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We report the detection of a clade 2.3.4.4b A(H5N1) reassortant virus with a neuraminidase surface protein derived from a North American lineage low-pathogenic avian influenza virus. This virus caused a widespread and ongoing outbreak across 45 poultry farms in British Columbia, Canada. Isolates from 8 farms reveal a mutation in the neuraminidase protein (H275Y) that is exceptionally rare among clade 2.3.4.4b viruses (present in 0.045% of publicly available clade 2.3.4.4b isolates). NA-H275Y is a well-known marker of resistance to the neuraminidase inhibitor oseltamivir. We demonstrate that this substitution maintains its resistance phenotype on the genetic background of H5N1 clade 2.3.4.4b viruses.

Senecavirus A (SVA) continues to cause vesicular lesions in swine in Canada and many regions worldwide. Since the vesicular lesions caused by SVA are similar to those caused by foot and mouth disease virus, swine vesicular disease virus and vesicular stomatitis virus, a foreign animal disease investigation must be initiated to rule out these diseases. SVA isolates from pigs displaying vesicular lesions in Canada from 2015 to 2023 were sequenced, and phylogeographic analysis was performed using the complete genome sequences. The results infer that SVA has spread between the United States and Canada several times. In addition, the results suggest that SVA spreads from different regions. SVA spread was inferred from Canada into Thailand, India and Mexico and inferred from the United States to Brazil, Columbia, Chile and China with ten separate introductions. Furthermore, recombination was observed in SVA genomes from Canada, the United States and China.

Wild birds are important hosts of influenza A viruses (IAVs) and play an important role in their ecology. The emergence of the A/goose/Guangdong/1/1996 H5N1 (Gs/GD) lineage marked a shift in IAV ecology, leading to recurrent outbreaks and mortality in wild birds from 2002 onwards. This lineage has evolved and diversified over time, with a recent important derivative being the 2.3.4.4b sub-lineage, which has caused significant mortality events in wild bird populations. An H5N1 clade 2.3.4.4b virus was transmitted into North America from Eurasia in 2021, with the first detection being in Newfoundland and Labrador in Atlantic Canada, and this virus and its reassortants then spread broadly throughout North America and beyond. Following the first 2021 detection, there have been three additional known incursions of Eurasian-origin strains into Atlantic Canada, a second H5N1 strain in 2022 and two H5N5 strains in 2023. In this study, we document a fifth incursion in Atlantic Canada that occurred in 2023 by another H5N5 strain. This strain spread throughout Atlantic Canada and into Quebec, infecting numerous species of wild birds and mammals. Genomic analysis revealed mammalian-adaptive mutations in some of the detected viruses (PB2-E627K and PB2-D701N) and mutations in the hemagglutinin (HA) and neuraminidase (NA) genes that are associated with enhanced viral fitness and avian transmission capabilities. Our findings indicate that this virus is continuing to circulate in wildlife, and confirms Atlantic Canada is an important North American entry point for Eurasian IAVs. Continued surveillance and genomic analysis of IAVs detected in the region is crucial to monitor the evolution of these viruses and assess potential risks to wildlife and public health., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

During the past 25 years, vesicular stomatitis virus (VSV) has produced multiple outbreaks in the US, resulting in the emergence of different viral lineages. Currently, very little is known about the pathogenesis of many of these lineages, thus limiting our understanding of the potential biological factors favoring each lineage in these outbreaks. In this study, we aimed to determine the potential phenotypic differences between two VSV Indiana (VSIV) serotype epidemic strains using a pig model. These strains are representative of the epidemic lineages that affected the US between 1997 and 1998 (IN98COE) and between 2019 and 2020 (IN0919WYB2), the latter responsible for one of the most extensive outbreaks in the US. Our initial genome analysis revealed the existence of 121 distinct mutations between both strains, including the presence of a 14-nucleotide insertion in the intergenic region between the G and L genes observed in IN0919WYB2. The levels of viral RNA in clinical samples between pigs infected with IN98COE or IN0919WYB2 were compared. Overall, higher and prolonged expression of viral RNA in pigs infected with IN98COE was observed. However, clinically, IN0919WYB2 was slightly more virulent than IN98COE, as well as more efficient at producing infection through contact transmission. Additionally, infectious virus was recovered from more samples when the pigs were infected with IN0919WYB2, as revealed by virus isolation in cell culture, indicating the increased ability of this virus to replicate in pigs. Sequence analyses conducted from isolates recovered from both experimental groups showed that IN0919WYB2 produced more variability during the infection, denoting the potential of this strain to evolve rapidly after a single infection-contact transmission event in pigs. Collectively, the results showed that epidemic strains of VSIV may represent disparate phenotypes in terms of virulence/transmissibility for livestock, a situation that may impact the intensity of an epidemic outbreak. This study also highlights the relevance of pathogenesis studies in pigs to characterize phenotypic differences in VSV strains affecting livestock in the field.

Bats are recognized as natural reservoirs for an array of diverse viruses, particularly coronaviruses, which have been linked to major human diseases like SARS-CoV and MERS-CoV. These viruses are believed to have originated in bats, highlighting their role in virus ecology and evolution. Our study focuses on the molecular characterization of bat-derived coronaviruses (CoVs) in Canada. Tissue samples from 500 bat specimens collected in Canada were analyzed using pan-coronavirus RT-PCR assays to detect the presence of CoVs from four genera: Alpha-CoVs, Beta-CoV, Gamma-CoV, and Delta-CoV. Phylogenetic analysis was performed targeting the RNA-dependent RNA polymerase (RdRP) gene. Our results showed an overall 1.4% CoV positivity rate in our bat sample size. Phylogenetic analysis based on the ~600 bp sequences led to the identification of an unclassified subgenus of Alpha-CoV, provisionally named Eptacovirus. The findings contribute to a better understanding of the diversity and evolution of CoVs found in the bat species of Canada. The current study underscores the significance of bats in the epidemiology of CoVs and enhances the knowledge of their genetic diversity and potential impact on global public health.

Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a tick-borne, risk group 4 pathogen that often causes a severe haemorrhagic disease in humans (CCHF) with high case fatality rates. The virus is believed to be maintained in a tick-vertebrate-tick ecological cycle involving numerous wild and domestic animal species; however the biology of CCHFV infection in these animals remains poorly understood. Here, we experimentally infect domestic sheep with CCHFV Kosovo Hoti, a clinical isolate representing high pathogenicity to humans and increasingly utilized in current research. In the absence of prominent clinical signs, the infection leads to an acute viremia and coinciding viral shedding, fever and markers for potential impairment in liver and kidney functions. A number of host responses distinguish the subclinical infection in sheep versus fatal infection in humans. These include an early reduction of neutrophil recruitment and its chemoattractant, IL-8, in the blood stream of infected sheep, whereas neutrophil infiltration and elevated IL-8 are features of fatal CCHFV infections reported in immunodeficient mice and humans. Several inflammatory cytokines that correlate with poor disease outcomes in humans and have potential to cause vascular dysfunction, a primary hallmark of severe CCHF, are down-regulated or restricted from increasing in sheep. Of particular interest, the detection of CCHFV RNA (including full-length genome) in a variety of sheep tissues long after the acute phase of infection indicates a widespread viral dissemination in the host and suggests a potentially long-term persisting impact of CCHFV infection. These findings reveal previously unrecognized aspects of CCHFV biology in animals.

Motivation: State-of-the-art tools for classifying metagenomic sequencing reads provide both rapid and accurate options, although the combination of both in a single tool is a constantly improving area of research. The machine learning-based Naïve Bayes Classifier (NBC) approach provides a theoretical basis for accurate classification of all reads in a sample., Results: We developed the multithreaded Minimizer-based Naïve Bayes Classifier (MNBC) tool to improve the NBC approach by applying minimizers, as well as plurality voting for closely related classification scores. A standard reference- and test-sequence framework using simulated variable-length reads benchmarked MNBC with six other state-of-the-art tools: MetaMaps, Ganon, Kraken2, KrakenUniq, CLARK, and Centrifuge. We also applied MNBC to the "marine" and "strain-madness" short-read metagenomic datasets in the Critical Assessment of Metagenome Interpretation (CAMI) II challenge using a corresponding database from the time. MNBC efficiently identified reads from unknown microorganisms, and exhibited the highest species- and genus-level precision and recall on short reads, as well as the highest species-level precision on long reads. It also achieved the highest accuracy on the "strain-madness" dataset., Availability and Implementation: MNBC is freely available at: https://github.com/ComputationalPathogens/MNBC., (© The Author(s) 2024. Published by Oxford University Press.)

Following the detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada, in late 2021, avian influenza virus (AIV) surveillance in wild birds was scaled up across Canada. Herein, we present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds during the first year (November 2021-November 2022) following the incursions of HPAIV from Eurasia. The key objectives of the surveillance program were to (i) identify the presence, distribution, and spread of HPAIV and other AIVs; (ii) identify wild bird morbidity and mortality associated with HPAIV; (iii) identify the range of wild bird species infected by HPAIV; and (iv) genetically characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central Flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live-captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole-genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic Flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds, to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in the presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across interagency partners., Importance: We present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds in the year following the first detection of highly pathogenic avian influenza virus (HPAIV) H5N1 on the continent. The surveillance program tested over 17,000 wild birds, both sick and apparently healthy, which revealed spatiotemporal and taxonomic patterns in HPAIV prevalence and mortality across Canada. The significant shift in the presence and distribution of HPAIV in Canada's wild birds underscores the need for sustained investment in wild bird surveillance and collaboration across One Health partners., Competing Interests: The authors declare no conflict of interest.

Highly pathogenic avian influenza (HPAI) viruses have spread at an unprecedented scale, leading to mass mortalities in birds and mammals. In 2023, a transatlantic incursion of HPAI A(H5N5) viruses into North America was detected, followed shortly thereafter by a mammalian detection. As these A(H5N5) viruses were similar to contemporary viruses described in Eurasia, the transatlantic spread of A(H5N5) viruses was most likely facilitated by pelagic seabirds. Some of the Canadian A(H5N5) viruses from birds and mammals possessed the PB2-E627K substitution known to facilitate adaptation to mammals. Ferrets inoculated with A(H5N5) viruses showed rapid, severe disease onset, with some evidence of direct contact transmission. However, these viruses have maintained receptor binding traits of avian influenza viruses and were susceptible to oseltamivir and zanamivir. Understanding the factors influencing the virulence and transmission of A(H5N5) in migratory birds and mammals is critical to minimize impacts on wildlife and public health., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

We describe an unusual mortality event caused by a highly pathogenic avian influenza (HPAI) A(H5N1) virus clade 2.3.4.4b involving harbor (Phoca vitulina) and gray (Halichoerus grypus) seals in the St. Lawrence Estuary, Quebec, Canada, in 2022. Fifteen (56%) of the seals submitted for necropsy were considered to be fatally infected by HPAI H5N1 containing fully Eurasian or Eurasian/North American genome constellations. Concurrently, presence of large numbers of bird carcasses infected with HPAI H5N1 at seal haul-out sites most likely contributed to the spillover of infection to the seals. Histologic changes included meningoencephalitis (100%), fibrinosuppurative alveolitis, and multiorgan acute necrotizing inflammation. This report of fatal HPAI H5N1 infection in pinnipeds in Canada raises concerns about the expanding host of this virus, the potential for the establishment of a marine mammal reservoir, and the public health risks associated with spillover to mammals.Nous décrivons un événement de mortalité inhabituelle causé par un virus de l'influenza aviaire hautement pathogène A(H5N1) clade 2.3.4.4b chez des phoques communs (Phoca vitulina) et gris (Halichoerus grypus) dans l'estuaire du Saint-Laurent au Québec, Canada, en 2022. Quinze (56%) des phoques soumis pour nécropsie ont été considérés comme étant fatalement infectés par le virus H5N1 de lignées eurasiennes ou de réassortiment eurasiennes/nord-américaines. Un grand nombre simultané de carcasses d'oiseaux infectés par le H5N1 sur les sites d'échouement a probablement contribué à la contamination de ces phoques. Les changements histologiques associés à cette infection incluaient : méningo-encéphalite (100%), alvéolite fibrinosuppurée et inflammation nécrosante aiguë multi-organique. Cette documentation soulève des préoccupations quant à l'émergence de virus mortels, à la possibilité d'établissement de réservoirs chez les mammifères marins, et aux risques pour la santé publique associés aux propagations du virus chez les mammifères.

Two cat nasal swabs from Canada's earliest confirmed SARS-CoV-2 positive domestic cats were sequenced to over 99% SARS-CoV-2 genome coverage. One cat had lineage A.23.1 SARS-CoV-2 not reported before in animals. Both sequences have multiple spike gene mutations and clustered closely with human-derived sequences in the global SARS-CoV-2 phylogenetic tree., Competing Interests: The authors declare no conflict of interest.

In this paper, we report the characterization of a genetically modified live-attenuated African swine fever virus (ASFV) field strain isolated from Vietnam. The isolate, ASFV-GUS-Vietnam, belongs to p72 genotype II, has six multi-gene family (MGF) genes deleted, and an Escherichia coli GusA gene (GUS) inserted. When six 6-8-week-old pigs were inoculated with ASFV-GUS-Vietnam oro-nasally (2 × 10 5 TCID 50 /pig), they developed viremia, mild fever, lethargy, and inappetence, and shed the virus in their oral and nasal secretions and feces. One of the pigs developed severe clinical signs and was euthanized 12 days post-infection, while the remaining five pigs recovered. When ASFV-GUS-Vietnam was inoculated intramuscularly (2 × 10 3 TCID 50 /pig) into four 6-8 weeks old pigs, they also developed viremia, mild fever, lethargy, inappetence, and shed the virus in their oral and nasal secretions and feces. Two contact pigs housed together with the four intramuscularly inoculated pigs, started to develop fever, viremia, loss of appetite, and lethargy 12 days post-contact, confirming horizontal transmission of ASFV-GUS-Vietnam. One of the contact pigs died of ASF on day 23 post-contact, while the other one recovered. The pigs that survived the exposure to ASFV-GUS-Vietnam via the mucosal or parenteral route were fully protected against the highly virulent ASFV Georgia 2007/1 challenge. This study showed that ASFV-GUS-Vietnam field isolate is able to induce complete protection in the majority of the pigs against highly virulent homologous ASFV challenge, but has the potential for horizontal transmission, and can be fatal in some animals. This study highlights the need for proper monitoring and surveillance when ASFV live-attenuated virus-based vaccines are used in the field for ASF control in endemic countries.

We conducted an in silico analysis to better understand the potential factors impacting host adaptation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in white-tailed deer, humans, and mink due to the strong evidence of sustained transmission within these hosts. Classification models trained on single nucleotide and amino acid differences between samples effectively identified white-tailed deer-, human-, and mink-derived SARS-CoV-2. For example, the balanced accuracy score of Extremely Randomized Trees classifiers was 0.984 ± 0.006. Eighty-eight commonly identified predictive mutations are found at sites under strong positive and negative selective pressure. A large fraction of sites under selection (86.9%) or identified by machine learning (87.1%) are found in genes other than the spike. Some locations encoded by these gene regions are predicted to be B- and T-cell epitopes or are implicated in modulating the immune response suggesting that host adaptation may involve the evasion of the host immune system, modulation of the class-I major-histocompatibility complex, and the diminished recognition of immune epitopes by CD8+ T cells. Our selection and machine learning analysis also identified that silent mutations, such as C7303T and C9430T, play an important role in discriminating deer-derived samples across multiple clades. Finally, our investigation into the origin of the B.1.641 lineage from white-tailed deer in Canada discovered an additional human sequence from Michigan related to the B.1.641 lineage sampled near the emergence of this lineage. These findings demonstrate that machine-learning approaches can be used in combination with evolutionary genomics to identify factors possibly involved in the cross-species transmission of viruses and the emergence of novel viral lineages.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible virus capable of infecting and establishing itself in human and wildlife populations, such as white-tailed deer. This fact highlights the importance of developing novel ways to identify genetic factors that contribute to its spread and adaptation to new host species. This is especially important since these populations can serve as reservoirs that potentially facilitate the re-introduction of new variants into human populations. In this study, we apply machine learning and phylogenetic methods to uncover biomarkers of SARS-CoV-2 adaptation in mink and white-tailed deer. We find evidence demonstrating that both non-synonymous and silent mutations can be used to differentiate animal-derived sequences from human-derived ones and each other. This evidence also suggests that host adaptation involves the evasion of the immune system and the suppression of antigen presentation. Finally, the methods developed here are general and can be used to investigate host adaptation in viruses other than SARS-CoV-2., Competing Interests: The authors declare no conflict of interest.

The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

White-tailed deer (WTD) are susceptible to SARS-CoV-2 and represent an important species for surveillance. Samples from WTD (n = 258) collected in November 2021 from Québec, Canada were analyzed for SARS-CoV-2 RNA. We employed viral genomics and host transcriptomics to further characterize infection and investigate host response. We detected Delta SARS-CoV-2 (B.1.617.2) in WTD from the Estrie region; sequences clustered with human sequences from October 2021 from Vermont, USA, which borders this region. Mutations in the S-gene and a deletion in ORF8 were detected. Host expression patterns in SARS-CoV-2 infected WTD were associated with the innate immune response, including signaling pathways related to anti-viral, pro- and anti-inflammatory signaling, and host damage. We found limited correlation between genes associated with innate immune response from human and WTD nasal samples, suggesting differences in responses to SARS-CoV-2 infection. Our findings provide preliminary insights into host response to SARS-CoV-2 infection in naturally infected WTD., Competing Interests: The authors declare no conflicts relevant to this article., (© 2023 The Authors.)

Wholly Eurasian highly pathogenic avian influenza H5N1 clade 2.3.4.4b virus was isolated from 2 free-ranging black bears with meningoencephalitis in Quebec, Canada. We found that isolates from both animals had the D701N mutation in the polymerase basic 2 gene, previously known to promote adaptation of H5N1 viruses to mammal hosts.

In December 2022 and January 2023, we isolated clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) viruses from six American crows ( Corvus brachyrhynchos ) from Prince Edward Island and a red fox ( Vulpes vulpes ) from Newfoundland, Canada. Using full-genome sequencing and phylogenetic analysis, these viruses were found to fall into two distinct phylogenetic clusters: one group containing H5N1 viruses that had been circulating in North and South America since late 2021, and the other one containing European H5N1 viruses reported in late 2022. The transatlantic re-introduction for the second time by pelagic/Icelandic bird migration via the same route used during the 2021 incursion of Eurasian origin H5N1 viruses into North America demonstrates that migratory birds continue to be the driving force for transcontinental dissemination of the virus. This new detection further demonstrates the continual long-term threat of H5N1 viruses for poultry and mammals and the subsequent impact on various wild bird populations wherever these viruses emerge. The continual emergence of clade 2.3.4.4b H5Nx viruses requires vigilant surveillance in wild birds, particularly in areas of the Americas, which lie within the migratory corridors for long-distance migratory birds originating from Europe and Asia. Although H5Nx viruses have been detected at higher rates in North America since 2021, a bidirectional flow of H5Nx genes of American origin viruses to Europe has never been reported. In the future, coordinated and systematic surveillance programs for HPAI viruses need to be launched between European and North American agencies.

Adenoviral hemorrhagic disease (AHD), caused by deer atadenovirus A (OdAdV-1), affects captive and free-ranging cervids across North America. Here we present a case of AHD in a 6-month-old female elk calf from a farm in Alberta. Histopathology revealed multisystemic vasculitis with endothelial intranuclear inclusion bodies, pulmonary hemorrhage, and small intestinal hemorrhage characteristic of the acute systemic form of AHD. Immunohistochemistry was positive for OdAdV-1, confirming the diagnosis. Whole-genome sequencing of the virus was conducted for phylogenetic comparison. This is the 1st reported case of AHD in a farmed elk in Canada and the 1st reported case in an elk in Alberta. Key clinical message: Adenoviral hemorrhagic disease (AHD) is an emerging disease that should be investigated as a top differential when diagnosticians and veterinarians encounter young cervids found dead with pulmonary edema or hemorrhage and/or hemorrhagic enteropathy., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

African swine fever (ASF) is a high-consequence transboundary hemorrhagic fever of swine. It continues to spread across the globe causing socio-economic issues and threatening food security and biodiversity. In 2020, Nigeria reported a major ASF outbreak, killing close to half a million pigs. Based on the partial sequences of the genes B646L (p72) and E183L (p54), the virus responsible for the outbreak was identified as an African swine fever virus (ASFV) p72 genotype II. Here, we report further characterization of ASFV RV502, one of the isolates obtained during the outbreak. The whole genome sequence of this virus revealed a deletion of 6535 bp between the nucleotide positions 11,760-18,295 of the genome, and an apparent reverse complement duplication of the 5' end of the genome at the 3' end. Phylogenetically, ASFV RV502 clustered together with ASFV MAL/19/Karonga and ASFV Tanzania/Rukwa/2017/1 suggesting that the virus responsible for the 2020 outbreak in Nigeria has a South-eastern African origin.

The complete genome of a novel torque teno virus species (Torque teno equus virus 2 (TTEqV2) isolate Alberta/2018) was obtained by high-throughput sequencing (HTS) of nucleic acid extracted from the lung and liver tissue of a Quarter Horse gelding that died of nonsuppurative encephalitis in Alberta, Canada. The 2805 nucleotide circular genome is the first complete genome from the Mutorquevirus genus and has been approved as a new species by the International Committee on Taxonomy of Viruses. The genome contains several characteristic features of torque teno virus (TTV) genomes, including an ORF1 encoding a putative 631 aa capsid protein with an arginine-rich N-terminus, several rolling circle replication associated amino acid motifs, and a downstream polyadenylation signal. A smaller overlapping ORF2 encodes a protein with an amino acid motif (WX 7 HX 3 CXCX 5 H) which, in general, is highly conserved in TTVs and anelloviruses. The UTR contains two GC-rich tracts, two highly conserved 15 nucleotide sequences, and what appears to be an atypical TATA-box sequence also observed in two other TTV genera. Codon usage analysis of TTEqV2 and 11 other selected anelloviruses from five host species revealed a bias toward adenine ending (A3) codons in the anelloviruses, while in contrast, A3 codons were observed at a low frequency in horse and the four other associated host species examined. Phylogenetic analysis of TTV ORF1 sequences available to date shows TTEqV2 clusters with the only other currently reported member of the Mutorquevirus genus, Torque teno equus virus 1 (TTEqV1, KR902501). Genome-wide pairwise alignment of TTEqV2 and TTEqV1 shows the absence of several highly conserved TTV features within the UTR of TTEqV1, suggesting it is incomplete and TTEqV2 is the first complete genome within the genus Mutorquevirus., (© 2023. Crown.)

Mosquitoes are important vectors for human and animal diseases. Genetic markers, like the mitochondrial COI gene, can facilitate the taxonomic classification of disease vectors, vector-borne disease surveillance, and prevention. Within the control region (CR) of the mitochondrial genome, there exists a highly variable and poorly studied non-coding AT-rich area that contains the origin of replication. Although the CR hypervariable region has been used for species differentiation of some animals, few studies have investigated the mosquito CR. In this study, we analyze the mosquito mitogenome CR sequences from 125 species and 17 genera. We discovered four conserved motifs located 80 to 230 bp upstream of the 12S rRNA gene. Two of these motifs were found within all 392 Anopheles (An.) CR sequences while the other two motifs were identified in all 37 Culex (Cx.) CR sequences. However, only 3 of the 304 non-Culicidae Dipteran mitogenome CR sequences contained these motifs. Interestingly, the short motif found in all 37 Culex sequences had poly-A and poly-T stretch of similar length that is predicted to form a stable hairpin. We show that supervised learning using the frequency chaos game representation of the CR can be used to differentiate mosquito genera from their dipteran relatives., (© 2022. Crown.)

A complete 30,616-nucleotide Cervid atadenovirus A genome was determined from the tissues of black-tailed deer that had died in 2020 in British Columbia, Canada. Unique, nonsynonymous single-nucleotide polymorphisms in the E1B, Iva2, and E4.3 coding regions and deletions totaling 74 nucleotides that were not observed in moose and red deer isolates were present.

Foot-and-Mouth Disease Virus (FMDV), the causative agent of Foot-and-Mouth Disease, is a highly feared, economically devastating transboundary pathogen. This is due to the virus' extremely contagious nature and its ability to utilize multiple transmission routes. As such, rapid and accurate diagnostic testing is imperative to the control of FMD. Identification of the FMDV serotype is necessary as it provides the foundation for appropriate vaccine selection and aids in outbreak source tracing. With the vast genetic diversity, there is a desperate need to be able to characterize FMDV without relying on prior knowledge of viral serotypes. In this study, the Neptune bioinformatics tool was used to identify genetic signatures specific to each Southern African Territories (SAT) 1, 2 and 3 genomes but exclusionary to the other circulating FMDV serotypes (A, O, Asia1, and the heterologous SAT1, SAT2 and/or SAT3). Identification of these unique genomic regions allowed the design of TaqMan-based real-time reverse transcriptase PCR (rRT-PCR) primer/probe sets for SAT1, SAT2 and SAT3 viruses. These assays were optimized using prototypic FMDV cell culture isolates using the same reagents and thermocycling conditions as the FMDV pan-serotype 3D rRT-PCR assay. Cross-reactivity was evaluated in tandem with the FMDV pan-serotype 3D rRT-PCR utilizing representative strains from FMDV serotypes A, O, Asia1, SAT1, SAT2 and SAT3. The SAT1, SAT2, and SAT3 primer/probe sets were specific for the homologous serotype and exclusionary to all others. SAT1 and SAT3 primer/probe sets were able to detect several topotypes, whereas the SAT2 assay was revealed to be specific for topotype VII. The SAT2 topotype VII specificity was possibly due to the use of sequence data deposited post-2011to design the rRT-PCR primers and probes. Each assay was tested against a panel of 99 bovine tissue samples from Nigeria, where SAT2 topotype VII viruses were correctly identified and no cross-reactivity was exhibited by the SAT1 and 3 assays. These novel SAT1, SAT3 and SAT2 topotype VII rRT-PCR assays have the potential to detect and differentiate circulating FMD SAT viruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chestley, Sroga, Nebroski, Hole, Ularamu, Lung and Nfon.)

From 2016 to 2020, high pathogenicity avian influenza (HPAI) H5 viruses circulated in Asia, Europe, and Africa, causing waves of infections and the deaths of millions of wild and domestic birds and presenting a zoonotic risk. In late 2021, H5N1 HPAI viruses were isolated from poultry in Canada and also retrospectively from a great black-backed gull ( Larus marinus ), raising concerns that the spread of these viruses to North America was mediated by migratory wild bird populations. In February and April 2022, H5N1 HPAI viruses were isolated from a bald eagle ( Haliaeetus leucocephalus ) and broiler chickens in British Columbia, Canada. Phylogenetic analysis showed that the virus from bald eagle was genetically related to H5N1 HPAI virus isolated in Hokkaido, Japan, in January 2022. The virus identified from broiler chickens was a reassortant H5N1 HPAI virus with unique constellation genome segments containing PB2 and NP from North American lineage LPAI viruses, and the remaining gene segments were genetically related to the original Newfoundland-like H5N1 HPAI viruses detected in November and December 2021 in Canada. This is the first report of H5 HPAI viruses' introduction to North America from the Pacific and the North Atlantic-linked flyways and highlights the expanding risk of genetically distinct virus introductions from different geographical locations and the potential for local reassortment with both the American lineage LPAI viruses in wild birds and with both Asian-like and European-like H5 HPAI viruses. We also report the presence of some amino acid substitutions across each segment that might contribute to the replicative efficiency of these viruses in mammalian host, evade adaptive immunity, and pose a potential zoonotic risk., (© Crown copyright 2022.)

We isolated a novel reassortant influenza A(H10N7) virus from a harbor seal in British Columbia, Canada, that died from bronchointerstitial pneumonia. The virus had unique genome constellations involving lineages from North America and Eurasia and polymerase basic 2 segment D701N mutation, associated with adaptation to mammals.

Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency's National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvβ6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly., (© 2021. The Author(s).)

This report describes the nucleotide sequences of eight Southern African Territories 2 (SAT2) serotype foot-and-mouth disease virus strains from 2017 to 2018 outbreaks in cattle in Nigeria. These viruses belong to topotype VII of SAT2 and were closely related to previous isolates from Nigeria and other West African countries.

In September 2019, high mortality in commercial rabbits was reported in the Greater Accra Region of Ghana. Rabbit hemorrhagic disease virus 2 phylogenetically related to isolates from 2015-2017 outbreaks in the Netherlands was confirmed as the causative agent. The virus has not yet been detected in native rabbits in Ghana.

Hantavirus cardiopulmonary syndrome (HCPS) is a severe respiratory disease caused by orthohantaviruses in the Americas with a fatality rate as high as 35%. In South America, Andes orthohantavirus ( Hantaviridae, Orthohantavirus , ANDV) is a major cause of HCPS, particularly in Chile and Argentina, where thousands of cases have been reported since the virus was discovered. Two strains of ANDV that are classically used for experimental studies of the virus are Chile-9717869, isolated from the natural reservoir, the long-tailed pygmy rice rat, and CHI-7913, an isolate from a lethal human case of HCPS. An important animal model for studying pathogenesis of HCPS is the lethal Syrian golden hamster model of ANDV infection. In this model, ANDV strain Chile-9717869 is uniformly lethal and has been used extensively for pathogenesis, vaccination, and therapeutic studies. Here we show that the CHI-7913 strain, despite having high sequence similarity with Chile-9717869, does not cause lethal disease in Syrian hamsters. CHI-7913, while being able to infect hamsters and replicate to moderate levels, showed a reduced ability to replicate within the tissues compared with Chile-9717869. Hamsters infected with CHI-7913 had reduced expression of cytokines IL-4, IL-6, and IFN-γ compared with Chile-9717869 infected animals, suggesting potentially limited immune-mediated pathology. These results demonstrate that certain ANDV strains may not be lethal in the classical Syrian hamster model of infection, and further exploration into the differences between lethal and non-lethal strains provide important insights into molecular determinants of pathogenic hantavirus infection. Importance: Andes orthohantavirus (ANDV) is a New World hantavirus that is a major cause of hantavirus cardiopulmonary syndrome (HCPS, also referred to as hantavirus pulmonary syndrome) in South America, particularly in Chile and Argentina. ANDV is one of the few hantaviruses for which there is a reliable animal model, the Syrian hamster model, which recapitulates important aspects of human disease. Here we infected hamsters with a human isolate of ANDV, CHI-7913, to assess its pathogenicity compared with the classical lethal Chile-9717869 strain. CHI-7913 had 22 amino acid differences compared with Chile-9717869, did not cause lethal disease in hamsters, and showed reduced ability to replicate in vivo Our data indicate potentially important molecular signatures for pathogenesis of ANDV infection in hamsters and may lead to insights into what drives pathogenesis of certain hantaviruses in humans., (© Crown copyright 2021.)

We describe for the first time the genetic and antigenic characterization of 18 avian avulavirus type-6 viruses (AAvV-6) that were isolated from wild waterfowl in the Americas over the span of 12 years. Only one of the AAvV-6 viruses isolated failed to hemagglutinate chicken red blood cells. We were able to obtain full genome sequences of 16 and 2 fusion gene sequences from the remaining 2 isolates. This is more than double the number of full genome sequences available at the NCBI database. These AAvV-6 viruses phylogenetically grouped into the 2 existing AAvV-6 genotype subgroups indicating the existence of an intercontinental epidemiological link with other AAvV-6 viruses isolated from migratory waterfowl from different Eurasian countries. Antigenic maps made using HI assay data for these isolates showed that the two genetic groups were also antigenically distinct. An isolate representing each genotype was inoculated in specific pathogen free (SPF) chickens, however, no clinical symptoms were observed. A duplex fusion gene based real-time assay for the detection and genotyping of AAvV-6 to genotype 1 and 2 was developed. Using the developed assay, the viral shedding pattern in the infected chickens was examined. The chickens infected with both genotypes were able to shed the virus orally for about a week, however, no significant cloacal shedding was detected in chickens of both groups. Chickens in both groups developed detectable levels of anti-hemagglutinin antibodies 7 days after infection.

The complete genome sequence of a novel circovirus (elk circovirus (ElkCV) Banff/2019) was determined via high throughput sequencing of liver tissue from a euthanized Rocky Mountain elk (Cervus canadensis nelsoni) from Alberta, Canada. The genome is circular and 1,787 nucleotides long, with two major ORFs encoding predicted proteins. Comparative genomic analysis to 4,164 publicly available complete and near complete circovirus genomes showed that ElkCV shares approximately 65% pairwise genome-wide nucleotide identity with the most closely related circovirus species, porcine circoviruses (PCV) 1 and 2 and bat-associated circovirus (BatACV) 11. ElkCV features a stem-loop within the origin of replication region characteristic of circoviruses. However, it differs from those found in PCV1, PCV2 and BatACV11 since it has a longer stem and contains hexamer repeats that overlap the stem in opposing orientations. Interestingly, stem-loop structures of similar length featuring repeats in a similar position and orientation are also seen in some avian circoviruses. Based on the demarcation threshold established by the International Committee on Taxonomy of Viruses (ICTV) for members of Circoviridae (80% pairwise genome-wide nucleotide identity), ElkCV represents a novel species and is the first complete circovirus genome reported from a cervid host.

The first Canadian H3N2 canine influenza A outbreak involving an Asian-origin H3N2 canine influenza virus (CIV) began in southwestern Ontario, Canada, in late December 2017. More H3N2 CIV cases were identified in central and eastern Ontario between March and October 2018. Based on epidemiological investigation, 5 clusters were identified (C1, C2, C3a, C3b, and C4); however, the origin of infection has only been revealed for epidemiological cluster C1. Here, we use phylogenetic analyses to unravel the links of virus transmission between the 5 epidemiological clusters and the origin of infection for all epidemiological clusters. Our results demonstrate that the Canadian H3N2 CIV sequences were grouped into four distinct phylogenetic clusters with minimal genetic diversity between these clusters. Large scale phylogenetic analysis of H3N2 CIV from around the globe showed that the Canadian CIVs formed a distinct new clade along with CIVs that have been circulating in the USA since 2017-2018 and in China since 2017. This clade shares a common ancestor of Asian origin. This study concludes that the H3N2 CIV outbreak in Ontario was driven by multiple introductions of South Korean/Chinese-origin H3N2 CIVs over 10 months.

Complete genome sequences of six Ambystoma tigrinum viruses (ATV) were determined directly from tail clips of western tiger salamanders ( Ambystoma mavortium ) from 2013 (high-mortality year) and 2014 (low-mortality year) in Alberta, Canada. The genome lengths ranged from 106,258 to 106,915 bp and contained 108 open reading frames encoding predicted proteins larger than 50 amino acids., (© Crown copyright 2019.)

The complete genome of a novel coronavirus was sequenced directly from the cloacal swab of a Canada goose that perished in a die-off of Canada and Snow geese in Cambridge Bay, Nunavut, Canada. Comparative genomics and phylogenetic analysis indicate it is a new species of Gammacoronavirus, as it falls below the threshold of 90% amino acid similarity in the protein domains used to demarcate Coronaviridae. Additional features that distinguish the genome of Canada goose coronavirus include 6 novel ORFs, a partial duplication of the 4 gene and a presumptive change in the proteolytic processing of polyproteins 1a and 1ab.

Rift Valley fever virus (RVFV) is a zoonotic arbovirus of the Phenuiviridae family. Infection causes abortions in pregnant animals, high mortality in neonate animals, and mild to severe symptoms in both people and animals. There is currently an ongoing effort to produce safe and efficacious veterinary vaccines against RVFV in livestock to protect against both primary infection in animals and zoonotic infections in people. To test the efficacy of these vaccines, it is essential to have a reliable challenge model in relevant target species, including ruminants. We evaluated two goat breeds (Nubian and LaMancha), three routes of inoculation (intranasal, mosquito-primed subcutaneous, and subcutaneous) using an infectious dose of 10⁷ pfu/mL, a virus strain from the 2006⁻2007 Kenyan/Sudan outbreak and compared the effect of using virus stocks produced in either mammalian or mosquito cells. Our results demonstrated that the highest and longest viremia titers were achieved in Nubian goats. The Nubian breed was also efficient at producing clinical signs, consistent viremia (peak viremia: 1.2 × 10³⁻1.0 × 10⁵ pfu/mL serum), nasal and oral shedding of viral RNA (1.5 × 10¹⁻8 × 10⁶ genome copies/swab), a systemic infection of tissues, and robust antibody responses regardless of the inoculation route. The Nubian goat breed and a needle-free intranasal inoculation technique could both be utilized in future vaccine and challenge studies. These studies are important for preventing the spread and outbreak of zoonotic viruses like RVFV and are supported by the Canadian-led BSL4ZNet network.

Senecavirus A (SVA), previously known as Seneca Valley virus, was first isolated in the United States in 2002. SVA was associated with porcine idiopathic vesicular disease in Canada and the USA in 2007 and 2012, respectively. Recent increase in SVA outbreaks resulting in neonatal mortality of piglets and/or vesicular lesions in sows in Brazil, the USA and Canada point to the necessity to study the pathogenicity and molecular epidemiology of the virus. Here, we report the analysis of the complete coding sequences of SVA from 2 clinical cases and 9 assembly yard environmental samples collected in 2015 in Canada, along with 22 previously released complete genomes in the GenBank. With this combined data set, the evolution of the SVA over a 12-month period in 2015/2016 was evaluated. These SVA isolates were characterized by a rapid accumulation of genetic variations driven mainly by a high nucleotide substitution rate and purifying selection. The SVA sequences clustered in clearly defined geographical areas with reported cases of SVA infection. No transmission links were identified between assembly yards, suggesting that point source introductions may have occurred. In addition, 25 fixed non-synonymous mutations were identified across all analyzed strains when compared to the prototype SVA strain (SVV-001). This study highlights the importance of monitoring SVA mutations for their role in increased virulence and impact on SVA diagnostics.