A Disease Outbreak in Beef Cattle Associated with Anaplasma and Mycoplasma Infections.

An outbreak of disease in a Swedish beef cattle herd initiated an in-depth study to investigate the presence of bacteria and viruses in the blood of clinically healthy (n = 10) and clinically diseased cattle (n = 20) using whole-genome shotgun sequencing (WGSS). The occurrence of infectious agents was also investigated in ticks found attached to healthy cattle (n = 61) and wild deer (n = 23), and in spleen samples from wild deer (n = 30) and wild boars (n = 10). Moreover, blood samples from 84 clinically healthy young stock were analysed for antibodies against Anaplasma phagocytophilum and Babesia divergens. The WGSS revealed the presence of at least three distinct Mycoplasma variants that were most closely related to Mycoplasma wenyonii. Two of these were very similar to a divergent M. wenyonii variant previously only detected in Mexico. These variants tended to be more common in the diseased cattle than in the healthy cattle but were not detected in the ticks or wild animals. The DNA of A. phagocytophilum was detected in similar proportions in diseased (33%) and healthy (40%) cattle, while 70% of the deer, 8% of ticks collected from the cattle and 19% of the ticks collected from deer were positive. Almost all the isolates from the cattle, deer and ticks belonged to Ecotype 1. Based on sequencing of the groEL-gene, most isolates of A. phagocytophilum from cattle were similar and belonged to a different cluster than the isolates from wild deer. Antibodies against A. phagocytophilum were detected in all the analysed samples. In conclusion, uncommon variants of Mycoplasma were detected, probably associated with the disease outbreak in combination with immune suppression due to granulocytic anaplasmosis. Moreover, A. phagocytophilum was found to be circulating within this cattle population, while circulation between cattle and deer occurred infrequently.

Characterization of a Novel Infectious Pancreatic Necrosis Virus (IPNV) from Genogroup 6 Identified in Sea Trout (Salmo trutta) from Lake Vänern, Sweden.

In November 2016, infectious pancreatic necrosis virus (IPNV) was isolated from a broodstock female of landlocked sea trout (Salmo trutta) in Lake Vänern in Sweden. VP2 gene sequencing placed the IPNV isolate in genogroup 6, for which pathogenicity is largely unknown. Lake Vänern hosts landlocked sea trout and salmon populations that are endangered, and thus the introduction of new pathogens poses a major threat. In this study we characterized the novel isolate by conducting an infection trial on three salmonid species present in Lake Vänern, whole genome sequencing of the isolate, and prevalence studies in the wild sea trout and salmon in Lake Vänern. During the infection trial, the pathogenicity of the Swedish isolate was compared to that of a pathogenic genogroup 5 isolate. Dead or moribund fish were collected, pooled, and analyzed by cell culture to identify infected individuals. In the trial, the Swedish isolate was detected in fewer sample pools in all three species compared to the genogroup 5 isolate. In addition, the prevalence studies showed a low prevalence (0.2-0.5%) of the virus in the feral salmonids in Lake Vänern. Together the data suggest that the novel Swedish IPNV genogroup 6 isolate is only mildly pathogenic to salmonids.

Genetic Characterization of Staphylococcus aureus From Subclinical Mastitis Cases in Dairy Cows in Rwanda.

Whole-genome sequencing was carried out on 30 Staphylococcus (S.) aureus isolates from dairy cows with subclinical mastitis from all five provinces of Rwanda. Twenty-five of the isolates produced enough sequence to be analyzed using core genome multilocus sequence typing (cg-MLST). The isolates group into three main clusters. The largest cluster contain isolates of sequence type (ST) 152 (n = 6) and the closely related ST1633 (n = 2). These sequence types have previously mainly been encountered in humans. The isolates of the second-largest cluster belong to ST5477 (n = 5),so far exclusively isolated from cows in Rwanda. The third cluster consists of isolates of ST97 (n = 4), which is a well-known bovine-adapted sequence type. These three clusters were all widespread over the country. Isolates of the usually human-adapted sequence types 1 (n = 2) and 5 (n= 1) were found and a single isolate of ST2430, previously found among humans in Africa. Finally, four isolates of novel sequence types were found: ST7108 (n = 2), ST7109 (n = 1), and ST7110 (n = 1). The blaZ penicillin resistance gene was found in 84% of the isolates and was in all cases corroborated by phenotypic resistance determination. Five (20%) of the isolates carried a tetracycline resistance gene, tet(K) or tetM, and three of these five also displayed phenotypic resistance while two isolates carried a tetM-gene but were yet tetracycline susceptible. Seven (28%) isolates carried the dfrG gene conferring resistance to trimethoprim. Four of these isolates indeed were resistant to trimethoprim while three isolates were sensitive. The str gene conferring resistance to aminoglycosides was found in three isolates; however, none of these displayed resistance to gentamycin. Our data revealed a high diversity of the sequence types of S. aureus isolates from cows with subclinical mastitis in Rwanda. Two major clusters of ST97 and ST5477 are likely to be bovine adapted and cause mastitis while the third cluster of ST152 usually have been found in humans and may signify a recent transmission of these types from human to cows, for example from hand milking. The high prevalence of this sequence type among dairy cows may pose zoonotic threat. The sequence types were widely distributed without any geographic correlation. Penicillin resistance, the most common type of resistance with a prevalence over 80%, but also tetracycline and trimethoprim resistance were displayed by several isolates.

Screening of Eurasian Tundra Reindeer for Viral Sequences by Next-Generation Sequencing.

Reindeer husbandry is essential for the livelihood and culture of indigenous people in the Arctic. Parts of the herding areas are also used as pastures for farm animals, facilitating potential transmission of viruses between species. Following the Covid-19 pandemic, viruses circulating in the wild are receiving increased attention, since they might pose a potential threat to human health. Climate change will influence the prevalence of infectious diseases of both humans and animals. The aim of this study was to detect known and previously unknown viruses in Eurasian tundra reindeer. In total, 623 nasal and 477 rectal swab samples were collected from reindeer herds in Fennoscandia, Iceland, and Eastern Russia during 2016-2019. Next-generation sequencing analysis and BLAST-homology searches indicated the presence of viruses of domesticated and wild animals, such as bovine viral diarrhea virus, bovine papillomavirus, alcephaline herpesvirus 1 and 2, deer mastadenovirus B, bovine rotavirus, and roe deer picobirnavirus. Several viral species previously found in reindeer and some novel species were detected, although the clinical relevance of these viruses in reindeer is largely unknown. These results indicate that it should be possible to find emerging viruses of relevance for both human and animal health using reindeer as a sentinel species.

RT-qPCR assay for detection of mink astrovirus in outbreaks of diarrhea on Danish mink farms.

Diarrhea in mink kits is a major cause of disease and mortality in the mink production. The etiology remains unknown in most outbreaks due to a lack of diagnostic assays. In the current study we present an RT-qPCR method to detect mink astrovirus in fecal samples from mink kits with diarrhea. All sampled animals were classified based on age and patoanatomical evaluation as having pre-weaning diarrhea, diarrhea in the growth period or as having no macroscopic signs of diarrhea. Fecal samples were analyzed for MiAstV with RT-qPCR, next generation sequencing and electron microscopy in parallel. Mink astrovirus was detected with RT-qPCR in 92 out of 203 samples. This detection was confirmed by next generation sequencing in a high proportion of samples (22/27), and by visualization of astrovirus particles with EM in some of the samples. Mink astrovirus was highly prevalent (68%) among kits in the outbreaks of pre-weaning diarrhea, in particular outbreaks from May, while less prevalent in outbreaks in June. Mink astrovirus was detected in outbreaks of diarrhea in the growth period, though in a much lesser extent than in the pre-weaning period. The role of mink astrovirus in the diarrhea disease complex of mink remain to be investigated, and for that purpose this sensitive and robust RT-qPCR can be a valuable tool in the future.

Visualization of intestinal infections with astro- and sapovirus in mink (Neovison vison) kits by in situ hybridization.

Clarification of the infection microbiology remains a challenge in the pre-weaning diarrhea (PWD) syndrome in farmed mink (Neovison vison). Duodenal, jejunal and colon sections from 36 mink kits with PWD were systematically examined by chromogen in situ hybridization targeting two incriminated viruses: mink astrovirus and mink sapovirus. Using the RNAscope® 2.5 HD Duplex Assay, astrovirus and sapovirus were visualized and simultaneously demonstrated in the gut tissue. Both viruses infect enterocytes in the small intestine with a specific localization pattern; astrovirus affects the two apical thirds of the villi, whereas sapovirus generally affects the basal parts of the villi. Furthermore, we demonstrated that astrovirus in mink does not target the goblet cells. This is the first time astro- and calicivirus have been visualized in mink kit gut tissue, and these findings might be important in clarification of the impact of these viruses in the PWD syndrome.

Detection and Genetic Characterization of Viruses Present in Free-Ranging Snow Leopards Using Next-Generation Sequencing.

Snow leopards inhabit the cold, arid environments of the high mountains of South and Central Asia. These living conditions likely affect the abundance and composition of microbes with the capacity to infect these animals. It is important to investigate the microbes that snow leopards are exposed to detect infectious disease threats and define a baseline for future changes that may impact the health of this endangered felid. In this work, next-generation sequencing is used to investigate the fecal (and in a few cases serum) virome of seven snow leopards from the Tost Mountains of Mongolia. The viral species to which the greatest number of sequences reads showed high similarity was rotavirus. Excluding one animal with overall very few sequence reads, four of six animals (67%) displayed evidence of rotavirus infection. A serum sample of a male and a rectal swab of a female snow leopard produced sequence reads identical or closely similar to felid herpesvirus 1, providing the first evidence that this virus infects snow leopards. In addition, the rectal swab from the same female also displayed sequence reads most similar to feline papillomavirus 2, which is the first evidence for this virus infecting snow leopards. The rectal swabs from all animals also showed evidence for the presence of small circular DNA viruses, predominantly Circular Rep-Encoding Single-Stranded (CRESS) DNA viruses and in one case feline anellovirus. Several of the viruses implicated in the present study could affect the health of snow leopards. In animals which are under environmental stress, for example, young dispersing individuals and lactating females, health issues may be exacerbated by latent virus infections.

Proficiency Testing of Metagenomics-Based Detection of Food-Borne Pathogens Using a Complex Artificial Sequencing Dataset.

Metagenomics-based high-throughput sequencing (HTS) enables comprehensive detection of all species comprised in a sample with a single assay and is becoming a standard method for outbreak investigation. However, unlike real-time PCR or serological assays, HTS datasets generated for pathogen detection do not easily provide yes/no answers. Rather, results of the taxonomic read assignment need to be assessed by trained personnel to gain information thereof. Proficiency tests are important instruments of validation, harmonization, and standardization. Within the European Union funded project COMPARE [COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe], we conducted a proficiency test to scrutinize the ability to assess diagnostic metagenomics data. An artificial dataset resembling shotgun sequencing of RNA from a sample of contaminated trout was provided to 12 participants with the request to provide a table with per-read taxonomic assignments at species level and a report with a summary and assessment of their findings, considering different categories like pathogen, background, or contaminations. Analysis of the read assignment tables showed that the software used reliably classified the reads taxonomically overall. However, usage of incomplete reference databases or inappropriate data pre-processing caused difficulties. From the combination of the participants' reports with their read assignments, we conclude that, although most species were detected, a number of important taxa were not or not correctly categorized. This implies that knowledge of and awareness for potentially dangerous species and contaminations need to be improved, hence, capacity building for the interpretation of diagnostic metagenomics datasets is necessary.

Identification and validation of internal reference genes for real-time quantitative polymerase chain reaction-based studies in Hyalomma anatolicum ticks.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by the orthonairovirus CCHF virus (CCHFV). Ticks of the genus Hyalomma are the viral reservoir and they represent the main vector transmitting the virus to their hosts during blood feeding. However, how CCHFV replicates in its natural arthropod host cells and the nature of virus/host interactions are still largely unknown. With the aim of developing tools for use in this field, we identified and validated expression of four candidate endogenous control tick genes in a Hyalomma anatolicum-derived cell line. These genes will be useful for normalization of viral/cellular transcripts in infection/expression studies or as internal controls in molecular epidemiology surveys of pathogens transmitted by Hyalomma ticks.

Seroprevalence of pestivirus in Eurasian tundra reindeer in Finland, Sweden, Norway, Iceland and Russian Federation.

Reindeer herding is of great importance for the indigenous people of the Fennoscandia peninsula and northern Russia. There are also free-ranging feral populations of reindeer in Finland, Iceland, Norway and Russian Federation. The genus Pestivirus contains several viral species that infect ungulates and often show capacity to transmit between different host species. Sera from 520 Eurasian tundra reindeer (Rangifer tarandus tarandus) from Finland, Sweden, Norway, Iceland and Russian Federation were analysed and the prevalence of pestivirus-specific antibodies was determined. Seropositivity proportion was 48.5% for Sweden and 41.2% for Norway, but only 1.6% for Iceland and 2.5% for Finland. All Russian reindeer investigated were seronegative. Pan-pestivirus RT-PCR of seronegative animals (n = 156) from seropositive herds confirmed their negative status. These results indicate unexpectedly non-uniform circulation of an as yet uncharacterised pestivirus in Eurasian reindeer populations. The high seroprevalence in some regions warrants further studies of pestivirus infection dynamics, effects on reindeer health and population dynamics.

Comparing the treatment effect of narrow spectrum antimicrobial, probiotic and fluid with amoxicillin in mink kits (Neovison vison) with pre-weaning diarrhea.

Pre-weaning diarrhea in mink kits (PWD), also known as "sticky kits" is a multifactorial syndrome of considerable concern in the mink production. Evidence based treatment protocols are not available, and treatment is therefore empirical and often based on the use of antimicrobials. The purpose of the study was to test the effect of 3 alternative treatments to a standard antibiotic treatment, to characterize the study groups microbiologically, and finally to compare the intestinal microbiota of the different treatment groups at the age of 42 days. In total, 226 one to three week old mink kits with PWD from 36 litters were treated with either 1) Lactobacillus reuteri, 2) benzylpenicillin, 3) Ringer lactate or 4) amoxicillin (controls). Effects of the treatments were measured as weight gain from day 0 to day 15 and mortality. Multivariable linear mixed model regression showed no significant difference in weight gain between probiotic-, penicillin or fluid-treated mink kits and the amoxicillin treated controls. There was also no significant difference in mortality risk between the treatment groups. Bacterial culture and next generation sequencing of the viral contents showed that the study groups were uniform with a high frequency of Staphylococcus intermedius group (SIG) bacteria, Escherichia coli, Enterococcus hirae, Mamastrovirus and Sapovirus which were representative for mink kits with PWD. 16S sequencing results of the bacterial microbiota, when the kits were 42 days old were dominated by clostridia in all groups and showed no clear differences in the bacterial composition between the different treatment groups.

Chlamydia pecorum Associated With an Outbreak of Infectious Keratoconjunctivitis in Semi-domesticated Reindeer in Sweden.

Infectious keratoconjunctivitis (IKC), the most common ocular disease in ruminants worldwide, has affected semi-domesticated Eurasian reindeer (Rangifer tarandus tarandus) for over 100 years, both as individual cases and in outbreaks affecting tens to hundreds of animals. Recurrent IKC outbreaks have been affecting a semi-domesticated reindeer herd in Östra Kikkejaure (Norrbotten county, Sweden) from 2014. The latest episode of these recurrent outbreaks, in winter 2016/2017, was investigated in this study. Clinical findings were in line with previous reports of IKC in semi-domesticated reindeer and the clinical signs displayed by the affected animals (n = 30) included increased lacrimation, follicular conjunctivitis, purulent secretions around the affected eyes and corneal edema. Laboratory analyses of the samples revealed the presence of Chlamydiaceae in most samples obtained from the clinically affected animals (98.3%, n = 60), but also a high seroprevalence of cervid herpesvirus 2 (CvHV2) antibodies (56.6%, n = 53). Moraxella bovoculi was isolated from nine IKC-affected animals during the outbreak (45.0%, n = 20). All affected animals were treated with long-acting antibiotics and recovered from the disease, testing negative for the presence of Chlamydiaceae DNA by PCR 16 days and 3 months after the initial treatment. For the first time, Chlamydia pecorum was identified in semi-domesticated reindeer, and the involvement of Chlamydiaceae in a clinical outbreak of IKC is reported. The CvHV2 seroprevalence (56.6%) and the data obtained from a previous outbreak in 2014 also suggest the involvement of the reindeer alphaherpesvirus in the recurrent outbreaks.

Investigation of the viral and bacterial microbiota in intestinal samples from mink (Neovison vison) with pre-weaning diarrhea syndrome using next generation sequencing.

Pre-weaning diarrhea (PWD) in mink kits is a common multifactorial syndrome on commercial mink farms. Several potential pathogens such as astroviruses, caliciviruses, Escherichia coli and Staphylococcus delphini have been studied, but the etiology of the syndrome seems complex. In pooled samples from 38 diarrheic and 42 non-diarrheic litters, each comprising of intestinal contents from 2-3 mink kits from the same litter, the bacterial populations were studied using Illumina Next Generation Sequencing technology and targeted 16S amplicon sequencing. In addition, we used deep sequencing to determine and compare the viral intestinal content in 31 healthy non-diarrheic and 30 diarrheic pooled samples (2-3 mink kits from the same litter per pool). The results showed high variations in composition of the bacterial species between the pools. Enterococci, staphylococci and streptococci dominated in both diarrheic and non-diarrheic pools. However, enterococci accounted for 70% of the reads in the diarrheic group compared to 50% in the non-diarrheic group and this increase was at the expense of staphylococci and streptococci which together accounted for 45% and 17% of the reads in the non-diarrheic and diarrheic group, respectively. Moreover, in the diarrheic pools there were more reads assigned to Clostridia, Escherichia-Shigella and Enterobacter compared to the non-diarrheic pools. The taxonomically categorized sequences from the virome showed that the most prevalent viruses in all pools were caliciviruses and mamastroviruses (almost exclusively type 10). However, the numbers of reads assigned to caliciviruses were almost 3 times higher in the diarrheic pools compared the non-diarrheic pools and Sapporo-like caliciviruses were more abundant than the Norwalk-like caliciviruses. The results from this study have contributed to the insight into the changes in the intestinal microbiota associated with the PWD syndrome of mink.

An unusual presentation of pseudocowpox associated with an outbreak of pustular ulcerative vulvovaginitis in a Swedish dairy herd.

Species Pseudocowpox virus (PCPV; family Poxviridae) is known to cause pustular cutaneous disease in cattle. We describe an outbreak of pseudocowpox with an unusual clinical picture in a free-stall dairy herd of ~80 cows. Approximately 90% of the cows had vesicles, erosions, papules, and scabs on the vulva and vaginal mucosa. Histologic analysis of biopsy tissues indicated a primary, although not specified, viral infection. Transmission electron microscopy revealed parapoxvirus particles in both tissue and vesicular materials. Deep sequencing analysis of extracted DNA from swabbed vesicle areas gave a contig of nearly 120,000 nucleotides, matching the PCPV strain VR 634 with 100% identity. Analyses confirmed the absence of other potential causes of pustular vulvovaginitis such as bovine herpesvirus 1 and Ureaplasma diversum. A rolling cow brush was suspected to be the fomite.

Multiple pathogen biomarker detection using an encoded bead array in droplet PCR.

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

Accurate Detection of Avian Respiratory Viruses by Use of Multiplex PCR-Based Luminex Suspension Microarray Assay.

A novel oligonucleotide suspension microarray (Luminex microsphere system) was developed for the rapid detection of avian respiratory viruses of major clinical importance. This test was optimized and validated with 70 clinical samples. The developed tool was accurate for high-throughput detection and differentiation of the most important avian respiratory viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV) in single- and mixed-virus infections. A multiplex reverse transcriptase PCR (RT-PCR), followed by a monoplex or a multiplex Luminex assays, were realized using a Luminex 200 analyzer instrument. The sensitivity, specificity, and reproducibility of the multiplex DNA suspension microarray system were evaluated. The results showed no significant differences in the median fluorescence intensity (MFI) value in monoplex and multiplex Luminex assays. The sensitivity and specificity proved to be completely concordant with monoplex real-time RT-PCR. We demonstrated that the multiplex DNA suspension microarray system is an accurate, high-throughput, and relatively simple method for the rapid detection of the main respiratory viruses of poultry.

Identification of combinatorial host-specific signatures with a potential to affect host adaptation in influenza A H1N1 and H3N2 subtypes.

BACKGROUND: The underlying strategies used by influenza A viruses (IAVs) to adapt to new hosts while crossing the species barrier are complex and yet to be understood completely. Several studies have been published identifying singular genomic signatures that indicate such a host switch. The complexity of the problem suggested that in addition to the singular signatures, there might be a combinatorial use of such genomic features, in nature, defining adaptation to hosts. RESULTS: We used computational rule-based modeling to identify combinatorial sets of interacting amino acid (aa) residues in 12 proteins of IAVs of H1N1 and H3N2 subtypes. We built highly accurate rule-based models for each protein that could differentiate between viral aa sequences coming from avian and human hosts. We found 68 host-specific combinations of aa residues, potentially associated to host adaptation on HA, M1, M2, NP, NS1, NEP, PA, PA-X, PB1 and PB2 proteins of the H1N1 subtype and 24 on M1, M2, NEP, PB1 and PB2 proteins of the H3N2 subtypes. In addition to these combinations, we found 132 novel singular aa signatures distributed among all proteins, including the newly discovered PA-X protein, of both subtypes. We showed that HA, NA, NP, NS1, NEP, PA-X and PA proteins of the H1N1 subtype carry H1N1-specific and HA, NA, PA-X, PA, PB1-F2 and PB1 of the H3N2 subtype carry H3N2-specific signatures. M1, M2, PB1-F2, PB1 and PB2 of H1N1 subtype, in addition to H1N1 signatures, also carry H3N2 signatures. Similarly M1, M2, NP, NS1, NEP and PB2 of H3N2 subtype were shown to carry both H3N2 and H1N1 host-specific signatures (HSSs). CONCLUSIONS: To sum it up, we computationally constructed simple IF-THEN rule-based models that could distinguish between aa sequences of avian and human IAVs. From the rules we identified HSSs having a potential to affect the adaptation to specific hosts. The identification of combinatorial HSSs suggests that the process of adaptation of IAVs to a new host is more complex than previously suggested. The present study provides a basis for further detailed studies with the aim to elucidate the molecular mechanisms providing the foundation for the adaptation process.

Genetic variation and dynamics of infections of equid herpesvirus 5 in individual horses.

Equid herpesvirus 5 (EHV-5) is related to the human Epstein-Barr virus (human herpesvirus 4) and has frequently been observed in equine populations worldwide. EHV-5 was previously assumed to be low to non-pathogenic; however, studies have also related the virus to the severe lung disease equine multinodular pulmonary fibrosis (EMPF). Genetic information of EHV-5 is scanty: the whole genome was recently described and only limited nucleotide sequences are available. In this study, samples were taken twice 1 year apart from eight healthy horses at the same professional training yard and samples from a ninth horse that was diagnosed with EMPF with samples taken pre- and post-mortem to analyse partial glycoprotein B (gB) gene of EHV-5 by using next-generation sequencing. The analysis resulted in 27 partial gB gene sequences, 11 unique sequence types and five amino acid sequences. These sequences could be classified within four genotypes (I-IV) of the EHV-5 gB gene based on the degree of similarity of the nucleotide and amino acid sequences, and in this work horses were shown to be identified with up to three different genotypes simultaneously. The observations showed a range of interactions between EHV-5 and the host over time, where the same virus persists in some horses, whereas others have a more dynamic infection pattern including strains from different genotypes. This study provides insight into the genetic variation and dynamics of EHV-5, and highlights that further work is needed to understand the EHV-5 interaction with its host.

A complete map of potential pathogenicity markers of avian influenza virus subtype H5 predicted from 11 expressed proteins.

BACKGROUND: Polybasic cleavage sites of the hemagglutinin (HA) proteins are considered to be the most important determinants indicating virulence of the avian influenza viruses (AIV). However, evidence is accumulating that these sites alone are not sufficient to establish high pathogenicity. There need to exist other sites located on the HA protein outside the cleavage site or on the other proteins expressed by AIV that contribute to the pathogenicity. RESULTS: We employed rule-based computational modeling to construct a map, with high statistical significance, of amino acid (AA) residues associated to pathogenicity in 11 proteins of the H5 type viruses. We found potential markers of pathogenicity in all of the 11 proteins expressed by the H5 type of AIV. AA mutations S-43(HA1)-D, D-83(HA1)-A in HA; S-269-D, E-41-H in NA; S-48-N, K-212-N in NS1; V-166-A in M1; G-14-E in M2; K-77-R, S-377-N in NP; and Q-48-P in PB1-F2 were identified as having a potential to shift the pathogenicity from low to high. Our results suggest that the low pathogenicity is common to most of the subtypes of the H5 AIV while the high pathogenicity is specific to each subtype. The models were developed using public data and validated on new, unseen sequences. CONCLUSIONS: Our models explicitly define a viral genetic background required for the virus to be highly pathogenic and thus confirm the hypothesis of the presence of pathogenicity markers beyond the cleavage site.