein was shown by the cd + t cells. the possible cooperation of cd + t cell and b cells in neutralizing ab producing was described previously by mitchison et al. [ ] , and the possibility of the enhanced reaction of plasma b cells, stimulated by cd + t cells, specific to the same protein, has also been suggested [ ] . several studies have documented the presence of antibodies generated against the n protein of sars-cov [ , ] and the high affinity of epitopic sites located in the n protein for forming peptide-antibody complexes in the serum of sars patients, particularly to days after the onset of infection. interestingly, vaccines containing sars-cov n protein failed to protect from homologous and heterologous challenges. in consequence, in the lungs of sars-cov-infected mice, the eosinophilic infiltrates were promoted and increased immunopathology was observed [ ] . the strongest humoral responses against s and n proteins were also detected in mers. although it was proven that mers-n-specific antibodies occurred later than s-specific antibodies [ ] , the vaccines containing mers-specific antibodies are still unknown. the main features of the humoral response in hcovs infections are presented in figure . despite the high range of sars-cov- infection, the severe course of the disease has mainly affected elderly patients, with children excluded from the risk group [ ] . moreover, despite the high rates of seropositivity of anti-receptor-binding domain (rbd) igg and igm ( % and %, respectively) and slightly lower rates of anti-n protein igg and igm measured after days of symptom onset, the disease was still active and clinical symptoms severe [ ] . to explain this phenomenon, antibody-dependent enhancement (ade) after previous exposure to other hcovs with a wide range of affinities has been proposed. in ade, infection is promoted through a virus binding to non-neutralizing antibodies from previous exposures to similar antigens. the virus-antibody immune complex binds to fc receptor (fcr) or complement receptors on the host cell surface, facilitating entry of the virus and sometimes enhancing its replication [ , ] . the results of ade are enhanced inflammatory process, overexpression and release of cytokines (cytokine storm) and multi-organ failure as a consequence of these processes. immune-mediated covs infections have been widely described. the vaccine against feline cov aggravated future disease via induction of infection-enhancing antibodies [ , ] . although the full-length sars-cov s protein stimulated protective immune response action in rodents, in human b cell lines it induced infection [ ] . in vitro studies have demonstrated, that anti-spike immune serum enhanced the infection of immune cells by sars-cov spike-pseudotyped lentiviral particles, as well as replication-competent sars-cov, via fcγ receptor ii (fcγrii), but not ace [ , ] . similarly, yip et al. [ ] documented that human macrophages may be infected by sars-cov via fcγrii. however, binding of an immune complex to fcγrii was not sufficient for ade induction, indicating that activation of the other signaling pathways downstream binding to fcγrii receptors is required [ ] . in a sars-cov infection of the promonocytic cell line expressing both fcγrii and ace , a high concentration of antibodies neutralized the virus, whereas a low content of antibodies induced ade [ ] . immunization of rhesus monkeys with a full length sars-cov s protein resulted in enhanced disease severity, with a dominant proinflammatory m -like macrophage profile in the lung tissue, increasing lung injury [ ] . moreover, macrophages produced a significantly greater amount of cytokines in the presence of deceased patients' serum and sars-cov in comparison with the virus alone [ ] . enhanced cytokine production was reduced after fcr blockade. on the contrary, in sars-cov the greatest neutralizing antibody titer was observed earlier in deceased patients in comparison with convalescents [ ] . however, a recent study showed a new mechanism for ade by engaging neutralizing antibodies. monoclonal neutralizing antibody (mab) binding to the mers-cov s protein induced changes in the s protein structure and mediated viral entry to the host cell via igg fcr [ , ] . moreover, ade of mers-cov admission depended on the mab dosage as well as the fcr expression on the cell surface [ ] . in humans, besides the immune cells (including monocytes) infiltrating lungs during pneumonia, epithelial cells of the lower respiratory tract also significantly expressed fcγr [ ] . in a severe course of sars and covid- substantial lung opacity was observed, indicating infiltration by monocytes [ , ] . the infection of human monocytes and macrophages by sars-cov- was also proven [ ] . furthermore, an early humoral response to sars-cov- and greater antibody titer were positively correlated with a delay in the viral clearance and, in consequence, with the severity of the disease [ ] . as mentioned above, great sequence identity and the presence of cross-reactive epitopes of sars-cov- and other hcovs were also documented [ , , ] . monocyte migration to the lungs and the presence of cross-reactive memory antibodies potentially promote the receptivity of elderly individuals to sars-cov- . the lack of immune memory of closely related hcovs (and the consequent inability of ade activation) might be responsible for the absence of clinical symptoms, as well as for the great frequency of undocumented sars-cov- infection, particularly in children [ ] . however, upregulation of ace which is significant component of the renin-angiotensin system (ras) has also been suggested as a cause of severe courses of hcovs infections. animal models demonstrated that angiotensin ii receptor type i (at r ) antagonists or ace inhibitors upregulated ace expression [ , ] . thus, the medicaments widely used in cardiac and hypertensive patients potentially promote the virus binding to the host cells. according to the above, an unequivocal assessment of ade presence in sars-cov- infection seems to be crucial in the vaccine development and antibody-based drug therapy. besides the application of specific anti-sars-cov- , the use of anti-ace with anti-fcγrii monoclonal antibodies to block ade activation and plasmapheresis for restraining cytokine storm elements in plasma has also been proposed as potentially the most effective method of covid- treatment [ ] . the animal model of sars-cov infection documented activation of the complement cascade in the lungs and showed that absence of complement significantly reduced the pathological changes in the respiratory tract, even though the viral load is the same. in the lungs of the transgenic mice deficient in c (c −/− ), which is the central component of the complement system, significantly lower neutrophils and inflammatory monocytes were presented than in infected controls [ ] . moreover, diminished cytokine and chemokine contents in both the lungs and serum of c −/− mice were detected, suggesting that inhibition of complement signaling might be an efficient therapeutic tool in the treatment of sars-cov infection. similarly, the complement system was inordinately activated in mers-cov-infected transgenic mice with human cd /dipeptidyl peptidase (hdpp ), which is a functional receptor for mers-cov. in response to mers-cov infection, enhanced contents of c a and c b- complement activation products in serum and lung tissues of hdpp -tg mice, respectively, were observed [ ] . inhibiting c a production by blocking its receptor (c ar) reduced lung damage and inflammatory responses [ ] . interestingly, the covid- non-survivors presented lower levels of c and c proteins at admission in comparison to patients who recovered [ ] . level of c protein was suggested as a predictor of mortality of covid- patients. recent research showed that mechanisms of responses against hcovs may also be enhanced by other elements of innate immunity. activation of human β-defensin (hbd ) resulted in the conjugation of this protein with the rbd of the mers-cov s protein (s rbd) [ , ] . in consequence, expression of chemokines able to recruit leukocytes (comprising monocytes, macrophages, natural killer cells, granulocytes, t cells, and dendritic cells) was promoted. moreover, enhanced expression of primary immune-inducing molecules (nod , tnf-α, il- β, and il- ) and antiviral factors (such as ifn-β, ifn-γ, mxa, pkr, and rnasel) were also detected, compared to treatment with s rbd alone. immunization of mice with hbd -conjugated s rbd enhanced the immunogenicity of s rbd and induced a stronger s rbd-specific neutralizing antibody response [ ] . the s rbd-hbd treatment also increased phosphorylation and activation of receptor-interacting serine/threonine-protein kinase and ifn regulatory factor . moreover, hbd promoted ccr -mediated nod signaling, inducing the production of type i ifns and an inflammatory response [ ] . although recognition of the relationship between sars-cov- infection and complement system activation is extremely important in defining the best path of treatment, the effect of sars-cov- on complement cascades is still unknown. li et al. [ ] analyzed the association between the cytokine pattern in acute infection and death in sars and suggested that the quality of immune response, rather than magnitude, may be critical in the progress of the disease. the investigation of innate, humoral, and t cell responses during the critical first - weeks may indicate whether they require immunosuppressing therapy or not. sars-cov- has induced the most widespread pandemic in recent decades. the presence of sars-cov- has resulted in almost . million covid- patients in countries and territories, including , fatalities (reported: june by john hopkins university). the differing severity of the covid- outbreak has affected different parts of the world for reasons which are still unclear. the epidemiological studies of a previous pandemic, sars, suggested that human-to-human transmission may enhance the immunogenicity of the virus and its virulence [ ] . as sars-cov- is the first hcov being transmitted directly among humans, it is highly probable that the wide range of the pandemic is a result of the way of transmission. although the phylogenetic similarity of sars-cov- concerned only sars-cov and mers-cov, but not common hcovs inducing mild infections of the respiratory system, the potential cross-reactivity of t cells and antibodies between these viruses and its potential impact on total immune responses and clinical outcomes cannot be excluded. moreover, the presence of mutations in the viral genome is also possible. the next danger is relatively late symptoms occurring in infected people and, in some cases, an asymptomatic course of infection, resulting in a lack of isolation in the early stage of the infection. on the other hand, the genetic similarity of sars-cov- , sars-cov, and to a lesser extent to mers-cov, the similarities between the structure of the epitopes and receptors, the course of the disease and the effects of the infection, allow undertaking a strategy against covid- based on experience gained during the previous pandemics until the mechanisms of covid- are better understood. the first studies related to covid- suggested a protective role of both cell-dependent and humoral immune responses in humans. similar to sars-cov and mers-cov, the sars-cov- infection primarily affected t lymphocytes, particularly cd + and cd + t cells, resulting in a reduction in their numbers and changes in cytokines secretion, including enhanced ifn-γ production by cd + t cells. several studies have also shown the diagnostic utility of serology in sars, mers, and covid- investigation. moreover, the correlation between the severity of the disease and potential immunological markers was documented, which may be a useful prognostic tool of the disease progression, and thereby, in the further course of the pandemic. based on previous experience, immune-informatic tools were used to define the structure of cytotoxic t lymphocyte and b cell epitopes. however, since sars-cov- antibody persistence and 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sars coronavirus are correlated with disease outcome of infected individuals a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein molecular mechanism for antibody-dependent enhancement of coronavirus entry the protein cell atlas webpage clinical characteristics of coronavirus disease in china sars: radiological features host-viral infection maps reveal signatures of severe covid- patients viral kinetics and antibody responses in patients with covid- antibody dependent enhancement due to original antigenic sin and the development of sars. front. immunol. , , effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme upregulation of angiotensin-converting enzyme (ace) in hepatic fibrosis by ace inhibitors anti-ace and anti-fcγrii monoclonal antibodies: a possible treatment for severe cases of covid- . drug des. dev blockade of the c a-c ar axis alleviates lung damage in hdpp -transgenic mice infected with mers-cov human β-defensin plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity human β-defensin is involved in ccr -mediated nod signal transduction, leading to activation of the innate immune response in macrophages funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -hm wd authors: helmy, yosra a.; el-adawy, hosny; abdelwhab, elsayed m. title: a comprehensive review of common bacterial, parasitic and viral zoonoses at the human-animal interface in egypt date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: hm wd egypt has a unique geographical location connecting the three old-world continents africa, asia and europe. it is the country with the highest population density in the middle east, northern africa and the mediterranean basin. this review summarizes the prevalence, reservoirs, sources of human infection and control regimes of common bacterial, parasitic and viral zoonoses in animals and humans in egypt. there is a gap of knowledge conerning the epidemiology of zoonotic diseases at the human-animal interface in different localities in egypt. some zoonotic agents are “exotic” for egypt (e.g., mers-cov and crimean-congo hemorrhagic fever virus), others are endemic (e.g., brucellosis, schistosomiasis and avian influenza). transboundary transmission of emerging pathogens from and to egypt occurred via different routes, mainly importation/exportation of apparently healthy animals or migratory birds. control of the infectious agents and multidrug resistant bacteria in the veterinary sector is on the frontline for infection control in humans. the implementation of control programs significantly decreased the prevalence of some zoonoses, such as schistosomiasis and fascioliasis, in some localities within the country. sustainable awareness, education and training targeting groups at high risk (veterinarians, farmers, abattoir workers, nurses, etc.) are important to lessen the burden of zoonotic diseases among egyptians. there is an urgent need for collaborative surveillance and intervention plans for the control of these diseases in egypt. zoonotic diseases (zd) are those infections that can be naturally transmitted from animals to humans with or without vector [ ] . in the past few decades, there has been a rise in the outbreaks of zoonotic diseases which have an enormous socioeconomic impact worldwide, for instance, all foodborne zoonoses occured in a single country costs about $ . billion annually [ ] . additionally, zd constitute % of all and cattle and buffaloes by b. abortus has been frequently reported [ , ] . in , the prevalence of animal and human brucellosis was % and . %, respectively, at a district located in the nile delta with high livestock density [ ] . in another serosurvey in east of egypt, . % and . % in private farms and individual cases, respectively, were found positive. brucella melitensis biovar was isolated from seroreactive animals [ ] . in the period between and , the prevalence of brucellosis among sheep, goats and cattle flocks in egyptian governorates were . %, . % and . %, respectively [ ] . in humans, up to persons per , population were found positive to brucella in and ; % were male with a median age of years. the infection was associated with close contact to animals and consumption of unpasteurized milk products [ ] . escherichia coli is gram-negative facultative bacteria that belongs to the family enterobacteriaceae. although it is normally commensal in nature and animals, many strains are food-and waterborne zoonotic pathogens. some strains like o and other enterohemorrhagic e. coli (ehec) cause no discernible disease in their animal reservoirs; however, diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome are not uncommon in humans [ ] . in egypt, many studies have shown a high prevalence of e. coli o strains in dairy and meat products in different locations [ , , ] . avian pathogenic escherichia coli (apec) infection is responsible for great economic losses in poultry industry. avian e. coli strains from broiler flocks in egypt were genetically similar to e. coli associated with human infection [ ] with prevalence rate in chicken visceral and human stool samples of . % and . %, respectively [ ] . enteropathogenic e. coli (epec), diffuse-adhering e. coli (daec) and enteroaggregative e. coli (eaec) are associated with infantile diarrhea in egyptian children where enteroadherent e. coli were identified from diarrheic children [ ] . in another study, e. coli was isolated from . % hospitalized patients and . % from outpatient clinics with high degrees of resistance to ciprofloxacin and co-trimoxazole [ ] . also, there is increasing incidence of carbapenemase-and extended-spectrum β-lactamase-producing enterobacteriaceae in dairy farms and hospital-acquired infections. this represents a major health problem because of the few therapeutic alternatives [ ] . listeriosis is one of the foodborne pathogens with high fatality rate. it is caused by listeria monocytogenes, a gram-positive bacterium which is able to grow at temperatures as low as • c challenging the control in human foodstuffs. l. monocytogenes can persist for long periods in the environment or as an asymptomatic infection in adult animals and birds. neurological disorders due to encephalitis are the most common clinical signs in ruminants, in addition to late abortion. consumption of raw milk, cheeses, raw-meat products, poultry and fish is the major sources of infection. human listeriosis is associated with serious invasive illness, particularly in elderly and immunocompromised patients, pregnant women, newborns and infants [ ] . in egypt, existence of the organism in water [ , ] , contaminated food [ ] , seafood [ ] , milk products and milk samples from apparently healthy buffaloes, cows, she-camels, goats and sheep has been reported [ ] [ ] [ ] [ ] . during , listeria spp. was detected in . % of poultry farm samples in egypt [ ] . the isolation rates of l. monocytogenes from different localities in egypt were % in beef burger, % in minced meat and % in luncheon meat; while sausage samples were all negative [ ] . in humans, l. monocytogenes is considered the most common lethal complication in egyptian patients with liver cirrhosis associated with ascites [ ] . detection of the organism in stool samples of hospitalized egyptians was reported in different localities [ , ] . q fever is a zoonotic bacterial disease with public health implications. the disease is caused by coxiella burnetii, a gram-negative bacteriumthat mostly affect ruminants. the bacterium causes abortion in sheep, goat and cattle and is excreted in infected animal feces, urine, milk, and birth products. people can get infected by inhalation of contaminated materials. c. burnetii causes febrile flu-like illness and pneumonia in poor hygiene settings. the epidemiology of q fever in africa is poorly understood [ , ] . the epidemiology of c. burnetii in egypt is not well-known [ ] . antibodies were detected in egyptian donkeys, goats, sheep, pigs, dogs and rats [ ] . q fever may be enzootic in cattle in northern egypt [ ] . in and , c. burnetii antibodies were detected in up to . %, . %, . % and % of sheep, goats, camels and cattle samples in different localities in egypt, respectively [ , , ] . however, all seropositive animals were negative for c. burnetii dna by pcr [ ] . in , the prevalence of c. burnetii in blood samples collected from domestic and imported livestock slaughtered at the abattoir in central egypt was % in buffalo, % in sheep, and % in camels [ ] . the prevalence of antibodies to c. burnetii among egyptians in various locations was to % particularly among cattle workers, veterinarians and veterinary assistants and those live in agricultural districts [ , , ] . schistosomiasis is the second most common parasitic infection globally after malaria [ ] and considered as one of the neglected tropical diseases (ntds) [ ] . schistosomiasis was reported in more than million people in countries and nearly million people are at risk of infection worldwide [ , ] . humans acquire the infection by contact with or drinking of contaminated water. there are three main species infecting humans including s. mansoni, s. haematobium and s. japonicum complex (s. japonicum and s. mekongi) [ ] . infection with schistosoma spp. results in skin rash or itching, flu-like illness and severe intestinal and urinary tract disorders [ , ] . chronic schistosomiasis can persist for years and lead to neurological complications and death [ , ] . schistosomiasis is one of the most endemic parasitic diseases in egypt. the earliest case of human schistosomiasis (s. haematobium) occurred more than years ago in an egyptian adolescent [ ] . in addition, s. haematobium calcified eggs were identified in two mummies aged and years old of the th dynasty [ , ] . between and , the infection rate was very high in school-age children and the infection can persist among adults [ , , ] . furthermore, working in agriculture is a risk factor for schistosoma spp. infection [ ] . the prevalence of s. haematobium and s. mansoni in egypt ranged between . % and % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the highest prevalences of s. haematobium were detected in lower egypt [ , ] , while the highest prevelance of s. mansoni was detected around central egypt [ , , ] . the overall prevalence of schistosomiasis in egypt declined year by year to reach % in , % in , . % in , . % in and - % in [ , [ ] [ ] [ ] . this continuous decrease was due to the control of schistosomiasis by using praziquantel in mass chemotherapy and due to snail control by using anti-bilharzial chemotherapy [ , , ] . fascioliasis is caused by a liver fluke belonging to genus fasciola (fasciola hepatica and f. gigantica). fasciola is one of the neglected foodborne trematodes that infect ruminants and humans worldwide [ ] [ ] [ ] . it was estimated that fascioliasis, associated with other diseases, especially schistosomiasis, affects at least . million people in more than countries [ ] [ ] [ ] . it causes gastrointestinal problems and chronic fascioliasis results in jaundice and inflammation of the liver, gallbladder and pancreas [ ] . fecal excretion of eggs from infected animals (e.g., cattle, sheep, buffaloes, donkeys and pigs) in fresh-water is the main source of infection. after hatching, larva lodge in a particular type of water snail (the intermediate host). carrier animals are infected by eating metacercaria encysted on leaves of water plants or vegetables [ ] . egypt is one of the endemic areas with fascioliasis in the world [ ] with annual loss in milk and meat being estimated to be % [ ] . the climatic factors influence the incidence of fascioliasis and all snail transmitted parasites in egypt [ , ] . f. gigantica is considered the endogenous fasciolid species in egypt with tropical and subtropical distribution [ ] while f. hepatica originated from europe and introduced to egypt through the importation of domestic animals [ , ] . fasciola spp. infected different animal species in egypt including; sheep, goats, cattle, buffaloes, horses, donkeys, camels and rabbits and the infection rates reach % in some areas [ , ] . the overall prevalence in egypt is unknown because reports show wide variations in infection rates [ ] . between and , the prevalence of fasciola spp. among different animal species in different localities in egypt ranged between % and % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in , fascioliasis has been reported in approximately all egyptian governorate where up to % of cattle and buffaloes and % of sheep were positive [ ] . since , human fascioliasis was reported in most of the egyptian governorates especially in the nile delta [ , ] . it was estimated that , humans are infected and million of persons are exposed to the risk of infection in egypt [ ] . between and , fascioliasis has been reported in humans in different localities in egypt and the prevalence rate was between % and % [ , , [ ] [ ] [ ] [ ] [ ] [ ] . from to , the prevalence of fascioliasis in the treated endemic areas was reduced from . to . % [ ] . this decrease in the prevalence was continued in to reach % [ ] . however, in , the prevalence increased again to reach % [ , ] . triclabendazole is the selective treatment of fascioliasis in specific high-risk age groups such as school children and villagers [ ] . cryptosporidiosis is a zoonotic protozoal disease caused by the genus cryptosporidium [ ] . cryptosporidium spp. was discovered by tyzzer in mice [ ] and the first human case was reported in [ ] . afterwards, more attention was given to cryptosporidiosis since it was determined to cause death in one acquired immune deficiency syndrome (aids) patient [ ] . cryptosporidia essentially entered veterinary medicine only in the early s with reports of cryptosporidium-associated neonatal calf diarrhea [ ] and established as a primary enteric pathogen [ ] . the importance of cryptosporidium as a public health problem began in , when more than , residents in milwaukee, wi, usa were affected by c. hominis due to the consumption of contaminated drinking water. this was reported as the largest world waterborne outbreak [ ] [ ] [ ] . out of cryptosporidium species, c. hominis and c. parvum are responsible for more than % of human cases of cryptosporidiosis [ ] , exhibiting acute self-limiting diarrhea in immunocompetent persons and life-threatening diarrhea in immunocompromised persons [ ] . it was estimated that to % of the developing countries' populations were infected with cryptosporidium particularly among -to- -year-old children and toddlers [ ] . reports on cryptosporidiosis in egypt are rare. the highest prevalence rates were reported in rural areas where there is close contact with animals. the accuracy of the reported prevalence rate depends on the used detection method [ ] . in animals, from to the prevalence rate of cryptosporidium infection ranged between % and % among different species including cattle, buffalo calves, camels, sheep, goats, lambs, dogs, wild rats and quails. the highest prevalence rates were reported in governorates that located close to the river nile [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in humans, between and cryptosporidium infection has been reported in almost all egyptian governorates with prevalence rates ranged between % and % or up to % in immunocompromised patients and diarrheic children [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . giardiasis is caused by giardia duodenalis (syn. g. intestinalis, g. lamblia) and considered one of the most common intestinal protozoal parasites affecting humans worldwide [ ] . it also infects other mammals, including pets and livestock [ ] . the high-risk group is children, especially those in daycare settings, orphanages and primary schools [ ] with . million annual cases in the developing countries [ ] . infection with giardia spp. results in gastrointestinal disorders [ ] . giardia cysts can be transmitted to humans through ingestion of contaminated water or food, but the parasite can be transmitted directly from infected individuals [ ] . g. duodenalis has been divided into eight different assemblages (a-h) that have varied host specificities [ ] . assemblages a and b have been found to infect humans and other mammals [ ] . in egypt, few studies have been done to identify the relation between different assemblages and the presence of symptoms, especially among children and animals [ , ] . between and , the prevalence of giardia spp. ranged between % and % among different animal species including ruminants, dairy cattle, stray cats, wild rats and fish [ , , [ ] [ ] [ ] . assemblage e was also more prevalent among ruminants and dairy cattle [ , ] . in humans, between and , giardiasis has been reported in different localities of egypt and the prevalence rates ranged between % and % [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the most prevalent g. duodenalis genotypes were assemblage a and b and to lesser extent assemblage e among diarrheic patients in egypt [ ] [ ] [ ] , , , [ ] [ ] [ ] [ ] . the prevalence of g. duodenalis was high in rural areas more than in urban areas which was attributed to the higher exposure to multiple risk factors such as poor water supply, poor sanitation, and presence of animals [ ] . interestingly, asymptomatic giardia infection can persist for months with . episodes per child/year in rural areas in egypt [ ] . toxoplasma gondii (t. gondii) is one of the most important human zoonotic protozoan parasites, infecting one third of the world's population [ ] . infection with t. gondii is prevalent in humans and animals, including poultry [ ] . cats are considered the key host in the transmission of t. gondii to humans and other animals as they excrete the environmentally resistant oocysts in their feces [ ] . human infections occur through ingestion of food or water containing viable cysts [ ] , congenitally, by blood transfusions or organs transplantation [ ] . the high risk groups are immunocompromised patients and fetuses whose mothers acquire acute infection during pregnancy [ ] . infection with t. gondii is asymptomatic in immunocompetent and primary infected pregnant women; however, severe complication and death have been also reported [ , [ ] [ ] [ ] . in egypt, indoor and outdoor cats are allowed to roam, where they hunt their own food or live on scraps of garbage. therefore, the environment is highly contaminated with oocysts excreted by these cats. this might affect livestock that will later be slaughtered for human consumption [ ] . in s, seroprevalence of the parasite ranged between % and % in stray and domestic cats [ ] [ ] [ ] while in , antibodies were reported in % of cats [ ] . recently, seroprevalence of t. gondii was up to %, indicating high environmental contamination with oocysts [ , , ] . furthermore, t. gondii antibodies were detected in % to % of ruminants, including cattle, sheep and goat and in equines, including horses and donkey [ ] [ ] [ ] [ ] [ ] [ ] . between and , seroprevalence of t. gondii in chickens, turkeys and ducks ranged between % and % in different localities in egypt [ ] [ ] [ ] [ ] [ ] [ ] . therefore, consumption of undercooked poultry meat may be considered a risk factor for toxoplamosis in humans or animals [ ] . in humans, there are limited data on the prevalence of t. gondii and associated risk factors in women during pregnancy. in different egyptian localities, between and , the prevalence of toxoplasma antibodies were ranged between % and % in pregnant women [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , % in cerebrospinal fluid of meningoencephalitis patients [ ] , . % in asymptomatic blood donors [ ] . influenza a viruses (iav), members of the rna family orthomyxoviridae, have up to subtypes according to the variation/combination of the surface glycoproteins hemagglutinin and neuraminidase. iav are further classified to human influenza, swine influenza (siv), bat influenza, equine influenza and avian influenza viruses (aiv). siv and aiv transmit from swine or birds to humans, respectively, mostly via direct contact with infected animals. the infection in humans ranges from mild self-limiting respiratory-like illness to death [ , ] . due to the very low pork production in egypt, swine influenza is not a major zd, although serosurveillance indicated infections of humans in - [ ] . on the contrary, aiv are very important zoonotic viruses in egypt. poultry industry in egypt was estimated in to be one billion birds with several millions of labors. in late , the asian highly pathogenic (hp) aiv of subtype h n was firstly detected in wild migratory birds in a northern egyptian wetland. in february , the virus transmitted to domestic commercial and backyard birds and in march the first human case was recorded. to date, the virus is endemic in egyptian birds causing tremendous economic impact despite the mass vaccination intervention strategy [ ] . another aiv subtype is h n which, in poultry, did not cause severe illness unless the infection is complicated by secondary bacterial infection or immunosuppression. the endemic h n in egyptian poultry was firstly detected in and vaccination is widely used to control the infection [ ] . in humans, egypt is the country with the highest recorded cases worldwide. the fatality rate in egypt is lower than the global rate. thus, lower virulence and subsequent adaptation of the virus in human has been assumed. many mutations that enhance virus binding to mammalian type receptors have been studied. subclinical infection in human has been reported as revealed by serological surveillance [ , ] . nevertheless, a recent study has shown that the virus has not yet acquired the aerogenic transmissibility as naïve ferrets cohoused with inoculated ferrets did not acquire infection [ ] . the egyptian h n viruses are highly susceptible to antiviral drugs (oseltamivir), but are resistant to amantadine. infection is usually acquired by intensive contact particularly with backyard birds. women and children are mostly affected. to date, there are human infections by h n viruses reported to the who [ ] . subclinical infection in poultry workers and co-infection of poultry and human with h and h in egypt have been reported. moreover, serosurvey revealed the presence of antibodies against h viruses in poultry [ ] and in humans [ ] , but no virus was isolated, so far [ , ] . the rabies virus (rabv) belongs to the genus lyssavirus of the rna family rhabdoviridae within the order mononegavirales. rabies is a widespread neurological zoonotic disease of all warm-blooded animals including humans. dogs are the most important host for rabv, however wild carnivores (e.g., fox) and bats are considered reservoirs. infection in humans occurs by direct contact with mucosal surfaces (e.g., bites) or possibly through contamination of wounds with infected materials (e.g., scratches). the virus has a relatively long incubation period (according to the site of bite) which may reach a few months to a year giving the opportunity for immediate vaccination/treatment. each year, about , fatal human infections are recorded worldwide [ , ] . in egypt, rabv was recognized before b.c. [ ] . although the notifiable disease is now endemic in many regions throughout the country, there are no recent reported outbreaks of rabv. it is worth mentioning that the canine population in egypt was estimated to exceed three million stray dogs and half a million owned dogs. vaccination of dogs in egypt was usually done by low-egg-passage of the flury strain, which has now been replaced by attenuated tissue culture vaccines [ ] . the disease transmitted to humans in egypt mainly by dog bites, however the virus has been detected in other animals: cats, ruminants (e.g., buffaloes), horses, donkeys, rodents and mongooses [ ] [ ] [ ] [ ] . in s, the virus was isolated from several rodents and wild mammals including gerbils, foxes and cats [ , ] . in , naval medical research unit three (namru- is a biomedical research laboratory of the us navy located in cairo, arab republic of egypt) isolated street rabv from dogs (n = ), cats (n = ), farm animals (n = ), gerbils (n = ) and jackal (n = ). in , an outbreak was reported to the oie. in - , there was a record for isolation of rabv from dogs in egypt [ ] . in - , two studies reported the detection of the virus in the brain samples of water buffaloes (bubalus bubalis) exhibiting fever and/or nervous signs. the infection was acquired mostly after being bitten by a fox. the virus was closely related to other street strains of dogs in egypt, israel and jordan [ , ] . in , rabv was isolated from brain tissue of an adult female dog. the dog developed hypersalivation, paralysis, and hyperesthesia consistent with rabies symptoms. the virus was genetically similar to canine rabv circulating in egypt [ ] . reports of human infection in egypt have been described from to s [ ] and to annual deaths due to rabv was reported [ , ] . in , a french woman died after corneal transplantation from an egyptian donor [ ] . in , namru isolated two street rabv from humans [ ] . genetically, the egyptian viruses from humans and animals are closely related to those isolated in israel and the me [ ] . lastly but not least, the huge number of stray dogs roaming freely with livestock and insufficient vaccination coverage of pets are among the most common hallmarks of the endemic status of rabv in egypt. rift valley fever (rvf) is a vector-borne zoonotic viral disease firstly reported among livestock in rift valley of kenya in the early s. the disease is caused by a rvf virus (rvfv), genus phlebovirus, a member of the rna family bunyaviridae and was first isolated in . the virus infects mosquitoes, which act as a reservoir, while animals and humans are considered amplifying hosts. rvfv infects a wide spectrum of mammals, causing abortion and mortality. in humans, symptoms range from mild fever, muscle pains and headaches to hepatic failure and death. direct contact to infected animal blood, aerosol, drinking unpasteurized contaminated milk, or the bite of infected mosquitoes are the major sources for human infection. vaccination of susceptible animals, restriction of movement (e.g., importation) and reduction or control of mosquitoes' population are the regular control measures. the disease is common in africa and the me [ , ] . in the last years, egypt reported five large outbreaks: in - , - , , and and the largest epizootic was in - [ ] [ ] [ ] [ ] [ ] [ ] . the primary introduction of rvfv into livestock in these outbreaks in egypt was mostly linked to importation of animals mainly camels from africa [ , , [ ] [ ] [ ] [ ] . the virus was isolated from various species of domestic animals (e.g., sheep, cows, buffaloes, camels, goats, horses, and rats) as well as humans [ , ] .the epizootics of rvf in egypt were reported every year round. the effects of rainfall and river discharge in addition to optimal constellation of interconnected hydrologic, entomologic (high mosquitoes' populations), and social conditions were incriminated in the spread of the virus [ , [ ] [ ] [ ] [ ] [ ] [ ] . moreover, anti-rvfv antibodies were detected in pigs in [ ] , domestic and imported cattle and buffaloes in [ ] and non-immunized dairy cattle from different localities in egypt in - [ ] . the reoccurrence of rvf outbreaks from time to time in animals in egypt challenges the importation control check points and the efficacy of the applied vaccination program [ , ] . in humans, in , rvfv in egypt caused huge outbreaks including , human infections and deaths [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the virus transmitted to eight swedish united nations emergency forces soldiers serving in egypt and the sinai peninsula [ ] . possible human-to-human transmission was described [ ] , however the infection mostly occurred by handling infected meat and inhaling natural virus aerosols [ , ] . in , in southern egypt, - human infections associated with ocular disease, fever and headache were reported [ , ] . serological surveillance indicated that workers at abattoir and sewage treatment plants in several governorates of egypt possessed rvfv antibodies without showing clinical signs [ , ] . moreover, in , rvfv antibodies were detected in~ % of veterinarians and their assistants, butchers and abattoir workers in pigs' abattoir [ ] . middle east respiratory syndrome (mers) caused by a newly emerging coronavirus (cov), designated lineage c of betacoronavirus, in the rna family coronaviridae. it was firstly reported in saudi arabia in a patient with respiratory illness in [ ] and transmitted to several countries not only in the middle east (me) but also in africa, asia and europe. the virus is usually transmitted from dromedary camels via direct contact or consumption of milk or medicinal use of camel urine [ ] . also, bats and alpacas were considered reservoir hosts [ , ] . however, limited human-to-human infections have been also reported. the infection can be mild or fatal in those patients with immune system disorders or chronic diseases [ ] . in egypt, out of swabs and serums collected in june-december from clinically healthy imported or locally reared camels in abattoirs, and positive samples were detected, respectively. attempts to isolate the virus in cell culture were not successful. none of samples collected from workers in these abattoirs were positive by rt-pcr [ ] . in another study, in june , all serum samples collected from humans, cows, water buffaloes, goats and sheep were negative for mers-cov antibodies. conversely, % of serum samples collected from dromedary camels were positive for mers-cov [ ] . from june to february , sera, nasal swabs, rectal swabs, milk samples, and urine samples were collected from camels in different sectors in egypt (importation quarantines, markets, abattoirs, free-roaming herds and farmed breeding herds). results revealed % seropositivity and % of other samples were positive by rt-pcr. seroprevalence was % in imported camels and % in locally raised camels, likewise rna detection rates were % and %, respectively. both juveniles and adult camels were positive by % and % seropositivity and similar rt-pcr detection rates of % and %, respectively [ ] . in humans, from to , none of nasopharyngeal and oropharyngeal swabs collected from returning egyptian pilgrims were positive for mers-cov [ ] . to january , only one human case was reported from egypt in april [ ] . crimean-congo hemorrhagic fever (cchfv) is a tick-borne virus from the bunyaviridae family. the virus causes severe hemorrhagic fever outbreaks, with a case fatality rate of up to %. it is endemic in some african, middle eastern and asian countries. farm animals like sheep, goats, cattle, camels and ostriches can be infected without showing any clinical signs. ticks of the genus hyalomma are the principal vector. humans and animals become infected after being bitten by ticks. also, animal to human transmission through contact with infected animal blood or tissues, particularly at the abattoirs are common. therefore, veterinarians, agricultural workers and slaughterhouse workers are most affected [ ] . in egypt in , the antibodies to cchfv detected in . % camels sera and in . % sheep sera but no antibodies were detected in sera from equines (donkeys, horses and mules), pigs, cows, and buffaloes [ ] . in - , % of serum samples collected from imported camels from sudan and kenya into egypt was positive for cchfv antibodies. native livestock including sheep and cows were negative [ ] . between september and august , . % of cattle, . % of water buffalo, . % of sheep and . % of goats were positive for cchfv antibodies [ ] . in july , ectoparasites removed from freshly slaughtered cattle, buffalo, sheep and camels imported from sudan and somalia were negative for cchfv except five camels were found to harbor ticks carrying rna from a new cchfv variant [ ] . in in a serosurvey in domestic and imported livestock, only one cow out of was positive while buffaloes, sheep, and camels were negative for cchfv antibodies [ ] . no antibodies were detected in sera from humans in [ ] . the only known human infection with cchfv in egypt happened in . an egyptian virologist died after mouth-pipetting a culture of a cchfv isolate that he had brought from iraq [ ] . west nile is a remerging zd caused by west nile fever virus (wnv), a flavivirus that belongs to the rna family flaviviridae. the virus was firstly identified in , in uganda. during the last decade, the incidence of wnv increased worldwide. the virus is transmitted by arthropod vectors and the mosquitoes of the genus culex are main reservoirs [ ] . the latter feed on birds especially passerines and thus birds become infected. migratory birds thought to be responsible for wide spread of wnv and reintroduction from enzootic to new regions [ ] . mosquitoes-birds-mosquitos' infection is the classical transmission cycle in nature. accidentally, human infections occur through biting or dealing with contaminated blood or tissues. the infection in birds is mostly subclinical, while humans exhibit mild, if any, illness to fatal west nile encephalitis particularly in at-risk individuals such as the elderly, immunocompromised and people with chronic illness [ ] . seasonal incidence of wnv outbreaks is linked to higher populations of culex mosquitoes during summer months in temperate regions and rainy seasons in the tropics. however, infections of humans have been also associated with other factors (e.g., blood transfusion, occupational exposure, etc.) [ ] . the egyptian climate is suitable for the spread of wnv [ ] where more than mosquito species and subspecies including culex species were identified [ , ] . the virus was firstly isolated from mosquitoes in early s [ ] . in the period from to , ( . %) out of , examined samples from mosquitoes and sand flies were positive for the wnv. interestingly, the virus transmitted from mosquitoes to sentinel chickens [ ] . it is worth mentioning that more than species of migratory birds visit egypt annually in addition to resident species of birds [ , ] . israeli-like wnv was isolated in white storks in egypt in - suggesting that migrating birds do play a crucial role in geographical spread of the virus [ ] . the first serological evidence for human infections with wnv in egypt were reported in in % of children and % of adults included in a study conducted by melnick et al. [ ] . in , . % of hospitalized febrile children were linked to wnv infection [ ] and out of patients suffering from aseptic meningitis or encephalitis possessed wnv antibodies [ ] . in , approximately half of male university students and % of patients in different localities had wnv antibodies [ , ] . between and , one out of patients with non-specific fever and myalgia was positive for wnv [ ] . in , % of school children aged - years [ ] and . % out of examined persons in the nile delta were positive for wnv antibodies [ ] . wnv had the highest prevalence ( . %) among other viruses in a serosurvey targeting workers in sewage treatment plants from january to october [ ] . in early s, a dutch -year-old female was infected during a holiday in egypt [ ] . in - , wnv was actively circulating in different areas in egypt causing febrile illness and up to % of human serum samples were positive [ ] . currently, the disease is mostly underestimated and scarce data are available, so far. in this review, some aspects of viral, bacterial and parasitic diseases with zoonotic importance in egypt were summarized. there is a gap of knowledge about the epidemiology of zoonotic diseases in different localities in egypt, which hinders accurate assessment of the human health burden. surveillance activity is high for some viral diseases such as influenza and mers but is still weak or neglected for others particularly at the human-animal interface. control of diseases in animals depends on the vaccination (e.g., against rvfv, aivs), anti-microbials for bacteria and antiprotozoal medication against parasitic infection. while some pathogens/diseases are exotic in egypt (e.g., mers-cov and rvfv acquired by importation of camels or wnv from wild birds), others are endemic (e.g., aiv, rabies, schistosoma, and fasciola). transboundary transmission of zoonotic agents from egypt to europe, asia and america occurred via different pathways. rabv was reported in the us via falsified vaccination certificate of dogs [ ] , in europe via corneal transplantation from an egyptian donor [ ] and in asia, probably by dog transfer [ ] . also, rvfv infected swedish soldiers on duty in egypt in and [ ] . egyptian aiv was reported in poultry in neighboring countries most likely due to smuggling of poultry or migratory birds [ ] . approximately , egyptians travel to saudi arabia in pilgrimage, every year, which is an important risk factor for possible introduction or spread of infections [ ] . there are several suggestions to improve the control of zoonoses in animals and humans in egypt. enhancing biosecurity and management in animal farms particularly in poultry sectors may reduce the risk of salmonellosis, campylobacteriosis, listeriosis and influenza viruses. multidrug-resistance of bacteria in animals, due to the misuse of antibiotics in the veterinary sector, is an increasing problem in egypt. therefore, regulations for antibiotic application in animals must be enforced to mitigate the serious public health hazard. vaccination as an alternative approach for the control of bacterial infections in animals, vaccination of stray dogs against rabv and regular investigation of cats for toxoplasmosis should be considered. longer quarantine periods or restriction of importation of animals, particularly camels, from endemic countries may be effective to reduce introduction of zoonotic viruses. control of vectors (e.g., mosquitoes), intermediate hosts (e.g., snails) and animal reservoirs (e.g., stray dogs, cats) should be key components in the intervention strategy of zoonoses in egypt. improving, providing and upgrading diagnostic techniques in both veterinary and human medicines are fundamental to early detect and contain zoonotic infections. last but not least, sustainable awareness, education and training targeting groups at high risk (veterinarians, farmers, abattoir workers, nurses, etc.) are of great importance to reduce the burden of zoonoses among egyptians. taken 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nervous system disorders in egypt prevalence of antibodies to arboviruses in egypt. results of a serologic survey among university students studies on arboviruses in egypt. i. hemagglutination-inhibition antibodies against arboviruses in human population of alexandria and abyss areas arboviral causes of non-specific fever and myalgia in a fever hospital patient population in cairo antibodies to certain arboviruses in humans from a flooded village in egypt west nile virus poliomyelitis after a holiday in egypt isolation and genetic characterization of a novel . . . a h n virus from a vaccinated meat-turkeys flock in egypt pandemic (h n ) and hajj pilgrims who received predeparture vaccination the authors declare no conflict of interest. key: cord- -lyxje u authors: m. najimudeen, shahnas; h. hassan, mohamed s.; c. cork, susan; abdul-careem, mohamed faizal title: infectious bronchitis coronavirus infection in chickens: multiple system disease with immune suppression date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: lyxje u in the early s, infectious bronchitis (ib) was first characterized as a respiratory disease in young chickens; later, the disease was also described in older chickens. the etiology of ib was confirmed later as being due to a coronavirus: the infectious bronchitis virus (ibv). being a coronavirus, ibv is subject to constant genome change due to mutation and recombination, with the consequence of changing clinical and pathological manifestations. the potential use of live attenuated vaccines for the control of ibv infection was demonstrated in the early s, but vaccine breaks occurred due to the emergence of new ibv serotypes. over the years, various ibv genotypes associated with reproductive, renal, gastrointestinal, muscular and immunosuppressive manifestations have emerged. ibv causes considerable economic impacts on global poultry production due to its pathogenesis involving multiple body systems and immune suppression; hence, there is a need to better understand the pathogenesis of infection and the immune response in order to help developing better management strategies. the evolution of new strains of ibv during the last nine decades against vaccine-induced immune response and changing clinical and pathological manifestations emphasize the necessity of the rational development of intervention strategies based on a thorough understanding of ibv interaction with the host. infectious bronchitis virus (ibv) belongs to the family coronaviridae and order nidovirales [ ] . ibv infects chickens and pheasants and induces a clinical disease known as infectious bronchitis (ib) [ ] . the infected chickens may appear depressed with various levels of breathing difficulty and have ruffled feathers [ ] [ ] [ ] [ ] . younger chickens show the most severe clinical manifestations [ ] . in addition, there is serological evidence that poultry workers may develop anti-ibv antibodies following exposure to infected birds, although there is no evidence of active infection in humans [ ] . however, experimentally, certain ibv strains (i.e., massachusetts (mass) and gray) have been found to be capable of replicating in human cell lines [ ] . the first ib case caused by the mass serotype was recorded from north dakota, usa [ ] . since then, hundreds of ibvs with heterogenous genomes have been emerging continuously as a result of mutations and recombination [ , ] . consequently, high ib-associated losses are recorded in spite of control attempts using live attenuated vaccines. different strains of ibv demonstrate varying properties with differences in pathogenesis, virulence, tissue and age tropisms and receptor figure . phylogenetic tree based on the s nucleotide sequences (from the atg start codon to the cleavage site of the spike protein). the phylogeny contains a total of infectious bronchitis virus (ibv) strains from different countries around the world. the ibv strain and genbank accession number are given for each strain. the sequences were aligned using clustal omega, and the phylogenetic tree was constructed using the maximum likelihood method available in raxml with bootstrap replicates for branch support (the numbers on the nodes represent the bootstrap values). the analyses were constructed using geneious ® v . . (https://www.geneious.com/). it is well established that, following the initial infection in the respiratory tract, the virus is disseminated to other tissues due to viremia [ , ] (figure ). however, the exact mechanisms by which ibv leads to viremia are not understood. recently, reddy et al. ( ) showed that a belgian nephropathogenic ibv strain, b , could infect blood monocytes and that these monocytes may facilitate the dissemination of ibv to visceral organs, including the kidney [ ] . in agreement with this finding, another study [ ] showed tropism of ibv towards monocytes by the mass , california phylogenetic tree based on the s nucleotide sequences (from the atg start codon to the cleavage site of the spike protein). the phylogeny contains a total of infectious bronchitis virus (ibv) strains from different countries around the world. the ibv strain and genbank accession number are given for each strain. the sequences were aligned using clustal omega, and the phylogenetic tree was constructed using the maximum likelihood method available in raxml with bootstrap replicates for branch support (the numbers on the nodes represent the bootstrap values). the analyses were constructed using geneious ® v . . (https://www.geneious.com/). ibv is typically transmitted to the host by inhalation, whereupon it attaches to the respiratory epithelium and enters by receptor-mediated endocytosis [ ] . once ibv enters the upper respiratory tract, it targets the epithelium, which is ciliated and includes mucus-secreting glands [ ] , causing ciliostasis and mucus accumulation. the virus replication in the respiratory epithelium peaks - days post-infection (dpi) depending on the infecting ibv strain [ , , ] . consequently, respiratory signs result within dpi and peak around dpi [ ] [ ] [ ] . these respiratory clinical manifestations include sneezing, gasping, coughing, tracheal rales, nasal discharge, conjunctivitis, and dyspnea [ ] . it is not uncommon to observe non-respiratory clinical manifestations such as depression, weight loss, lethargy and huddling together [ , , ] . the morbidity and mortality associated with respiratory tract ibv infection depend on the age of infection; young chickens are severely affected compared to adult chickens [ , ] . at post-mortem examination, hemorrhages and the accumulation of caseous, serous and catarrhal exudates in the trachea, nasal passage and sinuses [ , ] are evident, as well as gross changes in air sacs (i.e., the accumulation of foamy or cloudy exudates) [ ] . depending on the time of sampling, histological features include deciliation and dislodgment of epithelial cells, as well as mononuclear cell infiltration. these changes are visible around - dpi. further development of the lesions, with hyperplasia and hypertrophy of the epithelium and prominent mononuclear cell infiltration in lamina propria, are visible around - dpi. these stages are followed by recovery with repopulation of the mucosa with pseudostratified ciliated epithelium and goblet cells ( - dpi) [ , , ] . it is well established that, following the initial infection in the respiratory tract, the virus is disseminated to other tissues due to viremia [ , ] (figure ). however, the exact mechanisms by which ibv leads to viremia are not understood. recently, reddy et al. ( ) showed that a belgian nephropathogenic ibv strain, b , could infect blood monocytes and that these monocytes may facilitate the dissemination of ibv to visceral organs, including the kidney [ ] . in agreement with this finding, another study [ ] showed tropism of ibv towards monocytes by the mass , california (cal) , connecticut (conn) and iowa ibv strains. using the mass and conn ibv strains, amarasinghe et al. ( ) showed that ibv could infect low numbers of respiratory tract macrophages [ ] . it is also possible that ibv dissemination beyond the respiratory tract may involve the lymphatic system and infected macrophages, similar to marek's disease virus dissemination via infected lung macrophages [ ] . ) showed that ibv could infect low numbers of respiratory tract macrophages [ ] . it is also possible that ibv dissemination beyond the respiratory tract may involve the lymphatic system and infected macrophages, similar to marek's disease virus dissemination via infected lung macrophages [ ] . subsequent clinical manifestations of ibv infection related to the reproductive tract depend on the infecting ibv strain. for example, the m , aust t and qx-like strains are known to cause reproductive tract defects in long-lived chickens [ , , ] , leading to low egg production and quality, whereas ibv strains such as conn and iowa do not cause reproductive tract abnormalities [ ] . the mechanisms that lead certain ibv strains to establish reproductive tract infection are unknown. in different body systems. all the ibv strains primarily infect the respiratory tract, and based on the genotypes, the ibv infection can extend to various tissues, either persisting or leading to clinical and pathological manifestations. the solid arrows indicate paths that have been confirmed. the empty arrows indicate paths that have been suggested. the text boxes with continuous borders summarize histological changes, and the text boxes with discontinuous borders represent clinical manifestations. ses, shell-less egg syndrome; fls, false layer syndrome; git, gastrointestinal tract. subsequent clinical manifestations of ibv infection related to the reproductive tract depend on the infecting ibv strain. for example, the m , aust t and qx-like strains are known to cause reproductive tract defects in long-lived chickens [ , , ] , leading to low egg production and quality, whereas ibv strains such as conn and iowa do not cause reproductive tract abnormalities [ ] . the mechanisms that lead certain ibv strains to establish reproductive tract infection are unknown. however, ibv replication in reproductive mucosa has been documented in several studies [ , , ] . depending on the infecting ibv serotype, localization of ibv antigens in the oviduct can vary, and evidence of higher ibv replication has been observed in chickens infected at a younger age when compared to adults [ ] . this age difference in ibv replication is reflected in differences in clinical and pathological outcomes in chickens. for example, chickens infected with certain strains of ibv such as mass, qx-like strain or aust t at ages of - days develop cystic oviducts without impaired ovarian functions, which leads to false layer syndrome with no egg production [ , [ ] [ ] [ ] . such flocks with false layer syndrome do not reach peak production, with a consequence of premature culling [ , ] . infection with ibv in laying hens can negatively influence egg production, resulting in poor-quality eggs, such as misshapen, miscolored, thin, rough-shelled or shell-less eggs and eggs with watery albumin, meat or blood spots, which can peak at around two weeks post-infection [ , , ] . in addition, egg production in laying hens can drop by - % [ , ] . although production bounces back close to normal within nine weeks, there can be a - % decline relative to normal production [ , ] . ibv infection of the reproductive tract of laying hens leads to shorter, hypoglandular oviducts and regressed ovaries [ , ] . histologically, deciliation of the epithelium and a reduction in the height of the epithelial cells, along with epithelial desquamation and degeneration, are common in infected oviducts between and dpi [ ] with occasional follicular destruction and microbleeding [ , ] . fibroplasia and edema of infected lamina propria are also evident [ ] . in contrast to the development of false layers, chickens with patent oviducts were found to have an intact surface epithelium with localized hypoplasia of glandular areas [ ] . later, following around dpi, lymphoid nodules are seen in the oviduct [ ] . although the consequences of ibv targeting the reproductive tracts of female chickens is known, little attention has been given to changes corresponding to the reproductive tracts of male chickens, which may cause impacted sperm production, infertility and venereal transmission of ibv [ , ] . recent studies have found, depending on the infecting ibv strain, that ibv targets the testes and causes low sperm production and infertility [ , , ] . ibv replication in the efferent duct epithelium and the formation of epididymal stones lead to low sperm production and infertility [ ] . gallardo and colleagues observed ibv replication in cells of the seminiferous tubule (i.e., sertoli cells) infected with arkansas (ark) and mass ibv strains; moreover, they demonstrated ibv transmission to layers via infected semen, indicating the possibility of venereal transmission of ibv [ ] . ibv-induced changes including mononuclear cell and heterophil infiltration, necrosis and microbleeding of testes have been documented [ ] . the nephropathogenic ibv strains include b , aus t, qx-like, / , holte and gray [ , , , , ] . the ibv b strain is known to spread from the respiratory tissues via blood monocytes [ ] . following dissemination, ibv strains could infect ciliated cells of the nephron, including proximal, distal and collecting tubules, depending on the infecting ibv strain [ , , , ] . ibv strains that target the kidney have received increased attention due to their higher virulence in young chickens when compared to other ibv strains [ , , ] . apart from the general signs associated with ibv infection, nephropathogenic ibv strains result in weight loss, watery droppings, increased water consumption and an increase in the incidence of mortality [ , ] . the nephropathogenic ibv pathogenesis also varies according to the breed of the chickens; for example, the clinicopathological manifestations of nephropathogenic ibv are more severe in rhode island red chickens when compared to white leghorn chickens [ ] . at post-mortem examination, ibv-infected kidneys are pale, discolored and enlarged [ , ] . urate deposits are also commonly observed with tubular distention [ , , ] . histologically, tubules develop degenerative changes, ureters become distended with cellular debris and urate crystals are seen in the tubules; in addition, mononuclear cell recruitment in interstitial tissues in the medulla and cortex has been observed [ , , ] . although ibv has been isolated from cloacal swabs, there is no indication that ibv is transmitted via the fecal-oral route. it is possible that gastrointestinal infection follows respiratory infection, subsequent infection of monocytes and macrophages and the spread of ibv via the blood or lymphatics [ , ] . ibv strains such as qx-like strains, /b ( / ) and moroccan g are known to infect the gastrointestinal tissues, leading to clinical and pathological manifestations [ , [ ] [ ] [ ] . the qx-like strains are capable of targeting the proventriculus and ileum, leading to proventriculitis and, occasionally, diarrhea [ , ] . moroccan g ibv has also been shown to target the gastrointestinal tissues, such as the esophagus, jejunum, ileum and rectum [ ] . other experimental studies that used moroccan g ibv indicated that the virus targets the epithelial covering of the tips of villi of the ilium and rectum, leading to atrophy of the villi and desquamation of epithelial cells, with lymphocyte, macrophage and heterophil infiltration in the mucosa [ , ] . ibv /b (i.e., / ) has also been shown to replicate in the esophagus and ileum, leading to enteritis in young broiler chickens [ ] . in the early s, broiler chickens infected with ibv strain /b were found to develop bilateral pectoral myopathy [ ] . the disease was characterized by edema, due to a gelatinous material, followed by facial hemorrhage and mild separation of muscle fibers [ ] . although bilateral myopathy could not be reproduced with ibv /b, mild gross changes were observed with no indication of histological changes or muscle damage [ ] . further, a study that collected samples with bilateral pectoral myopathy from a slaughter plant in brazil could not establish an association of this condition with ibv infection, although ibv was detected in muscle tissues using molecular techniques [ ] . consequently, the muscle lesions were suggested to be caused by type iii hypersensitivity involving the deposition of immune complexes in the capillary walls of pectoral muscles, rather than lesions induced by viral replication [ ] . depending on the ibv strain, the virus can persist in the tissues of chickens for an extended period [ , , ] . for example, ibv (i.e., aust t strain) can persist, particularly in the cecal tonsils and kidney, for more than seven months [ ] . the mass ibv strain can persist in the cecal tonsils, spleen and kidney for about a month [ ] . although the period of ibv persistence is influenced by the age at infection [ ] , the length of persistence does not depend on the systemic anti-ibv antibody concentration [ ] . the implications of ibv persistence in chickens are twofold. first, persistently infected chickens are a source of infection for naïve chickens [ ] . second, persistent ibv infection promotes viral evolution [ ] . the ibv-induced potential immune-suppressive mechanisms are summarized in figure . ibv strains / , qx-like, strain g and mass infect various immune organs, such as the cecal tonsils [ ] , spleen [ ] , harderian gland and bursa of fabricius [ , ] . it is not known whether the tropism of ibv for these immune organs depends on the virulence of the infecting ibv strain or whether ibv replication in these immune organs impacts immune functions. it is very well documented that avian viruses that replicate in immune organs, such as marek's disease virus [ ] , chicken anemia virus [ , ] and infectious bursal disease virus [ ] , are immunosuppressive, impacting vaccine-mediated immune response and resulting in secondary bacterial infections [ ] . however, recent investigations provided molecular and cellular evidence that ibv, in fact, directly interferes with the host's innate response at various levels, potentially impacting the elicitation of adaptive host response. the toll-like receptors (tlrs) and are innate receptors and have a role in detecting ibv-associated molecular patterns, such as double-stranded (ds) and single-stranded (ss) ribonucleic acid (rna), respectively [ ] . certain brazilian strains of ibv are capable of inhibiting tlr signaling, leading to decreased proinflammatory cytokines and decreased mrna expression linked to the development of cell-mediated immune response, leading to increased pathology in the kidney [ ] . similarly, a conn strain of ibv has been shown to downregulate mrna expression of tlr , interleukin (il)- β and interferon gamma (ifn-γ), leading to increased ibv genome accumulation and more severe pathology in the respiratory tissues [ ] . certain strains of ibv can inhibit the expression of pathogen recognition receptors such as toll-like receptors (tlrs) or their signaling pathway, leading to reduced expression of proinflammatory cytokines, which eventually interferes with the innate immune response and induction of the adaptive immune response. ibv is also capable of replicating in respiratory tract macrophages, inhibiting their functions and inducing apoptosis. ibv is capable of incorporating cd molecules into its envelop during egress from the host cells, shielding it against lysis via complement-and antibody-dependent mechanisms. protection against ib is mediated by both antibody-and cell-mediated immune responses [ , ] . although the antibody-mediated immune response predominantly depends on a response , enabling it to persist in the host for a longer period. epithelial cells are the primary target sites of ibv replication, which results in deciliation and destruction, leading to inhibition of the mucociliary escalator mechanism. certain strains of ibv can inhibit the expression of pathogen recognition receptors such as toll-like receptors (tlrs) or their signaling pathway, leading to reduced expression of proinflammatory cytokines, which eventually interferes with the innate immune response and induction of the adaptive immune response. ibv is also capable of replicating in respiratory tract macrophages, inhibiting their functions and inducing apoptosis. ibv is capable of incorporating cd molecules into its envelop during egress from the host cells, shielding it against lysis via complement-and antibody-dependent mechanisms. one of the immune cell types that bridges innate and adaptive host responses is the macrophages, and the available data show that certain ibv serotypes (i.e., mass and conn) target respiratory tract macrophages and replicate within them, thus leading to a productive infection [ , ] . although the impact of immune cell targeting of ibv has not been studied completely, ibv replication in macrophages could decrease type ifns activity [ ] , similar to ibv's ability to hinder type ifns response in epithelial and fibroblast cells [ ] . type ifns are the main antiviral molecules synthesized in the host in response to viral replication, and, previously, it has been shown that ibv is sensitive to the antiviral activity of type ifns given as a treatment to prevent ib in chickens [ ] . although it is not known how ibv inhibits the function of type ifns in macrophages, one potential explanation is that the accessory proteins of ibv could play a role in this immune evasion strategy. in agreement with this view, the ability of ibv accessary proteins a and b to interfere with type ifns production in target cells other than macrophages has been shown [ , ] . other than this interference with the production of type ifns, ibv is capable of inhibiting the downstream signaling of type ifns, minimizing the expression of interferon-stimulating genes (isgs) by preventing the functioning of signal transducer and activator of transcription (stat ) [ ] . another implication of ibv replication in macrophages is the destruction of macrophages due to apoptosis. ibv could induce programmed cell death in avian macrophages via intrinsic and extrinsic routes involved in apoptosis [ ] . although the mechanisms of the destruction of macrophages by ibv require further investigation, evidence has shown that macrophage numbers increase at h and then decline in the trachea and lungs in response to ibv infection [ ] . it is important to understand whether this decline is related to the apoptosis of ibv-infected macrophages. the complement system functions as a part of the innate arm of the immune response and also plays a role in adaptive immune functions. the components of the complement pathway aid in the host immune response via complement-and antibody-mediated lysis of viruses [ ] [ ] [ ] [ ] . since host cells are shielded from lysis via this strategy due to the expression of cd in the host cell membrane, ibv is capable of incorporating cd into its envelop during exit from the host cells, negating lysis via complement-and antibody-dependent mechanisms [ ] . previous studies showed that ibv is vulnerable to attack by this strategy [ ] . ibv replication impacting the respiratory tract brings forth a defective clearance mechanism by the deciliation of the respiratory epithelium, thereby increasing the vulnerability of respiratory surfaces to secondary bacterial infections [ ] . the co-infection of respiratory pathogens with ibv causes complications and worsens the clinical and pathological manifestations [ , ] . the infection of chickens with mycoplasma gallisepticum followed by ibv infection can result in coryza, tracheitis and airsacculitis in the host [ ] . in addition, secondary infections with pathogenic escherichia coli can lead to prominent lesions in respiratory surfaces, pericarditis and death [ ] . in addition, the co-infection of haemophilus paragallinarum with ibv could result in severe lesion development and increased mortality rates [ ] . protection against ib is mediated by both antibody-and cell-mediated immune responses [ , ] . although the antibody-mediated immune response predominantly depends on a response to the ibv s protein, the main cell-mediated response, cd + cytotoxic t cell response is elicited by the ibv n protein [ ] . following ibv infection, memory b [ ] and t cells [ ] are formed and are present in peripheral blood and the spleen. following ib vaccination, the number of b and t cells increases in the harderian gland [ ] . increased recruitments of cd + and cd + t cells in the trachea have also been shown following application of live ib vaccines [ ] . vaccination is the primary choice for the control of ib in the poultry industry. the chickens receive multiple ib vaccinations, and the frequency of vaccination depends on the expected production life of the chicken. for broilers, day-old vaccination in the hatchery is commonly practiced followed by a booster vaccination at - weeks of age [ ] . multiple vaccinations with live attenuated vaccines are usually given to layers and breeders followed by a killed vaccine just before the onset of the laying period [ , ] . the live attenuated vaccines are administered via drinking water, eye drop, or coarse spray and inactivated vaccines are given parenterally. for example, in eastern canada, the layer and breeder pullets are vaccinated with various combinations of live attenuated ib vaccines starting at one day of age and then, at two, five and nine weeks followed by an inactivated ib vaccine given at fourteen weeks of age. during the lay, the chickens are not vaccinated. the goal of such layer and breeder vaccination strategies is to ensure the transfer of maternal antibodies to the offspring [ ] as well as provide the extended protection during the lay [ , ] . however, vaccination of day-old chickens is controversial for three reasons. first, maternal antibodies could be protective against potential ibv infection during the first few days of life while early vaccination could enhance the decay of the maternal antibodies [ ] . second, early vaccination (i.e., day of age) induces poor b cell and t cell responses when compared to later vaccination (i.e., day of age) [ ] . third, ib vaccination at the day of age can induce severe vaccine reactions [ ] . despite these reasons, live attenuated ib vaccination in the hatchery can be critical for the prevention of severe respiratory illness and cystic lesions in the oviduct that results from very early exposure to virulent ibvs [ , ] . since the introduction of vaccination against ib using live attenuated (mass serotype) vaccines, different vaccine serotypes such as / , ark, conn, d and d have been developed and made available commercially [ ] . based on the prevalence of the various ibv serotypes, and the availability of licensed vaccines in a geographical area, different areas within countries use different combinations of live attenuated and inactivated vaccines. in certain regions in europe, vaccines against mass and / serotypes are widely used, while, in certain states within usa, vaccines against mass, ark and conn serotypes are extensively used [ ] . the serological response to ibv is mainly induced by the s protein [ , ] and, consequently, cross-protection induced by ib vaccines against heterologous strains varies widely. the study of cook and colleagues indicated that the use of more than one vaccine serotype can produce better cross-protection against challenge with heterologous ibv serotypes [ ] . therefore, priming with more than one heterologous live vaccines and boosting with inactivated or live attenuated vaccines are practiced particularly by layer and breeder industries [ ] . it has also been shown that vaccination protocols that involve more than one serotype induced better immune cell recruitment in the respiratory mucosa when compared to vaccination with a single serotype vaccine [ ] . although control of ib relies on vaccination [ ] , several limitations of ib vaccination have been observed. the steady increase of ibv variants leading to frequent outbreaks in vaccinated flocks has become a concern increasingly [ , , ] . in addition, attenuated ibv strains used for vaccination can spread among individual birds within flocks [ ] , and changes in virulence during bird-to-bird passage can lead to production problems [ , ] . coinfection of host cells with live attenuated ib vaccines and wild type ibv can also result in genomic recombination contributing to the virus evolution [ , ] as well as mutations that can occur under the effect of immune pressure [ ] [ ] [ ] . although the global poultry industry practices hatchery vaccination against ib, day may not be the optimum time for vaccination in terms of inducing systemic and mucosal immune responses against ib [ ] because of the developing immune system [ ] . the availability of a limited number of licensed vaccines to choose in a geographical area is also a constraint. for example, in canada, various ibv variants including mass, conn, / , ca and dmv/ [ , , [ ] [ ] [ ] are circulating in poultry flocks and only live attenuated vaccines developed against mass and conn serotypes and inactivated vaccines developed against mass and ark serotypes are available for optimizing on farm ib vaccination strategies. given the limited efficacy of existing ib vaccination strategies, it is critical to establish an ibv surveillance system that characterizes the different ibv strains circulating in various geographical areas and that we understand the antigenic and/or genetic similarities between the circulating ibvs and the available ib vaccines. these data will lead to the optimization of ib vaccination strategies to prevent vaccine breaks. if the existing vaccines are not useful in optimizing vaccination strategies, it would also worth developing autogenous vaccines (inactivated) using characterized unique ibv isolates prevalent in a given geographical area in order to include in the existing vaccination regimes. given the available scientific evidence against ib vaccination at the hatchery [ , ] , it may also be appropriate to postpone the first ib vaccination to a barn vaccination done at seven days of age relying on maternal antibody response to protect the chickens during the first week of life [ ] . there are numerous issues surrounding the use of live attenuated ib vaccines [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and relying on inactivated vaccines can be an option, but they inherently lack the ability to induce mucosal immune response, which is critical for the control of ib [ ] [ ] [ ] . it is possible that inactivated vaccines can be used for the induction of mucosal immune response when combined with various nanoparticles [ ] . after decades of research into ibv and numerous studies on vaccine efficacy for the control of ib, this disease is still a major economic concern globally. although many studies have been conducted on ibv pathogenesis, there is little specific information on ibv receptors that determine macrophage tropism and tissue tropism, mechanisms that allow ibv dissemination from the respiratory tract to secondary tissues, ibv persistence in the cecal tonsils and kidney and the immunopathogenesis and immunosuppressive mechanisms of different strains of ibv. novel and sensitive assays and other necessary tools are now available for in-depth investigations of the mechanisms involved in these pathogenesis events. given the concern of vaccine breaks and the emergence of heterogeneous ibv strains, investigations leading to an in-depth understanding of the pathogenesis of ibv are necessary. studies of host-ibv interaction lead to the understanding of tropism of ibv for various body systems, severity of lesions produced in each of these tissues and ways the virus is shed to the environment [ , , , ] . for example, certain ibv variants impact tissues such as kidney and reproductive tract in addition to the respiratory tract [ , , , ] . we are not aware if the current vaccination protocols induce adequate mucosal immune responses in each of these tissues to minimize consequences of ibv replication. it is critical to optimize ib vaccination strategies that induce adequate immune responses in target body systems of these ibv variants minimizing the impact. in addition to secondary bacterial infections, vaccine induced immune response to any pathogen can be impacted by immune suppression induced by ibv. it is necessary to determine if ibv induced immune suppression impact the immune responses intended to be generated by a variety of vaccines used in chickens [ ] . 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bronchitis virus isolated in japan transmissibility of infectious bronchitis virus h vaccine strain among broilers under experimental conditions molecular epizootiology of infectious bronchitis virus in sweden indicating the involvement of a vaccine strain ion channel activity of influenza a virus m protein: characterization of the amantadine block genetic mutations in live infectious bronchitis vaccine viruses following single or dual in vitro infection of tracheal organ cultures different evolutionary trajectories of vaccine-controlled and non-controlled avian infectious bronchitis viruses in commercial poultry avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination hatchery vaccination against poultry viral diseases: potential mechanisms and limitations genotyping of infectious bronchitis viruses identified in canada between vaccine efficacy against ontario isolates of infectious bronchitis virus using phylogenetic analysis to examine the changing strains of infectious bronchitis virus infections in ontario over time vaccination against infectious bronchitis virus: a continuous challenge inactivated infectious bronchitis virus vaccine encapsulated in chitosan nanoparticles induces mucosal immune responses and effective protection against challenge etiology and immunology of infectious bronchitis virus comparison of the pathogenicity of qx-like, m and /b infectious bronchitis strains from different pathological conditions 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 conflict of interest. key: cord- -l jjb l authors: ojeda, nicolás; cárdenas, constanza; marshall, sergio title: interaction of the amino-terminal domain of the isav fusion protein with a cognate cell receptor date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: l jjb l the infectious salmon anemia virus (isav), etiological agent of the disease by the same name, causes major losses to the salmon industry. classified as a member of the orthomyxoviridae family, isav is characterized by the presence of two surface glycoproteins termed hemagglutinin esterase (he) and fusion protein (f), both of them directly involved in the initial interaction of the virus with the target cell. he mediates receptor binding and destruction, while f promotes the fusion process of the viral and cell membranes. the carboxy-terminal end of f (f( )) possesses canonical structural characteristics of a type i fusion protein, while no functional properties have been proposed for the amino-terminal (f( )) region. in this report, based on in silico modeling, we propose a tertiary structure for the f( ) region, which resembles a sialic acid binding domain. furthermore, using recombinant forms of both he and f proteins and an in vitro model system, we demonstrate the interaction of f with a cell receptor, the hydrolysis of this receptor by the he esterase, and a crucial role for f( ) in the fusion mechanism. our interpretation is that binding of f to its cell receptor is fundamental for membrane fusion and that the esterase in he modulates this interaction. infectious salmon anemia (isa) is an aggressive disease that primarily affects atlantic salmon (salmo salar), one of the most commercially relevant farmed fish. the disease has high mortality rates, with outbreaks occurring at later stages of the breeding process, seriously impacting the industry's sustainability [ ] . isa has affected all major salmon-producing countries, including canada, norway, scotland, the faroe islands, and chile [ ] [ ] [ ] [ ] [ ] . the etiological agent of isa is a relatively new virus within the orthomyxoviridae family and is the only member of the novel isavirus genus [ ] . the infectious salmon anemia virus (isav) shares similarities with other members of the family, including a segmented single-stranded negative-sense rna genome and a viral envelope [ , ] . the isav envelope contains two major glycoproteins that mediate binding, membrane fusion, and receptor destruction. one of them is a hemagglutinin esterase (he) which interacts with sialic acids on the susceptible cell surface via the hemagglutinin domain, promoting viral attachment; on the other hand, the esterase acts as a receptor-destroying enzyme (rde), allowing the release of new viral particles in the context of a productive infection [ , ] . the second isav envelope protein is known as fusion protein (f) and mediates the fusion process of the viral and cellular membranes. cleavage of f by extracellular proteases and a ph-mediated conformational change, once inside the endosome, are necessary for the activation of the fusion mechanism [ , ] . viral genome segments and code for he and f, respectively. virulence markers have been described on both genes, particularly deletions in a highly polymorphic region (hpr) on segment , corresponding to a region of the protein located near the transmembrane domain (i.e., stalk region), pathogens , , of and insertions near the cleavage site for f in segment [ , ] . nonvirulent strains of the virus carry a full-length he gene (hpr ) and have not been isolated in isav-permissive cell lines; on the other hand, highly virulent isav strains carry versions of the he gene with deletions in the hpr (hpr∆), suggesting that the hpr -type gene constitutes the ancestral he [ , , ] . both hpr and hpr∆ he types are fully functional in terms of receptor-binding and receptor-destroying activities, with a specific n- o acetylated sialic acid on the cell surface being identified as the viral receptor [ ] . nevertheless, the hpr length seems to have an influence on the fusion mechanism, with hpr he having lower fusion activity compared to the hpr∆ types [ ] . in influenza virus, receptor binding and membrane fusion are mediated by hemagglutinin (ha), with neuraminidase (na) being responsible for receptor destruction activity [ ] . regarding na, hydrolysis of sialic acids is not only related to liberation of new viral particles but also to cleavage of mucins from mucus, allowing access to target cells, and migration of virions to appropriated entry sites on polarized cells surfaces [ , ] . thus, equilibrium in the receptor-binding/receptor-destroying activities has an important role on the initial viral particle interactions with the cell. in influenza virus, variations in the length of the neuraminidase stalk region have been associated with modifications in tropism and infectivity, with deletions in this region of the na gene being recognized as virulence markers in avian strains [ , ] . a modification in the length of the na stalk region may affect the substrate accessibility of the enzyme, altering the receptor-binding/receptor-destroying activities equilibrium and ultimately affecting the viral infectivity [ ] . in isav, he contains both receptor-binding and -destroying activities, with no apparent influence from the hpr region over their equilibrium [ ] . interaction of ha with a cellular receptor is a key step for the activation of the fusion mechanism in influenza [ ] . in isav, no cellular receptor has been described for f, with he being regarded as solely responsible for viral adhesion to the cell surface [ , , ] . after proteolytic cleavage, f generates two subunits, linked by disulfide bridges [ ] . the carboxy-terminal f , contains structural motifs typically associated to fusion proteins [ ] . on the other hand, no function has been suggested for the amino-terminal f . interestingly, even though the fusion protein from isav seems incapable of binding red blood cells (rbcs) from salmon, it does retain its fusion activity in the absence of an he [ ] . initial interactions of viral particles with the cell are highly important for infection, with fusion of the viral and cellular membranes being a key step. viral surface proteins and cell glycome diversity are crucial elements regulating virulence and tropism [ , ] . receptor-binding, receptor-destroying, and membrane fusion activities have a unique distribution in isav, compared to other orthomyxovirus; moreover, he hpr length variation represents a novel mechanism for fusion activity and virulence regulation. in this context, interaction of f with a cell receptor, with regulation of the esterase activity over this receptor via hpr length, represents a feasible explanation for the differential activities described for the hpr and hpr∆ fusion systems. in this study, we aimed to determine the role of the f domain in the activity of the fusion protein. due to the lack of a crystal structure, and robust sequence homology to other species, we based our analysis on secondary structure homology and propose a hemagglutinin-like structure for f , suggesting a receptor-binding activity for this domain. we demonstrate the interaction of the f protein of isav with a cellular receptor, probably a carbohydrate motif, and the importance of f for the fusion process. interestingly, the he esterase has an effect over the interaction of f with its receptor, which further narrows down the chemical identity of the receptor and suggest a regulatory role for he not only in the adhesion but also in the fusion mechanism of isav. finally, we propose a model where the stalk length in he modulates the activity of the viral esterase over the putative f receptor and consequently over the fusion process. these results demonstrate the important role of viral surface proteins in the regulation of infectivity; in particular, they may further define the influence of hpr on the fusion activity in isav, suggesting a novel role for f (i.e., receptor interaction). finally, these results offer new insights into the isav fusion system, and the directly related infectivity, aiming to provide a better understanding of the viral behavior. the isav f carboxyl-end sequence contains canonical features of a type i fusion protein, and its characteristics have already been thoroughly analyzed [ , ] . furthermore, the structure of the fusion core has been resolved, allowing for robust definition of the two protein domains: a carboxy-terminal (f ), containing the transmembrane domain, a coiled coil region, and a fusion peptide, and an amino-terminal domain (f ), of undefined tertiary structure and function [ ] . due to the lack of a crystal structure and robust sequence homology to proteins from other species for the amino-terminal domain of f, we aimed to develop a structural model for this subdomain. a secondary structure analysis of the amino acid sequence for isav f revealed a high content of beta strands for the f subunit, with this topology being characteristic of hemagglutinin domains [ ] (figure ). considering that the f domain resembles the carboxy-terminal domain of influenza virus a ha and influenza virus c hemagglutinin-esterase-fusion hef, a hemagglutinin-like domain could constitute the amino-terminal structure of the protein. pathogens , , x for peer review of interaction). finally, these results offer new insights into the isav fusion system, and the directly related infectivity, aiming to provide a better understanding of the viral behavior. the isav f carboxyl-end sequence contains canonical features of a type i fusion protein, and its characteristics have already been thoroughly analyzed [ , ] . furthermore, the structure of the fusion core has been resolved, allowing for robust definition of the two protein domains: a carboxyterminal (f ), containing the transmembrane domain, a coiled coil region, and a fusion peptide, and an amino-terminal domain (f ), of undefined tertiary structure and function [ ] . due to the lack of a crystal structure and robust sequence homology to proteins from other species for the amino-terminal domain of f, we aimed to develop a structural model for this subdomain. a secondary structure analysis of the amino acid sequence for isav f revealed a high content of beta strands for the f subunit, with this topology being characteristic of hemagglutinin domains [ ] (figure ). considering that the f domain resembles the carboxy-terminal domain of influenza virus a ha and influenza virus c hemagglutinin-esterase-fusion hef, a hemagglutinin-like domain could constitute the amino-terminal structure of the protein. conservation of secondary structure among viral hemagglutinins. (a) secondary structure comparison for diverse viral hemagglutinins. psipred was used to calculate a secondary structure for infectious salmon anemia virus (isav) fusion protein (f) (aax . ), and experimentally defined secondary structures for influenza virus a hemagglutinin (ha) (inf ha), influenza virus c hef (inf hef), porcine torovirus hemagglutinin esterase (ptov he), and bovine coronavirus hemagglutinin esterase (bcov he) were obtained from their pdb entries ( xrs, flc, ilk, and cl , respectively). beta strands and alpha helices are indicated by red and blue arrows, respectively. boxed in green are the receptor-binding domains of each protein. there is a clear conservation of beta strands in all of the receptor-binding domains (b) tertiary structure of the receptor-binding domain for the selected viral hemagglutinins. beta strands constitute a swiss roll domain, with interstrand loops defining the specificity of interaction in each molecule. the corresponding cellular receptor is shown. a putative ligand-binding subdomain (f lb) was defined between positions to of the aax . sequence, considering the presence of theoretical alpha helices between residues - and - in f , typical of the carboxyl and amino ends of the receptor-binding domain of viral hemagglutinins. a theoretical d model for f lb was obtained using comparative modeling using the ptov he as a template, with a manual alignment based on the secondary structure of each sequence ( figure ). the isav f beta strands are arranged in an antiparallel fashion, forming a swiss roll structure, a putative ligand-binding subdomain (f lb) was defined between positions to of the aax . sequence, considering the presence of theoretical alpha helices between residues - and - in f , typical of the carboxyl and amino ends of the receptor-binding domain of viral hemagglutinins. a theoretical d model for f lb was obtained using comparative modeling using the ptov he as a template, with a manual alignment based on the secondary structure of each sequence ( figure ). the isav f beta strands are arranged in an antiparallel fashion, forming a swiss roll structure, similar to viral hemagglutinins. in canonical hemagglutinins, residues located at the loops connecting the beta strands are involved in ligand binding, with a hydrophobic pocket commonly interacting with the glycan [ ] . in our isav f model, two phenylalanine residues are located in this area and thus may play a role in the interaction with a potential receptor. pathogens , , x for peer review of similar to viral hemagglutinins. in canonical hemagglutinins, residues located at the loops connecting the beta strands are involved in ligand binding, with a hydrophobic pocket commonly interacting with the glycan [ ] . in our isav f model, two phenylalanine residues are located in this area and thus may play a role in the interaction with a potential receptor. to assess the direct interaction of isaf f, and he, with the cell surface, we used purified xflag-tagged soluble versions of isav he and f (rhe and rf) in lectin immunofluorescence assays, using ask cells as targets [ ] . indeed, both rhe and rf effectively interacted with the cell surface, as demonstrated by the fluorescent stain pattern (figure a , b). ask cells pretreatment with naoh (i.e., saponification) impeded the interaction of rf with the cell, suggesting that the receptor for f may correspond to a sialic acid, as has been already demonstrated for isav he (figure d ) [ ] . moreover, preincubation of the cells with soluble he blocked the adhesion of rf to the cell surface, and chemical pre-inactivation of the esterase on rhe by dcic treatment eliminated the blockage, suggesting that the initial effect was not due to ligand competition, but rather to the enzymatic activity of rhe (i.e., esterase) over the rf receptor (figure e ,f). these results suggest a direct interaction of f with the cell surface and, furthermore, with a sialic acid receptor, considering the results of the saponification and dcic assays. to confirm the interaction of he and f with their cell receptors, ask cells were pretreated with rhe and/or rf, prior to cell infection. after h, isav gene expression was used to evaluate the level of infection compared to mock-treated controls. figure shows the qrt-pcr inhibitory profiles obtained in each assay. the three treatments had a significant negative effect over the viral infection compared to the untreated control, with a pronounced effect for the rhe treatment ( % inhibition), a lower effect for rf ( . % inhibition), and the highest inhibitory effect for the treatment with a (a) secondary structure-based sequence alignment of the ptov he sequence ( ilk) and the isav f domain. the alignment was used to build a structural model using modeller [ ] . beta strands and alpha helices are indicated in red and blue arrows, respectively. the green arrowhead indicates the position of relevant phe residues (b) the theoretical structure for f displays a conserved swiss roll conformation. phe residues at positions and , possibly involved in receptor binding, are depicted in ball and stick representation. to assess the direct interaction of isaf f, and he, with the cell surface, we used purified xflag-tagged soluble versions of isav he and f (rhe and rf) in lectin immunofluorescence assays, using ask cells as targets [ ] . indeed, both rhe and rf effectively interacted with the cell surface, as demonstrated by the fluorescent stain pattern (figure a ,b). ask cells pretreatment with naoh (i.e., saponification) impeded the interaction of rf with the cell, suggesting that the receptor for f may correspond to a sialic acid, as has been already demonstrated for isav he (figure d ) [ ] . moreover, preincubation of the cells with soluble he blocked the adhesion of rf to the cell surface, and chemical pre-inactivation of the esterase on rhe by dcic treatment eliminated the blockage, suggesting that the initial effect was not due to ligand competition, but rather to the enzymatic activity of rhe (i.e., esterase) over the rf receptor (figure e ,f). these results suggest a direct interaction of f with the cell surface and, furthermore, with a sialic acid receptor, considering the results of the saponification and dcic assays. to confirm the interaction of he and f with their cell receptors, ask cells were pretreated with rhe and/or rf, prior to cell infection. after h, isav gene expression was used to evaluate the level of infection compared to mock-treated controls. figure shows the qrt-pcr inhibitory profiles obtained in each assay. the three treatments had a significant negative effect over the viral infection compared to the untreated control, with a pronounced effect for the rhe treatment ( % inhibition), a lower effect for rf ( . % inhibition), and the highest inhibitory effect for the treatment with a combination of the two lectins ( . % inhibition). notably, there was a significant difference between the double-lectin treatment and each one of the single-lectin treatments, suggesting an additive inhibitory effect on isav pathogens , , of infection for the rhe/rf combination ( figure ). these results confirm the interaction of both viral proteins with the cell surface. moreover, the interaction of each viral protein with a specific receptor is confirmed, which translates in the blockage of infection in this assay. pathogens , , x for peer review of combination of the two lectins ( . % inhibition). notably, there was a significant difference between the double-lectin treatment and each one of the single-lectin treatments, suggesting an additive inhibitory effect on isav infection for the rhe/rf combination ( figure ). these results confirm the interaction of both viral proteins with the cell surface. moreover, the interaction of each viral protein with a specific receptor is confirmed, which translates in the blockage of infection in this assay. combination of the two lectins ( . % inhibition). notably, there was a significant difference between the double-lectin treatment and each one of the single-lectin treatments, suggesting an additive inhibitory effect on isav infection for the rhe/rf combination ( figure ). these results confirm the interaction of both viral proteins with the cell surface. moreover, the interaction of each viral protein with a specific receptor is confirmed, which translates in the blockage of infection in this assay. to assess the role of the f domain on the fusion activity of f, different isav he and f combinations were evaluated in membrane fusion assays, using transfected chse/f cells expressing the viral he and f proteins and r -labeled salmon rbcs. after adhesion, cells were subjected to trypsin treatment and low ph to activate the fusion mechanism. fusion was evident as the transference of fluorescence from the rbc to the chse/f cell. we assessed the level of expression of every construct, using immunofluorescence assays, and found effective and comparable expression of all of them ( figures s -s ) . hemagglutinin esterase and fusion protein from the isav hpr strain were used as positive controls and initially compared to the he /f combination. as previously described, he is associated with a less efficient fusion system, with % of fusion, compared to the % of the positive control ( figure a ). to evaluate the role of phe and phe from f in the fusion mechanism, f mutants were developed, carrying phe to ile mutations (table ). both residues seem to have an important role in the protein functionality, with the phe ile mutation reducing the fusion activity to a . %, the phe ile mutation reducing fusion activity to a . %, and the double mutant completely eliminating the fusion activity ( figure b ). these results demonstrate an important role for these two residues, and the f domain, in the protein functionality and further suggest their participation in a receptor-binding site, with direct participation of these two amino acids. table . heterologous expression constructs developed to evaluate the fusion mechanism of isav. the combination of he and f from an hpr strain (he and f , respectively) was used as positive control. a combination of he from hpr (he ) and f was used to evaluate the influence of the stalk length on the system. mutants were designed to determine the influence of the f domain in f, and the esterase activity, accordingly. all wild type (wt) and mutant open reading frames (orfs) were cloned in the p xflag-cmv expression vector. all constructs were xflag-tagged at the carboxy-terminal end. fluorescent fusion proteins were developed for each construct, adding egfp or mcherry to the carboxy-terminal end of the hes and fs, respectively (not shown in table). considering our results, we hypothesized that the esterase in he has the capacity to hydrolyze the receptor for f and that this interaction is crucial for the fusion activity of f. to analyze the influence of the he hpr esterase activity over the fusion mechanism, an he ser ala mutant was developed, rendering the enzyme inactive [ ] . interestingly, the fusion mechanism with the he ∆est/f combination showed a level of % of fusion activity, times the activity displayed by the he /f system, implying a negative influence of the enzyme over the viral fusion mechanism (figure c ). this result coincides with our hypothesis, suggesting that the esterase in he has activity against the f receptor and that this activity has a detrimental effect over the membrane fusion mechanism. moreover, this result led us to the hypothesis that the diminished fusion activity associated with the he hpr -type may be connected to the esterase activity of this protein. pathogens , , x for peer review of mechanism. moreover, this result led us to the hypothesis that the diminished fusion activity associated with the he hpr -type may be connected to the esterase activity of this protein. esterase inactivation via ser ala mutation on he significantly augments the native fusion activity of the hpr system, from % to %. asterisks *, **, and *** indicate statistical significance with p < . , p < . , and p < . , respectively. the present study demonstrated a crucial role for the f domain of the isav f protein in membrane fusion, a key step in the viral infection process. f interacts with target cells, and its putative receptor may be hydrolyzed by the esterase present in the he viral protein. the enzyme has influence over the fusion phenomena, presumably hydrolyzing the f receptor. finally, we hypothesize that the activity of the enzyme over the f cellular ligand is regulated by the length of the hpr region, where an he with an extended region (i.e., hpr ) could be more active against this putative sialic acid. hydrolysis of the f receptor upon viral adhesion may impede f interaction with the cell, diminishing the membrane fusion process and the subsequent viral infection. these findings represent new data by which the functional basis of the isav hpr behavior and the virus infection mechanism may be understood. interaction with cell receptors is a key element for the activation of viral infective mechanisms, either directly or by conducting the viral particle to cell regions where these systems can be activated [ , ] . on the other hand, receptor destruction plays an important role in the release of the new viral particles. ultimately, differential expression of receptors in particular cells may regulate the pathogen's tropism [ ] . in the case of isav, adhesion to the target cells has been attributed to the viral he interaction with a sialic acid ( -n, -o acetylated) on the cell surface [ ] . the membrane fusion activity, on the other hand, is mediated by the so-called fusion protein (f), eventually promoting the transference of the viral content into the cell [ ] . finally, the release of the new virions is related to the enzymatic activity of the esterase in he, which acquires the receptor-destroying enzyme (rde) quality [ ] . in isav, this simplified scheme does not cover the real complexity of the system. one of the greatest questions surrounding isav is the relation between the nonvirulent hpr strains and their virulent counterparts. it has been proposed that the former are the ancestral strains, given that they have the genetic potential (i.e., full he gene) [ ] . certainly, the fact that hpr strains have not been isolated in culture has made the study of this potential relation very difficult. the particular mechanisms involved in the lack of virulence and the impossibility of in vitro isolation have not been fully resolved yet. the molecular characteristic that defines the isav hpr strains is the presence of an elongated version of the he gene, compared to the hpr∆ types. both hpr and hpr∆ hes are fully functional in terms of their receptor-binding activities, as proved by hemadsorption assays using salmon rbc. (c) esterase inactivation via ser ala mutation on he significantly augments the native fusion activity of the hpr system, from % to %. asterisks *, **, and *** indicate statistical significance with p < . , p < . , and p < . , respectively. the present study demonstrated a crucial role for the f domain of the isav f protein in membrane fusion, a key step in the viral infection process. f interacts with target cells, and its putative receptor may be hydrolyzed by the esterase present in the he viral protein. the enzyme has influence over the fusion phenomena, presumably hydrolyzing the f receptor. finally, we hypothesize that the activity of the enzyme over the f cellular ligand is regulated by the length of the hpr region, where an he with an extended region (i.e., hpr ) could be more active against this putative sialic acid. hydrolysis of the f receptor upon viral adhesion may impede f interaction with the cell, diminishing the membrane fusion process and the subsequent viral infection. these findings represent new data by which the functional basis of the isav hpr behavior and the virus infection mechanism may be understood. interaction with cell receptors is a key element for the activation of viral infective mechanisms, either directly or by conducting the viral particle to cell regions where these systems can be activated [ , ] . on the other hand, receptor destruction plays an important role in the release of the new viral particles. ultimately, differential expression of receptors in particular cells may regulate the pathogen's tropism [ ] . in the case of isav, adhesion to the target cells has been attributed to the viral he interaction with a sialic acid ( -n, -o acetylated) on the cell surface [ ] . the membrane fusion activity, on the other hand, is mediated by the so-called fusion protein (f), eventually promoting the transference of the viral content into the cell [ ] . finally, the release of the new virions is related to the enzymatic activity of the esterase in he, which acquires the receptor-destroying enzyme (rde) quality [ ] . in isav, this simplified scheme does not cover the real complexity of the system. one of the greatest questions surrounding isav is the relation between the nonvirulent hpr strains and their virulent counterparts. it has been proposed that the former are the ancestral strains, given that they have the genetic potential (i.e., full he gene) [ ] . certainly, the fact that hpr strains have not been isolated in culture has made the study of this potential relation very difficult. the particular mechanisms involved in the lack of virulence and the impossibility of in vitro isolation have not been fully resolved yet. the molecular characteristic that defines the isav hpr strains is the presence of an elongated version of the he gene, compared to the hpr∆ types. both hpr and hpr∆ hes are fully functional in terms of their receptor-binding activities, as proved by hemadsorption assays using salmon rbc. interestingly, when the assay was performed using rabbit rbcs, the esterase activity seemed to be more pronounced in the hpr -type, as manifested in the earlier release of this type of erythrocyte, as compared to a hpr∆ type [ ] . this suggests that the activity of the enzyme over the receptor pathogens , , of present in the rabbit rbcs may be regulated by the length of the hpr and that this ligand for he is different from the one located on salmon erythrocytes. on the other hand, the fact that salmon rbcs are not released in the hemadsorption assays suggests that the hpr-esterase regulation may not apply to the activity over the "primary ligand" ( n- o sialic acids) described as the main receptor for isav he. this stalk-length-mediated regulation of the rde activity phenomenon may have a parallel in influenza, where the length of the neuraminidase stalk region seems to be related to the viral tropism in avian strains [ , ] . interestingly, in isav, the fusion process is affected by the length of the hpr in he, with a full stalk being associated to a reduced fusion activity [ ] . interaction of the hemagglutinin (ha) with its receptor in influenza virus is a key step to the activation of the fusion mechanism, before the proteolytic cleavage and endosomal acidification [ ] . an analogous condition is true for paramyxovirus, were a structural change is promoted by the interaction of hn with its receptor, which in turn is transmitted to the f protein, triggering a rearrangement which ultimately leads to the membranes fusion [ ] . a similar model has been proposed for isav, where the direct interaction of he and f could be responsible for the activation of the latter and the concomitant dissociation upon receptor binding; although this model is robust, no conformational changes were detected on the isav he structure upon receptor binding, and thus, he is not likely the trigger to activate isav f [ , ] . on the other hand, there have been no reports of a direct interaction of the fusion protein of isav with the cell, prior to the conformational change and insertion of the fusion peptide. the equilibrium between the receptor-binding and -destroying activities is a key element for the infective process, not only in terms of the "input/output" ratio of viral particles, but also in the initial interaction of the pathogen with the cell surface [ , ] . in isav, if the primary receptor ( n- o sialic acids) binding via he is not affected by the hpr length/esterase activity, possibly a secondary viral protein-cellular ligand interaction is regulated by those elements. considering these findings, we postulated a putative interaction of f, prior to activation, with a cell receptor. there have been no reports of an experimental structure for the isav f subunit, and protein sequence homology to proteins from other species is very low, which makes it very difficult to project a model structure or function to this domain. hemagglutinin tertiary structures are relatively conserved among species, even though they share modest sequence homology [ , ] . furthermore, the f domain of isav f bears structural homology to the fusion domain of influenza virus ha, that is, a coiled coil structure and a hydrophobic fusion peptide, which suggest a conserved structure for the amino-terminal domain as well. effectively, a secondary structure prediction for the isav fusion protein revealed a high content of beta sheets, which is characteristic of hemagglutinin domains, forming a swiss roll type structure (figure ) [ ] . sequence alignment allowed for comparative modeling of a subdomain in f using the structure of the ptov he as template ( figure ) . although there is a low level of amino acid conservation between model and template ( . % identity), there is structural homology between the proteins, considering the presence of beta sheets in both and the location of particular hydrophobic residues [ ] . typically in hemagglutinins, hydrophobic amino acids located between the beta strands are directly involved in ligand binding [ , [ ] [ ] [ ] [ ] . using heterologous expression of isav he and f, we have previously obtained soluble, xflagtagged versions of the viral proteins [ ] . these were used to perform lectin immunofluorescence assays on ask cells, a fish cell line conventionally permissive to isav infection. the assays revealed a clear interaction of both rhe and rf with a cellular element, possibly a membrane-bound ligand (figure a,b) . interaction of rf with its putative receptor was hampered by preincubation of the cells with rhe, suggesting a competition for the cellular ligand between the two proteins ( figure e) . interestingly, treatment of rhe with dcic, an esterase inhibitor, reverted the effect, suggesting that the enzyme is responsible for the blockage of the interaction of rf with the cell (figure f) . isav esterase has shown activity against a variety of sialic acids, including -o-acetylated sialic acids, which are not necessarily recognized as receptors for he [ ] . these results imply that a sialic acid, acting as receptor for f, may be hydrolyzed by the esterase in he. moreover, saponification of the glyco-conjugates present on the ask cell membrane impeded the interaction of rf with its receptor, confirming the chemical nature of the molecule (figure e) . interaction of rhe and rf with target cells was further demonstrated by the inhibition of isav infection in lectin-treated ask cells. incubation of the cells with the soluble lectins, prior to cell infection, blocked the viral receptors on the cell surface, diminishing infection as reflected by a lower expression of the viral genomic segment as compared to the mock-treated controls (figure ) . preincubation of the cells with rhe had a more pronounced effect on viral infection inhibition than the rf treatment, this being probably related to the different strength of the interaction of he and f with their receptors. thus, the reported lack of hemadsorption capacity on f may be due to a weak interaction with its receptor on rbcs, where the low-strength binding of the sialic acids does not allow the adhesion and retention of the erythrocyte by the f-expressing cell [ ] . moreover, treatment of ask cells with a combination of both lectins resulted in the highest inhibition of viral gene expression/isav infection, with significant differences from the single-lectin-treated cells; accordingly, the result suggests the existence of independent receptors for he and f, manifested in an additive effect for the inhibition, and confirms the importance of the interaction of both proteins with the cell surface for the infective process. as previously reported, membrane fusion assays revealed a lower fusion activity in the hpr -type system, compared to a hpr∆ (figure a ) [ ] . similarly, we used this approach to evaluate the role of the f domain in the fusion mechanism. in particular, the importance of the aforementioned phenylalanine residues in the f lb was demonstrated by the phe ile and phe ile mutations, with the system showing a reduction in activity to . % and . %, respectively, compared to the control (figure b) . moreover, the double mutant abolished all fusion activity, proving the importance of these residues for the mechanism and a putative interaction of f with a cell receptor. both phe residues are highly conserved amongst isav f sequences reported in genbank, with only % of them showing a phe leu change (data not shown), which correlates with the importance of their function, as proven by these results. we suggest an influence of the hpr over the isav he esterase activity, considering the results previously reported for hemadsorption with rabbit rbcs, and that this activity may influence the interaction of f with its cell receptor [ ] . indeed, fusion assays using a ser ala mutated version of hpr he showed an increased fusion activity compared to the wt hpr he, demonstrating a negative influence of the enzymatic activity over the fusion mechanism. for the esterase inactive mutant, membrane fusion activity reached up to % (figure c ). these results coincide with the rhe-mediated impairment of the rf interaction with the cell and the recovery of such interaction with the dcic treatment (figure ), reinforcing the hypothesis of a direct relation of f with a sialic acid on the cell surface and the importance of this event for the activation of the fusion mechanism. we hypothesize that the combined roles of he and f define the fusion activity of the viral particle, where synergistic effects modulate this mechanism, to optimize viral replication. interestingly, even though salmon rbcs are effectively (and irreversibly) agglutinated by isav he and are capable of endocytosis, effective replication of isav on these cells has not been confirmed, and they are regarded as poor viral factories for other piscine viruses [ , [ ] [ ] [ ] [ ] . in this context, the virus may "select" an appropriate cell target, using a secondary receptor (i.e., an f receptor), independent of he. thus, the interaction of he with its receptor, as proved by hemadsorption and hemagglutination, does not suffice for the correct activation of the membrane fusion mechanism and cell infection. finally, a particular tropism may be related to the hpr behavior, where specific cells on the fish support viral replication for the strain (i.e., with high content or high exposure of the f receptor) at low rate and with no deleterious effect to the host. further studies should focus on resolving the specific identity of this sialic acid and its presence in particular tissues/cells of the fish. in conclusion, we have demonstrated a novel role for the f protein in isav, where interaction of the protein with a sialic acid on the cell surface, prior to activation, can be a key step for the membrane fusion process. highly conserved amino acids on the f subdomain are crucial for the protein function and possibly related to ligand binding. moreover, esterase activity in he hydrolyses the ligand for f, with the enzymatic activity being regulated by the length of the hpr. figure depicts the hypothetical model we propose for initial isav interaction with the cell surface, derived from our results and their interpretation. pathogens , , x for peer review of the ligand for f, with the enzymatic activity being regulated by the length of the hpr. figure depicts the hypothetical model we propose for initial isav interaction with the cell surface, derived from our results and their interpretation. the full-length stalk on the hpr -type augments the activity/accessibility of the esterase over a sialic acid, which acts as a receptor for f. lack of interaction of f (in purple) with the cell surface impairs the activation of the fusion mechanism and ultimately diminishes the infective capacity of these strains. on the hpr∆ types, the shortened stalk negatively regulates the enzymatic activity over the f receptor, allowing the interaction of f with this sialic acid and, finally, the successful activation of the fusion mechanism. atlantic salmon kidney (ask) cells (atcc crl ) [ ] and common bluegill embryo (chse/f) cells (formerly known as chse- , atcc ) [ ] were cultured in leibovitz's l- medium with mm glutamine (gibco) and supplemented with u/ml penicillin, µ g/ml streptomycin, . µ g/ml amphotericin, and % fetal bovine serum (gibco), at °c. cells were grown to % confluence and accordingly subcultured. field isolates of isav corresponding to the hpr and hpr b types were obtained from the laboratorio de genética e inmunología molecular strain collection. viral infection and propagation were performed using ask cells, where an % confluent cell monolayer was washed twice with l- medium and further covered with a viral dilution prepared in l- medium. after h of incubation, the viral inoculum was removed, and the cells were washed twice with l- medium and further cultured in l- medium supplemented with % fetal bovine serum and antibiotics, at °c. after days, the cell supernatant was recovered and filtered ( . μm), and viral aliquots were collected and stored at − °c. a plaque assay was used for virus tittering days post-infection (d.p.i.), as previously described [ ] . spodoptera frugiperda (sf ) cells were used for recombinant baculovirus production and recombinant protein expression. cells were grown in suspension culture using sf- iii medium the full-length stalk on the hpr -type augments the activity/accessibility of the esterase over a sialic acid, which acts as a receptor for f. lack of interaction of f (in purple) with the cell surface impairs the activation of the fusion mechanism and ultimately diminishes the infective capacity of these strains. on the hpr∆ types, the shortened stalk negatively regulates the enzymatic activity over the f receptor, allowing the interaction of f with this sialic acid and, finally, the successful activation of the fusion mechanism. atlantic salmon kidney (ask) cells (atcc crl ) [ ] and common bluegill embryo (chse/f) cells (formerly known as chse- , atcc ) [ ] were cultured in leibovitz's l- medium with mm glutamine (gibco) and supplemented with u/ml penicillin, µg/ml streptomycin, . µg/ml amphotericin, and % fetal bovine serum (gibco), at • c. cells were grown to % confluence and accordingly subcultured. field isolates of isav corresponding to the hpr and hpr b types were obtained from the laboratorio de genética e inmunología molecular strain collection. viral infection and propagation were performed using ask cells, where an % confluent cell monolayer was washed twice with l- medium and further covered with a viral dilution prepared in l- medium. after h of incubation, the viral inoculum was removed, and the cells were washed twice with l- medium and further cultured in l- medium supplemented with % fetal bovine serum and antibiotics, at • c. after days, the cell supernatant was recovered and filtered ( . µm), and viral aliquots were collected and stored at − • c. a plaque assay was used for virus tittering days post-infection (d.p.i.), as previously described [ ] . spodoptera frugiperda (sf ) cells were used for recombinant baculovirus production and recombinant protein expression. cells were grown in suspension culture using sf- iii medium (gibco) supplemented with % fetal bovine serum, at • c. suspension cells were seeded at . × cells/ml, agitated at rpm in ml flasks, and subcultured when reaching ( - ) × cells/ml. recombinant baculovirus were developed for the expression and purification of the extracellular domains of isav he and f proteins, using the baculovirus/sf cells expression system, as previously described by ojeda et al. [ ] . for recombinant protein production, a suspension culture of ml of sf cells at × /ml was infected using a multiplicity of infection (moi) of . after h culture at • c, sf cells were pelleted at × g, washed twice with grace's insect medium, and then suspended in lysis buffer ( mm tris-hcl ph . , mm nacl, mm edta, and % triton x- ). after a h incubation on ice, samples were sonicated using % of amplitude in five s repetitions, with s of cooling between each repetition. samples were then centrifuged at , × g for min to remove cell debris. the supernatant, containing the recombinant proteins, was filtered ( . µm) using a low-protein-binding filter (millipore, burlington, usa). recombinant hemagglutinin esterase and fusion proteins (rhe and rf) were purified using the anti-flag m magnetic beads system (sigma-aldrich, st. louis, missouri, usa) and the magnetight separator stand (novagen, darmstadt, germany) according to the manufacturer's instructions. protein concentration for each sample was measured using the bca assay kit and bsa standard (pierce, waltham, ma, usa). approximately µg of each protein was obtained from the infected sf cells. ask cells were seeded on mm glass bottom dishes ( × cells). following a h incubation, the cells were fixed with fresh % paraformaldehyde in phosphate-buffered saline (pbs) for min. then, cells were washed twice with pbs and treated with a blocking buffer ( % bsa and . % triton x- in pbs) for min. after the buffer was removed, ng of rhe or rf, diluted in blocking buffer, was added to the cells, followed by incubation for h. to analyze the influence of he over the interaction of f with a cell receptor, rhe was pretreated with µm dcic (sigma-aldrich, st. louis, missouri, usa) for min before incubating the cells with the recombinant lectin for h [ ] . after three pbs washes, ng of rf were added and incubated as described above; a dmso-treated aliquot of rhe was used as control. ask cells were treated with . m naoh for min for saponification. this procedure results in the de-o-acetylation of sialic acids, prior to the lectin incubation step, to evaluate the interaction of rf with sialic acids on the cell [ ] . as a negative control, ask cells were incubated with a protein extract from sf cells ( µg of total protein). after the lectin incubation steps, cells were washed three times with pbs. cells treated with single lectins were incubated with an anti-flag m antibody (sigma-aldrich, st. louis, missouri, usa) at a / dilution in blocking buffer. cells treated with rhe and rf were incubated with an anti-isav f ( b /a ) (bioschile, santiago, chile) at a / dilution. after h of incubation and three pbs washes, alexa fluor conjugated goat anti-mouse (thermo-fisher, waltham, ma, usa) was added to the cells at a / dilution in blocking buffer. following a final h incubation, cells were washed three times with pbs, and cell nuclei were stained with a solution of to-pro- ( µm in pbs) (thermo-fisher, waltham, ma, usa) for min. after three final washes with pbs, ml of the same buffer was added, and cells were examined by confocal microscopy using the tcssp ii confocal microscope (leica microsystems inc., wetzlar, germany). ask cells at % confluence, growing on a six-well plate ( . × cells per well), were washed two times with l- medium. then, each well was incubated with ng of sf -expressed rhe, rf, or a mixture of both in l- medium. as control, cells were treated with ng of total protein from sf cells. after a min incubation, cells were washed six times with l- medium. then, cells were infected with isav hpr b at an moi of . , as described above. total rna was extracted from the cells h post-infection (h.p.i.) using the trizol reagent (life technologies, carlsbad, california, usa) and suspended in nuclease-free water. a µl rna sample was used as template for qrt-pcr reactions using the stratagene qrt-pcr iii master mix according to the manufacturer's instructions. specific primers were used to analyze the expression of viral genes (viral segment ) [ ] and that of a cellular housekeeping gene (elongation factor α) used for normalization [ ] . the fold-change of viral gene expression relative to the control was assessed using the -∆∆ct method, as previously described [ ] (table ). table . primers used to amplify the viral orf corresponding to he and f proteins, to add restriction sites for cloning in p xflag-cmv , to mutate specific residues in both, and to evaluate the expression of viral and cellular genes. he was cloned between the not i and kpn i sites on the expression vector. for the he-egfp fusion, an xba i site was added to the and sites of he and egfp, respectively, due to a kpn i site present in the fluorescent protein gene. he and f fusion proteins were cloned between the not i/bam hi and eco ri/bam hi sites of the p xflag-cmv vector. a reference sequence (aax . ) for the isav fusion protein was retrieved from ncbi protein sequence database [ ] and was analyzed with the psipred method v . to predict secondary structure [ ] . comparison with the sequences and experimental secondary structures for hemagglutinins from porcine torovirus (ptov) ( i k), bovine coronavirus ( cl ) and influenza c virus ( flc) was performed, in order to define a putative ligand interacting domain for isav f [ , , ] . a d model of a partial f domain was obtained based on manual alignment with the ptov he structure sequence. comparative modeling was performed using the modeller interface in ucsf chimera [ , ] , using the ptov he structure as template. sequence alignment was performed using jalview [ ] . alignment and structural representations were obtained using clc genomic workbench (qiagen, hilden, germany). total rna was extracted from ask cells infected with isav hpr , as described before, using the trizol reagent (life technologies, carlsbad, california, usa) according to the manufacturer's instructions. a mg kidney sample from an isav hpr infected salmon was obtained from the laboratorio de patógenos acuícolas, and total rna was extracted using the trizol reagent, as described above. cdna was synthesized from µg of total rna with m-mlv reverse transcriptase (invitrogen, carlsbad, california, usa) using ng of oligo (dt) - , mm of each dntp, and u of m-mlv reverse transcriptase according to the manufacturer's instructions. the isav hpr he and f genes (he and f , respectively) and the isav hpr he gene (he ) were amplified through polymerase chain reaction (pcr) using the corresponding cdna as template and cloned in an appropriate expression vector. in detail, to analyze the influence of the esterase in hpr he and the influence of amino acids phe and phe in f over the fusion process, point mutations were added to the open reading frames (orfs), using overlap extension pcr (oe-pcr) [ ] . the orfs for egfp and mcherry were added to the end of the he and f orfs, respectively, to obtain fusion proteins and directly evaluate the construct's expression. an overview of the different constructs is presented in table . the wild type (wt), mutated, and fusion orfs were cloned in the p xflag-cmv vector (sigma-aldrich, st. louis, missouri, usa). accordingly, primers were designed for each process ( table ). the pcr reactions were performed in a µl total volume using a µl cdna sample as template, with x hf phusion buffer (neb, ipswich, ma, usa), µm dntps, nm of each primer, m betaine, and u of phusion dna polymerase (neb, ipswich, ma, usa). thermal conditions used for pcr reactions were as follows: min at • c, followed by cycles of s at • c, s at • c, and min at • c, with a final extension stage of min at • c. pcr products were cloned in the pjet . /blunt vector using the clonejet pcr cloning kit (thermo fisher, waltham, ma, usa) according to the manufacturer's instructions. after digestion with specific restriction enzymes (promega, madison, wisconsin, usa) and gel electrophoresis, gene fragments and the linearized p xflag-cmv vector were purified with the genejet gel extraction kit (thermo fisher, waltham, ma, usa). for cloning in the p xflag-cmv vector, a mixture containing ng of the digested expression vector and ng of the digested he or f gene inserts were ligated using the rapid dna ligation kit (thermo fisher, waltham, ma, usa) according to the manufacturer's instructions. recombinant plasmids (pdna) were isolated using the genejet plasmid dna kit (thermo fisher, waltham, ma, usa) from transformed dh α cells grown in lb broth. the cloned sequences and expression constructs were verified by sequencing (macrogen, seoul, korea) using the cmv f and cmv r primers. exponentially growing chse/f cells were seeded on glass bottom mm dishes and cultured overnight, as described above. cells were washed twice with pbs and then incubated in l- medium. a transfection mixture was prepared for each dish, diluting transfection reagent fugene (promega, madison, wisconsin, usa) in µl of l- medium and then adding pdna of the he and/or f expression constructs. here, ng of the he constructs and/or ng of the f constructs were added to the mixture [ ] . fugene was used in a reagent:pdna (µl:µg) ratio of : . after vortexing, the mixture was incubated for min and then added to the cells dropwise. after a h incubation at • c, the medium was removed and the cells washed three times with l- medium. cells were then incubated in supplemented l- medium for h, as described above, to evaluate protein expression and perform functional analyses. to evaluate the level of expression of the wt and mutant he and f, immunofluorescence assays were performed on transfected chse/f cells, using the anti-flag m antibody to detect the pflag tag on the end of each construct. after two pbs washes, cells were fixed with % paraformaldehyde and then treated with blocking buffer as described above. anti-flag m antibody was added to the cells at a / dilution in blocking buffer. after h incubation and three pbs washes, alexa fluor conjugated goat anti-mouse was added to the cells at a / dilution in blocking buffer. after a h incubation, cells were washed three times with pbs, adding a final ml of the same buffer, and then five representative fields of each transfected dish were analyzed. images were captured using the tcssp ii confocal microscope and then analyzed using the analyze tool of the fiji software package. corrected total cell fluorescence (ctcf) was calculated using ctcf = integrated density − (selected area × background fluorescence) and used as a measure of fluorescence intensity and protein expression level [ ] . in parallel, the expression of the he-egfp and f-mcherry fusion constructs was evaluated on the co-transfected cells to ensure the co-expression of both proteins. as described above, five fields of each transfected dish were evaluated by direct observation on the confocal microscope. blood samples ( µl) were obtained from pre-smolt salmo salar ( g) after appropriate sedation in . % benzocaine (centrovet, santiago, chile) for min. syringes and collection tubes were treated with an anticoagulant solution containing mm sodium citrate, prior to blood collection. purified red blood cells (rbc) were obtained via sedimentation of blood cells after centrifugation at × g for min. cells were washed three times with pbs and finally re-suspended in the same buffer to obtain a . % rbc suspension. for the hemadsorption assays, ml of a . % rbc suspension in pbs was added to chse/f cells expressing wt he or their mutants. cells were incubated for h and then washed six times with l- medium to remove unbound cells. bound erythrocytes were permeabilized by adding a solution of . m nh cl, followed by incubation for h; the hemoglobin-containing supernatant was then transferred to a -well plate for absorbance reading at nm to measure hemadsorption [ ] . for the membrane fusion assays, rbcs membranes were fluorescently labeled with octadecyl rhodamine b chloride (r ) (molecular probes, eugene, oregon, usa). briefly, µl of a mg/ml r solution was quickly added to ml of a % rbc suspension in pbs. the mix was incubated for min at rpm agitation. then, ml of supplemented l- medium was added, incubating for an additional min. erythrocytes were sedimented by centrifugation at × g and washed six times with ml of pbs to remove the excess of dye. rbcs were re-suspended in pbs to obtain a . % suspension [ ] . to evaluate the fusion activity of the different combinations of isav surface proteins, chse/f cells expressing wt or mutant he and f were washed two times with l- medium and then incubated with µg/ml trypsin for min. the enzyme was then inactivated by min incubation with supplemented l- medium. cells were washed three times with l- , and then ml of the r -labeled rbcs was added. after min, erythrocytes were removed, and the cells washed six times with l- medium. the fusion mechanism was activated by adding ml of ph . adjusted l- medium and incubating for min. the acid medium was removed and cells were fixed with % paraformaldehyde, as described above, after three washes with l- medium. cells were then analyzed using the confocal microscope, measuring the fusion activity as the percentage of membrane-fused cells (i.e., where fluorescence was transferred from the bound rbc to the chse/f cell) relative to the total number of chse/f cells with bound erythrocytes. ten fields of each dish, with comparable levels of bound rbcs, were analyzed [ ] . the results are presented as the means ± standard deviations of triplicate determinations. statistical significance of the data was determined by using a student's t-test. in all figures, p-values < . , < . and < . are indicated by *, ** and ***, respectively, and were considered significant as properly indicated. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : immunofluorescence assays and heterologous protein expression levels in chse/f cells. transfected cells were evaluated h post-transfection via immunofluorescence using the antiflag m antibody. proteins where detected in vesicles and membranous compartments of the cell, in accordance with their membrane-bound nature. all proteins had comparable levels of expression, as determined by fluorescence intensity measurements. hemagglutinin-esterase and fusion protein constructs appears in green and red, respectively; figure s : co-expression of he and f in transfected chse/f cells. fusion proteins for he and f where developed to assess the correct co-expression of proteins on co-transfected cells. as described above, both he and f where located at vesicles and membranous compartments of the cell, in accordance with their membrane-bound nature; figure s : hemadsorption levels for chse/f expressed hes. forty-eight hours post transfection, salmon rbcs were added to chse/f cells and hemadsorption levels were assessed measuring the hemoglobin released after erythrocytes were permeabilized, via 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distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- -lpn cqz authors: leguia, mariana; loyola, steev; rios, jane; juarez, diana; guevara, carolina; silva, maria; prieto, karla; wiley, michael; kasper, matthew r.; palacios, gustavo; bausch, daniel g. title: full genomic characterization of a saffold virus isolated in peru date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: lpn cqz while studying respiratory infections of unknown etiology we detected saffold virus in an oropharyngeal swab collected from a two-year-old female suffering from diarrhea and respiratory illness. the full viral genome recovered by deep sequencing showed % identity to a previously described saffold strain isolated in japan. phylogenetic analysis confirmed the peruvian saffold strain belongs to genotype and is most closely related to strains that have circulated in asia. this is the first documented case report of saffold virus in peru and the only complete genomic characterization of a saffold- isolate from the americas. saffold virus (safv) was first described in , after it was isolated from a stool sample taken in san diego, in , from an eight-month-old female with fever of unknown origin [ ] . safv belongs to the cardiovirus genus of the picornaviridae family. the cardiovirus genus is composed of two species, encephalomyocarditis virus, for which only one serotype has been reported, and theilovirus, for which distinct serotypes are known: safv - , theiler's murine encephalomyelitis virus, thera virus, and vilyuisk human encephalomyelitis virus [ ] . safvs are ubiquitous in populations around the world and have been linked to respiratory and gastrointestinal infections early in life [ ] [ ] [ ] [ ] . despite their extensive distribution, only a handful of safv genomic sequences from the americas have been described [ ] [ ] [ ] . furthermore, many of the sequences publicly available are not complete genomes, but rather very small fragments of partial vp sequences. aside from its original isolation in the usa, safv- has only been reported in bolivia [ ] . other serotypes, including safv- , , and , have been reported in bolivia, brazil, canada, and the usa [ ] [ ] [ ] . for the remaining serotypes, including safv- , , , , , , and , there are no known reports of isolates from the americas. here, we provide complete genomic characterization of a safv- isolate collected from a two-year-old female from the amazonian area of maynas, in loreto, peru. the safv strain described here was isolated from an oropharyngeal swab collected from a two-year-old female who presented with diarrhea, heart murmur, and symptoms of respiratory illness, including headache, sore throat, cough, rhinorrea, and dyspnea. the patient was identified during routine respiratory surveillance efforts carried out by investigators from the u.s. naval medical research unit no. in peru and neighboring countries, with institutional review board approval from all implicated partners (protocols nmrcd. . and nmrcd. . ). the sample was initially cultured in llc-mk , mdck, and vero-e cells, but by day cytopathic effect (cpe) had only been observed in llc-mk cells and no pathogens had been identified in the original sample using traditional pcr or elisa-based approaches. additional analyses of the cpe-positive llc-mk culture supernatant, using a highly multiplexed masstag pcr approach that can detect over respiratory pathogens simultaneously ( [ ] , also failed to identify any potential etiology. the sample then entered a pathogen discovery pipeline based on unbiased next-generation sequencing that eventually produced a match to a japanese isolate of safv- (accession #hq . ) with % identity at the nt level. the consensus sequence generated ( figure to further characterize our strain, we conducted phylogenetic analyses of both the full genome (orf only, nt, aa) and the complete viral protein (vp ) ( nt, aa). this latter region is expected to be under positive selection in order to avoid recognition by the host's immune system, and thus could potentially return different phylogenetic results when compared to the whole genome. to maintain sampling diversity as large as possible, trees were constructed using publicly available reference sequences that represent the totality of the diversity of safv strains in terms of genotype, year of isolation, and geographical origin ( table ). the full genome tree includes complete genome sequences covering serotypes - , whereas the full vp tree includes the same sequences used for the full genome tree plus additional available sequences containing complete vp genes. phylogenetic analyses of both full genome and complete vp sequences confirm that the peruvian safv strain collected in belongs to genotype and is most closely related to asian strains ( figure ). we also constructed an additional vp tree, this time containing partial vp sequences available from several bolivian isolates ( figure s ). although there are minor differences in the branching patterns of the two vp trees, the peruvian strain remains closely associated with asian strains within the safv- group despite the fact that a number of additional south american safv strains were included in the analysis. the fact that the peruvian isolate is most closely related to safv strains that have circulated in asia rather than in europe, for example, should not be surprising given the available information. specifically, there are no other reports of safv- from any country in the americas, indicating that information from additional american strains will be needed to establish more robust phylogenetic relationships in support of theories of safv movements throughout the world. as it stands, the information presented here can only be used to support grouping of the peruvian safv strain into group , with particular similarity to asian strains. nevertheless, it is worth noting that peru has historically received a large number of immigrants from asia, particularly from china and japan. along with further characterization of additional strains, this observation could be used to support a theory of introduction of safv into the americas directly from asia. each strain is labeled using standard identifiers, including virus type, country of isolation (using iso country codes), isolate name (if any), year of isolation, and genbank accession number. additionally, safv - serotypes are color-coded for easy viewing. the peruvian isolate described here is highlighted in bold and with an arrow. full genome trees were constructed using a total of publicly available complete genome sequences covering serotypes - . full vp trees were constructed using those same sequences plus additional sequences containing complete vp genes. scale bars represent the number of substitutions per site. penicillin-streptomycin (gibco, - . thermo fisher scientific: waltham, ma, usa). cultures were maintained at °c and % co until was observed. cpe-positive culture supernatants were extracted using the qiaamp cador pathogen mini kit (qiagen, . qiagen: valencia, ca, usa) according to the manufacturer's instructions and nucleic acids were analyzed by masstag pcr essentially as described [ , ] . briefly, rnas were reverse-transcribed using superscript ii (thermo fisher, - ) and random primers (thermo fisher, - ). reverse transcription products were amplified using a panel of primers labeled with photocleavable mass codes (agilent, custom. agilent technologies: santa clara, ca, usa) targeting influenza viruses a and b, respiratory syncytial viruses a and b, human parainfluenza viruses - , human metapneumovirus; coronavirus oc and e, enterovirus, rhinovirus, and adenovirus. upon removal of unincorporated primers, tags were released by uv irradiation and analyzed using a series single quadrupole lc/ms system (agilent technologies). cpe-positive culture supernatants were preserved in trizol ls (thermo fisher, - ) and rna was extracted using direct-zol™ rna miniprep kit (zymo research, r ) according to the manufacturer's instructions. rnas were converted to cdna and amplified using sequence-independent single primer amplification as described [ ] with the following modifications. to enhance coverage of the terminal ends, an oligo containing three rgtp at the ′ end (gccggagctctgcagatatcggccattatggccrgrgrg) and the fr rv-t primer [ ] were added during first-strand synthesis and the reverse transcriptase was changed to maxima h minus (thermo fisher, ep ), which has terminal transferase activity that enables addition of the rgtp containing oligo to the ′ end during cdna synthesis. amplicons were sheared to ~ bp and used as starting material for truseq libraries (illumina fc- - . illumina, inc.: hayward, ca, usa) prepared according to the manufacturer's instructions. sequencing was performed on a miseq using a cycle kit (illumina ms- - ). cutadapt [ ] and prinseq-lite [ ] were used to trim primers and remove poor quality reads, respectively. reads were assembled into contigs using ray meta [ ] and annotation was done by blast in combination with custom scripts. a number of available safv sequences were downloaded from genbank to serve as references (table ) . to cover as many genotypes, years, and geographical regions as possible, we considered complete genomes (at least nt covering all aa of representative safv - isolates, including the peruvian isolate), complete vp sequences ( nt covering all aa), and seven additional partial vp sequences (at least nt) specifically from the americas. theiler's murine encephalomyelitis virus and theilers-like virus genomes were used as outgroups in the analysis. alignments were constructed using muscle and trees were generated using the maximum likelihood algorithm with bootstrap replicates in mega . [ , ] . genetic distances were calculated using a general time reversible gamma distributed model. table . sequences used for phylogenetic analysis. the peruvian isolate is highlighted in gray. sequences from isolates with complete genomes were used for both full genome and vp phylogenetic trees. sequences from isolates with partial vp sequences were used for an additional vp tree that is almost identical to the complete vp tree ( figure s ). abbreviations: safv-saffold virus; tmev-theiler's murine encephalomyelitis virus. our case report constitutes the first isolation of safv in peru and the only complete genomic characterization of a safv- isolate from the americas. although the characteristics of the patient from whom the specimen was isolated agree with previous reports that the virus affects young children and that it is linked to both respiratory and gastrointestinal infections, little is known about the prevalence of safv infections in south american populations. at least two serological studies in peru have shown the presence of neutralizing antibodies to closely related viruses of the cardiovirusgenus [ , ] . one of these studies reported that % of the population of the amazonian city of iquitos had neutralizing antibodies to encephalomyocarditis virus [ ] . interestingly, the authors also reported elevated cross-reactivity rates ( . %), suggesting that many sero-conversions could be due to the presence of closely related members of the cardiovirus genus, including safv. upcoming studies looking specifically at safvs should help elucidate both the prevalence and virulence of these pathogens, particularly in vulnerable populations. in turn, these may allow more robust characterizations of safv evolution in the americas. the views expressed in this article are those of the authors and do not reflect the official policy or position of the department of the navy, department of defense, or the u.s. government. we are military service members or employees of the u.s. government. the work was prepared as part of our official duties. title u.s.c. § provides that "copyright protection under this title is not available for any work of the united states government." title u.s.c. § defines u.s. government work as a work prepared by military service members or employees of the u.s. government as part of that person's official duties. the work was supported by work unit number . . gb.b . the study protocol was approved by namru- 's irb in compliance with all applicable federal regulations governing the protection of human subjects. discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin the encephalomyocarditis virus genetic diversity of circulating saffold viruses in pakistan and afghanistan is there still room for novel viral pathogens in pediatric respiratory tract infections? saffold viruses in pediatric patients with diarrhea in thailand saffold virus, a human theiler's-like cardiovirus, is ubiquitous and causes infection early in life new saffold cardioviruses in children circulation of lineages of a novel saffold cardiovirus in humans diversity of picornaviruses in rural bolivia masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during multiplex masstag-pcr for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade viral genome sequencing by random priming methods cutadapt removes adapter sequences from high-throughput sequencing reads quality control and preprocessing of metagenomic datasets ray meta: scalable de novo metagenome assembly and profiling multiple sequence alignment with high accuracy and high throughput molecular evolutionary genetics analysis version . prevalence and risk factors for encephalomyocarditis virus infection in peru. vector borne zoonotic dis human febrile illness caused by encephalomyocarditis virus infection we thank the local health authorities, particularly diresa-loreto, for support with this and other ongoing studies. the authors declare no conflict of interest. key: cord- - fhg o authors: mull, nathaniel; jackson, reilly; sironen, tarja; forbes, kristian m. title: ecology of neglected rodent-borne american orthohantaviruses date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: fhg o the number of documented american orthohantaviruses has increased significantly over recent decades, but most fundamental research has remained focused on just two of them: andes virus (andv) and sin nombre virus (snv). the majority of american orthohantaviruses are known to cause disease in humans, and most of these pathogenic strains were not described prior to human cases, indicating the importance of understanding all members of the virus clade. in this review, we summarize information on the ecology of under-studied rodent-borne american orthohantaviruses to form general conclusions and highlight important gaps in knowledge. information regarding the presence and genetic diversity of many orthohantaviruses throughout the distributional range of their hosts is minimal and would significantly benefit from virus isolations to indicate a reservoir role. additionally, few studies have investigated the mechanisms underlying transmission routes and factors affecting the environmental persistence of orthohantaviruses, limiting our understanding of factors driving prevalence fluctuations. as landscapes continue to change, host ranges and human exposure to orthohantaviruses likely will as well. research on the ecology of neglected orthohantaviruses is necessary for understanding both current and future threats to human health. due to their direct noticeable impacts on humans, certain viruses tend to receive relatively large amounts of research attention. members of the coronaviridae (sars-cov, mers-cov, and now sars-cov- ), filoviridae (ebola and marburg virus), flaviviridae (west nile and zika virus), lyssaviridae (rabies), and paramyxoviridae (hendra and nipah virus) families contain several dangerous human pathogens that have emerged in recent decades and have resulted in extensive research attention. while studying such viruses is important, there are an untold number of other pathogens that persist among humans and wildlife that receive little to no attention [ ] . even in high-profile viral groups, a disproportionate amount of attention is given to the viruses that are known to cause disease in humans, highlighted by the current global response to sars-cov- . due to unforeseeable circumstances, such as host-switching events (e.g., influenza virus, human immunodeficiency virus [ ] ), exposure to new viruses via landscape encroachment (e.g., hendra virus [ ] , nipah virus [ ]), and changes in host or virus geographic range due to climate change, species introduction, or migration events (e.g., zika virus [ ] , west nile virus [ ] ), less-significant viruses can quickly become significant human health concerns. therefore, viruses that are disproportionately under-studied require research focus, and they may ultimately aid understanding of related viruses and increase awareness of current and future threats. a key example of research bias within a virus group is the hantavirus family (bunyavirales: hantaviridae). recent taxonomic restructuring of hantaviruses was necessitated by the discovery of non-rodent-and non-mammal-borne viruses [ , ] . however, mammals, particularly rodents, are still the most common natural hosts of hantaviruses, encompassing viruses in the largest subfamily (mammantavirinae) and genus (orthohantavirus) [ ] , and only rodent-borne orthohantaviruses have been linked to human disease [ ] . human infections caused by spillover of old world and new world orthohantaviruses can result in hemorrhagic fever with renal syndrome (hfrs) or hantavirus cardiopulmonary syndrome (hcps or hps), respectively [ ] . the international committee on taxonomy of viruses (ictv) lists unique orthohantaviruses distributed throughout the world, with distinct viruses within virus species endemic to north and south america [ ] [ ] [ ] . although the first known american orthohantavirus, prospect hill virus (phv), was described in [ ] , most viruses were found shortly after the outbreak of sin nombre virus (snv) [ ] in north america and the cases of andes virus (andv) [ ] in south america (table ) . new orthohantaviruses and genotypes continue to be identified via broad surveillance. some discovered genotypes are suggested to be distinct viruses, but a lack of sequence data and virus isolation prevents formal taxonomic placement. for example, phylogenetic analyses show up to distinct branches within the andes orthohantavirus clade [ , ] , but only four strains meet all ictv criteria as distinct viruses (tables and a ) [ ] [ ] [ ] . despite an increasing number of described hantaviruses, andv and snv are disproportionately studied when compared to other orthohantaviruses in the americas (table ) . such bias may be the reason for inadequate information to discriminate between potentially different viruses, and the lack of distinction may discourage the collection of additional data, creating a negative feedback loop. muleshoe virus (mulv), for instance, is a genotype of black creek canal virus (bccv), and evidence supports mulv being a separate virus based on genetic differences [ ] . however, the necessary ictv criterion of mulv isolation has not been accomplished, which keeps mulv from being distinguished as a distinct virus strain and may limit the amount of research conducted on this genotype. until more virus-specific information is known, we must infer characteristics of under-studied orthohantaviruses using other available information. in this review, we summarize current knowledge on neglected orthohantaviruses and highlight areas where future research is necessary. to determine the potential range of these viruses, we report evidence regarding the rodent hosts of each american orthohantavirus and the potential for various host-virus relationships and communities based on existing evidence. information regarding transmission for well-studied orthohantavirus systems is used to postulate the transmission characteristics of neglected american orthohantaviruses, including direct transmission routes, environmental persistence, and spillover risk to humans. as the number of described orthohantaviruses increases, so does the number of suggested reservoir hosts (table a ) . reservoir hosts typically have asymptomatic and persistent infections [ , ] , although there is evidence of negative effects associated with orthohantavirus infection on the survival of young animals [ ] and possibly decreased weight gain in newly-infected individuals [ ] . most studies that identify orthohantavirus infections in rodents have not evaluated the pathological or demographic consequences of infections. the ability of rodents to be infected with an orthohantavirus without noticeable effects does not alone implicate them as a reservoir. virus isolation is generally deemed the gold-standard evidence to support a reservoir role, followed by positive polymerase chain reaction (pcr) results. while orthohantavirus isolation from rodent hosts is rare-even for well-established virus-host relationships (e.g., snv and peromyscus maniculatus [ ] )-recent advances in establishing rodent cell cultures, such as those of the bccv host sigmodon hispidus, may aid future isolations [ ] . in contrast, positive rt-pcr results for a particular virus in multiple rodent species are common (table a ) . orthohantavirus infections are generally considered single-host-single-virus systems [ ] [ ] [ ] , and viruses tend to co-diverge with their hosts [ ] . the term "primary host" is sometimes used for the most common reservoir host [ , ] , but this wording retracts from the idea that orthohantaviruses could persist in multiple hosts with the same propensity. evidence increasingly suggests that some american orthohantaviruses do not follow the single-host-single-virus paradigm as strictly as their old world counterparts (table a ). for example, the reservoir for lechiguanas virus (lechv) is considered to be oligoryzomys flavescens, but results from a recent study found lechv-positive reverse transcriptase pcr (rt-pcr) samples from oligoryzomys nigripes in argentina, while all o. flavescens samples were seronegative [ ] . similarly, a study in texas found snv-positive rt-pcr samples from five seropositive peromyscus attwateri, four p. leucopus, one p. laceiarus, and one reithrodontomys fulvescens, but all of the sampled p. maniculatus, the reservoir of snv, were seronegative except a single rt-pcr negative individual [ ] . it is unknown whether such instances are caused by frequent spillover events or the persistence of the virus within or among multiple species. multiple-host systems are also more common than generally acknowledged when considering virus genotypes that are not classified as separate viruses by the ictv. in such cases, the reservoirs for a virus strain would be the combination of reservoirs for all genotypes. for example, limestone canyon virus (lscv) is a genotype of snv that is associated with peromyscus boylii and other peromyscus species [ , ] instead of p. maniculatus, the reservoir of snv; isla vista virus (islav) is a genotype of phv that is associated with microtus californicus [ ] instead of m. pennsylvanicus, the reservoir of phv; and rio mearim virus (rimev) and anajatuba virus (anajv) are genotypes of rio mamoré virus (riomv) that are associated with holochilus sciureus and oligoryzomys fornesi, respectively [ ] , instead of o. microtis, the reservoir of riomv (table a ). in some circumstances, distinct orthohantavirus genotypes are also host subspecies-dependent. for instance, oryzomys couesi is suggested to be the reservoir for catacamas virus (catv) and playa de oro virus (orov), a genotype associated with a clade composed of catv, bccv, and bayou virus (bayv), but orov and catv are associated with different subspecies of o. couesi [ , ] . choclo virus (chov) and maporal virus (mapv) are both associated with oligoryzomys fulvescens, although a distinction in the mitochondrial cytochrome-b gene suggests that these viruses are host subspecies-specific, infecting o. f. costaricensis and o. f. delicatus, respectively [ ] . another technical issue to consider is the concept of host-switching events among orthohantaviruses. evidence of historical host switching has resulted in hantavirus lineages among disparate mammal taxa [ ] [ ] [ ] . more recent host-switching is supported by several mismatches in the cophylogeny of orthohantaviruses and rodent hosts. for example, monongahela virus (mglv) is an orthohantavirus genotype primarily carried by p. maniculatus nubiterrae, a subspecies of the mouse associated with snv, despite mglv often showing a closer phylogenetic relationship to new york virus (nyv), which is associated with p. leucopus [ ] [ ] [ ] (although the true relationship is still pathogens , , of unknown (figure ) , and mglv has also been reported in p. leucopus [ ] ). additionally, orov and catv are often found in the same species, o. couesi, despite orov being more closely-related to bccv, which is associated with s. hispidus. minimal range overlap between o. couesi and s. hispidus and minimal genome sequencing prevent conclusion of a host-switch event among these viruses. additional host-switching events have been proposed as the reason for the multitude of reported hosts in american orthohantaviruses (table a ) , such as from oligoryzomys flavescens to o. nigripes for lechv [ ] . however, while reports of orthohantaviruses infecting multiple species supports multiple hosts for many orthohantaviruses and, therefore, a plethora of host switching events, there is a shortage of research examining the competence of many putative hosts and therefore the classification of true reservoirs. information regarding the relative transmissibility of virus from each host to humans and other wildlife is also lacking, with the exception of several case studies involving focused trapping around areas of assumed exposure (e.g., [ , ] ). additionally, no orthohantavirus has been isolated from more than one rodent species (table a ) , although few studies have reported such attempts. further research is, therefore, necessary to determine if frequent documentation of american orthohantaviruses in multiple species represents host switches or spillover. phylogenetic tree demonstrating relatedness among american rodent-borne orthohantaviruses. the evolutionary history was inferred using the maximum likelihood method implemented in mega . the percentage of trees in which the associated taxa clustered together is shown next to the branches; values over % are shown. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. triangular branches represent multiple closely-related sequences. the analysis involved orthohantavirus s segment nucleotide sequences retrieved from genbank. additional host-switching events have been proposed as the reason for the multitude of reported hosts in american orthohantaviruses (table a ) , such as from oligoryzomys flavescens to o. nigripes for lechv [ ] . however, while reports of orthohantaviruses infecting multiple species supports multiple hosts for many orthohantaviruses and, therefore, a plethora of host switching events, there is a shortage of research examining the competence of many putative hosts and therefore the classification of true reservoirs. information regarding the relative transmissibility of virus from each host to humans and other wildlife is also lacking, with the exception of several case studies involving focused trapping around areas of assumed exposure (e.g., [ , ] ). additionally, no orthohantavirus has been isolated from more than one rodent species (table a ) , although few studies have reported such attempts. further research is, therefore, necessary to determine if frequent documentation of american orthohantaviruses in multiple species represents host switches or spillover. in addition to the potential for multiple hosts, the number of sympatric viruses must also be considered. propensity for coexistence of different orthohantaviruses within a rodent community appears to vary spatially and temporally. in one texas study, viruses, and even virus genomes, appear to segregate at the county level [ ] . similar results were found in mexico, with most states containing only one orthohantavirus [ ] , although another study examining a smaller portion of the same mexican region found viruses to commonly coexist [ ] . sympatric riomv genotypes, anajv and rimev, were also found in the same area but in distinct host species [ ] . in california and nevada, elmcv, phv, and snv were also found in the same area, indicating that viruses hosted by diverse rodents can exist in sympatry [ ] . thus, multiple orthohantaviruses may exist together in rodent communities, but separation based on habitat type and species distributions likely play a role in structuring their presence. in the absence of data on orthohantavirus presence in a particular area, host distributions may be useful as proxies, as rodent ranges and habitat types are often well-documented [ , , ] . several orthohantaviruses have been found throughout large extents of their host range, including bccv [ , ] , bayv [ , ] , and others, indicating that orthohantaviruses have the potential to be present throughout the entire range of host species. however, the use of virus genotypes causes confusion when determining the range of orthohantaviruses. for example, bccv is used in florida, united states [ ] while mulv is used in texas [ ] . similarly, chov is used in panama [ ] while its genotype jabora virus (jabv) is used in brazil [ ] . until such genotypes are considered distinct viruses by taxonomists (i.e., ictv), acknowledgement of these relationships may be helpful in minimizing confusion and aiding understanding of orthohantavirus distributions. without analyzing positive samples throughout species ranges for new viruses, incorrect assumptions may also be made regarding orthohantavirus distributions. for example, riomv infects oligoryzomys microtis throughout most of its range in south america [ ] , so hcps cases in french guiana were thought to be riomv [ ] . however, virus sequencing from an hcps case in french guiana found that maripa virus (marv), a then-new virus closely-related to riomv found in o. fulvescens and zygodontomys brevicauda, was the responsible agent [ ] [ ] [ ] . difficulty in estimating virus range via host range also increases when one species can host several viruses. both necocli virus (necv) [ ] and marv [ ] have been found in z. brevicauda via positive rt-pcr, but the range of each particular virus is unknown. a similar situation was found for o. longicaudatus, the most common host of andv and also the host of oran virus (ornv), although the increased attention given to andv revealed which populations of o. longicaudatus host which virus [ ] . therefore, host distribution can be useful in estimating virus distribution, but caution should be applied. hantaviruses are likely to spread to new areas and vanish from existing areas due to changes in rodent host distribution and abundance. changes in grassland habitats caused by land-use changes and climate change [ ] [ ] [ ] have been strongly associated with rodent distributional changes. for example, range expansion of a north american grassland rodent species, baiomys taylori, was recently found in new mexico, united states, likely due to an increase in grassland areas, particularly along roadsides, due to climate change and habitat disturbance [ ] . thus, the grassland rodents that host orthohantaviruses may show similar patterns in the future. much of what we know about the transmission of american orthohantaviruses among conspecific rodent hosts is derived from studies of andv and snv [ ] . both viruses are primarily shed in saliva, occasionally in urine, and apparently not in feces, suggesting that behaviors such as grooming and biting are the primary routes of transmission [ , , ] . such transmission contrasts with old world orthohantaviruses such as puumala virus (puuv), which are commonly shed in feces as well [ , ] . older males are more commonly infected with orthohantaviruses than other much of what we know about the transmission of american orthohantaviruses among conspecific rodent hosts is derived from studies of andv and snv [ ] . both viruses are primarily shed in saliva, occasionally in urine, and apparently not in feces, suggesting that behaviors such as grooming and biting are the primary routes of transmission [ , , ] . such transmission contrasts with old world orthohantaviruses such as puumala virus (puuv), which are commonly shed in feces as well [ , ] . older males are more commonly infected with orthohantaviruses than other demographic groups [ ] , including snv [ , ] , laguna negra virus (lanv) [ ] , lechv [ ] , and bccv [ ] . compounding more exposure opportunities for older individuals, higher prevalence in older males is assumed to result from increased aggression and competition, primarily for access to mates [ , ] . associations of bayv-infected male o. palustris with receptive females and non-infected males with non-receptive females [ ] further supports the concept of reproductive behaviors as a primary driver of orthohantavirus transmission among wild rodents. thus, some females likely become infected via allogrooming during copulative behaviors common in rodents (e.g., [ , ] ). the occasional shedding of the virus in urine may be important for transmission among conspecifics, and perhaps heterospecifics as well. urine is used by rodents for various reproductive and territorial behaviors [ ] , creating ample opportunities for exposure of virus in aerosolized urine via oropharyngeal routes. however, information pertaining to virus persistence outside the host in american orthohantaviruses is limited to circumstantial evidence regarding spillover infections, and such transmission may be mitigated by uncommon virus shedding in urine. relatively frequent rodent spillover events (i.e., transmission from one species to another) [ ] suggests that other variables, including overlap in habitat use such as shared runways, burrows, and nests, is necessary for transmission among species. during the breeding season, many rodents compete for mates, food, space, and protection of offspring, so there is little overlap in space use by conspecifics, and often congeners [ , ] . however, in the non-breeding season, these territories break down and space overlap increases [ , ] . during this time, many rodents also share burrows within [ , ] and occasionally among [ ] species. during warmer months, some species may also use the burrows of other species who have since vacated [ , ] . burrow-sharing behavior in rodents has been associated with the spread of several other diseases, including plague (yersinia pestis) [ ] , tick-borne relapsing fever (borellia spp.) [ ] , and possibly valley fever (coccidioides spp.) [ ] , and the stable cool, humid microclimates of burrows [ , ] may allow orthohantaviruses to persist in the environment. this phenomenon would also help explain why multiple species can be infected by the same orthohantavirus, potential opportunities of spillover to non-muroid rodents [ ] , and original host-switching events to other rodents, shrews, and moles ( figure ). further research is necessary to determine the role of habitat overlap on conspecific orthohantavirus infection via competition, excrement exposure, and other potential sources of virus shedding and routes of transmission. regardless of the routes of transmission, population density appears to play a role in orthohantavirus maintenance. experimental modeling of snv prevalence in peromyscus maniculatus populations and hcps cases indicates that climate-mediated fluctuations in host abundance are linked to orthohantavirus outbreaks [ , ] . high seroprevalence in p. maniculatus is found after a time lag following high rainfall events, particularly those associated with the el niño-southern oscillation (enso) [ , ] . although this phenomenon has been relatively well-studied in snv, data demonstrating similar patterns among other american orthohantaviruses is lacking. however, such lag times in other systems may explain why less-abundant species in a rodent community may occasionally be the primary carriers of orthohantavirus [ , ] , as population sizes could have been larger in a recent season. theoretical models indicate that orthohantavirus transmission among rodents also has aspects of frequency-dependent transmission. infection prevalence is greatly influenced by contact rates [ ] , which increase as population density increases. however, increases in prevalence are greater in males than in females [ ] , likely due to increased competitive encounters among males, but not females, at higher densities. high seroprevalence among overwintering animals [ , ] are assumed to be caused by persistently infected animals infecting susceptible individuals. population sizes generally crash during this time period [ ] (although p. maniculatus populations remained stable prior to the hcps outbreak of - [ ] , likely due to a strong enso event), suggesting that winter infections may be caused by frequent interactions despite low host density. it is unclear how the stable winters of tropical regions impact orthohantavirus transmission systems in northern south america and central america. further attempts to imitate such systems in a controlled environment are necessary to better understand how orthohantaviruses persist and proliferate through rodent populations. most american orthohantaviruses have been associated with at least one human case of hcps ( / ) , and approximately half ( / ) were discovered following an hcps case (table ) . practically all hcps cases are thought to be caused by spillover events from rodents to humans [ , ] . the exception comes from andv in argentina and chile where some evidence supports transmission from infected patients to family members and medical workers [ ] [ ] [ ] . however, these instances are limited to outbreaks in small, rural communities, and regional medical staff that cared for hcps patients had similar seroprevalence to the general population [ , ] . several orthohantaviruses were originally discovered through broad surveillance of rodent tissues but were later implicated with human disease. for example, riomv was originally discovered while studying the host associations of andes orthohantavirus strains in [ ] , and was connected to hcps eight years later [ ] . other hcps cases were attributed to the incorrect orthohantavirus until the actual virus was described, such as marv cases originally diagnosed as riomv, as mentioned previously [ ] . similarly, due to regional variation in virus prevalence, elmcv was suggested to be the etiological agent of several hcps cases ascribed to snv, but the virus in these cases was never tested [ ] . without verification via sequencing of hcps cases, elmcv is considered to not cause disease in humans. thus, certain orthohantaviruses may be infectious to humans but incorrectly dismissed due to a lack of sequencing. conversely, additional orthohantaviruses or viral genotypes that are pathogenic to humans may exist that have not yet been linked to any hosts, such as tunari virus (tunv), which was discovered following an hcps case but the reservoir is still unknown [ ] . while understanding host ecology may help explain the maintenance of orthohantaviruses in wild rodent populations, it can also inform spillover threats to humans. snv and andv are both found most commonly in generalist rodent species that can be locally abundant. these host characteristics allow viruses to be present in most habitats throughout a large geographical range, increasing the likelihood of encounters between infected rodents and susceptible humans. however, due to the large number of described orthohantaviruses and their hosts, most regions and habitats have the capacity to contain multiple viruses of human health concern. on the other hand, some species and their viruses are common in a variety of habitat types. for example, in west virginia, united states, where peromyscus are the dominant muroid rodents, hcps cases were attributed to exposure of airborne particulates of p. maniculatus secretions within cabins [ , ] . such cases indicate an infection risk in seasonally-used buildings in rural areas in the northeast, similar to initial assessments in the southwestern united states [ ] . therefore, these generalist species appear to be capable of transmitting virus to humans regardless of habitat. urban areas may pose a risk for human exposure to orthohantaviruses and their hosts as well. for example, in addition to their abundance in forested habitats, peromyscus mice are common in green urban spaces, such as the park system in new york city [ , ] , and nyv was discovered on shelter island near new york city [ ] . notably, homeless residents may be at increased risk, as sleeping near rodent activity was associated with european orthohantavirus infections [ ] , although empirical evidence is lacking for american viruses. due to limited migration of wild rodents throughout urban areas [ , , ] , green spaces may also be protected from orthohantavirus invasion. orthohantaviruses carried by invasive rodents, such as seoul virus (seov) in rattus norvegicus, may pose a risk as well. seov has been documented in the united states and canada due to the pet trade [ ] , while wild rats can also carry this virus. one study found a seroprevalence for orthohantavirus in r. norvegicus of . % overall and - . % in green spaces in baltimore, maryland [ ] . although broader documentation of orthohantaviruses in urban areas is lacking, these findings as well as observations of a range of other disease-causing pathogens in urban rodents (e.g., [ , ] ) suggest that this may be an area of major human health concern. while current threats would likely be documented already, misdiagnoses, failure to seek medical attention, and the potential for future outbreaks warrant attention. it appears that hcps risk is greatest in areas where humans infiltrate rodent habitats, rather than vice versa, such as areas of landscape fragmentation and encroachment caused by urbanization and development. in uruguay, oligoryzomys flavescens infected with lechv were more common in disturbed habitats than in undisturbed habitats [ ] . relationships between habitat encroachment and infection risk occur for other zoonotic diseases, such as nipah virus [ ] and ebola virus [ , ] , suggesting a possible pattern in orthohantaviruses other than lechv as well. many forms of habitat encroachment can increase risk of exposure to orthohantaviruses. ranching and farming activity in the midwest united states prairies, such as construction of new barns and field plowing, could expose individuals to sigmodon hispidus (bccv); construction of rice fields and other encroachments into marsh habitat in the southern united states could expose individuals to oryzomyz palustris (bayv); the creation of edge habitat via development in the amazon basin provides additional habitat for oligoryzomys microtis (riomv) and increases contact with humans. all of these rodent species carry orthohantaviruses that cause disease in humans (table ) [ , , ] . estimating the risk of exposure to most orthohantaviruses with various human activities in south america is more difficult due to minimal information about the ecology of the rodent hosts; although some evidence indicates that habitat disturbance, particularly construction of domiciles in rural areas, appears to increase the risk of human exposure to hantaviruses there as well [ , ] . interestingly, ornv-positive oligoryzomys longicaudatus were found in orán, argentina, outside of the reported distribution range of o. longicaudatus (figure ) [ ] , showing the ability for agricultural development to expand orthohantavirus presence. despite the discovery of at least different new world orthohantaviruses carried by rodents, most orthohantavirus studies in the americas focus on andv and snv. while the majority of hcps cases are attributed to these viruses [ , ] , recent evidence suggests that such statistics may be skewed due to misdiagnosis of either the causative orthohantavirus or of the disease itself [ , ] . we show that despite having many similar characteristics, american orthohantaviruses differ from their old world counterparts and from each other in several ways. in the absence of empirical data, we shed light on the diversity, transmission, and risk of spillover for neglected american orthohantaviruses and viral genotypes using the ecology of their hosts and information on andv and snv. additionally, comparisons were occasionally made to old world orthohantaviruses. the ecological approach from this review may also be useful in implicating transmission and spillover risk of old world orthohantaviruses not yet examined. a key constraint to inferring information about each orthohantavirus system is the complexity between the taxonomy of orthohantaviruses and their hosts. related viruses appear to interact with hosts similarly, as shown by the comparable phylogenies of orthohantaviruses and their natural rodent hosts [ ] , their affinity to cause disease in humans (table ) , and frequent spillover or multiple related hosts (table a ) . however, confusion in orthohantavirus taxonomy and the number of distinct virus strains limits further conclusions. in particular, surveillance of related rodent species may produce additional genetic samples that allow clearer orthohantavirus phylogenies to be constructed. additional information regarding mole-borne orthohantaviruses, such as oxbow virus (oxbv) and rockport virus (rkpv) [ , ] , and shrew-borne genotypes, such as ash river virus (arrv), camp ripley virus (rplv), and jemez springs virus (jmsv) [ , ] , which have similar taxonomical issues due to minimal research and have some overlap in rodent phylogeny (figure ), may aid in understanding rodent-borne orthohantaviruses. ultimately, broader surveillance will aid in understanding which genotypes constitute distinct viruses and which represent genetic diversity of single orthohantaviruses. in addition to the controversy over viral taxonomy, the ability for multiple orthohantaviruses and their hosts to persist in the same environment and region [ , ] (figure ) further limits conclusions on orthohantavirus samples that are not sequenced, whether rodent or human. since multiple rodent species are commonly found rt-pcr positive for particular american orthohantavirus strains (table a ) , virus-host relationships are unclear. although orthohantaviruses are difficult to isolate, attempts to isolate these viruses from rodent samples is necessary to determine which rodents are reservoirs and which species experience frequent spillover events. these results will aid in determining whether american orthohantaviruses follow a single-host system like their old world counterparts. empirical data on the ecology of neglected american orthohantaviruses are crucial to understanding transmission and persistence of such viruses and threats to human health. few studies have examined the impacts of new world orthohantaviruses on rodent populations, with the exceptions of variation in prevalence between sexes and age classes [ ] [ ] [ ] , survivorship of age classes [ ] , and reproduction-dependent spatial variation [ ] . additional information regarding transmission routes and environmental persistence is also necessary, as the minimal data currently available using snv and andv show mixed results [ , , ] . although hcps cases are often associated with snv and andv, changes in the landscape, climate, and host switching may cause particular orthohantaviruses to increase in severity. each orthohantavirus may have the capability to become more significant to human health in the future, and insight into each virus is necessary for adequate preparation. various viral families have existed amongst humans with little to no impact until recent decades. therefore, research regarding neglected american orthohantaviruses is crucial for a holistic understanding of orthohantavirus epidemiology and to enable preparation for future risks. the authors declare no conflict of interest. table a . evidence supporting natural infections of orthohantaviruses in american rodents. bolded viruses represent strains accepted as distinct by the international committee for taxonomy of viruses (ictv), and non-bolded viruses indicate genotypes not accepted as distinct viruses by ictv. genotype placements are based on published phylogenetic analyses. all studies first found rodents to be seropositive for orthohantavirus antibodies and then performed reverse transcriptase polymerase chain reaction (rt-pcr) prior to sequencing or virus isolation (except for phv, where isolation was attempted without rt-pcr). orthohantaviruses have tri-segmented genomes-s, m, and l segments. studies including only seropositive rodents without additional diagnostic evidence of infection were not included in this a strategy to estimate unknown viral diversity in mammals cross-species 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cases presented in wuhan, china. they were caused by a previously unknown coronavirus. all patients had been associated with the wuhan wholefood market, where seafood and live animals are sold. the virus spread rapidly and public health authorities in china initiated a containment effort. however, by that time, travelers had carried the virus to many countries, sparking memories of the previous coronavirus epidemics, severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), and causing widespread media attention and panic. based on clinical criteria and available serological and molecular information, the new disease was called coronavirus disease of (covid- ), and the novel coronavirus was called sars coronavirus- (sars-cov- ), emphasizing its close relationship to the sars virus (sars-cov). the scientific community raced to uncover the origin of the virus, understand the pathogenesis of the disease, develop treatment options, define the risk factors, and work on vaccine development. here we present a summary of current knowledge regarding the novel coronavirus and the disease it causes. coronaviruses, named for the crown-like spikes on their surface (latin: corona = crown), are positive-sense rna viruses that belong to the coronvirinae subfamily, in the coronaviridae family of the nidovirales order [ ] . they have four main subgroups-alpha, beta, gamma, and delta-based on their genomic structure. alpha-and betacoronaviruses infect only mammals, usually causing respiratory symptoms in humans and gastroenteritis in other animals [ , ] . until december of , only six different coronaviruses were known to infect humans. four of these (hcov-nl , hcov- e, hcov-oc and hku ) usually caused mild common cold-type symptoms in immunocompetent people and the other two have caused pandemics in the past two decades. in - , the severe acute respiratory syndrome coronavirus (sars-cov) caused a sars epidemic that resulted in a % mortality. similarly, the middle east respiratory syndrome coronavirus (mers-cov) caused a devastating pandemic in with a % mortality rate. in late , a cluster of pneumonia cases in wuhan city, hubei province, china were identified as with a novel betacoronavirus, first called the novel coronavirus ( -ncov) and often referred to as the wuhan coronavirus. when the genomics of the -ncov was sequenced, it shared . % of the genetic sequence of the sars-cov that caused the - pandemic [ ] and the international committee on taxonomy of viruses renamed the -ncov as sars-cov- [ ] . patients began to present in november and december with various degrees of respiratory distress of unknown etiology and treated at the time as possible influenza infections. as it became apparent that most cases had a shared history of exposure to the huanan seafood wholesale market (the so-called "wet market"), the wuhan local health authority issued an epidemiologic alert on december and the wet market was closed. about a week later, on january , chinese researchers shared the full genetic sequence of the novel coronavirus, now called sars-cov- [ ] . since the novel coronavirus was recognized, the disease it caused was termed coronavirus disease (covid- ) , and several reports on the clinical presentation, epidemiology, and treatment strategies have been published [ ] [ ] [ ] [ ] . in addition, several websites have been setup to track the epidemic and the case detection rate, which are being updated as often as hourly [ ] [ ] [ ] [ ] . on january , the world health organization (who) declared the covid- outbreak to be a global public health emergency, sixth after h n ( ), polio ( ), ebola in west africa ( ), zika ( ) and ebola in the democratic republic of congo ( ), and on march , the who characterized covid- as a pandemic [ ] . the timeline of events is summarized in figure . all coronaviruses that have caused diseases to humans have had animal origins-generally either in bats or rodents [ ] . previous outbreaks of betacoronaviruses in humans involved direct exposure to animals other than bats. in the case of sars-cov and mers-cov, they were transmitted directly to humans from civet cats and dromedary camels respectively ( figure ). animal origins of human coronaviruses. severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) and were transmitted to humans from bats by civet cats and dromedary camels, respectively. the sars-cov- was likely transmitted to humans through pangolins that are illegally sold in chinese markets [ , ] . the sars-related coronaviruses are covered by spike proteins that contain a variable receptor-binding domain (rbd). this rbd binds to angiotensin-converting enzyme- (ace- ) receptor found in the heart, lungs, kidneys, and gastrointestinal tract [ ] thus facilitating viral entry into target cells. based on genomic sequencing, the rbd of sars-cov- appears to be a mutated version of its most closely related virus, ratg , sampled from bats (rhinolophus affinis) [ ] . it is, therefore, believed that the sars-cov- also originated from bats and, after mutating, was able to infect other animals. the mutation increased the rbd affinity to ace- in humans, but also other animals such as ferrets and malayan pangolins (manis javanica; a long-snouted, ant-eating mammal sold illegally for use in traditional chinese medicine), but also decreased the rbd affinity to ace- found in rodents and civets. the pangolin is believed to be the intermediate host of sars-cov- [ ] . there was some early speculation that sars-cov- emerged from a manmade manipulation of an existing coronavirus, but there is no evidence to support such a theory. in fact, anderson et al. suggest that the particular mutation that was found in the rbd of sars-cov- is different to what would have been predicted based on previously used genetic systems. the authors, however, stated that "it is currently impossible to prove or disprove the other theories of [the sars-cov- ] origin [ ] ". since sars-cov and sars-cov- are so similar, the biochemical interactions and the pathogenesis are likely similar. binding of the sars-cov to the angiotensin-converting enzyme (ace- ) receptors in the type ii pneumocytes in the lungs triggers a cascade of inflammation in the lower respiratory tract [ ] . it has been demonstrated that when the sars spike protein binds to the ace- receptor ( figure a ), the complex is proteolytically processed by type transmembrane protease tmprss leading to cleavage of ace- and activation of the spike protein ( figure b ) [ , ] , similar to the mechanism employed by influenza and human metapneumovirus, thus facilitating viral entry into the target cell ( figure c ). it has been suggested that cells in which ace- and tmprss are simultaneously present are most susceptible to entry by sars-cov [ ] . early indications are that sars-cov- virus also requires ace- and tmprss to enter cells [ ] . in the first published review of the clinical presentation of patients admitted to hospital with covid- [ ] , % of patients had a fever, % had a cough, and % had shortness of breath on admission. however, those admitted may have had less severe symptoms for to days prior to presentation, during which they were likely contagious. by the time patients developed shortness of breath, they had been sick for an average of eight days. once admitted to the hospital, all patients developed clinical pneumonia supported by chest ct findings, and of the patients ( %) developed hypoxic respiratory failure necessitating icu admission. four patients ( %) required mechanical ventilation, two of which received extracorporeal membrane oxygenation due to refractory hypoxia. in total, six patients died, giving a case fatality rate (cfr) of % and triggering panic that quickly spread worldwide. while early media reports suggested that deaths were more likely in patients with comorbid conditions, of the patients described in the chinese review, only % had comorbid conditions and the average age was . as of march , gmt, there were , confirmed cases, about half of which ( , cases, . %) were within mainland china. about % of ill people had severe disease, and . % had mild disease and a total of tested-positive cases were asymptomatic [ , ] . while initially confined to china among those who visited the wuhan wet market, over the course of about months the sars-cov- has to date been confirmed in countries and one cruise ship [ ] . the chinese cdc published the epidemiologic characteristics of the covid- outbreak as of february (table ) [ ] . initial data suggests that the majority of patients ( %) were over age years, and that the risk of death increases with age. no deaths were reported in patients younger than years old, and only . % of the total fatalities were in patients younger than years of age. [ ] . [ , ] . due to aggressive containment strategies in china, including a mass quarantine of the entire million population of wuhan, the acceleration of new cases in china has slowed whereas that outside of china has increased. as of march nd, the number of daily new cases outside of china was nine times higher than those within china. many countries have instituted travel bans and/or quarantine procedures for incoming travelers. closures of public schools and social gatherings have been instituted in many countries in an effort to contain the spread of covid- and decrease the public health burden [ , ] and the cdc has released recommendations on school closure criteria [ ] . in comparison, the sars pandemic, which also originated in china, resulted in people infected and deaths ( . %). on the other hand, the mers pandemic infected people causing deaths ( . %). therefore, although mers and sars had higher mortality, the much larger number of people infected with sars-cov- , and the rate at which the number is increasing, raises red epidemiologic flags. to assess the magnitude of the risk posed by the sars-cov- , we review four parameters that we believe important: the transmission rate, the incubation period, the case fatality rate (cfr), and the determination of whether asymptomatic transmission can occur. the reproduction number, or "r naught" (r ), is a mathematical term that defines contagiousness [ ] . specifically, it is the number of people that one sick host can infect. if the r is less than one the disease will disappear. if the r ≥ then the disease will spread between people. estimates of the r of sars-cov- have ranged from . to as high as . [ ] although the world health organization estimates it is between . and . [ ] . for the purposes of comparison, the mean r for seasonal influenza is between . and . (variable by region and immunization rates), whereas for sars was between and . . the slightly higher r for sars-cov- may be because it has a longer prodromal period, increasing the period during which the infected host is contagious. coronaviruses are generally thought to be spread most often by respiratory droplets, not to be confused with airborne transmission [ ] . droplets are larger and tend to fall to the ground close to the infected host and only infect others if the droplet is intercepted by a susceptible host prior to landing. droplet transmission is typically limited to short distances, generally less than m. however, the airborne route involves much smaller droplets that can float and move longer distances with air currents. under certain humidity and temperature environments, airborne droplets can remain in flight for hours. generally, pathogens that are transmissible via the airborne route have higher r , because infected particles can remain in the air long after the infected individual has left the premises. this airborne route occurs, for example, in measles (r between and [ ] ) and chicken pox (r s between . and . [ ] ). once infected droplets have landed on surfaces, their survivability on those surfaces determines if contact transmission is possible. based on our current understanding from other betacoronaviruses, including sars and mers, coronaviruses can survive, and remain infectious, from h up to days on inanimate surfaces such as metal, glass, or plastic, with increased survival in colder and dryer environments [ ] [ ] [ ] . for this reason, the chinese government has been reported to be disinfecting and even destroying cash in an effort to contain the virus [ ] . reassuringly, cleansing of surfaces with common biocidals such as ethanol and sodium hypochlorite is very effective at inactivation of the coronaviruses within min of exposure [ ] . the timing of maximum infectivity is currently being assessed. a small study of patients showed that nasal viral load peaks within days of symptom onset, suggesting that transmission of disease is more likely to occur early in the course of infection [ ] . understanding incubation periods is very important as it allows health authorities to introduce more effective quarantine systems for suspected cases. the best current estimates of the sars-cov- infection range from to days. analysis of the first cases of covid- in wuhan a mean incubation period of . days [ ] . a later report, based on cases, reported a mean incubation period of . days [ ] . yet another report, on cases who traveled to wuhan between and january, had incubation period ranges from . to . days, with a mean of . days [ ] . to calculate the case fatality rate (cfr) of an infection, one must divide the mortality number (m) by all those who were infected. the total number of those infected includes those who were infected and recovered without presentation (i r ), infected and presented to a health care facility (i p ), and infected and died (i d ). the cfr would be m/(i r + i p + i d ). clearly, one must have an accurate estimation of each of these parameters to accurately determine the cfr of covid- . while the (m) is generally easier to count, and a focus of media, the denominator can take much longer to calculate. during the early phases of a deadly epidemic, the number of those who were infected and recovered (i r ) is not yet known, since only those who were infected and became seriously ill are recognized and tested. in addition, because this is a novel virus, there were no existing detection methods, so early deaths due to clinical entities such as influenza, for example, may have been mis-attributed to covid- . the viral genome was published about weeks after the start of the outbreak, and pcr analysis was quickly used to diagnose suspected cases [ ] . public health officials can now test suspected cases, especially close contacts of known cases, and others with mild symptoms, but the testing capabilities can become saturated, potentially limiting the ability to get an accurate estimation of i p . for example, the initial ability of the wuhan health authority was limited to tests per day, but that number has grown to tests per day [ ] . the combination of these factors leads to a gross underestimation of the denominator of the cfr calculation, and thus an exaggeration of the mortality. until we are able to accurately represent i r and i p , it is currently impossible to precisely estimate the cfr of sars-cov- . however, during the course of a potentially fatal pandemic, an accurate estimation of cfr is important. while it is tempting to estimate the cfr by dividing the number of known deaths by the total number of confirmed cases, the resulting number may be off by orders of magnitude, especially since infected individuals at one point in time may die x days later. using the lag period approach and dividing the current number of deaths to the number of cases x days ago may be a more acute estimator of cfr. nucleuswealth.com applied this method by using the number of deaths at any particular day and dividing by number of cases , , or days prior. as seen in figure , as time progresses, whether whichever number of days is used for x, the cfr seems to converge at just under % for cases within hubei, and about . % for cases outside of hubei [ ] . the higher mortality in wuhan may be overestimated because early in the course of this epidemic, viral testing was limited to only the severe cases. however, the china national health commission admits that wuhan has a relative lack of medical resources, which may have contributed to the higher mortality rate. pathogens , , x for peer review of and dividing the current number of deaths to the number of cases x days ago may be a more acute estimator of cfr. nucleuswealth.com applied this method by using the number of deaths at any particular day and dividing by number of cases , , or days prior. as seen in figure , as time progresses, whether whichever number of days is used for x, the cfr seems to converge at just under % for cases within hubei, and about . % for cases outside of hubei [ ] . the higher mortality in wuhan may be overestimated because early in the course of this epidemic, viral testing was limited to only the severe cases. however, the china national health commission admits that wuhan has a relative lack of medical resources, which may have contributed to the higher mortality rate. infection transmission by asymptomatic individuals can make control of disease spread challenging. since late january, sars-cov- transmission from infected but still asymptomatic individuals has been increasingly reported [ , ] . assessment of the viral loads in symptomatic individuals not only showed that the viral loads peak within the first few days of symptoms, but also that asymptomatic patients can have a similarly high viral load without showing symptoms [ ] . it was suggested that viral testing should no longer be limited to symptomatic individuals, but also include those who have traveled to affected areas [ ] . at such an early phase of the covid- pandemic, it is difficult to accurately describe the populations most at risk, especially when teasing out risk factors for infection from risk factors for death from disease. early on, it became clear that those who have visited the wuhan wet market were most at risk of infection, but the population visiting the market is not an accurate reflection of the general population. the chinese cdc published the epidemiologic characteristics of the covid- outbreak along with associated risk factors for death [ ] . the largest risk factor for death is age. other risk factors include male sex and the presence of comorbid conditions (table ). however, in addition to real age-specific mortality, the age-based risk could reflect underlying comorbidities among the elderly and the distribution of the underlying population in wuhan, where the outbreak initiated. infection transmission by asymptomatic individuals can make control of disease spread challenging. since late january, sars-cov- transmission from infected but still asymptomatic individuals has been increasingly reported [ , ] . assessment of the viral loads in symptomatic individuals not only showed that the viral loads peak within the first few days of symptoms, but also that asymptomatic patients can have a similarly high viral load without showing symptoms [ ] . it was suggested that viral testing should no longer be limited to symptomatic individuals, but also include those who have traveled to affected areas [ ] . at such an early phase of the covid- pandemic, it is difficult to accurately describe the populations most at risk, especially when teasing out risk factors for infection from risk factors for death from disease. early on, it became clear that those who have visited the wuhan wet market were most at risk of infection, but the population visiting the market is not an accurate reflection of the general population. the chinese cdc published the epidemiologic characteristics of the covid- outbreak along with associated risk factors for death [ ] . the largest risk factor for death is age. other risk factors include male sex and the presence of comorbid conditions (table ) . however, in addition to real age-specific mortality, the age-based risk could reflect underlying comorbidities among the elderly and the distribution of the underlying population in wuhan, where the outbreak initiated. table . fatality rate by age, sex, and pre-existing medical conditions. the death rate represents the probability (%) of the corresponding group of dying from sars-cov- [ ] . with what we know about the pathogenesis of the sars-cov virus, it seems reasonable to assume that those with higher levels of ace- receptors may be at greatest risk. there was some speculation that the expression of ace- receptors may be linked to race, specifically after an early report suggested that asian males had higher ace- -expressing cell ratios than white and african americans [ ] . however, the sample size contained only eight different individuals (five african americans, two whites, and one asian) and extrapolating those findings to a whole race is impractical. yet, in another study assessing ace- receptor expression in tissues of patients with lung cancer, there were no significant disparities in ace- gene expression between racial groups (asian vs. caucasian), age groups (older or younger than years old), or gender groups (male vs. females) [ ] . ace- gene expression was, however, significantly elevated in smokers suggesting that smoking history should be considered in identifying susceptible populations. since smoking in china is predominantly a male attribute ( % of men, . % of women) [ ] , this may help to explain the gender difference seen in the hospitals in china. early in the covid- epidemic, it appeared that children were a protected group, but this may have been because they were less likely to have frequented the wuhan wet market, or because they were more likely to have asymptomatic or mild disease and thus less likely to have been tested. covid- has affected infants as young as month of age [ ] , most with mild or asymptomatic disease. there have been no reported cases of adverse infant outcomes for mothers who developed covid- during pregnancy. second to the hubei population, the other population at increasing risk is healthcare workers. as of february , , total of healthcare workers in china have been infected, five of whom fatally [ ] . the current best strategy of treatment of patients with covid- is purely supportive. clinicians and intensive care specialists are applying much of what they have learned during the sars epidemic to guide current therapy of covid- . recommendations for admission to critical care units, guidelines for infection control, and procedures to minimize nosocomial transmission are being established [ ] . however, there are several fronts that are being studied to develop targeted treatments. the most efficient approach to the treatment of covid- is to test whether existing antiviral drugs are effective. in previous betacoronavirus epidemics, several antiviral drugs, such as ribavirin, interferon, lopinavir-ritonavir, and darunavir/cobicistat (prezcobix) were tested, with some showing promising in vitro results [ ] . remdesivir, an adenosine analog used against rna viruses (including sars and mers-cov), was a candidate ebola treatment with promising in vitro results but disappointing in vivo effects against ebola [ , ] . there is currently in vitro evidence that remdesivir may be effective in controlling sars-cov- infection [ ] . in fact, compassionate use of remdesivir was employed in the treatment of the first covid- case in the united states, during a period of rapid clinical deterioration, and within one day there was dramatic improvement of the clinical condition [ ] . randomized double-blinded, placebo-controlled clinical trials are currently underway in china and usa to evaluate the efficacy of remdesivir and initial results are expected by the end of april [ , ] . other existing drug candidates include chloroquine and camostat mesylate. chloroquine is a widely used anti-malarial drug that is known to block virus-cell fusion and has been shown to interfere with the glycosylation of sars-cov and ace- cellular receptors, rendering the ace- -sars-cov interaction less efficient [ ] . there is also in vitro evidence that chloroquine may be effective in preventing sars-cov- cellular entry [ ] . camostat mesylate, also known as foy [ ] , was initially developed and currently approved for the treatment of chronic pancreatitis in japan [ , ] . camostat mesylate targets the tmprss protease, theoretically preventing viral entry. researchers in germany showed that camostat mesylate reduced the amount of sars-cov- viral replication [ ] . a simple but very effective treatment modality is the use of convalescent plasma, or serum from patients who have recovered from the virus, to treat patients. patients with resolved viral infection will have developed a specific antibody response which may be helpful in neutralizing viruses in newly infected individuals. this modality was successfully employed during the - ebola outbreak [ , ] . however, the use of convalescent sera is of limited benefit in an outbreak situation since the exponential growth of infected patients exceeds the ability of previous patients to provide donor plasma. the recent finding that sars-cov- binds to the same ace- receptors targeted by the sars-cov [ ] opens up the possibility of using the previous research on the sars epidemic and applying it to covid- . the first strategy would be to employ either a small receptor-binding domain (rbd) or a neutralizing antibody targeting the ace- receptor, thus blocking the binding of s protein and preventing virus entry into cells. initial in vitro results have shown promising results [ , ] and specific monoclonal antibodies are being contemplated as candidates for treatment [ , ] . the main limitation of using rbds or antibodies is that the treatment must be given within a specific time window, before the initiation of viral replication [ ] . in addition, the side effects of ace- blockade, especially since ace- is also present in non-pulmonary tissue, must be understood and minimized before implementation. in addition, finally, the turnover of ace- receptors would influence how often the therapeutic rbd or antibody would have to be administered. a second strategy is to create an ace- -like molecule that would bind to the s protein of the coronavirus itself. again, research in to the sars virus demonstrated that soluble ace- proteins blocked the sars virus from infecting cells in vitro [ , ] . the additional benefit to using this strategy lies in the possible prevention of s protein-mediated ace- shedding that has been shown to induce the pulmonary edema characteristic of sars [ , ] . a phase ii clinical trial of recombinant ace- in ards reported significant modulation of inflammatory proteins, but no significant differences in respiratory parameters [ ] . further research is necessary to assess if the animal studies will translate to clinical benefit. there are currently more than clinical trials to test a variety of potential sars-cov- treatments [ ] . the long-term goal of sars-cov- research is developing an effective vaccine to yield neutralizing antibodies. the national institutes of health in the us, and baylor university in waco, texas, are working on a vaccine based on what they know about the coronavirus in general, using information from the sars outbreak. in addition, the recent mapping of the sars-cov- spike protein may pave the way for more rapid development of a specific vaccine [ ] . of interest is the use of a relatively new vaccine technology, rna vaccines that have the ability to elicit potent immune responses against infectious diseases and certain cancers [ , ] . traditional vaccines 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doc_id: cord_uid: hteott the emergence of severe acute respiratory syndrome coronavirus (sars-cov- ) has resulted in more than million infections and more than , deaths worldwide. there is an urgent need to develop a safe and effective vaccine against sars-cov- . currently, several strategies are being pursued to develop a safe and effective sars-cov- vaccine. however, each vaccine strategy has distinct advantages and disadvantages. therefore, it is important to evaluate multiple vaccine platforms to select the most efficient vaccine platform for sars-cov- . in this regard, newcastle disease virus (ndv), an avian virus, has several well-suited properties for development of a vector vaccine against sars-cov- . here, we elaborate on the idea of considering ndv as a vaccine vector for sars-cov- . the ongoing pandemic of coronavirus disease (covid- ) has already resulted in more than million infections and more than , deaths, and has devastated the livelihoods of people globally. the causative agent is a coronavirus that has sequence similarity with the severe acute respiratory syndrome (sars) coronavirus of (sars-cov) [ ] [ ] [ ] [ ] . this new virus is named sars-cov- . currently, no vaccine or antiviral is available against sars-cov- . a safe and effective vaccine is urgently needed to control the current pandemic and any potential future pandemic. coronaviruses are enveloped, positive-sense, single-stranded rna viruses belonging to the family coronaviridae. these viruses mostly infect animals, including avian species and mammals. in humans, they generally cause mild respiratory infections [ ] . however, some zoonotic coronaviruses, which include the sars-cov, middle east respiratory syndrome coronavirus (mers-cov), and sars-cov- , have caused severe respiratory disease in humans [ , , ] . the genome of coronavirus is approximately kb long and encodes several structural and nonstructural proteins. proteins that form the structure of coronavirus are the spike (s), envelope (e), membrane (m), and nucleocapsid (n). the s gene with a size of . kb in length encodes the s protein. the s protein is the largest of all coronavirus structural proteins with a size of kda to kda. the s protein is heavily glycosylated and forms a crown-like structure on the envelope of the virion. the s protein mediates attachment and entry of the virion into the cell, and is the major protective antigen [ ] . from studies of sars, mers, and avian coronaviruses, it is well known that a successful vaccine must incorporate the whole s protein [ ] [ ] [ ] . the major neutralizing antigenic sites on this protein are highly conformation-dependent. therefore, the s protein, to be effective as a vaccine antigen, must be presented to the host in its native conformation. currently, several different approaches are being pursued at a rapid speed to develop a safe and effective vaccine against sars-cov- [ ] . among these strategies, a viral vector vaccine offers a number of advantages over other vaccines. first of all, it provides a live-virus vaccine approach that does not require involvement of the complete pathogen, which avoids the handling of live sars-cov- . a live viral vector vaccine mimics the natural infection of the pathogen against which the vaccine is made. the vector infects cells in the target host and expresses the foreign antigen intracellularly, inducing both innate and adaptive immunity. the in vivo expression of the foreign antigen in target host cells presents the protective epitopes in native conformation, which is particularly important for the s protein of sars-cov- . replication of the vector in the host enhances the magnitude of the immune response and often requires a low dose of the vaccine. some viral vectors are respirotropic, which are more suited for immunization against a respiratory pathogen like sars-cov- because they can induce local as well as systemic immunity. another advantage of viral vector vaccines is that they can be produced rapidly and cheaply. vector vaccines are not associated with the risk of reversion to virulence, a concern with live-attenuated vaccines, or with the risk of incomplete inactivation of inactivated vaccines. however, viral vector vaccines also have some challenges. most viral vector vaccines are replication-competent in vivo; therefore, they must be non-pathogenic to the target host, and have sufficient replication and antigen expression to elicit a protective immune response. sometimes, it is difficult to achieve both with a replication-competent vector. however, these challenges can be avoided by choosing a naturally attenuated virus vector that has a good track record of safety as a live vaccine in other species. a viral vector vaccine for sars-cov- must be highly safe and capable of inducing a protective immune response. currently, a number of dna and rna virus vector platforms are under evaluation for a sars-cov- vaccine, including attenuated vaccinia virus, replication-defective adenovirus, vesicular stomatitis virus, human parainfluenza viruses, and alphavirus replicons. however, each viral vector has some limitations that may or may not be possible to overcome. for example, the immunogenicity of some vaccinia virus vector vaccines has not been satisfactory, replication-defective adenovirus vector vaccines require a high dose and may not induce good local immunity, the safety of vesicular stomatitis virus vector vaccine in humans is questionable, human parainfluenza virus vector vaccines may not be effective in adults due to pre-existing immunity to the vector, and alphavirus vector vaccines are difficult to manufacture in large scale. keeping these limitations in mind, we think newcastle disease virus (ndv), as avian virus, has a number of characteristics that make it suitable for use as a vaccine vector for sars-cov- . ndv is an enveloped, negative-sense, single-stranded rna virus in the family paramyxoviridae [ ] . the genome is either , , , , or , nucleotides long [ ] [ ] [ ] [ ] and contains six transcription units encoding nucleocapsid (n), phosphoprotein (p, v, and w), matrix (m), fusion (f), hemagglutinin-neuraminidase (hn), and large polymerase (l) proteins [ ] . the l protein is an rna-dependent rna polymerase that associates with the n and p proteins to form the viral replication complex [ ] . this complex is responsible for transcription and replication of the viral genome. the f and hn proteins are involved in virus attachment and entry. the m protein is involved in the virus assembly process. two additional proteins, v and w, are produced via rna editing of the p gene [ ] . the v protein is an interferon antagonist [ ] . the function of the w protein is not established. the viral genes are ordered '-n-p/v/w-m-f-hn-l- ' and are separated by short intergenic regions. the genes are flanked at the ' and ' ends by extragenic regions called the leader and trailer, respectively. the leader and trailer regions are involved in transcription and replication of the viral genome. the beginning and end of each gene contains conserved transcription signals known as the gene-start (gs) and gene-end (ge), respectively [ ] . ndv strains have been classified into three pathotypes based on the severity of disease produced in chickens: low virulence (lentogenic), moderately virulent (mesogenic), and highly virulent (velogenic). lentogenic strains cause mild or subclinical infection and are often used as live vaccines in the poultry industry. a major determinant of ndv virulence is the cleavability of the f protein into f and f subunits, which is necessary for virus infectivity. in velogenic and mesogenic strains, the f protein cleavage site contains multiple basic residues, which is cleaved by ubiquitous intracellular furin-like proteases. the f cleavage site of lentogenic strains contains fewer basic residues that cannot be cleaved by furin-like proteases but is cleaved by trypsin-like secretory proteases found only in the respiratory and enteric tracts, which restricts their replication to those sites [ ] . ndv has several advantages over other viral vectors for the development of a vector vaccine against sars-cov- . the effectiveness of ndv-vectored vaccines has already been evaluated against sars-cov in monkeys [ ] , against mers-cov in camels [ ] , and against avian infectious bronchitis virus (ibv) in chickens, a natural host challenge model [ ] . the lentogenic ndv strains such as lasota or b used as a vaccine vector are highly attenuated and safe in poultry. these strains have been used for more than years as live virus vaccines in the poultry industry with a good track record of safety and efficacy [ ] . this feature makes lentogenic ndv strains unique among all live viral vaccines. in contrast, other currently used live viral vaccines in humans and animals are not naturally attenuated. therefore, these ndv strains are not likely to cause disease in any wild or domestic avian species. studies have shown that insertion of foreign genes into the genomes of ndv results in reduced rather than increased pathogenicity in birds. therefore, the vaccine construct of ndv containing the s gene of sars-cov- will not pose any environmental or agricultural risk. ndv is an avian virus and does not naturally infect non-avian species. it is highly attenuated in all non-avian species, including humans, due to natural host-range restriction. the safety and immunogenicity of ndv-vectored vaccines have been extensively studied in monkeys, a non-human primate model that is closely related phylogenetically and anatomically to humans, and which is a good model for pre-clinical studies of human vaccines [ , [ ] [ ] [ ] [ ] [ ] . in one study, intranasal and intratracheal inoculation of african green and rhesus monkeys with × . plaque forming units (pfu) per site with either a lentogenic strain (lasota) or a mesogenic strain (beaudette c) of ndv did not cause any clinical disease signs, and there was little or no virus shedding in throat swabs or tracheal lavage. analysis of necropsy specimens showed only low-level replication of the virus in the upper respiratory tract and lungs, and no virus was detected in the blood and other organs [ ] . in another study, african green monkeys were immunized through the respiratory tract with two doses of pfu of ndv expressing the s glycoproteins of sars-cov. the vaccinated monkeys developed robust sars-cov-specific neutralizing antibodies. the immunized animals were challenged with a high dose of sars-cov, and tissue samples were collected at the peak of viral replication (day ). viral assay of the lung samples showed that there was a substantial reduction of challenge virus titer in the immunized animals compared with control animals [ ] . these results suggest that ndv is highly attenuated in non-human primates and its replication is restricted only to the respiratory tract. there have been reports of ndv infection in humans involving poultry farmers and laboratory researchers [ ] . in rare cases, it causes disease, which is usually mild, with conjunctivitis, laryngitis, and flu-like symptoms. these symptoms are self-limiting and disappear in to days [ , ] . in a study in india, % of poultry workers were found to be seropositive for ndv compared with less than % of the general population, indicating that most ndv infections in poultry workers are asymptomatic [ ] . in another study in the united states, % of poultry workers also were found to be seropositive for ndv [ ] , again showing that most ndv infections in poultry workers are asymptomatic [ ] . moreover, there is no evidence for human-to-human transmission. ndv has been investigated as an oncolytic agent due to its selective replication in tumor cells and associated cell death eliciting innate and adaptive antitumor immune responses [ ] . several clinical trials have been conducted utilizing various pathotypes of ndv in human cancer patients [ ] [ ] [ ] . direct inoculation of ndv into thousands of patients by intranasal or parenteral routes has resulted in only mild side effects. the common side effects have been mild flu-like symptoms, conjunctivitis, and laryngitis [ , ] . in a dose-escalation trial, cancer patients were administered one or more increasing doses of a mesogenic ndv strain. the ndv strain was well tolerated in doses of at least × infectious units by the intravenous route and at least × infectious units by the intratumoral route. the infected patients displayed mild flu-like symptoms and approximately % of the patients had transient low-level virus shedding [ ] . in another clinical study, inhalation of approximately × pfu of ndv was associated with only one day of low-grade fever in % of patients [ ] . to date, there has been no report on accumulative toxicity associated with repeated vaccination with ndv [ ] . overall, the safety of ndv strains as anticancer agents has been consistently high with mild side effects, suggesting that ndv is highly safe in humans. ndv strains mimic restricted natural infection in the respiratory tract of avian and non-avian host cells resulting in the inducement of innate and adaptive immunity. ndv potentially expresses the protective epitopes of the foreign protein in native conformation in host cells that is crucial in order for the s protein of sars-cov- to induce a protective immune response. it also induces good local immunity in the respiratory tract where pathogens like sars-cov- enter the body. the restricted replication of ndv in the respiratory tract of humans would lead to the induction of a balanced immune response. development of a balanced immune response is less likely to lead to the antibody-dependent enhancement of disease, which is sometimes associated with vaccination. ndv vector vaccines have shown promising results against sars-cov and ibv [ , ] . ndv possesses strong immunostimulatory properties through the induction of large amounts of type interferon (ifn) and by its inability to block type ifn response in human cells. the viral surface proteins (f and hn) also play an important role for the immunostimulatory properties of the virus by upregulating major histocompatibility complex (mhc) and cell-adhesion molecules and facilitating lymphocytes and antigen-presenting cells through their expression at the surface of infected cells. [ ] . ndv encodes only six major proteins. therefore, the antigenic competition between the foreign protein and the vector proteins is much less than other dna viral vectors, whose genomes encode large numbers of proteins. ndv is an avian virus, which avoids the problem of pre-existing immunity to the vector. this is a potential problem with viral vectors of common human viruses or viral vectors that are antigenically related to common human viruses. serological studies have indicated that more than % of the human population is seronegative for ndv [ ] . therefore, the entire human population is amenable to ndv-vectored vaccines. foreign genes in ndv are stable. ndv does not undergo the genetic recombination or genetic reassortment observed for certain rna viruses. ndv is an acute cytoplasmic rna virus, which precludes concerns of long-term infection or integration into host cell dna. modular organization of the ndv genome facilitates the insertion of foreign genes. infectious ndv can be produced from cloned cdnas by a method called reverse genetics. this involves co-transfecting cells with four t polymerase driven plasmids. one plasmid encodes a positive-sense copy of the genome (positive-sense is used instead of negative-sense to avoid hybridization of the plasmid expressed rna with viral mrnas) and three plasmids encode the n, p, and l proteins of the viral polymerase complex. [ , ] . the t rna polymerase inside the cell is provided either by a recombinant vaccinia virus, from a co-transfected t expression plasmid, or a cell line that expresses t polymerase. this creates an artificial viral replication cycle inside the cell and leads to the production of infectious ndv. this method allows for insertion of a foreign gene into the viral genome. to construct the ndv-vectored sars-cov- vaccine, the open reading frame encoding the s protein of sars-cov- , the codon optimized for higher expression in human cells, will be engineered to contain the ndv-specific gene-start and gene-end sequences at the beginning and end of the orf, respectively (figure ). the s gene transcription cassette is then inserted into a 'non-coding region of an ndv gene as an additional transcription unit [ , ] . owing to polar gradient transcription, foreign genes are expressed more efficiently when placed closer to the '-end of the genome [ ] . a foreign gene can be placed between any two genes of ndv, but the insertion site between the p and m genes has been found to be optimal for efficient expression of the foreign gene replication of ndv [ ] [ ] [ ] [ ] . ndv can accommodate foreign sequences of at least . kb with a good degree of stability [ ] . therefore, ndv will not have any problem in accommodating the s gene of sars-cov- , which is approximately . kb in length. pathogens , , x for peer review of precludes concerns of long-term infection or integration into host cell dna. modular organization of the ndv genome facilitates the insertion of foreign genes. infectious ndv can be produced from cloned cdnas by a method called reverse genetics. this involves co-transfecting cells with four t polymerase driven plasmids. one plasmid encodes a positive-sense copy of the genome (positive-sense is used instead of negative-sense to avoid hybridization of the plasmid expressed rna with viral mrnas) and three plasmids encode the n, p, and l proteins of the viral polymerase complex. [ , ] . the t rna polymerase inside the cell is provided either by a recombinant vaccinia virus, from a co-transfected t expression plasmid, or a cell line that expresses t polymerase. this creates an artificial viral replication cycle inside the cell and leads to the production of infectious ndv. this method allows for insertion of a foreign gene into the viral genome. to construct the ndv-vectored sars-cov- vaccine, the open reading frame encoding the s protein of sars-cov- , the codon optimized for higher expression in human cells, will be engineered to contain the ndv-specific gene-start and gene-end sequences at the beginning and end of the orf, respectively (figure ). the s gene transcription cassette is then inserted into a 'non-coding region of an ndv gene as an additional transcription unit [ , ] . owing to polar gradient transcription, foreign genes are expressed more efficiently when placed closer to the '-end of the genome [ ] . a foreign gene can be placed between any two genes of ndv, but the insertion site between the p and m genes has been found to be optimal for efficient expression of the foreign gene replication of ndv [ ] [ ] [ ] [ ] . ndv can accommodate foreign sequences of at least . kb with a good degree of stability [ ] . therefore, ndv will not have any problem in accommodating the s gene of sars-cov- , which is approximately . kb in length. infectious newcastle disease virus (ndv) can be produced from cloned cdnas by a method called reverse genetics. infectious rndv expressing sars-cov- is generated by co-transfecting cells by four t polymerase driven plasmids, which include one plasmid encoding a positive-sense copy of the genome of ndv containing the sequence of the s gene of sars-cov- flanked between p and m genes of ndv, and three plasmids encoding the n, p, and l genes, and a recombinant vaccinia virus expressing t polymerase. infectious newcastle disease virus (ndv) can be produced from cloned cdnas by a method called reverse genetics. infectious rndv expressing sars-cov- is generated by co-transfecting cells by four t polymerase driven plasmids, which include one plasmid encoding a positive-sense copy of the genome of ndv containing the sequence of the s gene of sars-cov- flanked between p and m genes of ndv, and three plasmids encoding the n, p, and l genes, and a recombinant vaccinia virus expressing t polymerase. there is a dire need for the development of an effective vaccine against sars-cov- . however, the development of a safe and effective vaccine against sars-cov- is a challenging task. prior studies from other pathogenic coronaviruses of humans (sars and mers) have indicated the potential of ndv as a vaccine vector. ndv has several features that merit attention with respect to vaccine safety and efficacy. excellent data exist on the safety of ndv in humans that establish and monitor safety, and also define correlates and the durability of protection. development of an ndv-vectored vaccine may 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of the replication, pathogenicity, and immunogenicity of avian paramyxovirus (apmv]) serotypes , , , , , and in rhesus macaques immunization of primates with a newcastle disease virus-vectored vaccine via the respiratory tract induces a high titer of serum neutralizing antibodies against highly pathogenic avian influenza virus newcastle disease virus as a vaccine vector for humans respiratory tract immunization of non-human primates with a newcastle disease virus-vectored vaccine candidate against ebola virus elicits a neutralizing antibody response recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates human health implications of avian influenza viruses and paramyxoviruses an outbreak of conjunctivitis due to newcastle disease virus (ndv) occurring in poultry workers human conjunctivitis due to the newcastle-disease virus of fowls newcastle disease virus antibodies in human sera reactions of human sera to avian 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immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses p and m gene junction is the optimal insertion site in newcastle disease virus vaccine vector for foreign gene expression this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license author contributions: s.k.s. and e.s.; writing. all authors have read and agreed to the published version of the manuscript.funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -yaeduwvb authors: james, claire d.; roberts, sally title: viral interactions with pdz domain-containing proteins—an oncogenic trait? date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: yaeduwvb many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis b and c, human t cell leukaemia virus type , kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type ), encode in their limited genome the ability to target cellular proteins containing psd / dlg/zo- (pdz) interaction modules. in many cases (but not always), the viruses have evolved to bind the pdz domains using the same short linear peptide motifs found in host protein-pdz interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. what is striking is that the diverse viruses target a common subset of pdz proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. this review focuses on the oncogenic viruses and the role of targeting pdz proteins in the virus life cycle and the contribution of virus-pdz protein interactions to virus-mediated oncogenesis. we highlight how many of the viral associations with pdz proteins lead to deregulation of pi k/akt signalling, benefitting virus replication but as a consequence also contributing to oncogenesis. psd /dlg/zo- (pdz) domains are one of the most widely distributed protein-protein interaction domains; the human genome encodes over pdz domain-containing proteins [ ] . the domain itself is an evolutionarily conserved domain of approximately amino acids folded into six β-sheets (βa-βf) and two α-helices (αa-αb), named for its presence in psd , dlg, and zo- proteins. the domains are often found in combination with a number of other protein-protein interaction modules, for example members of the membrane-associated guanylate kinase (maguk) family include an sh and inactive guanylate kinase domain, as well as multiple pdz modules. the presence of modular domains within a single protein allows pdz domain-containing proteins (herein referred to as pdz proteins) to form interactions with many partner proteins simultaneously and act as a scaffold; thus these proteins are involved in myriad of functions, including organizing multiprotein signalling complexes at the immunological and neurological synapses, forming cell-cell junctions, cell proliferation and survival, intracellular trafficking, apico-basal and planar polarity. perhaps not surprisingly considering the wide remit of pdz protein functions, deregulation of a number of pdz proteins has been linked to tumourigenesis [ ] . however, it is often not clear whether to define some pdz proteins as oncogenes or tumour suppressors, as their roles in tumourigenesis is context-dependent [ , ] . whilst abundance and/or cellular localization is a contributing factor in pdz protein function, tissue specificity is also of importance; this is the case for the epithelial polarity protein scrib, which has a tumour suppressor role in epithelia but a pro-oncogenic role in myc-driven lymphomas [ , ] . binding to the pdz domain is mediated by short peptide sequences-pdz-binding motifs (pbm)-often but not always found at the carboxy terminus of proteins. pdz domains are grouped into specificity classes based on the consensus sequence of the pbm recognized. early studies defined three main classes of specificity; class i s/t-x-ϕ-cooh, class ii ϕ-x-ϕ-cooh and class iii d/e-x-ϕ-cooh, where ϕ is an hydrophobic residue, but more recent analyses using protein arrays and peptide libraries have defined at least distinct specificity classes (the reader should refer to the references for details of the consensus sequences) [ , ] . in binding, the pbm sits into the hydrophobic groove of the pdz domain formed between one of the β sheets (βb) and the α helix αb, with a highly conserved carboxylate binding loop at the base. amino acids outside the pbm contribute to pdz substrate specificity of binding, and phosphorylation of the pbm can act as a negative or positive regulator of pdz binding [ , ] . likewise, phosphorylation within the pdz domain itself is also a regulatory mechanism of ligand binding [ ] . a number of diverse but highly pathogenic viruses encode proteins that target pdz domain containing proteins, often but not always, using a pbm. pathogenicity of both the sars coronavirus (sars-cov), a positive strand rna virus that causes severe acute respiratory infections, and the neurotropic rabies virus, are linked to pdz-binding functions of their envelope proteins. recombinant sars-cov expressing the envelope e protein lacking the c-terminal pbm had no effect on viral growth in a murine infection model, but caused less lung damage and mortality than in mice infected with the wild type virus [ , ] . the e protein pbm binds to the tight junction associated pdz protein pals , leading to disturbed tight junction formation of polarized epithelia. it can also induce proinflammatory cytokine expression, possibly through pbm binding of the pdz protein syntenin- . both of these pbm functions could be determinants of the lung pathobiology associated with sars-cov infection [ , ] . survival of rabies infected neuronal cells is associated with the ability of the viral envelope g protein to interact with the pdz domain-containing serine threonine kinase mast , leading to the disruption of the mast -pten complex that is intimately involved in the inhibition of neuronal survival [ ] . in an attenuated strain, a mutation within the g protein pbm confers an expansion of the pdz proteins bound, and leads to apoptosis of neuronal cells [ ] . also, the canonical class i pbm at the c-terminus of the ns protein of over % type a influenza viruses is a good target for attenuation of these pathogenic viruses [ ] [ ] [ ] [ ] . removal of the pbm decreases efficacy of influenza h n virus transmission, as well as virus replication, and increases interferon expression in infected cells [ , ] . moreover, switching the h n pbm for the sequence of an avian-specific strain increased virus virulence [ ] . the ns pbm of the highly pathogenic strain h n interacts with the pdz domains of several pdz proteins, including the epithelial polarity regulators dlg and scrib and the tight junction protein magi- [ ] [ ] [ ] . using models of influenza a infection, this motif has been shown to contribute to the pathogenesis of these viruses by disruption of tight junction integrity of polarized epithelia, also disruption of cell junctions may benefit viral dissemination within the host, and/or spread to other hosts [ ] . furthermore, relocalization of scrib to cytoplasmic puncta in infected cells is associated with the abrogation of the polarity protein's pro-apoptotic function and may therefore protect infected cells from viral induction of apoptotic pathways, thus supporting viral propagation [ , ] . targeting pdz protein function is also a common function amongst viruses classified as human carcinogens by the world health organization [ ] . as well as having roles in virus infectivity, the virus-pdz protein associations are important in virus entry, replication and for some of these viruses, cell transformation. these include the hepatitis viruses b and c, kaposi sarcoma herpesvirus, human t cell leukaemia virus type , high-risk human papillomaviruses, and human immunodeficiency virus type . in this review, we examine the interactions between these cancer-causing viruses and pdz proteins and discuss their contribution to pathogenesis of this important group of viruses. whilst hiv- is defined as a group i carcinogen, it contributes indirectly to carcinogenesis via immunosuppression and infected individuals have higher incidence rates of tumours driven by other cancer-causing viruses, and so it is also included in the review. interactions with pdz proteins are also pivotal in the formation of mammary tumours in mice infected by the human adenovirus type and although this virus is not linked to human cancers, the study of ad interactions with pdz proteins has given valuable insight in viral carcinogenesis. the different virus-pdz associations are listed in table and common pdz targets are shown in figure . table for full list) yes (class i, s/txv/l) degradation via the proteasome, transcriptional downregulation, altered subcellular localization, complex formation to alter function see table high-risk alpha-hpv e * dlg , magi- , patj, scrib no degradation via the proteasome [ , ] alter function high-risk alpha-hpv e * dlg , magi- , patj, scrib no degradation via the proteasome [ , ] interactions between virus proteins and pdz domains/proteins have also been identified from peptide/protein screens but they are not included in this table [ ] [ ] [ ] ; hpv e * protein is an e isoform that lacks the pdz-binding motifs (pbm). (?) function/effect not proven. a common risk factor for hepatocellular carcinoma is chronic infection with hcv. the virus primarily infects the highly polarized epithelium in the liver and depolarization of this tissue by the virus is critical for virus entry and cell-to-cell transmission of virus. entry of hcv occurs at tight junctions and most likely requires disruption of these structures, contributing to depolarization of the tissue [ ] . the tight junction protein claudin and the major receptor for high-density lipoproteins, sr-b (class b scavenger receptor) are important entry factors for hcv and both proteins encode pbm at the c-termini. the interaction between sr-b and a pdz domain-containing adaptor partner pdzk facilitates virus entry [ ] . expression of the hcv core protein disrupts the apicobasal polarity of polarized epithelia and leads to deregulation of components of the scribble polarity complex dlg and scrib at cell-cell contacts. dlg protein expression is decreased and there is mislocalisation of scrib from cell-cell contacts in liver epithelial cells [ ] . the core protein does not target the polarity pdz proteins directly, but subverts the phosphoinositide (pi) phosphastase ship , leading to a decrease in production of the pi phosphatidylinositol , bisphosphate (pip ) which has a role in the targeting of dlg and scrib to the basolateral membrane ( figure ). such deregulation a common risk factor for hepatocellular carcinoma is chronic infection with hcv. the virus primarily infects the highly polarized epithelium in the liver and depolarization of this tissue by the virus is critical for virus entry and cell-to-cell transmission of virus. entry of hcv occurs at tight junctions and most likely requires disruption of these structures, contributing to depolarization of the tissue [ ] . the tight junction protein claudin and the major receptor for high-density lipoproteins, sr-b (class b scavenger receptor) are important entry factors for hcv and both proteins encode pbm at the c-termini. the interaction between sr-b and a pdz domain-containing adaptor partner pdzk facilitates virus entry [ ] . expression of the hcv core protein disrupts the apicobasal polarity of polarized epithelia and leads to deregulation of components of the scribble polarity complex dlg and scrib at cell-cell contacts. dlg protein expression is decreased and there is mislocalisation of scrib from cell-cell contacts in liver epithelial cells [ ] . the core protein does not target the polarity pdz proteins directly, but subverts the phosphoinositide (pi) phosphastase ship , leading to a decrease in production of the pi phosphatidylinositol , bisphosphate (pip ) which has a role in the targeting of dlg and scrib to the basolateral membrane ( figure ). such deregulation of the polarity pathways regulated by the scribble components may be a contributing factor in hcv driven oncogenesis [ ] . also, it has been reported that the carboxyl sequence of the poorly characterized non-structural protein ns b encodes a class i pbm-like sequence (x-t-x-c); however, as yet there is no experimental evidence that supports interaction between ns b and pdz proteins [ ] . nevertheless, propagation of other rna viruses within flaviviridae (tick borne encephalitis virus, west-nile virus and dengue virus) is dependent upon pbms within the non-structural ns proteins, and have been shown to bind to various pdz proteins, including those involved in polarity control and tight junction assembly and maintenance; and mutations in these pbm negatively affect viral replication [ , ] . as yet there is no experimental evidence that supports interaction between ns b and pdz proteins [ ] . nevertheless, propagation of other rna viruses within flaviviridae (tick borne encephalitis virus, west-nile virus and dengue virus) is dependent upon pbms within the non-structural ns proteins, and have been shown to bind to various pdz proteins, including those involved in polarity control and tight junction assembly and maintenance; and mutations in these pbm negatively affect viral replication [ , ] . hbv is another virus whose infection of the liver causes acute and chronic hepatitis with an increased risk of the development of hepatocellular carcinoma. like the hcv core, the activities of the core protein of hbv (hbvc) are linked to viral pathogenicity. yeast two hybrid screening for hbv core partners identified the pdz protein gipc (gaip-interaction protein, c-terminus, family member , also known as tip- ) as a robust binding partner. the interaction is mediated by the core's pbm, which has sequence similarities with other ligands that bind gipc (x-x-c-cooh, a nontypical pbm [ ] ), and deletion of the carboxyl cysteine from the hbvc was sufficient to block the interaction [ ] . the consequences of this hbvc activity in hbv pathogenesis is unknown, but as gipc is a scaffold protein important in the endocytic trafficking of multiple signal transduction receptors, the association may have an important role in facilitating movement of the hbv capsids to the nucleus [ ] . it is possible that the association between the core and gipc is linked to the virus's role in carcinogenesis via disruption of interactions of cellular substrates with the pdz domain of gipc . pertinent to this is the interaction between lysophosphatidic acid (lpa) and the pdz domain of gipc , which is necessary to attenuate lpa signalling ( figure ). loss of this control leads to increased cell proliferation and survival through the resulting upregulation of the akt pathway [ ] . this activity may function synergistically with the virus's hbx activation of akt signalling to block apoptosis of infected hepatocytes, thus enabling persistence of this viral carcinogen [ ] . hbv is another virus whose infection of the liver causes acute and chronic hepatitis with an increased risk of the development of hepatocellular carcinoma. like the hcv core, the activities of the core protein of hbv (hbvc) are linked to viral pathogenicity. yeast two hybrid screening for hbv core partners identified the pdz protein gipc (gaip-interaction protein, c-terminus, family member , also known as tip- ) as a robust binding partner. the interaction is mediated by the core's pbm, which has sequence similarities with other ligands that bind gipc (x-x-c-cooh, a non-typical pbm [ ] ), and deletion of the carboxyl cysteine from the hbvc was sufficient to block the interaction [ ] . the consequences of this hbvc activity in hbv pathogenesis is unknown, but as gipc is a scaffold protein important in the endocytic trafficking of multiple signal transduction receptors, the association may have an important role in facilitating movement of the hbv capsids to the nucleus [ ] . it is possible that the association between the core and gipc is linked to the virus's role in carcinogenesis via disruption of interactions of cellular substrates with the pdz domain of gipc . pertinent to this is the interaction between lysophosphatidic acid (lpa) and the pdz domain of gipc , which is necessary to attenuate lpa signalling ( figure ). loss of this control leads to increased cell proliferation and survival through the resulting upregulation of the akt pathway [ ] . this activity may function synergistically with the virus's hbx activation of akt signalling to block apoptosis of infected hepatocytes, thus enabling persistence of this viral carcinogen [ ] . the hbvc can also bind via its pbm to the non-receptor protein tyrosine phosphatase ptpn ; however the precise role of this association in hbv pathogenesis is not known. ptpn has been shown to have a suppressive role on hbv gene expression, but this control is independent of the pbm activity of the hbvc [ ] . kshv (also known as human herpesvirus ) is the aetiological agent of kaposi sarcoma, primary effusion lymphoma and multicentric castleman disease and kshv induced cancers are particularly common among patients with acquired immunodeficiency syndrome (aids). the route to carcinogenesis is not fully understood, but as with all cancer-causing viruses it involves dysregulation of signalling pathways important for host proliferation and survival. notably, a critical mechanism is the constitutive activation of the nuclear factor κb (nf-κb) and signal transducer and activator of transcription (stat ) signalling pathways. in normal signalling, the tumour suppressor pdz-lim domain-containing protein (pdlim , the lim domain is a protein:protein interaction domain composed of two contiguous zinc fingers) regulates turnover of rela (p ), one of the components of the nf-κb, and stat by ubiquitination and degradation via the proteasome [ ] . this normal mechanism of turnover of stat and nf-κb is deregulated in kshv pathogenesis by the virus repressing pdlim transcription by increased dna methylation of the promoter region [ ] . hiv- is a lentivirus that infects several different types of immune cells, but primarily cd + positive t cells, and causes aids, a condition that is characterized by immune suppression, and aids patients are at high-risk of opportunistic microbial infections, including cancer-causing viruses. interactions between hiv- and host pdz proteins have been reported to take place at early and late stages of virus infection. the hiv- gag polyprotein interacts with pdz proteins, although the interactions are not mediated by a classical pbm. the cytoskeletal protein pdzd interacts with gag involving those regions of the polyprotein associated with early post-entry events; knockdown of this cellular cofactor inhibits hiv- infection and negatively affects viral capsid stability, whereas pdzd overexpression enhances infection by hiv- [ , ] . pdzd is a poorly understood protein that has been shown to regulate microtubule stability through an interaction with moesin and microtubule stability at the viral synapse is likely to be important for hiv- infection [ , ] . however, recent findings using cell lines in which pdzd expression has been eliminated by the crispr-cas system indicated that hiv- infection is not affected by loss of this host protein [ ] . the other known pdz target of gag is dlg [ ] . binding to dlg is mediated through the nucleocapsid domain of gag and the viral protein associates with an isoform of dlg that preferentially locates to the plasma membrane, where the two proteins colocalize [ ] . the gag-dlg association regulates hiv-infectivity, such that depletion of dlg enhanced infectivity and dlg overexpression reduced particle infectivity [ ] . loss of dlg from infected cells leads to the redistribution of env and gag from predominantly plasma membrane sites to intracellular vesicle-like structures that function as specialized platforms for the generation of infectious particles and suggests that dlg regulates plasma membrane dynamics in hiv- infected t cells [ ] . dlg is not the only pdz protein to act antagonistically in hiv- infectivity; syntenin- is recruited to the plasma membrane during hiv- attachment by the viral envelope glycoprotein (env) and associates with the main hiv- receptor cd [ ] . over expression of syntenin- inhibits hiv production and silencing increases viral entry, most likely by inducing actin polymerization and accumulation at the viral attachment site to inhibit virus entry [ ] . hiv- transmission occurs at mucosal epithelial surfaces and one of the consequences of hiv- infection is increased permeability of the intestinal tract leading to an enhancement in microbial infections. in polarized epithelial cell cultures, hiv- infection causes a displacement of the pdz protein zo- from the tight junctions in the apical region of the membrane, followed by a marked reduction in the transcription of zo- and other tight junction proteins [ ] . the disruption of tight junction integrity is mediated by the hiv- glycoprotein , which induces upregulation of the inflammatory cytokine tnf-α, a known disruptor of tight junctions [ , ] . htlv- is a retrovirus that is the aetiological agent of the highly aggressive adult t cell leukaemia and primarily replicates in cd + t cells. transmission of the virus occurs predominantly by direct contact between infected and target cells; the site of contact forms a virological synapse where the gag glycoprotein and envelope (env) protein, together with the viral rna become concentrated. the env protein contains at its c-terminus a class i pbm and the activity of this motif contributes to the ability of env to trigger cell-to-cell fusion as judged by analysis of syncytium formation-a marker of cell-to-cell fusion [ ] . there is also evidence that the pbm regulates env trafficking since disruption of the motif causes accelerated endocytosis and degradation of the env protein [ ] . thus, binding to pdz proteins may be important for the proper localization of env-directing it to specific microdomains in the plasma membrane. to date, only one pdz protein, dlg has been identified as binding to env in a pbm dependent manner. env and dlg colocalize at sites characteristic of virological synapses on htlv- -infected t cells [ ] . moreover, depletion of dlg reduced syncytium formation, although env expression was not affected suggesting that additional pdz proteins might be involved in regulating env trafficking [ , ] . since there is a partial colocalization between dlg and the major htlv- receptor glut- (glucose transporter type ) at intercellular contacts formed between infected and target cells, a possible function for this interaction may be to aid efficient clustering of glut- at the polarised synapse [ ] . the htlv- tax transcription factor also contains a canonical class i pbm at its c-terminus. tax is the major oncogenic determinant of htlv- ; when expressed alone it induces the transformation of rat fibroblasts in culture and is capable of promoting leukaemia in transgenic mice [ ] [ ] [ ] . studies of the contribution of the htlv- motif to the transformation functions of tax have shown that deletion of the motif reduces the transformation potential of tax in rat fibroblasts [ ] . the domain is also necessary for t cell overproliferation and reconstruction of a tax pbm deletion mutation in the context of whole htlv- virus showed that the motif was critical for induction of proliferation of human lymphocytes following viral infection [ , ] . using this same model of htlv- pathogenesis, the mutant tax pbm deletion virus, whilst not necessary for htlv- driven immortalization, was shown to be a requirement for acquisition of host genomic instability as judged by micronuclei formation, and the mutant virus was severely compromised in its ability to establish persistent infection in rabbits [ ] . taken together, the function of the tax pbm plays a significant role in those characteristics of the virus necessary to induce t cell transformation. in further support of the role of the pbm in htlv- driven transformation, a pbm is absent from the tax protein of htlv- , a virus that is not leukemogenic. interestingly, as for cellular pbm-pdz interactions, the function of the tax pbm is susceptible to regulation by phosphorylation [ ] . the protein kinase casein kinase phosphorylates the threonine residue within the tax pbm and negatively regulates binding to pdz proteins, suggesting that timing of pdz targeting may be important in the htlv- replication cycle and host transformation [ ] . but what are the physiological targets of this domain? to date, scribble polarity complex components dlg and scrib have been identified as robust binding partners of the tax pbm and both polarity proteins have been shown to have critical roles in t cell signalling, including the formation and organization of the immunological synapse [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . the subcellular localization of both proteins is affected by tax expression; in htlv- infected t cells, and in cells overexpressing tax, dlg and scrib are relocated from cell membranes to cytoplasmic puncti or granules and this is concomitant with an increase in the insolubility of the polarity proteins, effects that are dependent on an intact tax pbm [ , , , ] . the relocalization of dlg and scrib is a likely mechanism of inactivation of their normal function. indeed, the ability of dlg to block cell cycle progression of fibroblasts is inhibited by tax overexpression and is pbm dependent [ ] . the inhibition of cell cycle progression is associated with an increase in phosphorylation of dlg and it is well known that cell cycle mediated phosphorylation of dlg regulates dlg localization and function [ , ] . so, is dlg inactivation necessary for t cell transformation by tax? whilst silencing expression of dlg in tax expressing t cells augments both growth and transformation of a mouse t cell line, depletion of dlg does not complement transformation in the absence of the tax pbm, altogether suggesting that htlv- transformation does not depend solely on tax binding and inactivating dlg [ ] . correct localization of dlg and scrib is important for the formation of signalling complexes at specific sites in the cell and tax pbm mediated effects on these proteins is likely to lead to a disruption of these signalling pathways. two critical signal transduction pathways for t cell signalling are the t-cell receptor (tcr)-induced activation via the transcription factor nuclear factor of activated t cells (nfat), and the akt pathway. in t cells, dlg is required for p -mediated activation of nfat and stabilization of pten to inhibit akt-mediated signalling ( figure ) [ , ] . moreover, scrib may also organize receptor signalling through the nfat pathway since overexpression of scrib attenuates nfat activity and in tax expressing t-cells, this activity of scrib was inhibited, whereas scrib orchestrated inhibition of nfat still occurred when the tax pbm was mutated [ ] . in addition, in immortalized t cells, tax stimulates akt activating phosphorylation in a pbm dependent manner by a mechanism that involves decreased membrane localization of the phosphatases pten and phlpp (ph domain and leucine rich repeat protein), a critical requisite for both phosphatases to negatively regulate akt activity. in both cases, overexpression of membrane forms of the phosphatases overcame the effects of tax on akt phosphorylation. tax-mediated mislocalization of these phosphatases from the plasma membrane may involve tax disruption of dlg and scrib scaffolding functions; scrib is necessary for membrane localization of phlpp [ ] and pten is a binding partner of dlg [ ] . how dlg regulates pten activity is not altogether clear, but dlg increases pten stability and augments pten's inhibition function of the akt pathway [ ] . interestingly, tax attenuates binding between pten and dlg in t cells and this requires the activity of the pbm [ ] . the htlv- tax protein has been shown to bind to many other pdz proteins besides dlg and scrib (table ), but as yet the physiological relevance of these interactions to htlv- infection and transformation is unknown. singling out one of these, the t-cell specific interleukin- precursor (pro-il- ) binds to tax via the pbm in human t-cells, where both proteins colocalize in the nucleus [ ] . pro-il- is highly expressed in all t cells and is the precursor to il- . the presence of an intact pbm enables tax-expressing cells to overcome pro-il- driven cell cycle arrest, suggesting a role for the interaction in overcoming the growth inhibitory activity of pro-il- and promoting cell cycle progression [ ] . such progression is favourable for viral propagation and is likely to contribute to the overproliferative phenotype of htlv- transformed cells. whilst not human carcinogens, ad are potent carcinogens in rodent cells and are provocative agents for the study of carcinogenesis. this review will focus on ad type (ad ); a human virus commonly associated with benign eye infections, but causative of mammary tumours in rats in the presence of high levels of the hormone oestrogen [ ] . unlike other human ad which drive carcinogenesis by the actions of the products of the e a and e b genes, ad 's oncogenicity is determined by the e region orf gene (e orf ) function alone [ ] . three regions of e orf are important in cell transformation, including a c-terminal class i pbm, which is known to bind to a number of pdz domain-containing proteins, including dlg and tight junction proteins, magi- , mupp , patj and zo- [ ] [ ] [ ] [ ] (table ). in fact, in polarized epithelial cells, the expression of e orf blocks localization of the tight junction proteins to the intercellular junctions and many of the proteins become sequestered to e orf -containing structures in the cytoplasm. the disruption of tight junction function and the associated polarity defects are entirely dependent on an intact viral pbm [ ] . in contrast to the effects upon the tight junction proteins, dlg is not sequestered into the cytoplasm but driven to accumulate at the plasma membrane. a second intriguing function of the e orf pbm is the activation of pi k pathway at the plasma membrane, a function that is dependent on the e orf pbm localizing the viral protein to sites in the membrane and triggering activation of downstream targets, including akt/pkb [ ] . importantly, overexpression of individual pdz targets of e orf , including dlg , suppressed e -orf mediated akt stimulation, strongly suggesting a link between this activity and pdz targeting. since this function of the e orf pbm was not required for tight junction disruption, this represents a distinct activity mediated largely (but not entirely [ ] ) by the pbm. all together, these studies suggest that the different e orf functions-disruption of tight junctions and apicobasal polarity, and activation of the pi k signalling pathway-cooperate to transform mammary cells. the next part in this story went on to understand the mechanism of pi k activation, and this led to a series of very elegant studies from the javier laboratory. an important finding was that the increase in pi k activity was not a consequence of inactivation of dlg function as first thought, but that e orf exerts a gain-of-function upon dlg , by binding dlg and promoting a conformational change allowing binding to the plasma membrane to direct pi k activation [ ] . this led to the idea that dlg , whilst widely regarded as a tumour suppressor can acquire oncogenic characteristics in a different cellular context [ ] . the summation of these studies has shown that e orf forms complexes with pi k and dlg in the cytoplasm; complex formation leads to increased stabilization of pi k and increasing pi k catalytic activity and activation of akt. the complex is then recruited to the plasma membrane through the induction of changes to dlg conformation [ ] . deregulated activation of akt thereby promoting cellular survival and metabolism, which are important for virus replication, but also necessary for adenovirus-mediated transformation ( figure ) . indeed, the formation of the e orf /pi k/dlg ternary complex was necessary for anchorage independent cell growth-a robust indicator of cell transformation. in addition, the complex of e orf and dlg also deregulates epidermal growth factor receptor signalling leading to activation of nuclear myc; an action thought to aid virion production in infected cells, although this action is not solely driven by the function of the e orf pbm [ ] . the ability of e orf to activate pi k in a dlg dependent fashion was found to be a conserved function between human ad subgroups a through to d, including those members used as e orf -encoding vectors in human gene therapy and vaccination, raising possible safety concerns of their use in humans [ ] . whilst some hpv types have been identified, only a small subset ( types) of these are defined as carcinogens, including some as possible or probable carcinogens [ ] . these viruses-referred to as high-risk types-infect the mucosal surfaces of the anogenital and oropharyngeal tracts and are associated with cancers at these sites (cervix, vulva, vagina, penile, anal, tonsil and base of tongue). hpv types and are the most common high-risk types, present in~ % of cervical cancers which has a nearly % association with high-risk hpv infection, and hpv is found in over % of hpv driven oropharyngeal carcinomas. the e and e early viral proteins are the main drivers of oncogenesis and all express an e protein that contains a class i pbm at the c-terminus of the protein. the conservation of this motif amongst the high-risk viruses is a strong indication that a pdz targeting function is important in their life cycle and as an oncogenic signature. intriguingly, a recent analysis of the ability of different hpv types to target pdz proteins has revealed that some of the non-cancer causing types can degrade pdz proteins (e.g., hpv can degrade magi- ), suggesting that the ability to degrade pdz proteins was acquired prior to the oncogeneic trait and that the ancestor of both high-risk and low-risk types acquired this new trait [ ] . the authors of this study propose a model in which this new trait allowed these viruses to colonize new cellular niches (e.g., the cervical transformation zone), but for viral fitness and survival adapted to this site by acquiring additional functions such as htert activation and p degradation, functions that are necessary for cell transformation [ ] . several systems have been used to demonstrate the importance of this e domain to hpv pathogenesis. life cycle studies based on human keratinocytes transfected with whole hpv genomes in which the e pbm has been deleted have shown that pbm function is critical for the vegetative phase of the life cycle, with the mutant genome unable to support viral genome amplification or expression of the viral late proteins [ ] . a role for the e pbm in viral episome maintenance was also identified in the life cycle models [ ] [ ] [ ] . the various life cycle defects correlated with a marked reduction in proliferation of the mutant genome-containing cells, indicating that the e pbm regulates proliferation of the viral genome-containing and that this is important for multiple phases of hpv dna replication [ , ] . interestingly, although e also mediates the degradation of the tumour suppressor p in infected cells, further depletion of p protein by using p -targetted sirnas, or by the introduction of a dominant negative , into cells harbouring e -pbm mutant hpv genome-containing cells, rescues maintenance of the viral genomes, suggesting that there is mutual cooperation between these two e functions in viral genome maintenance during the virus life cycle [ ] . in agreement with the life cycle studies, expression of e in mammalian keratinocytes showed that the e pbm promotes cell proliferation, but also the acquisition of phenotypes linked to invasive and metastatic growth of tumours (epithelial mesenchymal transition and anchorage independent cell growth) [ ] [ ] [ ] [ ] [ ] . in addition, the motif is necessary for the morphological transformation and induction of tumourigenesis of rodent cell lines and contributes to tumour development in a transgenic mouse model of cervical carcinogenesis [ , ] . however, the motif was not required for the immortalization of keratinocytes suggesting that the e pbm function is of more importance in the later stages of hpv driven carcinogenesis [ ] . the link between this hpv function and cell transformation is further supported by the discovery that the e oncoprotein of the rhesus papillomavirus type virus (rhpv , also known as macaca mulatta pv [mmpv ]), which drives mucosal neoplasia, including cervical cancer in its host (rhesus macaques), encodes a functional c-terminal pbm whose integrity is required for transformation of rodent cell lines [ ] . but what of the targets of this virally encoded pbm? seventeen pdz proteins that interact with the e proteins have been identified to date and in the majority of cases (but not all) binding leads to degradation of the pdz protein via the proteasome [ , ] (table ). the pdz substrates are largely involved in defining cell polarity, cell-cell attachment, and organizing cell signalling pathways associated with these functions (table ) . notably, components of the three apico-basal polarity complexes, crumbs, par and scribble are e targets, suggesting that deregulation of pathways controlled by these complexes is an important function (figure ). the scribble component dlg was the first pdz protein identified as an e binding partner and is targeted for degradation [ , ] . during epithelial differentiation, the localization of dlg changes; in the basal proliferating compartment it is cytoplasmic and nuclear but in more differentiated cells present at the cell periphery, indicating location specific functions [ , ] . it is the nuclear phospho-dlg forms that are preferentially targets for proteasomal degradation by hpv e [ , ] , suggesting that nuclear dlg may encode tumour suppressor activity [ ] . but proteasomal degradation of dlg is perhaps not the only outcome of e binding since several studies have identified functional e /dlg complexes. in hpv -positive cervical cancer cells, rhog activity is upregulated and e contributes to this upregulation by forming a complex with dlg and a rhog guanine exchange factor (sgef) [ ] . moreover, the e -dlg -sgef complex contributes to invasive phenotype of the cells, indicating that dlg acquires oncogenic functions in the presence of the viral oncoprotein [ ] , as has been noted with ad e orf (see section on adenoviruses). in hpv positive cervical cancers, the intercellular communication channels known as gap junctions are lost and e has been found to be in complex with dlg and the gap junction component connexin protein cx in cervical cancer cells, and this complex may act to inhibit normal trafficking of connexin to the cell membrane [ , ] . gap junctions and their components, particularly cx play an important role in cell invasion and the e -dlg -cx complex may contributes to metastatic properties of cervical cancer cells (figure ). therefore, in hpv infections the tumour suppressor forms of dlg that are involved in the negative regulation of cell proliferation might be the initial target of the e pbm, but during disease progression dlg , either through mislocalization and/or the stabilization of specific pools, acquires oncogenic functions mediated by interaction with e [ , ] . the other scribble component targeted by e is scrib and this interaction disturbs scrib localization at the periphery of epithelial cells and induces loss of tight junction integrity [ ] . e also targets several other pdz proteins associated with tight junctions, including magi- , mupp , par and patj (table ; figure ). recent findings have shown that introduction of a mutant form of magi- that cannot be targeted by e for degradation (as it contains a point mutation in the critical residue in pdz to which e binds) into hpv positive cervical cancer cells enhances the ability of these cells to form junctional complexes [ ] . these studies also identified roles for magi- in the negative control of cell proliferation and as a regulator of apoptosis, indicating that e may target different functional pools of the pdz protein; selective targeting of these may be relevant at different stages of the virus life cycle and/or disease progression [ ] . protection of hpv infected cells against apoptosis also involves e pdz dependent activation of the pi k-akt and nf-κb signalling pathways [ , ] ; e targets the na + /h + exchange regulatory factor (nherf ), a pdz protein known to attenuate pi k signalling through the negative regulator pten (figure ). hpv e is known to stimulate the key regulator of cellular metabolism mtorc leading to activation of cap-dependent translation, and the e pbm function has been shown to contribute to the enhancement of translation [ ] (figure ) . one of the most difficult questions to answer is which of e pdz substrate(s) in the growing list are physiologically relevant targets? in metastatic cervical cancers, the reduction in expression of the polarity proteins dlg and scrib could be e mediated via the proteasome [ , , ] . indeed, levels of both proteins are rescued upon silencing e expression in hpv-positive cervical cancer cell lines, although the most marked change was noted for magi- , with membrane bound and nuclear pools of the protein restored and re-localization of magi- to tight junctions [ ] . pard , an e substrate that is not degraded but relocalized from apical domain tight junctions to the nucleus, is restored to the junctions upon e silencing in the cancer cells [ ] . somewhat surprisingly therefore, the level of expression and localization of endogenous dlg and scrib proteins remain unaltered in normal human keratinocytes expressing e (e alone, or e and e together, or viral oncoprotein expression driven from intact viral genomes) and there are no changes in the expression or localization of these proteins when the e pbm is deleted [ , , ] . therefore, it is likely that there is differential regulation of pdz substrates by e at the different stages of hpv pathogenesis. since the activity of the e pbm is regulated by phosphorylation this may be one layer of control. the threonine or serine embedded in the e pbm is phosphorylated by protein kinases pka and akt and negatively regulates binding to pdz domains, including those of dlg and magi- [ ] . conversely, e pbm phosphorylation confers the ability to interact with a number of - - isoforms [ , ] . - - proteins have a wide range of cellular functions, regulating key proteins involved in signalling pathways associated with transcription, cell cycle control and apoptosis, although the function of - - binding to e is unclear at this stage. however, expression of a mutant form of hpv e that cannot be phosphorylated within the pbm (and therefore cannot bind - - proteins), but can bind pdz substrates enhanced the e mediated morphological transformation of human keratinocytes [ ] . when this same mutation was embedded in intact hpv genomes and transfected into primary keratinocytes, there was a marked increase in hyperproliferation of the epidermal keratinocytes [ ] . thus, modulation of the pka and/or akt signalling pathways in hpv infections is likely to impact on e function and may be a contributing factor to tumour promotion and progression [ , ] . the mechanism of hpv targeting of pdz substrates is complicated further by finding that e *, a spliced isoform of e which lacks a pbm, is able to target dlg , patj, magi- , and to a lesser extent scrib, for proteasomal degradation [ , ] (figure ) . moreover, hpv e and e cooperate to induce the degradation of nherf . hpv e promotes the accumulation of phosphorylated forms of nherf through activation of cyclin-dependent kinases and the modified forms of the pdz protein are the preferential degradation targets of the e pbm [ ] . thus, multiple cooperative mechanisms exist in hpv infections to control pdz protein expression and function. targeting the various pdz proteins is not equal amongst the different high-risk hpv types and substrate specificity involves the pbm sequence as well as sequences upstream of the pbm [ , , , , ] . for example, both hpv and hpv e proteins are able to target dlg and scrib, but exhibit different preferences in binding the pdz proteins; hpv e preferentially binds scrib over dlg and vice versa for hpv [ , ] . the pbm of these two oncoproteins only differs by a single amino acid (the last amino acid) and, notably, the specificity for dlg and scrib can be switched within the context of the whole e protein by switching the last residue [ ] . whether this has relevance to the carcinogenic strength of the virus (hpv is the most prevalent high risk type in cancers) is not clear, but it might indicate that the ability to target at least one protein of the polarity scribble complex is a key function. differential targeting of pdz proteins may also have relevance to the pathogenesis of the virus at different anatomical sites. one such target is ptpn , a non-receptor tyrosine phosphatase (hpv and hpv e pbm also target another family member ptpn [ , ] ). hpv is associated with over % of the hpv positive oropharnygeal tumours (tonsil and base of tongue). in human tonsil keratinocytes, the hpv e pbm contributes to anchorage independent cell growth and synergizes with the small gtpase ras for invasion in vivo, and these malignant hallmarks were abrogated by reintroduction of ptpn [ ] . interestingly, up to % of hpv-negative head and neck cancers contain disrupting mutation in the ptpn gene. disruption of ptpn regulation of the ras/raf/mek/erk signalling pathways linked through ephrin b receptor-mediated signalling is likely to be important in both hpv positive and negative head and neck tumours [ , ] . the list of e pbm targets is still growing with the use of various types of peptide and proteomic-based screening methods; for example the ubiquitin e ligase pdzrn was recently identified as a potential novel target of hpv e pbm in two separate studies [ , ] . the interaction has been confirmed as a degradation target of the hpv and hpv e in a pbm dependent mechanism [ ] . the e mediated loss of pdzrn was linked to activation of stat β and this pathway is known to be important in amplification of the viral genomes in the upper layers of the infected epithelia by activating the atm mediated dna damage response [ ] . thus, the e pbm targeting of pdzrn may function synergistically with e to activate stat and support viral amplification. other potential novel targets of the e pbm include α and β syntrophins, snta and sntb , whose functions include regulating the activity of the gtpase rac during tight junction assembly, and sorting nexin (snx ), important in endosomal recycling pathways [ ] , but whether these are genuine targets of e , and the function of these interactions in the hpv life cycle and in hpv driven cancers requires further investigation. intriguingly, papillomaviruses may also target pdz proteins by blocking their transcription. the e protein of hpv , a betapapillomavirus implicated in the development of squamous carcinomas of the skin repressed transcription of the pdz protein syntenin- , and hpv e was also able to repress syntenin- transcription but to a much lesser extent than the hpv protein [ ] . loss of syntenin- expression was shown to inhibit differentiation and promote proliferation of human keratinocytes; possibly by deregulation of pip mediated nuclear signalling, a signal transduction pathway linked to epithelial cell growth and differentiation [ , ] . it will be important to establish if other pdz proteins are similarly deregulated at a transcriptional level in hpv infected and transformed cells. one of the striking features of this group of viruses is the commonality in pdz targets ( figure ). many are components of the polarity complexes and the deregulation of the cellular pathways they control is involved in virus infection, transmission, replication, survival and persistence in the host, and formation of new progeny. for many of the viruses, these interactions also play a part in their oncogenic actions and indicates that the deregulation of the pdz protein controlled pathways is a finely balanced process in the virus life cycle and once this balance is perturbed this can lead, or contribute to oncogenesis. this perhaps is not so surprising when it is known that many of the components of the polarity complexes behave as tumour suppressors in different systems, although this behaviour can be context dependent. the viruses deregulate the pdz proteins in many different ways-inducing cellular mislocalization, proteasomal degradation, or by binding to the protein to compete with host cell partners, and intriguingly, some viral-pdz protein associations lead to the pdz protein acquiring oncogenic functions. interestingly, many of the virus-pdz associations discussed here affect the pi k/akt signalling pathway that is fundamental to cell growth and survival ( figure ) and is thus an obvious target for the viruses to exploit for replication. indeed, many other viruses that are not oncogenic use an array of strategies to interfere with this cell signalling pathway and perhaps some of these may be relevant to the viruses discussed in this review [ ] . the use of high-throughput proteomics and construction of large libraries to screen protein-protein interactions is leading to the identification of further pdz targets for these viruses. thus, one area to tackle is to confirm those interactions which are physiologically relevant, a problem particularly significant to the high risk hpv e proteins which already has a list of seventeen targets and is growing with addition of new potential targets from the large screening arrays; the use of hpv life cycle models will be an important asset in these investigations. it is important that we understand the intricacies of these interaction; when and where they occur during the life cycle and what interactions are involved in oncogenesis, because these interactions are attractive therapeutic targets that can be targeted using disrupting molecules [ , ] . pdz domains and their binding partners: structure, specificity, and modification pdz domains: the building blocks regulating tumorigenesis the pdz protein discs-large (dlg): the "jekyll and hyde" of the epithelial polarity proteins differential regulation of cell-cell contact, invasion and anoikis by hscrib and hdlg in keratinocytes scribble acts as an oncogene in emu-myc-driven lymphoma mislocalization of the cell polarity protein scribble promotes mammary tumorigenesis and is associated with basal breast cancer a specificity map for the pdz domain family the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis the sars coronavirus e protein interacts with pals and alters tight junction formation and epithelial morphogenesis interference with the pten-mast interaction by a viral protein leads to cellular relocalization of pten attenuation of rabies virulence: takeover by the cytoplasmic domain of its envelope protein large-scale sequence analysis of avian influenza isolates species-specific contribution of the four c-terminal amino acids of influenza a virus ns protein to virulence amelioration of influenza virus pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated ns gene the ns gene contributes to the virulence of h n avian influenza viruses the pdz-binding motif of the avian ns protein affects transmission of the influenza a(h n ) virus truncation of the ns protein converts a low pathogenic avian influenza virus into a strong interferon inducer in duck cells a new influenza virus virulence determinant: the ns protein four c-terminal residues modulate pathogenicity analysis of the pdz binding specificities of influenza a virus ns proteins the avian influenza virus ns esev pdz binding motif associates with dlg and scribble to disrupt cellular tight junctions pdlim selectively interacts with the pdz binding motif of highly pathogenic avian h n influenza a virus ns the esev pdz-binding motif of the avian influenza a virus ns protein protects infected cells from apoptosis by directly targeting scribble a review of human carcinogens-part b: biological agents gassama-diagne, a. ship regulates epithelial cell polarity through its lipid product, which binds to dlg , a pathway subverted by hepatitis c virus core protein interaction of hepatitis b virus core protein with human gipc suppression of hepatitis b viral gene expression by protein-tyrosine phosphatase ptpn oncovirus kaposi sarcoma herpesvirus (kshv) represses tumor suppressor pdlim to persistently activate nuclear factor kappab (nf-kappab) and stat transcription factors for tumorigenesis and tumor maintenance pdzd is a novel gag-interacting factor that promotes retroviral infection human discs large is a new negative regulator of human immunodeficiency virus- infectivity the pdz-adaptor protein syntenin- regulates hiv- entry exposure to hiv- directly impairs mucosal epithelial barrier integrity allowing microbial translocation human dlg protein binds to the envelope glycoproteins of human t-cell leukemia virus type and regulates envelope mediated cell-cell fusion in t lymphocytes the c-terminus of the htlv- tax oncoprotein mediates interaction with the pdz domain of cellular proteins tax oncoprotein of htlv- binds to the human homologue of drosophila discs large tumor suppressor protein, hdlg, and perturbs its function in cell growth control human t-cell leukemia virus type tax protein interacts with and mislocalizes the pdz domain protein magi- human t-cell leukemia virus type tax oncoprotein induces and interacts with a multi-pdz domain protein interactions of the pdz-protein magi- with adenovirus e -orf and high-risk papillomavirus e oncoproteins link of the unique oncogenic properties of adenovirus type e -orf to a select interaction with the candidate tumor suppressor protein zo- multi-pdz domain protein mupp is a cellular target for both adenovirus e -orf and high-risk papillomavirus type e oncoproteins viral oncoprotein-induced mislocalization of select pdz proteins disrupts tight junctions and causes polarity defects in epithelial cells oncogenic function for the dlg mammalian homolog of the drosophila discs-large tumor suppressor human papillomavirus type e oncoprotein inhibits transcription of the pdz protein syntenin- human and primate tumour viruses use pdz binding as an evolutionarily conserved mechanism of targeting cell polarity regulators the human papillomavirus (hpv) e * proteins from high-risk, mucosal hpvs can direct degradation of cellular proteins in the absence of full-length e protein patj, a tight junction-associated pdz protein, is a novel degradation target of high-risk human papillomavirus e and the alternatively spliced isoform e large-scale interaction profiling of pdz domains through proteomic peptide-phage display using human and viral phage peptidomes the human pdzome: a gateway to psd -disc large-zonula occludens (pdz)-mediated functions interpreting cancer genomes using systematic host network perturbations by tumour virus proteins hepatitis c virus infection reduces hepatocellular polarity in a vascular endothelial growth factor-dependent manner the sr-bi partner pdzk facilitates hepatitis c virus entry interaction networks of hepatitis c virus ns b: implications for antiviral therapy two pdz binding motifs within ns have roles in tick-borne encephalitis virus replication flavivirus ns associates with host-cell proteins zonula occludens- (zo- ) and regulating synaptic membrane exocytosis- (rims ) via an internal pdz binding mechanism emerging theme: cellular pdz proteins as common targets of pathogenic viruses gipc binds to the human lutropin receptor (hlhr) through an unusual pdz domain binding motif, and it regulates the sorting of the internalized human choriogonadotropin and the density of cell surface hlhr the pdz protein gipc regulates trafficking of the lpa receptor from appl signaling endosomes and attenuates the cell's response to lpa the hepatitis b virus (hbv) hbx protein activates akt to simultaneously regulate hbv replication and hepatocyte survival pdlim -mediated termination of transcription factor nf-kappab activation by intranuclear sequestration and degradation of the p subunit contribution of pdxd to stabilization of the human immunodeficiency virus type capsid histone deacetylase regulates human immunodeficiency virus type infection pdzd is a novel moesin-interacting cytoskeletal regulatory protein that suppresses infection by herpes simplex virus type efficient human immunodeficiency virus (hiv- ) infection of cells lacking pdzd tnf-alpha modulation of intestinal epithelial tight junction barrier is regulated by erk / activation of elk- opposing effects of a tyrosine-based sorting motif and a pdz-binding motif regulate human t-lymphotropic virus type envelope trafficking knockdown of synapse-associated protein dlg reduces syncytium formation induced by human t-cell leukemia virus type pdz domain-binding motif of human t-cell leukemia virus type tax oncoprotein augments the transforming activity in a rat fibroblast cell line oncogenic transformation by the tax gene of humant-cell leukemia virus type in vitro development of leukemia in mice transgenic for the tax gene of human t-cell leukemia virus type pdz binding motif of htlv- tax promotes virus-mediated t-cell proliferation in vitro and persistence in vivo scaffold protein dlgh coordinates alternative p kinase activation, directing t cell receptor signals toward nfat but not nf-kappab transcription factors the pleiotropic protein kinase ck phosphorylates htlv- tax protein in vitro, targeting its pdz-binding motif (dlg ) complexes in lymphocyte activation a network of pdz-containing proteins regulates t cell polarity and morphology during migration and immunological synapse formation the pdz domain-binding motif of the human t cell leukemia virus type tax protein induces mislocalization of the tumor suppressor hscrib in t cells binding of human virus oncoproteins to hdlg/sap , a mammalian homolog of the drosophila discs large tumor suppressor protein dlgh is a negative regulator of t-lymphocyte proliferation akt pathway activation by human t-cell leukemia virus type tax oncoprotein the pdz domain binding motif (pbm) of human t-cell leukemia virus type tax can be substituted by heterologous pbms from viral oncoproteins during t-cell transformation cdk phosphorylation of the discs large tumour suppressor controls its localisation and stability inactivation of tumor suppressor dlg augments transformation of a t-cell line induced by humant-cell leukemia virus type tax protein scaffold protein disc large homolog is required for t-cell receptor-induced activation of regulatory t-cell function scribble-mediated membrane targeting of phlpp is required for its negative regulation of akt threonine phosphorylation of the mmac /pten pdz binding domain both inhibits and stimulates pdz binding a functional network of the tumor suppressors apc, hdlg, and pten, that relies on recognition of specific pdz-domains binding of htlv- tax oncoprotein to the precursor of interleukin- , a t cell pdz domain-containing protein human adenovirus type -induced rat mammary tumors requirement for the adenovirus type e region in production of mammary tumors selective pdz protein-dependent stimulation of phosphatidylinositol -kinase by the adenovirus e -orf oncoprotein a new crucial protein interaction element that targets the adenovirus e -orf oncoprotein to membrane vesicles the human adenovirus e -orf protein subverts discs large to mediate membrane recruitment and dysregulation of phosphatidylinositol -kinase adenovirus e -orf dysregulates epidermal growth factor and insulin/insulin-like growth factor receptors to mediate constitutive myc expression hijacking dlg for oncogenic phosphatidylinositol -kinase activation in human epithelial cells is a conserved mechanism of human adenovirus e -orf proteins degradation of human pdz-proteins by human alphapapillomaviruses represents an evolutionary adaptation to a novel cellular niche the role of protein kinase a regulation of the e pdz-binding domain during the differentiation-dependent life cycle of human papillomavirus type role of the pdz domain-binding motif of the oncoprotein e in the pathogenesis of human papillomavirus type stabilization of hpv e protein by pdz proteins, and potential implications for genome maintenance papillomavirus e pdz interactions can be replaced by repression of p to promote episomal human papillomavirus genome maintenance requirement of pdz-containing proteins for cell cycle regulation and differentiation in the mouse lens epithelium the pdz ligand domain of the human papillomavirus type e protein is required for e 's induction of epithelial hyperplasia in vivo deletion of the pdz motif of hpv e preventing immortalization and anchorage-independent growth in human tonsil epithelial cells activity of the human papillomavirus e pdz-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes degradation of tyrosine phosphatase ptpn (ptph ) by association with oncogenic human papillomavirus e proteins binding of high-risk human papillomavirus e oncoproteins to the human homologue of the drosophila discs large tumor suppressor protein the human papillomavirus e oncogene dysregulates the cell cycle and contributes to cervical carcinogenesis through two independent activities binding of pdz proteins to hpv e proteins does neither correlate with epidemiological risk classification nor with the immortalization of foreskin keratinocytes hpv e degradation of p and pdz containing substrates in an e ap null background the role of the ubiquitin ligase e -ap in human papillomavirus e -mediated degradation of pdz domain-containing proteins oncogenic human papillomavirus e proteins target the discs large tumour suppressor for proteasome-mediated degradation the invasive capacity of hpv transformed cells requires the hdlg-dependent enhancement of sgef/rhog activity hpv e controls the gap junction protein cx in cervical tumour cells human scribble (vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus e proteins and the e ap ubiquitin-protein ligase oncogenic human papillomavirus e proteins target the magi- and magi- proteins for degradation the pdz protein tip- is a gain of function target of the hpv e oncoprotein human papillomavirus type e protein binds the cellular pdz protein tip- /gipc, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome e ap-dependent degradation of dlg /psd by high-risk human papillomavirus type e protein protein tyrosine phosphatase h is a target of the e oncoprotein of high-risk genital human papillomaviruses human papillomavirus type e protein interacts with cystic fibrosis transmembrane regulator-associated ligand and promotes e -associated protein-mediated ubiquitination and proteasomal degradation the pdz binding motif of human papillomavirus type e induces ptpn loss, which allows anchorage-independent growth and synergizes with ras for invasive growth e and e from human papillomavirus type cooperate to target the pdz protein na/h exchange regulatory factor human papillomavirus (hpv)- e oncoprotein interferes with the epithelial cell polarity par protein pdzrn /lnx is a novel target of human papillomavirus type (hpv- ) and hpv- e changes in localization of human discs large (hdlg) during keratinocyte differentiation are [corrected] associated with expression of alternatively spliced hdlg variants hpv e specifically targets different cellular pools of its pdz domain-containing tumour suppressor substrates for proteasome-mediated degradation the high-risk hpv e oncoprotein preferentially targets phosphorylated nuclear forms of hdlg a functional interaction between the maguk protein hdlg and the gap junction protein connexin in cervical tumour cells changes in expression of the human homologue of the drosophila discs large tumour suppressor protein in high-grade premalignant cervical neoplasias restoration of magi- expression in human papillomavirus-positive tumor cells induces cell growth arrest and apoptosis human papillomavirus type e activates nf-kappab, induces ciap- expression, and protects against apoptosis in a pdz binding motif-dependent manner activation of cap-dependent translation by mucosal human papillomavirus e proteins is dependent on the integrity of the lxxll binding motif differential expression of the human homologue of drosophila discs large oncosuppressor in histologic samples from human papillomavirus-associated lesions as a marker for progression to malignancy analysis of the expression and localisation of a lap protein, human scribble, in the normal and neoplastic epithelium of uterine cervix a systematic analysis of human papillomavirus (hpv) e pdz substrates identifies magi- as a major target of hpv type (hpv- ) and hpv- whose loss accompanies disruption of tight junctions roles of the pdz domain-binding motif of the human papillomavirus type e on the immortalization and differentiation of primary human foreskin keratinocytes two distinct activities contribute to human papillomavirus e 's oncogenic potential differential regulation of human papillomavirus e by protein kinase a: conditional degradation of human discs large protein by oncogenic e cancer-causing human papillomavirus e proteins display major differences in the phospho-regulation of their pdz interactions high-risk human papillomavirus e oncoproteins interact with - - zeta in a pdz binding motif-dependent manner the hscrib/dlg apico-basal control complex is differentially targeted by hpv- and hpv- e proteins impaired ptpn phosphatase activity in spontaneous or hpv-induced squamous cell carcinomas potentiates oncogene signaling through the map kinase pathway the jak-stat transcriptional regulator, stat- , activates the atm dna damage pathway to induce hpv genome amplification upon epithelial differentiation pdz domain-phosphoinositide interactions in cell-signaling make yourself at home: viral hijacking of the pi k/akt signaling pathway targeting the two oncogenic functional sites of the hpv e oncoprotein with a high-affinity bivalent ligand an rna aptamer targets the pdz-binding motif of the hpv e oncoprotein acknowledgments: claire d. james was supported by the medical research council's doctoral training partnership awarded to the college of medical and dentistry (university of birmingham). we are grateful to all members of the roberts laboratory, past and present, and the funding agencies (wellcome trust and cancer research uk). we apologize to those authors we were unable to cite due to space constraints.author contributions: claire d. james and sally roberts wrote the manuscript. the authors declare no conflicts of interest. key: cord- -b fp d authors: segura, mariela; aragon, virginia; brockmeier, susan l.; gebhart, connie; de greeff, astrid; kerdsin, anusak; o’dea, mark a; okura, masatoshi; saléry, mariette; schultsz, constance; valentin-weigand, peter; weinert, lucy a.; wells, jerry m.; gottschalk, marcelo title: update on streptococcus suis research and prevention in the era of antimicrobial restriction: th international workshop on s. suis † date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: b fp d streptococcus suis is a swine pathogen and a zoonotic agent afflicting people in close contact with infected pigs or pork meat. sporadic cases of human infections have been reported worldwide. in addition, s. suis outbreaks emerged in asia, making this bacterium a primary health concern in this part of the globe. in pigs, s. suis disease results in decreased performance and increased mortality, which have a significant economic impact on swine production worldwide. facing the new regulations in preventive use of antimicrobials in livestock and lack of effective vaccines, control of s. suis infections is worrisome. increasing and sharing of knowledge on this pathogen is of utmost importance. as such, the pathogenesis and epidemiology of the infection, antimicrobial resistance, progress on diagnosis, prevention, and control were among the topics discussed during the th international workshop on streptococcus suis (held in montreal, canada, june ). this review gathers together recent findings on this important pathogen from lectures performed by lead researchers from several countries including australia, canada, france, germany, japan, spain, thailand, the netherlands, uk, and usa. finally, policies and recommendations for the manufacture, quality control, and use of inactivated autogenous vaccines are addressed to advance this important field in veterinary medicine. streptococcus suis is considered one of the most important bacterial swine pathogens leading to important economic losses to the porcine industry worldwide. s. suis has been reported globally in both traditional and intensive swine operations [ ] . control is based on an alarming overuse of antimicrobials, leading to a dramatic increase of the risk related to antimicrobial resistance. it is also an agent of disease in humans and considered in most oecd (organisation for economic co-operation and development) countries as an occupational disease affecting mostly swine industry workers. in asia, this pathogen affects the general population and represents a significant public health concern [ ] . after a deadly chinese human outbreak, research teams worldwide turned their attention to s. suis with an explosion of published articles ( figure ). streptococcus suis is considered one of the most important bacterial swine pathogens leading to important economic losses to the porcine industry worldwide. s. suis has been reported globally in both traditional and intensive swine operations [ ] . control is based on an alarming overuse of antimicrobials, leading to a dramatic increase of the risk related to antimicrobial resistance. it is also an agent of disease in humans and considered in most oecd (organisation for economic cooperation and development) countries as an occupational disease affecting mostly swine industry workers. in asia, this pathogen affects the general population and represents a significant public health concern [ ] . after a deadly chinese human outbreak, research teams worldwide turned their attention to s. suis with an explosion of published articles ( figure ). in , the st international workshop on s. suis was organized with the aim to increase international collaborations. since then, the upsurge of studies certainly contributed to our understanding of bacterial-host interactions. however, the use of different research models resulted in misconceptions and complicated diagnostics and vaccine development [ , ] . eight years after the st workshop and after two other workshops ( and ), the th international workshop on s. suis was organized to strengthen scientific knowledge and provide, through international in , the st international workshop on s. suis was organized with the aim to increase international collaborations. since then, the upsurge of studies certainly contributed to our understanding of bacterial-host interactions. however, the use of different research models resulted in misconceptions and complicated diagnostics and vaccine development [ , ] . eight years after the st workshop and after two other workshops ( and ), the th international workshop on s. suis was organized to strengthen scientific knowledge and provide, through international cooperation, relevant scientific information, and advice that will have a direct influence on the decisions made by the swine industry. cooperation, relevant scientific information, and advice that will have a direct influence on the decisions made by the swine industry. the diagnosis and epidemiology of the infection in humans and pigs; different aspects of the pathogenesis of the disease; antimicrobial resistance, prevention and control; and finally autogenous vaccine policy were addressed during the meeting and are further discussed below. s. suis is an encapsulated pathogen, and the capsular polysaccharide (cps) antigen is the basis of s. suis classification into serotypes [ ] . originally, serotypes were identified. however, phylogenetic and/or sequence analyses showed that the reference strains of serotypes , , , , , and should be taxonomically removed from the s. suis species. serotypes and were reclassified as streptococcus orisratti. serotypes , , and were proposed as streptococcus parasuis, while serotype was classified as streptococcus ruminantium [ ] [ ] [ ] . nevertheless, more extensive studies using a higher number of s. suis and s. suis-like strains will provide additional insights into the classification of these strains and make the species boundaries clearer [ ] . in fact, all serotypes of s. suis (or s. suis-like strains) (with the exception of serotype ) are isolated from diseased pigs [ ] , and from the clinical point of view, many diagnostic laboratories still identify the serotypes. the tools for molecular epidemiology of s. suis have been recently reviewed in reference [ ] . the worldwide distribution of major s. suis serotypes involved in swine clinical cases is schematically represented in figure . among those reported, serotype is considered the most common cause of infections in piglets worldwide and a major zoonotic agent [ ] . nevertheless, other serotypes are increasing in importance in different countries, as is the case of serotype , particularly in some countries of western europe. by means of novel animal models and diagnostic tools, de greeff et al. (appendix a) epidemiologically determined the population genetics of s. suis serotype in the netherlands [ ] . obtained data using comparative genome hybridization and whole genome sequencing suggest that clinical serotype swine isolates are genetically very similar whereas serotype isolates carried by healthy pigs are more heterogeneous. a few carriage isolates clustered together with clinical isolates; these carriage isolates probably reflect clinical isolates that are not causing any clinical outbreaks on the farms but do have virulent potential. by infecting caesarean derived colostrum deprived (cdcd) piglets intravenously with a high dose of bacteria, it was shown that, within s. suis serotype , the virulence of clinical and tonsillar carriage isolates differs significantly (appendix a). interestingly, a recent study indicated that s. suis isolates associated with disease in pigs comprise predominantly of serotypes , , and / (appendix b), which is consistent with reports from other pig producing countries [ ] . in figure , it can also be appreciated that the epidemiological situation in north america is different from other countries. in this part of the globe, multiple serotypes are found in swine clinical cases [ ] . indeed, recent works confirmed that a variety of s. suis serotypes can be found in commercial swine production systems in usa and canada, with serotypes / , , , , , and commonly isolated from systemic infection sites, the frequencies depending on the study [ , ] . besides this classification based on cps antigen/gene locus, s. suis is genetically differentiated into sequence types (st) by multilocus sequence typing (mlst) [ ] . the distribution of most important sts within serotype is represented in figure . pathogens , , x for peer review of in figure , it can also be appreciated that the epidemiological situation in north america is different from other countries. in this part of the globe, multiple serotypes are found in swine clinical cases [ ] . indeed, recent works confirmed that a variety of s. suis serotypes can be found in commercial swine production systems in usa and canada, with serotypes / , , , , , and commonly isolated from systemic infection sites, the frequencies depending on the study [ , ] . besides this classification based on cps antigen/gene locus, s. suis is genetically differentiated into sequence types (st) by multilocus sequence typing (mlst) [ ] . the distribution of most important sts within serotype is represented in figure . . most important sequence types (sts) of streptococcus suis serotype as determined by multilocus sequence typing (mlst): st serotype strains are mostly associated with disease in both pigs (where data are available) and humans in europe, asia, africa, and south america. the situation is different in north america, where fewer clinical st cases of infection in pigs and only one human st case has been described. st , a single locus variant of st , is endemic to mainland china. interestingly, japan and thailand are the only countries reporting st human cases [ ] . "?" means no data available from swine clinical cases. it is important to note that epidemiological data is missing for several countries; for example, information relating to s. suis in the australian pig herd is limited [ , ] . to fill this gap, o'dea et al. (appendix b) analysed and characterised a significant number of s. suis isolates from diseased pigs obtained across multiple production sites over a seven year period [ ] . the most prominent mlsts were, in decreasing order, st , st , st , st , and st . analysis of serotype and mlst combinations showed a high proportion of isolates as serotype st , serotype st , and serotype st . a deeper phylogenetic comparison of the s. suis isolates from australian pigs against a global collection of s. suis strains showed that australian clones of s. suis associated with clinical disease in pigs have maintained a stable core genome, mirroring the international seed-stock from which they were derived (mainly from the uk and north america). while australian serotype st isolates could be distinguished from those of usa and canada, only small single nucleotide polymorphism . most important sequence types (sts) of streptococcus suis serotype as determined by multilocus sequence typing (mlst): st serotype strains are mostly associated with disease in both pigs (where data are available) and humans in europe, asia, africa, and south america. the situation is different in north america, where fewer clinical st cases of infection in pigs and only one human st case has been described. st , a single locus variant of st , is endemic to mainland china. interestingly, japan and thailand are the only countries reporting st human cases [ ] . "?" means no data available from swine clinical cases. it is important to note that epidemiological data is missing for several countries; for example, information relating to s. suis in the australian pig herd is limited [ , ] . to fill this gap, o'dea et al. (appendix b) analysed and characterised a significant number of s. suis isolates from diseased pigs obtained across multiple production sites over a seven year period [ ] . the most prominent mlsts were, in decreasing order, st , st , st , st , and st . analysis of serotype and mlst combinations showed a high proportion of isolates as serotype st , serotype st , and serotype st . a deeper phylogenetic comparison of the s. suis isolates from australian pigs against a global collection of s. suis strains showed that australian clones of s. suis associated with clinical disease in pigs have maintained a stable core genome, mirroring the international seed-stock from which they were derived (mainly from the uk and north america). while australian serotype st isolates could be distinguished from those of usa and canada, only small single nucleotide polymorphism (snp) differences are notable across the core genome. despite the limited number, the characterisation of serotype / st clones is significant, a finding also recently observed in north america. indeed, gebhart et al. (appendix c) characterised the diversity of a contemporary collection of s. suis isolates across north america (mainly in usa) by serotyping and mlst to address the limited information on current s. suis strains circulating within this country [ ] . the predominant st was st , followed by st , st , and st among multiple sts identified, illustrating the high diversity among s. suis isolates. importantly, this study revealed the predominance of serotype / st from clinically affected pigs in usa. in addition, the study also reported st strains, containing the three classical virulence-associated genes (epf, sly, and mrp), which are considered highly virulent and potentially zoonotic, an unexpected finding based on previous epidemiological data [ ] . epidemiological surveillance of these strains in north america is recommended [ ] . s. suis is a zoonotic disease of increasing awareness. sporadic cases of human infection have been described worldwide since ; however, multiple outbreaks in some asian countries highlight the public health importance of this infection [ , [ ] [ ] [ ] (appendix d). small skin wounds are the main route of entry in humans in western countries, although in some cases no wound is evidenced [ ] . in some asian countries, such as vietnam and thailand, human s. suis is considered among the most frequent causes of bacterial meningitis in adults [ , ] . cultural differences between asian and western countries likely impact the epidemiology of s. suis including lifestyle; common use of backyard production systems; close contact of humans with pigs; and, in countries such as vietnam, laos and thailand, the common practice of consuming raw pork products [ ] [ ] [ ] . it has been clearly demonstrated that the oral route of infection is the principal cause of human infection in these countries [ , ] . as aforementioned, serotype is the main zoonotic serotype; however, serotype has also been isolated from humans many times in thailand and uk and uncommonly in france, australia, and canada [ , ] . after serotype , serotype is the third serotype most commonly found from humans [ ] . sporadic cases due to other serotypes have also been reported [ ] ; among them, serotype , one of the most important serotype recovered from diseased pigs in europe, has been recently shown to have zoonotic potential [ ] . within serotype , sts involved in human cases are represented in figure and parallel those found in swine clinical cases, highlighting their epidemiological link. kerdsin et al. (appendix d) have been extensively studying the epidemiological situation in thailand [ , ] . to date, four outbreaks of s. suis infections in humans have been recorded in that country, with case fatality rates varying from . % to . %. the food safety campaign implementation in phayao province during - showed a marked decrease of the disease incidence proportion and stresses the need for health policies to reduce the burden of this infection [ ] . microbiological characterisation of s. suis in thailand showed that serotype is the main serotype for human infections, followed by serotypes , , , , , and . of note, st and st (serotype ) are predominant in thai human infections. interestingly, the latter is almost exclusively found in that country. on the other hand, st is the main st among serotype isolates from human cases. kerdsin et al. also revealed that thai serotype isolates are of st (appendix d) [ ] , also commonly affecting pigs in some european countries [ , ] . schultsz et al. (appendix e) have been studying the emergence of zoonotic s. suis infections in the netherlands. zoonotic s. suis serotype strains of st , isolated from human patients, were shown to be highly genetically related to serotype strains of st [ ] . it has been shown that the cps is a major virulence factor (see section ) and that the cps gene locus can be exchanged between s. suis of different serotypes. interestingly, a study from the netherlands [ ] showed that such cps switch may lead to an increase in zoonotic potential. in addition to cps locus acquisition, loss or acquisition of other genes or loci may contribute to changes in zoonotic potential. for example, the s. suis st strains (typically found in the netherlands) acquired not only the serotype cps locus but also a type restriction-modification (r-m) system. schultsz ability to adapt to specific niches through the acquisition of a r-m system that can regulate expression of cps and/or other (surface exposed) molecules. besides serotype st strains, which present high zoonotic potential worldwide, serotype st is only endemic to china, and it was responsible for human outbreaks in and in this country [ ] . in a recent study addressing the evolution of st strains in china, it was shown that, of sporadic st s. suis strains, which mostly caused sepsis, serotype was the most frequent, followed by serotype . compared to the genome of the epidemic strain (serotype , st ), the major differences in the genomes of sporadic st strains were the absence of the kb pathogenicity island specific to the epidemic strain and insertion of mobile elements that play a significant role in the horizontal transfer of antimicrobial resistance genes [ ] . this is the first study addressing the evolution of the st strains and reporting serotype st isolates, highlighting the need to increase the surveillance of this human life-threatening lineage of s. suis. in addition to the "classical" described serotypes, a novel variant (serotype chz) and strains carrying novel capsular polysaccharide loci (ncl - ) have been identified recently [ ] [ ] [ ] [ ] [ ] . these findings expand the views of the genetic diversity of s. suis cps loci. novel cps loci are continually being found, and their discovery should eventually reduce the number of untypeable strains recovered from diseased animals. nevertheless, their virulence potential and the role of these ncls in the pathogenesis of the disease remain to be evaluated. in pigs, s. suis usually colonises the upper respiratory tract, in particular the pharyngeal and palatine tonsils, but alimentary and genital tracts can also be colonisation sites. in fact, piglets are first colonised by s. suis from birth as soon as they pass through the birth canal, since s. suis is found in the sow vagina. in humans, s. suis nasopharyngeal colonisation has been reported in people working in close contact with infected animals, such as butchers and abattoir workers [ , , ] . the bacteria may also colonise the gastrointestinal tract in people consuming fresh/raw contaminated pork meat [ ] . consequently, the mucosal barrier is the first line of defence of the host against this pathogen. in swine, colonisation of the upper respiratory tract by s. suis may lead to an asymptomatic carriage but is also considered the first step for the development of an invasive disease, particularly in the context of coinfections with porcine respiratory viruses or polymicrobial infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . indeed, s. suis belongs to the "porcine respiratory disease complex", a term used to describe a common multifactorial respiratory disease in swine that occurs as a result of polymicrobial infections, environmental stressors, and host factors such age and immunological status. brockmeier et al. (appendix f) have been analysing the complexity of these polymicrobial infections, which can be the result of multiple viruses, bacteria, or a combination of viruses and bacteria infecting the pig at the same time or in close succession. for example, the majority of cases where porcine reproductive and respiratory syndrome virus (prrsv) or swine influenza virus (swiv) was determined to be the primary aetiology, also presented with secondary bacterial pneumonia caused by s. suis, among other bacterial species. experimental data suggest that coinfections with viruses, such as prrsv, swiv, porcine circovirus, and porcine respiratory coronavirus (prcv), generally result in a more severe clinical disease outcome. there are several mechanisms that might contribute to increased susceptibility to secondary infection and the enhanced disease that often occurs with coinfections, including disruption of the epithelial barrier or alteration of the innate or adaptive immune responses by the primary pathogen, direct interactions between or among the pathogens such as the formation of multispecies biofilms, disturbances to the ecological niche such as the upper respiratory microbiota, or enhanced transmission. brockmeier et al. (appendix f) and others [ , [ ] [ ] [ ] have shown that coinfection with prrsv, swiv or prcv and bacteria such as bordetella bronchiseptica, s. suis or glaesserella (haemophilus) parasuis results in a greater incidence of disease, greater percentage of lungs affected, more severe lesions, and slower resolution of such lesions than occurs with infection with a single pathogen alone and that this is often correlated with an amplified local pro-inflammatory cytokine response. in the absence of appropriate models of polymicrobial infections typically seen in swine production facilities, mechanistic understanding of the underlying complex molecular and cellular interactions remains limited. the transition between s. suis colonisation of the mucosal barriers and systemic infection is only partially understood. in the studies performed by wells et al. [ ] , a hypothetical model to explain systemic infection of piglets with s. suis based on the identification of s. suis in the tonsillar lymphoid tissue and presence of large numbers of cd + macrophages was proposed. in this model, cd + macrophages might act as reservoirs for replication of virulent strains of s. suis, whereby environmental and physical stressors that reduce competition from the tonsil microbiota lead to increased s. suis in the tonsil and escape into the bloodstream through the many small blood vessels permeating the lymphoid tissue. such a model would also be compatible with the finding that serotype strains interacting with the cd siglec receptor through their sialylated cps are more commonly associated with invasive disease. wells et al. also proposed that subversion of the innate functions of cd + macrophages by coinfecting viruses such as prrsv or swiv may also contribute to the survival of s. suis and spread to the bloodstream. in this context, the potential mechanisms used by s. suis serotypes without sialic acid in their cpss are largely unknown. it is also possible that survival and or replication capacity of s. suis in this particular subset of macrophages is a characteristic associated with disease-causing strains. in this regard, research performed by valentin-weigand et al. (appendix g) has significantly contributed to our understanding of viral-bacterial interactions during airway coinfections. one important suggested virulence factor is the s. suis cholesterol-dependent cytotoxin, named suilysin, produced by many virulent strains. this toxin has multiple actions towards a variety of cells (reviewed in reference [ ] ), including attachment of s. suis to the surface of the respiratory epithelium and/or direct lysis of epithelial cells [ , ] , probably facilitating bacterial breach of this mucosal barrier. however, suilysin-negative clinical isolates are also found frequently in swine populations worldwide and especially in north america [ , ] . valentin-weigand et al. found that, during secondary bacterial infection, suilysin contributes to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas cytotoxic effects induced by swiv became prominent at later stages of infection. besides suilysin, it has been shown that a prior infection by swiv enhances the adherence to and colonisation of porcine airway epithelial cells by s. suis in a sialic-acid dependent manner and facilitates invasion in a suilysin-independent fashion [ ] [ ] [ ] . these findings might explain why suilysin-negative strains can also cause clinical infections (appendix g). as aforementioned, the potential mechanisms used by s. suis serotypes without sialic acid in their cpss remain to be discovered. in humans, the intestinal route of infection seems to be an important port of entry after consumption of fresh/raw contaminated pork meat in some asian countries. in pigs, this route of infection has also been investigated by direct inoculation in the intestine or oral capsule-mediated delivery of virulent strains of s. suis into the small intestine of piglets [ , ] . in view of the inoculum size, stress-inducing conditions, and the small proportion of challenged animals which developed invasive disease in those studies, translocation from the intestine appears to be a possible but not a very efficient route of infection. the conditions leading to sufficient passage of s. suis through the stomach are still unclear and might differ in neonatal, suckling, or weaning periods. more studies are required on oro-gastrointestinal s. suis infections in piglets to confirm the relevance of intestinal colonisation vs. infection (translocation followed by systemic dissemination) in swine clinical cases [ ] . to enlighten this controversial aspect of s. suis disease pathogenesis, de greeff et al. (appendix a) studied the putative role of intestinal colonisation for serotype and showed that, during a s. suis serotype outbreak, tonsils of piglets were colonised with different serotypes. the highest percentage of colonisation was found for serotype , followed by serotypes and (appendix a, table a ). similarly, serotype was the major serotype colonising the intestine (appendix a, table a ). the data provided by this study suggest that serotype is a better coloniser of the porcine intestine than the other serotypes. interestingly, previous in vitro studies have shown that serotype isolates can adhere to porcine intestinal cells more efficiently than the other serotypes [ ] . similarly, serotype strains were shown to adhere better to a human intestinal epithelial cell line than serotype strains, suggesting a relationship with the high zoonotic potential of the former serotype (appendix e). in this in vitro model, the cps was shown to interfere with these first steps of s. suis colonisation and invasion, as reported in other in vitro studies with epithelial cells [ , ] . recent developments in stem cell research allowed intestinal organoids to be used as infection models for s. suis. organoids contain most cell types present in the proximal and distal intestinal mucosa and can be dissociated and grown as monolayers on semipermeable membranes [ , ] . a zoonotic s. suis serotype strain was shown to translocate in this model, which makes it a promising in vitro model for future study of intestinal infection with s. suis (appendix e). the lack of a well-standardized in vivo mucosal infection model slowed down research progress in this area. to develop such a model, de greeff et al. (appendix a) applied several mucosal infection routes to piglets from different backgrounds. it was shown that intranasal inoculation in combination with oral inoculation result in a reproducible colonisation of the tonsils and the intestine of piglets; however, this mild mucosal colonisation model failed to replicate clinical disease. different stressors facilitate and increase colonisation by s. suis but are not strict requirements for colonisation. a harsher mucosal challenge can be achieved by combining oral inoculation with intratracheal inoculation. in this case, the level of intestinal colonisation is lower, but more clinical symptoms like lameness, central nervous signs, or septicaemia were induced in the piglets (appendix a). further optimization of the oro-gastrointestinal infection model will definitively impact research aimed to dissect how s. suis breaches the mucosal barriers. encapsulated extracellular s. suis is a highly invasive pathogen. after penetration of host mucosal barriers, it can reach and survive in the blood and finally invades multiple organs including spleen, liver, kidney, lung, and heart. in addition, s. suis is able to cross the brain microvascular endothelial cells (bmecs) and/or the epithelial cells of the choroid plexus at the blood-brain barrier (bbb) and/or the blood-cerebrospinal fluid barrier to gain access to central nervous system (cns) [ , , ] . septicaemia, meningitis, endocarditis, pneumonia, and arthritis are the most common forms of s. suis invasive disease. the cps of s. suis is without doubt the major virulence factor allowing this bacterium evasion of immune-clearance mechanisms [ , ] . however, almost all of the studies on the role of cps used serotype strains, and little is known on the role of cpss of other serotypes as a virulence factor. therefore, it is unknown whether differences in serotype themselves (i.e., differences in cps structure/composition) directly affect s. suis virulence. to answer this question, for the first time, okura et al. (appendix h) experimentally generated serotype switched mutants. six serotype switched mutants were generated using the serotype reference strain p / (p / cps to , p / cps to , p / cps to , p / cps to , p / cps to , and p / cps to ) by exchanging the cps synthesis gene cluster for those of serotypes , , , , and , respectively. their virulence was compared in mice and pigs. only a serotype switch to cps type or to type showed a marked and consistent impact of bacterial virulence traits. the cps conferred to s. suis a hyper-virulent phenotype, whereas the cps conferred to s. suis a non-virulent character. serotype switch from cps to cps or to cps had restricted impact, and serotype switch from cps to cps or to cps had no significant effect on s. suis virulence (appendix h). taken together, these findings suggest that serotype switching can differentially modulate s. suis virulence depending on the cps expressed and demonstrate its importance on s. suis pathogenesis and clinical disease. based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, ferrando et al. [ ] proposed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs, and blood. in the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. several virulence factors involved in adherence to host cells, degradation of connective tissue (spreading factors), and avoidance of phagocytic killing, including suilysin, are upregulated when glucose is diminished. as discussed above, suilysin may pathogens , , of facilitate dispersion of bacteria in mucosal tissues due to loss of barrier integrity. once s. suis reaches the bloodstream, metabolism is adapted for optimal growth on glucose and the expression of virulence factors is reduced. in infected organs, glucose levels are lower than in the blood and are further reduced by inflammation and utilization by s. suis, leading to upregulation of suilysin and other virulence factors. these studies have important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition. to further understand the genetic basis of disease in s. suis, weinert et al. [ ] studied the genomic signatures of human and pig clinical isolates from the united kingdom and vietnam. isolates associated with disease were shown to contain substantially fewer genes than nonclinical isolates but are more likely to encode virulence factors. human disease isolates are limited to a single-virulent population, originating in the s, when pig production was intensified, but no reliable genomic differences between pig and human isolates were observed, suggesting lack of consistent genomic adaptation of s. suis to the human population. the authors also reported little geographical clustering of different s. suis subpopulations and high rates of recombination in s. suis worldwide, implying that an increase in virulence anywhere in the world could have a global impact over a short timescale [ ] . s. suis infections are one of the main causes of antimicrobial usage in piglets. indeed, the incidence of the disease may be as high as %, although it is usually kept lower than % in the field due to the extensive and routine prophylactic and/or metaphylactic use of antimicrobials. data from antimicrobial resistance of s. suis worldwide are alarming, and restriction of antimicrobials as a preventive measure must be a primary concern [ ] . in addition, the most effective drugs against s. suis are those in categories and (critically or highly important). the industry is trying to reduce the use of these drugs given their importance in human medicine. indeed, s. suis is considered a niche for antimicrobial resistance and represents a high risk of transmission of resistance to other pathogens [ ] . surprisingly, despite the worldwide use of beta-lactams in pigs for over years, the majority of clinical s. suis remains sensitive to these antibiotics. however, beta-lactam resistant strains do exist and are primarily found in commensal sites (appendix i, figure a ). o'dea et al. (appendix b) reported clinical resistance to penicillin g, albeit at a relatively low level in . % of isolates. in addition, they reported similar levels of antimicrobial resistance in australian strains compared to those overseas with regards to tetracycline ( . %), erythromycin ( . %), and trimethoprim/sulfamethoxazole ( . %). resistance to florfenicol was . %, while all isolates were clinically susceptible to enrofloxacin, likely due to this being banned from use in food producing animals in australia. therefore, this is an aspect of s. suis in australia that must be carefully monitored from both an animal and public health point of view. in a recent work, libante et al. [ ] made a comprehensive in silico search and analysis of integrative conjugative elements (ices) and integrative and mobilizable elements (imes) and extensive identification of antimicrobial resistance genes present in s. suis draft genomes. almost antimicrobial resistance genes were detected in the genomes analysed. a huge amount of ices, imes, and derived elements were detected at various chromosomal sites. high diversity was observed in recombination but also in conjugation/mobilization modules. besides ices, imes appear to be major vehicles of antimicrobial resistance genes. the authors concluded that further studies are needed to evaluate such gene fluxes inside and between ecosystems and the contribution of ices and imes in these gene transfers keeping in mind a one health global perspective. as s. suis is a very early coloniser of piglets, it cannot be eliminated by early weaning [ ] . to reduce antimicrobial use, s. suis disease prevention should concentrate on management of predisposing factors and, mainly, vaccination. despite intensive research leading to different vaccine-candidate antigens [ ] , no universally efficacious s. suis vaccine has been commercialised so far. the dream of having a universal cross-protective vaccine is highly challenging due to the high genomic diversity of s. suis. as discussed in the work performed by weinert et al. (appendix i), considerable genetic diversity of s. suis exists. nonetheless, they have shown many trends in genetic and phenotypic differences between strains isolated from the upper respiratory tract of pigs without s. suis clinical signs (referred as "nonclinical") and strains isolated from the lungs or systemic sites of pigs with s. suis clinical signs (referred as "clinical") (appendix i, figure a ). overall, the genetic diversity of clinical s. suis is less than that of nonclinical strains, implying that a design of a universal vaccine to control s. suis clinical infection might be possible. over subunit vaccine candidates have been reported [ , ] and new ones are continuously being characterised, yet homologous protection is still controversial and cross-protection (either against other serotypes or at least using heterologous strains) was evaluated in few of these studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] . lack of well-standardized animal models for immunization and challenge, especially for serotypes other than , is one of the reasons of limited progress towards a subunit vaccine, which also results in contradictory results for a same vaccine candidate [ ] . amongst other confounding factors are the use of mouse models (for pre-screening) without confirmation in swine trials, number of vaccine doses, and different adjuvants (which are not always compatible with a future use in swine medicine). more research, with better standardized protocols, would certainly advance subunit vaccine development. one common vaccine target is, without doubt, the cps because the majority of clinical strains have a capsule (appendix i, figure a ). nevertheless, the vaccine-induced protection will be serotype-restricted. multiple different polysaccharide epitopes should be selected in a vaccine to target different serotypes, and vaccination-driven strain replacement of the population might be expected. finally, in addition to their diversity and as aforementioned, cps loci are often recombination "hot-spots" and this gives rise to cps "switching" between strains. highly conserved proteins might help overcome these limitations and fight strain replacement/evolution. as s. suis is part of the commensal flora, any preventive/control strategy should eliminate clinical isolates without altering the precious balance of the pig mucosal microbiota. albeit possible disease-protective limitations, a systemic vaccination approach is to be prioritized in this context (appendix i). due to the young age of the piglets affected and the lack of an effective commercial vaccine (as discussed above), many farms use metaphylactic perinatal antimicrobials to control s. suis disease. besides the public health concern related to antimicrobial use in production animals and the increase in antimicrobial resistance, an additional problem of antimicrobial usage is that these treatments can also affect the beneficial bacteria of the microbiota. in an attempt to understand the harmful effect of overuse of antimicrobials at early age, aragon et al. (appendix j) have evaluated the outcome of perinatal antimicrobial treatment on the nasal microbiota at weaning. elimination of perinatal antimicrobials resulted in an increase in bacterial diversity in the nasal microbiota at weaning. furthermore, management of lactation without perinatal antimicrobials had a beneficial impact later in life in terms of animal health and productivity in the nursery phase. albeit more detailed studies are required, aragon et al. also observed that perinatal ceftiofur administration might favour colonisation with potentially clinically relevant serotypes of s. suis. as such, the precise effects of antimicrobial metaphylaxis are difficult to predict (appendix j). the only available vaccines used in the field are autogenous, which consist of killed bacteria ("bacterin") from the predominant strain(s) recovered in an affected farm, produced by licenced laboratories and given back to the same farm only. however, there are very few scientific studies demonstrating whether the use of such vaccines in the field correlates with a reduced mortality and curative antimicrobial use. indeed, field peer-reviewed reports on autogenous vaccines are almost nonexistent (only published papers in the last years) [ , ] ; others are incomplete and, in most of them, a control (non-vaccinated) group is missing, which may preclude any scientifically sound conclusions [ ] . on the other hand, controlled experimental (laboratory) studies have shown contradictory results concerning bacterin-induced protection [ , ] . that controversial protective response could be attributed, among others, to a loss of antigenicity caused by the killing procedure and/or production of antibodies against antigens not associated with protection. moreover, autogenous vaccines are "manufacturer-related" (each licenced laboratory uses different protocols, antigen concentration, adjuvants, etc.) and "farm-specific" (useful against homologous-same strain-challenge only) [ ] . finally, the correct diagnosis of s. suis as a primary cause of disease may complicate the choice of the strain(s) to be included in the autogenous vaccine. there is still an unresolved issue concerning isolates recovered from lungs, which are considered by many researchers as secondary invaders and may or may not (depending on the laboratory) be included in the vaccine as antigens. definitively, more studies are required to generate scientific knowledge to improve this important preventive tool and to help reduce the use of antimicrobials. the wide use of autogenous vaccines and cross-border movement of animals vaccinated with autogenous vaccines is now a common practice within europe. therefore, harmonising quality of these products has been considered necessary by the coordination group for mutual recognition and decentralised procedure-veterinary (cmdv) and the national competent authorities for veterinary medicinal products (vmps) in europe. m. saléry discussed in appendix k the regulatory framework of the manufacture, control, and use of autogenous vaccines within the european union. recently, a new legislation on vmps has been adopted in european union, applicable in january . in this new regulation, autogenous vaccines have an updated definition and fall under european regimen. article ( ) of this regulation requires that some articles of the regulation apply to "inactivated vmp manufactured from pathogens and antigens obtained from an animal or animals in an epidemiological unit and used for the treatment of that animal or those animals in the same epidemiological unit or for the treatment of an animal or animals in a unit having a confirmed epidemiological link". the introduction of the concept of epidemiological link allows now the use of autogenous vaccines in parental lines or for animals prior their introduction in fattening sites where they will be in contact with new pathogens. in addition, different laboratories working under different conditions presently exist in europe. from january , all autogenous vaccines will have to be done under good manufacturing practices (gmp) or gmp-like requirements. the manufacturers will have to be authorized, and the compliance with the requirements will be controlled by inspection. as a consequence, autogenous vaccines will be produced under similar quality conditions. however, among others, differences in antigen preparation, antigen concentration, method of killing, and type and concentration of the adjuvants will still exist. in appendix k, m. saléry summarized key points from the cmdv' recommendation paper [ ] , which deals with the manufacture, the control, and the use of viral and bacterial autogenous vaccines in europe. in the framework of the new regulation, the cmdv' recommendations will be updated in the coming years. the current and future regulatory framework ensures those products are of high quality. nevertheless, safety and efficacy are not regulated. safety and, more importantly, efficacy of s. suis autogenous vaccines need to be better demonstrated through field use and laboratory research. intensification of animal food production and emergence of new production systems (such as raised without antibiotics or organic systems as well as "humane" animal farming) has resulted in emergence or reemergence of pathogens. in this context, coinfections are more likely to occur, increasing the incidence and/or enhancing clinical disease with certain pathogens, including s. suis. continued development of suitable models for polymicrobial infections and a better understanding of the underlying pathological mechanisms are required to develop effective intervention strategies to prevent the effects of these diseases on swine production. mucosal infection models still require optimization and have the potential to improve our knowledge of the pathogenesis of s. suis-induced disease. these models would significantly contribute to vaccine development as well. research on mucosal infection and immunity will also help address the potential use of nutrition management and/or microbiota enhancement to improve swine health and, consequently, s. suis control strategies. early medication with antimicrobials has to be carefully considered since antimicrobials may interfere with the establishment of the microbiota and, in consequence, with immune maturation and other microbiota functions, which may have a lasting health effect later in life. s. suis importance as a zoonotic pathogen is continuously increasing due to, at least in part, the pressure to reduce antimicrobial use in livestock. this public health threat is enhanced by the lack of universally effective vaccines that might reduce the infection load in swine and thus the risk for humans. global strengthening of swine trade amongst countries has the potential to facilitate exchange of genetic material through recombination and mobile genetic elements, which may result in selective advantage and niche adaptation. it is crucial to understand the factors that are involved in the ability to cause zoonotic infection and to monitor if these may eventually result in s. suis strains that are more human adapted. indeed, a better worldwide surveillance system of s. suis-related disease in swine and humans would improve our understanding of the epidemiological evolution of this pathogen. new molecular tools have been developed; however, due to the diversity of s. suis and lack of appropriate markers to differentiate virulent strains from commensal ones, alternative techniques might still be required to achieve a comprehensive understanding of the s. suis bacterial community, including virulence-associated gene profiling. the development of such tools that might eventually allow prediction of virulence potential of a strain might be compromised by sampling strategies and the rich s. suis community in the upper respiratory tract of healthy animals. in the era of antimicrobial restrictions and new social meat consumption trends, improvement and/or development of vaccination strategies is of utmost importance. in spite of decades of research on s. suis vaccines, autogenous bacterins are the only control strategy available that swine producers have access to. due to intensive use of these vaccines in europe, new policies are in place that would improve and normalize their manufacturing. the implantation of similar policies worldwide, including north america, where the use of autogenous vaccines is also widespread, would be expected or sought after. nevertheless, more field studies are essential to scientifically validate their protective effect and, consequently, their cost-benefit impact for swine producers. 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; or in the decision to publish the results. streptocccus suis serotype infection: novel animal models and diagnostic tools streptococcus suis is a highly diverse organism; currently serotypes have been described [ , ] . historically, serotype was the predominant serotype causing infections in piglets worldwide. however, recently, a shift towards serotype has occurred, in particular in western europe [ ] . in recent years, however, incidence of serotype infections in pigs increased in south east asia [ ] and to a lesser extent in north america [ ] . in thailand, serotype was isolated from a human patient with severe disease symptoms [ ] , emphasizing the zoonotic potential of serotype isolates, that was already suggested [ ] . in the netherlands, all farms are colonised by serotype . colonisation with s. suis serotype is not directly correlated with s. suis disease. obviously, additional risk factors are determinative in causing disease. unfortunately, autovaccination against serotype is less effective than against serotype , making it difficult to combat the infection [ , ] . under experimental conditions, s. suis serotype is hardly virulent [ ] , whereas in practice, many piglets develop severe invasive disease after s. suis serotype infection. there could be different explanations for this discrepancy. first of all, additional risk factors occurring under field conditions may play a role in disease development like existing viral or bacterial coinfections, management factors, or housing conditions. alternatively, serotype isolates in the field might differ in virulence from the isolates used in experimental infections. it has been described that the s. suis serotype population is genetically very diverse [ , , , ] . in addition, it has been described that differences in virulence exist within the serotype population [ ] , similar to that described for serotype [ ] . taken together, s. suis serotype has been emerging for the last decade and is spreading around the world. due to the large genetic and phenotypic heterogeneity in the serotype population, it is a complex pathogen to study. to get more insight in the genetic population of s. suis serotype in the netherlands, an epidemiological study was performed [ ] . two populations of serotype isolates were included for genotyping analysis: clinical isolates and isolates from healthy carrier piglets. seventy-eight isolates from clinical cases of s. suis were included in the study and were isolated from the cns of animals with invasive s. suis disease (meningitis). the isolates from healthy carrier pigs were isolated from independent farms that did not have clinical outbreaks caused by s. suis for at least two years (n = ). tonsils of healthy sows and piglets were sampled, and s. suis serotype isolates were isolated after plating. genotyping of the isolates using comparative genome hybridization (cgh) revealed that the two populations were genetically very distinct. cgh split the population in two clusters: one cluster only contained carriage isolates, whereas the other cluster contained all clinical isolates plus a few carriage isolates. these carriage isolates probably reflect clinical isolates that are not causing any clinical outbreaks on the farms but do have virulent potential. to further analyse the genetic differences between the isolates, all serotype isolates were subjected to whole genome sequencing. a systematic analysis of the data showed that the clinical isolates clustered mainly together in one group whereas the carriage isolates were distributed across different groups. this suggested that virulent serotype isolates are genetically very similar, whereas the carriage isolates are more heterogeneous [ ] , as was described before [ , , , ] . based on our data, the heterogeneity was only found for the carriage isolates. our data suggest that at least two virulence phenotypes of serotype isolates exist: clinical isolates that are virulent and avirulent carriage isolates. to confirm there are differences in virulence among serotype isolates, four isolates were tested for virulence under experimental conditions. two carrier isolates, one clinical isolate, and one laboratory strain that clustered among the clinical isolates were tested for virulence by infecting cdcd piglets intravenously with a high dose of each of the isolates. the survival curves clearly showed there was a significant difference in virulence between carriage isolates and clinical isolates. although the carriage isolates could induce s. suis specific disease symptoms like lameness, %- % of the piglets survived the infection, whereas the piglets infected with the clinical isolates all died within days postinfection. in conclusion, it was shown that, within s. suis serotype , the virulence of clinical isolates and tonsillar carriage isolates differed significantly. due to the zoonotic s. suis outbreaks in south east asia, a lot of attention has been paid to putative risk factors for human s. suis infections [ , ] . one of the risk factors that has been identified is occupational exposure, which is the main risk factor for human infections in the western world as well [ , ] . however, a new risk factor that was identified due to the outbreak in asia is consumption of raw or undercooked pig products like raw blood soup. by ingesting contaminated pork products, s. suis infections can occur. this insight directed research into the role of intestinal translocation in porcine s. suis infections. it is known that s. suis colonises the gastrointestinal tract of piglets in high numbers, in particular after weaning [ ] . in addition, under experimental conditions, invasive s. suis serotype infections could be induced by intestinal inoculation of piglets [ ] . to study the putative role of intestinal colonisation for serotype , an epidemiological study was performed at the wageningen university and research experimental farm in sterksel, the netherlands. it was shown that, during a s. suis serotype outbreak, tonsils of piglets were colonised with different serotypes. the highest percentage of colonisation was found for serotype , followed by serotype and (table a ) . only a few animals were colonised by serotype . subsequently, colonisation in s. suis specific organs and the intestine was studied in s. suis diseased animals and healthy litter mates. it was shown that specific organs were exclusively colonised by serotype , as was expected since there was a serotype disease outbreak on the farm. however, up to % of the healthy controls and % of the diseased piglets were also colonised by serotype in the intestine, whereas the other serotypes did not colonise at all in the intestine or only in a limited number of animals (table a ) . faecal samples were also screened for presence of serotypes , , , and . whereas none of the piglets were positive for serotypes or , a few animals were positive for serotype and a considerable number of animals were positive for serotype in faeces. although only one farm was sampled in this study, the data suggest that serotype is a better coloniser of the porcine intestine than the other serotypes. this suggestion was supported by the data published by ferrando et al. [ ] that showed that serotype isolates can adhere to porcine intestinal cells more efficiently that the other serotypes. furthermore, it was shown that translocation of s. suis serotype via the intestine can cause severe invasive disease in piglets. the putative role of the intestine in invasive translocation of s. suis opens up new possibilities in the prevention of s. suis disease. if the intestine plays an important role in the pathogenesis of disease, feed interventions might contribute to prevention of disease. feed components can either strengthen the intestinal barrier thus preventing translocation, they can exhibit antimicrobial effects and kill s. suis, or they can affect microbiota composition in such a way that s. suis cannot colonise anymore (competitive exclusion). to evaluate the effect of this kind of interventions, an experimental mucosal infection model is required. at the moment, only an intravenous model is available that leads to severe invasive disease symptoms. a more subtle infection model following mucosal challenge route would be very interesting. to develop such a model, several mucosal infection routes were applied to piglets from a different background. it was shown that intranasal inoculation in combination with oral inoculation will result in a reproducible colonisation of the tonsils and the intestine of piglets, even when they are endemically colonised by serotype . to mimic the weaning stress piglets experience under field conditions, several stressors were applied to the piglets, like transport stress, social stress (no acclimatization), or pretreatment of the nasal mucosa with acetic acid. these stressors facilitate and increase colonisation by s. suis but are a not strict requirement for colonisation. this mild mucosal colonisation model does not lead to clinical disease. a harsher mucosal challenge can be achieved by combining oral inoculation with intratracheal inoculation. in this case, the level of intestinal colonisation is lower, but more clinical symptoms like lameness, central nervous signs, or septicaemia were induced in the piglets. before applying any of those models, some critical parameters need to be determined. first of all, the background of the piglets is of utmost importance for the outcome of the experiment. piglets from the field with maternal antibodies and a natural exposure to endemic pathogens are more resilient to experimental s. suis infection. this is particularly true for animals from farms with a relative high hygiene standard and good management practices. piglets from high health farms with a specific pathogen free status are more susceptible to infection. cdcd piglets are very sensitive to all infections due to their complete naive immune system. another important parameter is the application of a stressor. especially when more resilient piglets are used, a stressor is very helpful to increase susceptibility to colonisation and disease. several stressors can be used; in our studies, social stress, transport stress, and acid exposure were applied. in conclusion, it was shown that s. suis serotype is a very heterogeneous pathogen that has at least two pathotypes. carriage isolates are very diverse and have a low virulence, whereas clinical isolates are genetically less diverse and more virulent (though they are less virulent than mrp+ef+ information relating to streptococcus suis in the australian pig herd is limited [ , ] . given the paucity of data available on s. suis associated with disease in australian pigs, the aim of this study was to analyse and characterise a significant number of s. suis isolates from diseased pigs obtained across multiple production sites and spanning a seven-year period. a total of swabs from archived clinical isolates spanning the years to were subjected to antimicrobial susceptibility testing via broth microdilution; whole genome sequencing via illumina nextseq; and detailed genomic interrogation for presence of antimicrobial resistance elements, putative virulence genes, serotype, and mlst. in addition, core genome phylogenetic analysis of australian isolates and previously published international isolates was performed. genomic analysis of the isolates revealed that isolates belonged to previously identified mlsts, with isolates belonging to one of analysis of the capsular genes resulted in two major serotypes being detected: serotype ( / ) and serotype ( / ). the other main serotypes were / ( / ), ( / ), ( / ), ( / ), and ( / ). analysis of serotype and mlst combinations showed a high proportion of isolates as serotype st ( . %), serotype st ( . %), and serotype st ( . %). the proportions of isolates carrying the main virulence factors mrp, epf, or sly across all isolates were . % (cps / , , , and ), . % (cps / , , and ), and . % (cps / , , , , , , , , , , , , , , , and ), respectively. only a single isolate carried both mrp and sly, while . % of isolates carried both mrp and epf and . % carried mrp, epf, and sly. the isolates carrying all three genes belonged to serotypes / (n = ), (n = ), and , and all belonged to st , with a significant association between st and the carriage of all three genes (fisher's exact test p-value < . ). detailed examination of the virulence gene profiles demonstrated that the australian isolates could be grouped in multiple clades based on putative virulence gene carriage. of most significance was a single clade containing a conserved block of putative virulence genes, which was the only group to contain the genes dppiv, epf, mrp, sbp , oppa, and lde. this group consisted predominantly of serotype / and serotype isolates, and all were st . on average, the total gene content of systemic isolates from brain and joint was , significantly lower than that from respiratory isolates which carried (mann-whitney u test p-value = . ). when the total gene content of isolates was compared to the number of putative virulence genes, a trend was observed based upon st. this was most apparent in the st isolates which had a significantly higher number of putative virulence genes compared to the other sts and the lowest average total gene content of (p-value < . ). phylogenetic comparison of the s. suis strains isolated from australian pigs against a global collection of s. suis strains resulted in the identification of four major clades. the strains from this study were present in all four clades. the majority of the australian strains ( / ) clustered in clade , which was comprised entirely of strains from the uk, north america, and canada. only eight strains were present in clade , which was made up of vietnamese and uk isolates. while australian serotype st isolates could be distinguished from north america and canadian strains, the small snp difference was notable; as aside from a single isolate, canadian and australian isolates differed by a maximum of only snps across the core genome. a high proportion of the isolates were resistant to both tetracycline ( . %) and erythromycin ( . %). low levels of resistance were observed for florfenicol ( . %), penicillin g ( . %), ampicillin ( . %), and trimethoprim/ sulphamethoxazole ( . %). none of the isolates were resistant to enrofloxacin. in this study, we present the key findings of the virulence potential of st clones and the limited evolution of australian clones from their global seed strains. in terms of virulence genes, the mrp+/epf +/sly+ combination was carried by all st isolates, and these were consistently the isolates with the largest array of putative virulence factors. in addition, a conserved constellation of virulence genes, a number of which are associated with adhesion and which were not present in other mlsts, provides some insight into the virulence of st clones. when this gene block was investigated in st clones from overseas, it was also found to be highly conserved across isolates. taken together, these factors suggest that both australian and international st clones have a distinct fitness advantage in terms of binding ability towards host target cells, and st bacterial adhesion factors may be a promising vaccine target. antimicrobial resistance of australian strains was similar to levels reported overseas with regards to tetracycline ( . %), erythromycin ( . %), and trimethoprim/sulfamethoxazole ( . %). resistance to florfenicol was . %, while all isolates were clinically susceptible to enrofloxacin, likely due to this being banned from use in food producing animals in australia. of concern was the observed clinical resistance to penicillin g, albeit at a relatively low level in . % of isolates. therefore, this is an aspect of s. suis in australia that must be carefully monitored from both an animal and public health point of view. in conclusion, our findings demonstrate that australian clones of s. suis associated with clinical disease in pigs have maintained a stable core genome, mirroring the international seed-stock from which they were derived. australian clones associated with disease in pigs consist predominantly of serotypes , , and / , which is consistent with reports from other pig producing countries [ ] . despite the limited number, the characterisation of serotype / st clones is significant, as all displayed distinctive factors associated with highly virulent s. suis. in the upper respiratory tract of pigs, and pathogenic s. suis strains are associated with meningitis, arthritis, endocarditis/epicarditis, polyserositis, and septicaemia in piglets and growing pigs [ , ] . identifying major disease-causing strains can promote the development of treatment and control plans. mlst is a widely used method for subtyping strains, but information on the subtype distribution of s. suis strains in the usa is limited or outdated. a study investigated the serotype distribution of porcine s. suis strains in minnesota and reported the prevalence of serotypes - and , of which serotype was the predominant serotype associated with neurological disease [ ] . a study identified serotypes - and / in naturally infected pigs, with serotype being the predominant serotype in usa [ ] . a large usa study in investigated the serotype distribution of s. suis strains collected from to from states. this study illustrated that the distribution of strains was similar to that in canada in which serotypes / , , , , and were most prevalent in diseased pigs [ , ] and dissimilar to the distribution in europe in which serotype occurs at a considerably higher percentage of isolates than in north america [ ] . on the other hand, studies addressing mlst for s. suis largely focus on common sts of serotype . st and st are more common among strains recovered from diseased animals in north america, while st strains are more prevalent in europe and asia [ , , ] . the objective of this study was to characterise the diversity of s. suis isolates collected between to across north america (mainly the usa) by serotyping and mlst to address the limited information on current s. suis strains circulating within the usa [ ] . isolates were also characterised by pathotype, based on clinical information and site of isolation, as pathogenic (from neurologic or systemic tissues, n = ), possibly opportunistic (from lung samples, n = ), or commensal (from healthy pigs, n = ). the subtype distributions were further analysed by pathotype to determine prevalent subtypes recovered from diseased pigs. serotyping identified different serotypes and the predominant serotypes were / (n = ) and (n = ). fifty-eight different sts were identified and were newly identified sts ( - , - , and ; n = ). the predominant st was st (n = ), followed by st (n = ), st , and st (n = each). fifteen of the serotypes identified contained multiple sts, with the number of different sts within a single serotype ranging from to . the subdivision of s. suis serotypes by st further illustrated the high diversity among the isolates. furthermore, the isolates were selected from different states, representing the major swine producing states in the usa. geographic distribution of the s. suis serotypes in our study identified serotype / in of the states, with a concentration in five. we investigated associations between subtype (serotype and mlst) and pathotype classifications using odds ratio (or) and phylogenetic analyses. between %- % of isolates belonging to serotypes , / , , , , and were classified as the pathogenic pathotype, and these associations were supported by or analysis. twelve sts, including sts , , , and , contained over % of isolates classified as pathogenic, which demonstrated the same associations by or. interestingly, many of the novel sts that occurred as singletons were identified as the commensal pathotype. commensal isolates are not generally subjected to subtyping by mlst, which may explain the large number of novel sts. to investigate genetic relationships among the samples and possible associations among serotype, genotype, and pathotype classifications, the mlst allelic sequences were clustered. mlst clustering analysis demonstrated three clades with an association to pathotype. the first clade represents mostly the pathogenic pathotype and includes largely serotype (with some serotype and ) and st isolates. the second clade represents mostly the pathogenic pathotype and contains serotype / and st isolates, the predominant subtypes in our usa sample set. the third clade represents mostly the commensal pathotype and a variety of serotypes and sts (most of them novel sts). these results demonstrated the use of serotyping and mlst to differentiate pathogenic from commensal isolates and to establish links between pathotype and subtype, thus increasing the knowledge on s. suis strains circulating in the usa. however, due to the diversity of s. suis, alternative subtyping techniques should be explored to consistently differentiate pathogenic from commensal strains. alternative subtyping techniques include virulence-associated gene (vag) profiling of s. suis. identification of the virulence factors of s. suis has been previously explored for understanding the pathogenesis of s. suis infection, and there are currently over putative virulence factors reported [ , , , ] . however, information on the distribution of these factors in usa isolates is limited. we characterised the same set of isolates by vag profiling to investigate the distribution of the vags in usa isolates. presence/absence of these vags was used to perform clustering analysis, which also demonstrated three clades with an association to pathotype; these clades were further investigated by serotype and st. the first clade represents the pathogenic pathotype containing serotypes and and st , consistent with publications associating st with high virulence [ , ] . the second clade largely represents the pathogenic pathotype and contains isolates subtyped as serotype / and st . the third clade contains isolates of the commensal and possibly opportunistic pathotypes belonging to numerous serotypes and sts. the three classical vags of s. suis serotype strains (epf, sly, and mrp) were found in the st clade but not in the st clade. several other vags were identified that may potentially differentiate the pathogenic from the commensal pathotypes and may be better predictors of pathogenicity for usa strains. in this study, s. suis isolates from north america were characterised by serotyping, mlst, and vag profiling. these findings will contribute to the knowledge on the population structure of s. suis strains currently circulating in the usa. we demonstrated the use of serotyping and mlst for the subtyping of strains and for investigating associations between subtypes and pathotype to identify pathogenic strains. the identification of pathogenic strains is important for enhancing strain selection for preventive strategies. the subtype distributions in our study elucidated the predominance of serotype / st and st isolates from clinically affected pigs in the usa, which may inform new diagnostic approaches and virulence studies. we also determined the diversity of the isolates by mlst and vag clustering analyses, both of which illustrated one commensal and two pathogenic clades [ ] . in thailand, s. suis infection was first described in , with cases of meningitis [ ] . since the largest outbreak of human s. suis infection occurred in sichuan province, china in [ ] , the importance of this disease has been increasingly recognized in thailand. up to the present, outbreaks of s. suis infections in humans have been recorded in thailand. the first outbreak including laboratory confirmed cases and deaths, occurring in phayao province during may of [ ] . a second outbreak was recognized in chiang mai and lamphoon provinces during june-july, , including confirmed cases, suspected cases, and fatal cases with septic shock (http://www.boe.moph.go.th/annual/annual% /wesr /wk _ /wk _ .pdf). the third outbreak was found in phetchabul province in april, ; this outbreak had confirmed cases with fatal (http://www.boe.moph.go.th/annual/aesr /wesr_ /wk _ .pdf). the fourth outbreak has been reported in uttaradit province in may, with cases (https://ddc.moph.go.th/uploads/files/ f dfba d ca d c a a .pdf). consequently, a total of cases and fatal cases of s. suis infection in thailand have been reported by the bureau of epidemiology, ministry of public health since -august, (http://www.boe.moph.go.th/boedb/surdata/disease.php?dcontent=situation&ds= ). of note, case fatality rate was . % in contrast with retrospective studies that revealed case fatality rates of . % and . %, respectively [ , ] . however, a prospective study demonstrated a case fatality rate of . % and an incidence rate of . per , in the general population. the main route of s. suis infection in thailand was consumption of traditional raw pork or pig's blood products [ , [ ] [ ] [ ] . food safety campaign implementation in phayao province during - showed a marked decrease of the incidence proportion [ ] . the annual incidence proportion significantly decreased from . / , persons in (before implementation) to . / , persons in , to . / , persons in , and to . / , persons in . this revealed a . / , persons decrease in the trend of incidence proportion after campaign (p < . ) [ ] . microbiological characterisation of s. suis in thailand showed that serotype ( . %) was the main serotype in human infections in thailand, followed by serotype ( . %), ( . %), ( . %), ( . %), ( . %), and ( . %), respectively [ , , , [ ] [ ] [ ] [ ] . mlst classified serotype into clonal complexes (cc) that were cc , cc , cc , cc , and cc / . while serotype was classified to only cc by mlst, st was the major st of serotype found in cc , whereas st was the main st in cc of serotype . st , st , st , and st were the main sts in cc , cc , cc , and cc / of serotype [ , , , ] . of note, st and (serotype ) are predominant sts in thai human infections. in contrast, serotypes and and some isolates of serotype were classified in cc / , with the majority belonging to st . it is also interesting that cc , cc / , and cc / are exclusively found in thailand only. mlst determined that the thai serotype isolates were of st , which belongs to clonal complex [ ] . serotype is the most common serotype affecting pigs in european countries, especially st which is found in ≥ % of cases in the netherlands [ , ] . streptococcus suis is an emerging zoonotic pathogen. although many countries perform systematic surveillance of vaccine-preventable and other common causes of meningitis, s. suis meningitis is not a notifiable disease, with the exception of northern thailand between and . the burden of human s. suis infections is likely to be underestimated as indicated by case reports from an increasing number of countries [ , [ ] [ ] [ ] [ ] [ ] [ ] , and human s. suis infections have now been reported from all continents. a recent study estimated the burden of disease caused by s. suis in vietnam and reported annual disease incidence ranging from . to . per , population, in the years - [ ] . risk factors for human infection include consumption of raw pork blood or products; direct exposure to pigs, particularly in the presence of skin lesions; pig related occupations; and male sex [ , , ] . human-to-human transmission has not been demonstrated to occur to date. s. suis serotype is the main serotype causing zoonotic infections. serotype is known to also contribute significantly [ ] . other serotypes have been reported in single cases, including serotypes , , , , , and . the serotype is determined by the antigenic properties of the cps of s. suis, which is encoded by multiple genes located in a single locus. the cps gene locus can be exchanged between s. suis of different serotypes, and in a study from the netherlands, it was shown that such cps switch may lead to an increase in zoonotic potential. zoonotic s. suis serotype strains of st , isolated from human patients, were shown to be genetically highly related to serotype strains of st . s. suis st isolates have never been isolated from human patients but are highly implicated in invasive disease in pigs in the netherlands [ ] . the serotype cps appears to induce very little to no protective immunity, as was illustrated by a case of reinfection with s. suis serotype in a dutch slaughterhouse worker [ ] . in addition to cps locus acquisition, loss or acquisition of other genes or loci may contribute to changes in zoonotic potential. for example, the s. suis st strains acquired not only the serotype cps locus but also a prophage carrying a type restriction-modification (r-m) system (ssucc p) [ ] . such r-m systems have previously been shown to regulate gene expression through changes in methylation, driven by the phase variation of the specificity unit of the system [ ] . further analysis of this system in our lab indicates that it is indeed phase variable and that this variation results in differences in colony morphology. this observation suggests that s. suis st strains may have acquired the ability to adapt to specific niches through the acquisition of a r-m system that can regulate cps and/or other surface exposed molecules gene expression (manuscript submitted). ingestion of contaminated food items is an important route of infection with s. suis, especially in southeast asia. we have previously shown that the cps modulates the interaction of s. suis with intestinal epithelial cells in vitro. using human caco- cells and porcine ipec cells, we observed that serotype strains adhered better to the human cells than serotype strains, whilst the opposite was observed for the porcine cells to which serotype strains adhered better. in addition, non-encapsulated mutant strains showed increased adherence, invasion, and translocation across caco- cells, clearly indicating a role for the cps in intestinal infection in this experimental model [ ] . to further study s. suis intestinal translocation, models are needed that represent the intestinal tract better than colon carcinoma cells in continuous culture. recently, developments in stem cell research have made it possible to grow intestinal organoids, which contain most cell types present in the proximal and distal intestinal mucosa ex vivo. in addition, these organoids can now be grown in two-dimensional structures, thus allowing study of translocation from lumen into mucosa, replicating intestinal translocation. initial experiments with listeria monocytogenes, another gram-positive pathogen known to cause meningitis after intestinal translocation to the blood, demonstrated invasion and translocation patterns similar to those observed in mouse models and mouse organoids. the zoonotic s. suis serotype strain bm was shown to translocate in this model as well, which makes it a promising model for future study of intestinal infection with s. suis. s. suis is likely to continue to emerge as a zoonotic pathogen, given the pressure to reduce antimicrobial use in livestock because of the global threat of antimicrobial resistance. in addition, a universally effective vaccine that can be used in pigs is not available. the global export of pigs that carry multiple serotypes of s. suis will facilitate exchange of genetic material through recombination and mobile genetic elements that may result in selective advantage and niche adaptation. it is crucial to understand the factors that are involved in the ability to cause zoonotic infection and to monitor if this may eventually result in s. suis strains that are human adapted and have become human-to-human transmissible. the role of concurrent infections in predisposing to streptococcus suis and other swine diseases susan l. brockmeier usda, ars, national animal disease center, ames, ia although we know that pathogens such as streptococcus suis can be primary pathogens, causing disease on their own, experimental and anecdotal evidence from the field indicate that the incidence and severity of disease with certain pathogens like s. suis can increase when concurrent infections with additional pathogens occur. the last national animal health monitoring survey conducted by the animal and plant health inspection service of the united states department of agriculture indicated that respiratory disease is the primary cause of both nursery and grower/finisher deaths in swine and that meningitis is the second most common cause of nursery deaths, which is primarily caused by s. suis and glaesserella (haemophilus) parasuis. the top ten reported primary etiologies of porcine respiratory disease cases at the iowa state university diagnostic laboratory from to pathogens , , of were prrsv, swiv, mycoplasma hyopneumoniae, pasteurella multocida, s. suis, unspecified bacteria, actinobacillus suis, g. parasuis, actinobacillus pleuropneumoniae, and bordetella bronchiseptica, in that order. from the cases where swiv was identified as the primary etiology, % had secondary bacterial pneumonia as well, with s. suis, p. multocida, g. parasuis, b. bronchiseptica, and escherichia coli being the most prevalent bacteria isolated (courtesy of dr. phillip gauger, iowa state university). these results were similar for cases where prrsv was determined to be the primary aetiology. the term porcine respiratory disease complex has been used to describe this common multifactorial respiratory disease in swine that occurs as a result of polymicrobial infections, environmental stressors, and host factors such age and immunological status. coinfections can be the result of multiple viruses, bacteria, or a combination of viruses and bacteria infecting the pig at the same time or in close succession. numerous examples of coinfections with common swine viruses such as prrsv, swiv, porcine circovirus (pcv), and prcv have been detected in the diagnostic lab and investigated experimentally, and coinfections with these viruses generally result in a more severe clinical disease outcome [ ] [ ] [ ] [ ] . similarly, coinfections with the viruses mentioned above and common swine bacterial pathogens have often shown that these viruses are potent initiators of secondary bacterial infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . primary bacterial pathogens can also predispose to secondary bacterial colonisation and disease. common examples of this in swine are enzootic pneumonia, where m. hyopneumoniae predisposes to secondary pneumonia with p. multocida, and atrophic rhinitis, where b. bronchiseptica predisposes to secondary infection with toxigenic strains of p. multocida [ ] [ ] [ ] . our lab has also demonstrated that b. bronchiseptica can increase colonisation with other bacteria such as g. parasuis [ ] . coinfections are not limited to only two pathogens and can be complicated in nature. for example, we have shown that prrsv alone predisposes pigs to pneumonia with b. bronchiseptica but not with p. multocida whereas b. bronchiseptica can predispose to nasal colonisation with p. multocida [ ] . however, if the pig is infected with both prrsv and b. bronchiseptica, this can lead to pneumonia with p. multocida as well [ ] . there are several mechanisms that contribute to increased susceptibility to secondary infection and the enhanced disease that often occurs with coinfections, including disruption of the epithelial barrier or alteration of the innate or adaptive immune responses by the primary pathogen, direct interactions between or among the pathogens such as the formation of multispecies biofilms, disturbances to the ecological niche such as the upper respiratory microbiota, or enhanced transmission. an example of mechanical disruption would be the epithelial damage caused by pathogens such as swiv or b. bronchiseptica resulting in decreased cilial clearance and potentially in exposure of new attachment sites and systemic invasion [ , ] . decreased clearance of bacteria by professional phagocytes such as alveolar macrophages and neutrophils can lead to secondary bacterial infections. viruses such as prrsv and swiv and bacteria such as b. bronchiseptica have been shown to affect phagocyte clearance though such mechanisms as being directly cytotoxic, altering functions such as phagocytic or bactericidal ability, or inhibition of recruitment of phagocytes to the site of infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these actions can be due to direct interactions of the pathogens with the phagocytes or via altered cytokine production. often, there is an altered cytokine response to coinfections compared to the response seen with a single pathogen resulting from signalling through multiple pattern recognition receptors that recognize pathogen-associated molecular patterns. frequently, this results in an amplified inflammatory response with coinfections that lead to more severe clinical signs and enhanced lesions. we and others have shown that coinfection with prrsv, swiv, or prcv and bacteria such as b. bronchiseptica, s. suis, or g. parasuis result in a greater incidence of disease, greater percentage of lung affected, more severe lesions, and slower resolution of lesions than occuring with infection with the single pathogen alone, and this is often correlated with an amplified pro-inflammatory cytokine response [ , , [ ] [ ] [ ] (brockmeier, unpublished data) . direct interactions among pathogens can lead to enhanced infection with secondary pathogens as well. for example, it has been shown that swiv can promote adherence, colonisation, and invasion of s. suis due to interaction with the capsular sialic acid moiety present on some serotypes [ , , ] . it has also been demonstrated that certain species of bacteria such as bordetella can enhance colonisation of other bacterial species by a process called piracy of adhesins, whereby one bacterial species can utilize adhesins secreted by another bacterial species to attach to host cells [ ] . there has been a great deal of interest lately in examining the microbiome of the digestive and respiratory tract and in determining if and how the local community of microbes might influence gut and respiratory tract health and/or predisposition to infection. studies currently or recently performed have shown that infection with pathogens such as swiv, prrsv, and b. bronchiseptica results in significant changes to the nasal microbial composition and significant increases in abundance of certain genera, such as actinobacillus and streptococcus (brockmeier, unpublished data). in addition, antimicrobial usage typically used in swine production has been shown to result in similar changes to the microbiota [ ] . further studies will be needed in order to determine whether these changes in composition result in increased incidence of disease with pathogens such as s. suis. development of animal models that recapitulate the polymicrobial infections typically seen in swine production facilities can be challenging but are needed in order determine the best intervention strategies to prevent the clinical disease seen with coinfections. many factors such as the virulence of the pathogens, immune status, age of the pig, order and timing of exposure to the pathogens, and possibly microbiome can all affect the success of developing a model that reproduces the polymicrobial diseases that are seen on the farm. once a model is developed, intervention strategies such as vaccination, antimicrobials, or other biotherapeutics can be tested for their effectiveness at preventing or treating disease outbreaks. for instance, with coinfection models that we have developed, we have demonstrated that, for certain strains of prrsv, vaccination can reduce the incidence of secondary bacterial pneumonia [ ] . others have shown that a previous low dose challenge with s. suis used to vaccinate pigs, but not previous prrsv vaccination, resulted in a lower incidence of streptococcal disease in a prrsv/s. suis coinfection model [ ] . in conclusion, the intensification of animal agriculture has resulted in the circulation of numerous potential pathogens at any given time on swine farms, and coinfections are more likely the norm rather than the exception. this can result in increased incidence and enhanced disease with certain pathogens including s. suis. we are just beginning to determine the pathogenesis of these polymicrobial infections and to develop suitable models for their study. however, continued study in these areas is needed to be able to develop effective intervention strategies to prevent the effects of these diseases on swine production. coinfections with swiv may affect the course of s. suis infections, as has been shown recently in a precision-cut lung slice model and in immortalized cells [ , ] . however, the molecular mechanisms underlying these coinfections are poorly understood. so far, it has been shown that the interaction of swiv hemagglutinins with capsular sialic acids of s. suis results in the binding of streptococci to virus-infected cells [ , ] . this adherence mechanism seems to be even more efficient than binding mediated by bacterial adhesins [ ] . the enhanced adherence and colonisation efficiencies of s. suis observed after coinfection with swiv affect also the subsequent tissue invasion. furthermore, a coinfection with swiv provides an additional advantage because virus infection induces apoptosis of virus-infected cells, which are mainly ciliated cells. it has been proposed previously that the loss of these specialized cells enables bacterial pathogens to get access to subepithelial cells and, thus, to spread to other parts of the host organism [ , ] . so far, the mechanisms that enable s. suis to invade mucosal surfaces are not well known. a streptococcal protein that contributes to bacteria-host cell association is suilysin, though it is not a typical adhesin as it is not cell-bound but secreted [ ] . this cholesterol-dependent cytotoxin can induce apoptosis, resulting in epithelial damage. in addition, it is able to mediate attachment of s. suis to the surface of the respiratory epithelium [ , ] . it has been suggested that suilysin facilitates the colonisation and the establishment of the initial stages of infection in pigs by promoting host cell lysis and invasion of s. suis through the epithelium when bacteria have colonised the upper respiratory tract [ , ] . furthermore, s. suis lacking the suilysin-gene has been shown to be avirulent in a mouse infection model when applied via the intraperitoneal route but virulent in pigs after intravenous infection, which indicates that suilysin is not essential for s. suis to cause systemic infection in pigs once the bacteria have entered the blood vessels [ ] . nevertheless, suilysin-negative clinical isolates are also found frequently in swine populations worldwide [ , ] . the prevalent ratios of suilysin-positive and suilysin-negative strains worldwide are varying, and not all virulent strains of s. suis produce suilysin [ , ] . therefore, analyzing the biological role of suilysin in different stages of bacterial infection may help to better understand the pathogenesis of non-cytotoxic s. suis strains. in a recent study, we applied highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface conditions to analyse the contribution of swiv to the virulence of s. suis with a special focus on its cytolytic toxin suilysin [ ] . we found that, during secondary bacterial infection, suilysin of s. suis contributes to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas cytotoxic effects induced by swiv became prominent at later stages of infection [ ] . prior infection by swiv enhanced the adherence to and colonisation of porcine airway epithelial cells by the wildtype s. suis strain and a suilysin-negative s. suis mutant in a sialic-acid dependent manner. a striking difference was observed with respect to bacterial invasion. after bacterial mono-infection, only wild-type s. suis showed an invasive phenotype whereas the mutant remained adherent. when the epithelial cells were preinfected with swiv, also the suilysin-negative mutant showed invasion capacity [ ] . as mentioned above, suilysin is known to be a virulence-associated factor that facilitates s. suis invasion as shown for both immortalized cells and differentiated airway epithelial cells [ , ] . the cytolytic activity of suilysin contributes significantly to this effect, as shown in our previous study, in which a mutant expressing a point-mutated suilysin lacking cytolytic activity was significantly less invasive than the wild-type streptococci [ ] . nevertheless, there was a significant difference between the invasion rates of two mutants lacking only the cytotoxic activity or lacking the whole protein [ ] . this finding suggests that there may be invasion-promoting effects of suilysin which do not depend on the cytolytic activity. taken together, suilysin-negative s. suis strains can become invasive in a coinfection scenario with swiv, which might explain why suilysin-negative strains can cause clinical infections. finally, these results may contribute to our understanding of viral-bacterial interactions on airway coinfections. streptococcus suis is an important zoonotic pathogen causing various diseases in pigs and humans [ ] . strains of s. suis can be classified into different serotypes on the basis of antigenic differences in cps [ , ] . among more than serotypes, serotype is the most frequently associated with clinical cases in both pigs and humans [ ] . meanwhile, cps of s. suis is known to be a major virulence factor contributing to protection against phagocytosis by host cells [ ] . in addition, several studies demonstrated attenuated virulence of the isogenic non-encapsulated mutants by in vivo murine and porcine models [ ] . however, almost all of the studies used serotype strains, and little is known on the roles of cpss of other serotypes as a virulence factor. therefore, it is unknown whether differences in serotype themselves (i.e., differences in cps structure) directly affect s. suis virulence. to answer this question, we experimentally generated six serotype-switched mutants using the serotype strain p / (p / cps to , p / cps to , p / cps to , p / cps to , p / cps to , and p / cps to ) by exchanging the cps synthesis gene cluster for those of serotypes , , , , , and , respectively, and compared their virulence in mice and pigs. in mice, s. suis strain/mutants were intraperitoneally inoculated at a final dose of × cfu to groups of - six-week-old c bl/ mice for survival ( days postinfection) and blood bacterial burden ( , , and h postinfection) evaluation. p / cps to , expressing serotype cps, showed higher mortality and blood bacterial load than serotype strain p / , with all infected mice succumbing within h from septic shock. the virulence of p / cps to , p / cps to , and p / cps to were equivalent to that of p / (survival: approximately % of infected animals), whereas the mortality of mice inoculated with p / cps to and p / cps to was remarkably reduced compared with that of p / , with % and % of survived animals, respectively. in pigs, one hour after intranasal administration of % acetic acid, groups of - crossbreed specific pathogen-free piglets aged weeks were intranasally inoculated with approximately × cfu of respective s. suis strain/mutants to monitor for clinical signs for days postinoculation and to evaluate bacterial recovery from infected animals. clinical signs such as lameness, shivering, and dysstasia were observed only with p / , p / cps to , and p / cps to strains. inoculated strains were recovered from several major organs and blood from the pigs showing clinical signs infected with p / and p / cps to , while p / cps to was recovered only from brain. in addition, p / cps to was also recovered from several major organs of experimentally infected but clinically healthy pigs. except for one of the pigs infected with p / cps to , in which the inoculum was recovered from several major organs and blood, recovery of infected bacteria from blood or more than two of major organs was not observed in infected pigs that showed no clinical signs during the experiment period. taken together, our data indicate that serotype switch in s. suis has different impacts on the virulence in mice and pigs depending on switched serotypes. this suggests that cps structure itself is an important factor in determining levels of s. suis virulence, although further studies are needed to elucidate the mechanisms behind the observed phenomena. bacterial pathogens can and do evolve against treatments. the most obvious of such change is the acquisition of resistance to antimicrobials. even by the early s, large numbers of s. suis isolates from pigs had become resistant to tetracycline and macrolide classes of antibiotics [ ] , and more recently, some isolates have become resistant to all classes of antibiotics used in pigs [ , ] . resistance also arises against vaccines [ ] . s. suis bacterin vaccines consist of strains isolated from a herd, which are then inactivated and administered as treatment. there has been no consistent evidence that bacterin vaccines give cross-protection against different strains of the bacterium, and so vaccinated animals can develop s. suis clinical disease [ ] . the failure of these s. suis vaccines is therefore different from acquired resistance. however, treating only a subpopulation of s. suis could lead to vaccination-driven strain replacement or to vaccination-driven bacterial evolutionary change (acquired vaccine resistance). these processes will decrease vaccine effectiveness over time. given our knowledge of the ecology and genetics of s. suis, it is important to consider how s. suis might respond and evolve against new and existing treatments and whether we can use this information to design treatments that are more "evolution-proof". the lack of cross-protection of bacterin vaccines speaks to the considerable genetic diversity of s. suis, which makes it a challenge to design a universal vaccine. a universal vaccine would need to contain an antigen that is highly conserved at the protein level across the whole s. suis species. unfortunately, less than half the proteins found in an average s. suis strain are conserved across the species at more than % protein identity [ ] . this is without considering that lineages of "diverse s. suis", which cause typical s. suis disease, are found colonising the same niches and freely recombine with s. suis [ ] . their limited conservation with "normal s. suis" at the amino acid level substantially decreases the number of species-wide conserved proteins [ ] . it is doubtful that a suitable universal antigen exists, but if it did and was able to induce mucosal immunity (i.e., immunity against carriage of s. suis), we could eliminate s. suis from pig populations and deliver herd immunity effects, protecting pigs that had not been vaccinated. s. suis is, however, an extremely common commensal, found in the tonsils of most and perhaps all pigs. wiping out a ubiquitous part of the microbiome may have unforeseen effects on other common opportunistic pathogens inhabiting the upper respiratory tract of pigs, such as haemophilus parasuis and actinobacillus pleuropneumoniae. another more feasible strategy would be to selectively target strains that are more prone to causing clinical disease. our lab and others have shown many trends in genetic and phenotypic differences between strains isolated from the upper respiratory tract of pigs without s. suis clinical signs (hereafter "nonclinical") and strains isolated from the lungs or systemic sites of pigs with s. suis clinical signs (hereafter "clinical") ( figure a ) [ , , , ] . overall, the genetic diversity of clinical s. suis is less than that of nonclinical strains, implying that we need not design a universal vaccine to eliminate the majority of s. suis disease. one obvious candidate for a vaccine targeting clinical strains is the cps outer layer of s. suis. this is because the majority of clinical strains have a capsule ( figure a ), which helps them to survive in the blood [ ] . indeed, polysaccharide capsules are widely used or considered in treatments for other streptococcal pathogens. however, capsules evolve rapidly. because they are genetically diverse, multiple different polysaccharides are needed in a vaccine to target different strains. as with bacterin s. suis vaccines, there will be vaccine failure if we do not target all cps types. not only that, we may get vaccination-driven strain replacement of the population, and this can reduce the overall vaccine effectiveness over time as non-vaccine types rise in frequency. this has been suggested as a reason for the rise in non-vaccine strains in invasive pneumococcal disease [ ] . in addition to their diversity, capsule loci are often recombination "hot-spots", and this gives rise to frequent and easy capsule "switching" between strains [ ] . this means that highly invasive strains can quickly evolve non-vaccine cps types, leading to acquired vaccine resistance. for these reasons, it would be desirable to identify alternative virulence candidates. proteins with higher conservation and in regions of the genome with greater syntenic preservation and less recombination would be obvious choices. there may also be inherent problems with targeting only clinical strains. despite the worldwide use of beta-lactams in pigs for over years, the majority of clinical s. suis remain sensitive to these antibiotics. however, beta-lactam resistant strains do exist and are primarily found in commensal sites ( figure a ). these resistant strains could spread if the susceptible (clinical) strains were wiped out. the problems discussed above-strain replacement/evolution-will be reduced if we exclusively induce systemic immunity (i.e., immunity against systemic infection that has no effect on colonisation). this is because s. suis is an opportunistic pathogen and so commensal sites (and not clinical sites) are the site of transmission of s. suis to other pigs. if the s. suis flora in commensal sites remain unaffected by vaccination, transmission dynamics remain unchanged. we would still get vaccine failure for any strains that were not targeted by the vaccine, but the effectiveness of this vaccine will not decrease over time. unfortunately, systemic immunity may be less successful in protecting against s. suis disease than mucosal immunity. for example, pneumococcal vaccines triggering systemic immunity with protection against invasive disease appear to be less effective against pneumonia [ ] . vaccine failure for any strains that were not targeted by the vaccine, but the effectiveness of this vaccine will not decrease over time. unfortunately, systemic immunity may be less successful in protecting against s. suis disease than mucosal immunity. for example, pneumococcal vaccines triggering systemic immunity with protection against invasive disease appear to be less effective against pneumonia [ ] . figure a . general trends in genetic and phenotypic differences between nonclinical strains of s. suis taken from the upper respiratory tract of pigs versus clinical strains of s. suis taken from the brain and lungs [ , , , ] : mlst = multi-locus sequence type; each mlst is defined by displaying the same allelic variant at housekeeping genes. the pictures have been reproduced under a creative commons . licence from servier medical art (www.smart.servier.com). to summarise, existing data on s. suis allow us to draw some important conclusions. first, if we do not have a universal target against all s. suis, we cannot rule out strain replacement/evolution, leading to decreased vaccine effectiveness over time, with potential downstream effects on antimicrobial effectiveness. second, and more positively, s. suis strain replacement/evolution should not be a problem with vaccines that induce systemic but not mucosal immunity. these are considerations that we can bear in mind in designing new vaccines but are minor in view of the many other existing challenges in bringing an effective s. suis vaccine to market. streptococcus suis is an early coloniser of the upper respiratory tract of piglets, which are commonly colonised by s. suis from birth. in fact, piglets can get colonised as soon as they pass through the birth canal, since s. suis is found in the sow vagina. virulence of the s. suis strains and the immunity of the animals influence the outcome of the infection. s. suis infections are one of the figure a . general trends in genetic and phenotypic differences between nonclinical strains of s. suis taken from the upper respiratory tract of pigs versus clinical strains of s. suis taken from the brain and lungs [ , , , ] : mlst = multi-locus sequence type; each mlst is defined by displaying the same allelic variant at housekeeping genes. the pictures have been reproduced under a creative commons . licence from servier medical art (www.smart.servier.com). to summarise, existing data on s. suis allow us to draw some important conclusions. first, if we do not have a universal target against all s. suis, we cannot rule out strain replacement/evolution, leading to decreased vaccine effectiveness over time, with potential downstream effects on antimicrobial effectiveness. second, and more positively, s. suis strain replacement/evolution should not be a problem with vaccines that induce systemic but not mucosal immunity. these are considerations that we can bear in mind in designing new vaccines but are minor in view of the many other existing challenges in bringing an effective s. suis vaccine to market. perinatal antimicrobials: friend or foe? virginia aragon streptococcus suis is an early coloniser of the upper respiratory tract of piglets, which are commonly colonised by s. suis from birth. in fact, piglets can get colonised as soon as they pass through the birth canal, since s. suis is found in the sow vagina. virulence of the s. suis strains and the immunity of the animals influence the outcome of the infection. s. suis infections are one of the main causes of antimicrobial usage in piglets. due to the young age of the piglets affected and the lack of an effective commercial vaccine, many farms use metaphylactic perinatal antimicrobials to control of s. suis disease. a similar situation is found with the diseases produced by other early colonisers of piglets, such as haemophilus parasuis and mycoplasma hyorhinis. nowadays, public and private institutions are urging for a significant reduction in antimicrobial use, since the emergence of drug resistances has become a major health problem. an additional problem of antimicrobial usage is that these treatments can also affect the beneficial bacteria of the microbiota. microbiota is the community of microorganisms that live on different tissues on all multicellular organisms. pathogen exclusion is an important role of the microbiota [ ] , and it can be achieved by competition or by induction of the correct maturation of the immune system. thus, the microbiota is essential for health, and this has changed our current view on antimicrobials, since they can have deleterious effects on favourable bacterial populations [ ] . however, the use of metaphylactic antimicrobials at early stages of life is still in practice in porcine production. clinical observations indicate that overuse of antimicrobials at an early age can have a harmful effect later in the pigs' health [ ] . in an attempt to understand this phenomenon, the outcome of perinatal antimicrobial treatment on the nasal microbiota at weaning was studied in two farms [ ] . these two farms reported polyserositis cases in the nursery phase and used antimicrobials during the lactation phase for disease control. the nasal microbiota was determined when antimicrobial treatment was used early in life and later when antimicrobial treatments were eliminated during the lactation period. elimination of perinatal antimicrobials resulted in an increase in bacterial diversity in the nasal microbiota at weaning. the specific analysis of the relative abundance of streptococcus showed a limited reduction of this genus at weaning after removal of antimicrobials in both farms. however, interpretation of this information is not straightforward since the genus streptococcus includes several species, some of which cannot be considered pathogenic. furthermore, s. suis strains display a wide variety of virulence capacity, complicating the interpretation of the result on streptococcal abundance with respect to health consequences. further analysis of the microbiota composition found that the elimination of antimicrobials produced an increase in the relative abundance of prevotella and lactobacillus and a decrease in moraxella and bergeyella in both farms. while prevotella and lactobacillus are considered important components of a healthy microbiota, moraxella and bergeyella have been reported to have pathogenic potential. in agreement, these changes in microbiota composition were accompanied by an improvement of the piglets' health and a higher productivity in the nursery phase. mortality (in one of the farms) and medication cost in the nursery (in both farms) were reduced, indicating that management of lactation without perinatal antimicrobials had a beneficial impact later in life. thus, the changes observed after elimination of antimicrobials can be explained by the consequent reduction of pathogenic genera and the simultaneous increase of beneficial bacterial genera, together with the general increase in microbiota diversity, which has been widely associated with improved health status. a different study showed that perinatal ceftiofur administration delayed the colonisation by s. suis and the pathogen load. however, a detailed analysis of the s. suis strains showed that different strains were selected by the antimicrobial drug and that serovars associated with virulence were more abundant in the nasal cavities of the treated piglets (unpublished results). while these results need deeper analysis, they indicate that the precise effects of antimicrobials are difficult to predict. early medication with antimicrobials has to be carefully considered since antimicrobials interfere with the establishment of the microbiota and, in consequence, with immune maturation and other microbiota functions, which may have a lasting health effect later in life. controlling s. suis in the field may require the use of autogenous vaccines since no other vaccines are nowadays licensed in europe. obviously, the wide use of autogenous vaccines and cross-border movement of animals vaccinated with autogenous vaccines is now a common practice within europe. therefore, harmonising quality of these products has been considered necessary by the coordination group for mutual recognition and decentralised procedure-veterinary (cmdv) and the national competent authorities for veterinary medicinal products (vmps) in europe. the current european directive / [ ] that regulates vmps defines autogenous vaccines but does not provide any other requirements. article ( ) states that the directive shall not apply for "inactivated immunological vmps which are manufactured from pathogens and antigens obtained from an animal or animals from a holding and used for the treatment of that animal or the animals of that holding in the same locality". article , about non-inactivated autogenous vaccines, says that the member states (ms) may provide that the directive shall not apply. in other words, the inactivated autogenous vaccines fall under national legislation whereas live autogenous vaccines may be under european regimen (so should be authorized as any other licensed vaccines) or national regimen according to the ms decision. in january a new legislation on vmps has been adopted in the european union, the regulation / [ ] , applicable in january . in this new regulation, autogenous vaccines have an updated definition and fall under european regimen. article ( ) of this regulation requires that some articles of the regulation apply to "inactivated vmp manufactured from pathogens and antigens obtained from an animal or animals in an epidemiological unit and used for the treatment of that animal or those animals in the same epidemiological unit or for the treatment of an animal or animals in a unit having a confirmed epidemiological link". in , the cmdv started a work on autogenous vaccines and overviewed the different situations and regulations in europe [ ] . the outcome was that european ms had various experiences and backgrounds with respect to the autogenous vaccines manufacture and use. as an example, in france, where only bacterial autogenous vaccines are allowed, the manufacturer should be authorized to prepare the autogenous vaccine before marketing the autogenous vaccine. the french agency authorizes the preparation based on four pillars: - the preparation site (called establishment), authorized based on inspection and a technical dossier - the qualified person, mentioned in the authorization as the person who prepares and releases the product -a positive list of bacteria and animal species, in annex of the license given to the manufacturer and based on the lists of disease for which no authorized vaccines with marketing authorization is available as an alternative. in cases when autogenous vaccine is needed when a vaccine is already authorized but in lack of efficacy situation, the manufacturer should seek a derogation. the french market is displayed in figure a . the product -a positive list of bacteria and animal species, in annex of the license given to the manufacturer and based on the lists of disease for which no authorized vaccines with marketing authorization is available as an alternative. in cases when autogenous vaccine is needed when a vaccine is already authorized but in lack of efficacy situation, the manufacturer should seek a derogation. -a positive list of adjuvants, based on the adjuvants used by the manufacturer and in compliance with the maximum residues limit regulation. the french market is displayed in figure a . recommendations paper and new veterinary regulation: then, the cmdv has prepared a recommendation paper [ ] which deals with the manufacture, the control, and the use of viral and bacterial autogenous vaccines in europe. in the framework of the new regulation, this paper will be updated in the coming years. the same locality has been defined as the same and single rearing site, farm where the pathogen is present or multiple sites having an epidemiological link. it has been clarified that groups of animals have an epidemiological link when one of them is to be put in contact with pathogens it has never met before but which are present in other group of animals raised in another rearing site or farm. the movement between sites should be considered. it reflects the actual situation in europe and current field practices in integrated systems for poultry, fishes, and pigs especially. the introduction of the concept of epidemiological link allows now the use of autogenous vaccines in parental lines or, for animals, prior introduction in fattening sites where they will be in contact with new pathogens. in the new regulation, the concept is now directly in the definition, article ( ). the paper states the following: -autogenous vaccines should be used only to solve an exceptional epidemiological situation, not in replacement of the regular authorized vmp. it should be shown that no licensed immunological vmp is available under the cascade prescriptions or that licensed immunological vmp have been proved to be not efficacious for the specific situation. -specific pathogen should have been isolated in the locality, during an outbreak of the disease. - the veterinarian should have made the diagnosis and is responsible of the administration. they are also obliged to report any quality defects and adverse events as regulated by the provisions for the pharmacovigilance. -manufacture should be done under good manufacturing practices (gmp) or gmp-like requirements. the manufacturers should have been authorized, and the compliance with the requirements is controlled by inspection. a qualified person should be designated to ensure the quality of each batch. transmissible spongiform encephalopathies' (tse) guidance should be followed for the starting materials. -a veterinarian should make the diagnosis, collect the antigen, and ensure the traceability whereas an authorized competent site (a laboratory) should ensure the isolation and the identification. -starting materials should comply with the european pharmacopoeia (ph. eur.) requirements; must be sterile; and, if of biological origin, must comply with tse and extraneous agents requirements. seed material should be pure, and inactivation should be validated. -batches should be controlled for sterility, complete inactivation, and endotoxin, if relevant. all these disposals are partially now in articles , , ( ) , and of the new regulation / , whereas adds the provisions that manufacture should be in accordance with the principles of gmp and that guidelines for gmp should be developed for the autogenous vaccines. the autogenous vaccines in eu are a very interesting therapeutic tool to fight against diseases, especially s. suis. the current and future regulatory framework ensure high quality for those products. safety and efficacy are not regulated. however, safety and efficacy of these products are better demonstrated 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medicinal products and repealing directive autogenous vaccines in europe: national approaches to authorisation switching of capsular type impacts on streptococcus suis virulence in mice and pigs masatoshi okura , jean-philippe auger , tomoyuki shibahara , , guillaume goyette-desjardins , marie-rose van calsteren , fumito maruyama , , makoto osaki , mariela segura , marcelo gottschalk , and daisuke takamatsu , treatments for streptococcus suis in the light of evolution lucy a. weinert manufacture, control, and use of autogenous vaccines within european union-the regulatory framework mariette saléry