Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle.

The highly pathogenic avian influenza (HPAI) H5N1 virus clade 2.3.4.4b has caused the death of millions of domestic birds and thousands of wild birds in the USA since January 2022 (refs. 1-4). Throughout this outbreak, spillovers to mammals have been frequently documented5-12. Here we report spillover of the HPAI H5N1 virus to dairy cattle across several states in the USA. The affected cows displayed clinical signs encompassing decreased feed intake, altered faecal consistency, respiratory distress and decreased milk production with abnormal milk. Infectious virus and viral RNA were consistently detected in milk from affected cows. Viral distribution in tissues via immunohistochemistry and in situ hybridization revealed a distinct tropism of the virus for the epithelial cells lining the alveoli of the mammary gland in cows. Whole viral genome sequences recovered from dairy cows, birds, domestic cats and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiological and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a premise in a different state. These results demonstrate the transmission of the HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface, underscoring the ability of the virus to cross species barriers.

A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

White-tailed deer (Odocoileus virginianus) may serve as a wildlife reservoir for nearly extinct SARS-CoV-2 variants of concern.

The spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (WTD) and its ability to transmit from deer to deer raised concerns about the role of WTD in the epidemiology and ecology of the virus. Here, we present a comprehensive cross-sectional study assessing the prevalence, genetic diversity, and evolution of SARS-CoV-2 in WTD in the State of New York (NY). A total of 5,462 retropharyngeal lymph node samples collected from free-ranging hunter-harvested WTD during the hunting seasons of 2020 (Season 1, September to December 2020, n = 2,700) and 2021 (Season 2, September to December 2021, n = 2,762) were tested by SARS-CoV-2 real-time RT-PCR (rRT-PCR). SARS-CoV-2 RNA was detected in 17 samples (0.6%) from Season 1 and in 583 samples (21.1%) from Season 2. Hotspots of infection were identified in multiple confined geographic areas of NY. Sequence analysis of SARS-CoV-2 genomes from 164 samples demonstrated the presence of multiple SARS-CoV-2 lineages and the cocirculation of three major variants of concern (VOCs) (Alpha, Gamma, and Delta) in WTD. Our analysis suggests the occurrence of multiple spillover events (human to deer) of the Alpha and Delta lineages with subsequent deer-to-deer transmission and adaptation of the viruses. Detection of Alpha and Gamma variants in WTD long after their broad circulation in humans in NY suggests that WTD may serve as a wildlife reservoir for VOCs no longer circulating in humans. Thus, implementation of continuous surveillance programs to monitor SARS-CoV-2 dynamics in WTD is warranted, and measures to minimize virus transmission between humans and animals are urgently needed.

The SARS-CoV-2 Spike is a virulence determinant and plays a major role on the attenuated phenotype of Omicron virus in a feline model of infection.

The role of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.1 Spike (S) on disease pathogenesis was investigated. For this, we generated recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 S gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 strain genome. The recombinant viruses were characterized in vitro and in vivo. Viral entry, cell-cell fusion, plaque size, and the replication kinetics of the rWA1-Omi-S virus were markedly impaired when compared to the rWA1-D614G virus, demonstrating a lower fusogenicity and ability to spread cell-to-cell of rWA1-Omi-S. To assess the contribution of the Omicron BA.1 S protein to SARS-CoV-2 pathogenesis, the pathogenicity of rWA1-D614G and rWA1-Omi-S viruses was compared in a feline model. While the rWA1-D614G-inoculated cats were lethargic and showed increased body temperatures on days 2 and 3 post-infection (pi), rWA1-Omi-S-inoculated cats remained subclinical and gained weight throughout the 14-day experimental period. Animals inoculated with rWA1-D614G presented higher infectious virus shedding in nasal secretions, when compared to rWA1-Omi-S-inoculated animals. In addition, tissue replication of the rWA1-Omi-S was markedly reduced compared to the rWA1-D614G, as evidenced by lower viral load in tissues on days 3 and 5 pi. Histologic examination of the nasal turbinate and lungs revealed intense inflammatory infiltration in rWA1-D614G-inoculated animals, whereas rWA1-Omi-S-inoculated cats presented only mild to modest inflammation. Together, these results demonstrate that the S protein is a major virulence determinant for SARS-CoV-2 playing a major role for the attenuated phenotype of the Omicron virus. IMPORTANCE: We have demonstrated that the Omicron BA.1.1 variant presents lower pathogenicity when compared to D614G (B.1) lineage in a feline model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There are over 50 mutations across the Omicron genome, of which more than two-thirds are present in the Spike (S) protein. To assess the role of the Omicron BA.1 S on virus pathogenesis, recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 Spike gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 were generated. While the Omicron BA.1 S promoted early entry into cells, it led to impaired fusogenic activity and cell-cell spread. Infection studies with the recombinant viruses in a relevant naturally susceptible feline model of SARS-CoV-2 infection here revealed an attenuated phenotype of rWA1-Omi-S, demonstrating that the Omi-S is a major determinant of the attenuated disease phenotype of Omicron strains.

Infection Dynamics, Pathogenesis, and Immunity to SARS-CoV-2 in Naturally Susceptible Animal Species.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, presents a broad host range. Domestic cats and white-tailed deer (WTD) are particularly susceptible to SARS-CoV-2 with multiple variant strains being associated with infections in these species. The virus replicates in the upper respiratory tract and in associated lymphoid tissues, and it is shed through oral and nasal secretions, which leads to efficient transmission of the virus to contact animals. Robust cell-mediated and humoral immune responses are induced upon infection in domestic cats, which curb the progression of clinical disease and are associated with control of infection. In WTD, high levels of neutralizing Abs are detected early upon infection. In this review, the current understanding of the infection dynamics, pathogenesis, and immune responses to SARS-CoV-2 infection in animals, with special focus on naturally susceptible felids and WTD, are discussed.

Antibodies to Influenza A(H5N1) Virus in Hunting Dogs Retrieving Wild Fowl, Washington, USA.

We detected antibodies to H5 and N1 subtype influenza A viruses in 4/194 (2%) dogs from Washington, USA, that hunted or engaged in hunt tests and training with wild birds. Historical data provided by dog owners showed seropositive dogs had high levels of exposure to waterfowl.

From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection/exposure rates approaching 30-40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans, at the human-WTD interface during hunting or venison farming, and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2 B.1 lineage, WTD fawns (~8-month-old) shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD (3-4 years of age). Infectious virus was detected up to day 6 pi in nasal secretions, and from various respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field.

Continuing evolution of H5N1 highly pathogenic avian influenza viruses of clade 2.3.2.1a G2 genotype in domestic poultry of Bangladesh during 2018-2021.

We characterized 15 H5N1 HPAI viruses from different small- and medium-scale poultry flocks across Bangladesh during 2018-2021 based on their complete genome sequences. The antigenic relatedness of H5N1 HPAI viruses from different timepoints was analysed. During 2020-2021, 42.11% of the flocks tested positive for at least one of the respiratory infections, with 15.79% showing influenza A virus, of which 8.77% tested positive for HPAIV H5N1. Co-infections with two to four pathogens were detected in 15.8% of flocks. Phylogeny and gene constellation analyses based on complete genome sequences of 15 HPAI viruses revealed the continuing circulation of H5 clade 2.3.2.1a genotype G2 viruses. In the HA protein of the study isolates, functionally meaningful mutations caused the loss of an N-linked glycosylation site (T156A), a modified antigenic site A (S141P), and a mutation in the receptor binding pocket (E193R/K). Consequently, antigenic analysis revealed a significant loss of cross-reactivity between viruses from different host species and periods. Most viruses displayed oseltamivir resistance markers at positions V96, I97, S227, and N275 (N1 numbering) of the NA protein. In addition, for the PB2, M1, and NS1 proteins, significant mutations were noticed that have been associated with polymerase activity and increased virulence for mammals in all study isolates. These results highlight the need for intensified genomic surveillance of HPAI circulating in poultry in Bangladesh and for establishing appropriate control measures to decrease the circulation of these viruses in poultry in the country.

Age-Related Susceptibility of Ferrets to SARS-CoV-2 Infection.

Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the outcome of coronavirus disease 2019 (COVID-19) have been linked to underlying health conditions and the age of affected individuals. Here, we assessed the effect of age on SARS-CoV-2 infection using a ferret model. For this, young (6-month-old) and aged (18- to 39-month-old) ferrets were inoculated intranasally with various doses of SARS-CoV-2. By using infectious virus shedding in respiratory secretions and seroconversion, we estimated that the infectious dose of SARS-CoV-2 in aged animals is ∼32 PFU per animal, while in young animals it was estimated to be ∼100 PFU. We showed that viral replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets compared to young animals. Similar to observations in humans, this was associated with higher transcription levels of two key viral entry factors, ACE2 and TMPRSS2, in the upper respiratory tract of aged ferrets. IMPORTANCE In humans, ACE2 and TMPRSS2 are expressed in various cells and tissues, and differential expression has been described in young and old people, with a higher level of expressing cells being detected in the nasal brushing of older people than young individuals. We described the same pattern occurring in ferrets, and we demonstrated that age affects susceptibility of ferrets to SARS-CoV-2. Aged animals were more likely to get infected when exposed to lower infectious dose of the virus than young animals, and the viral replication in the upper respiratory tract and shedding are enhanced in aged ferrets. Together, these results suggest that the higher infectivity and enhanced ability of SARS-CoV-2 to replicate in aged individuals is associated, at least in part, with transcription levels of ACE2 and TMPRSS2 at the sites of virus entry. The young and aged ferret model developed here may represent a great platform to assess age-related differences in SARS-CoV-2 infection dynamics and replication.

The Omicron Variant BA.1.1 Presents a Lower Pathogenicity than B.1 D614G and Delta Variants in a Feline Model of SARS-CoV-2 Infection.

Omicron (B.1.1.529) is the most recent SARS-CoV-2 variant of concern, which emerged in late 2021 and rapidly achieved global predominance by early 2022. In this study, we compared the infection dynamics, tissue tropism, and pathogenesis and pathogenicity of SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), and Omicron BA.1.1 (B.1.1.529) variants in a highly susceptible feline model of infection. Although D614G- and Delta-inoculated cats became lethargic and showed increased body temperatures between days 1 and 3 postinfection (pi), Omicron-inoculated cats remained subclinical and, similar to control animals, gained weight throughout the 14-day experimental period. Intranasal inoculation of cats with D614G- and the Delta variants resulted in high infectious virus shedding in nasal secretions (up to 6.3 log10 TCID50.Ml-1), whereas strikingly lower level of viruses shedding (<3.1 log10 TCID50.Ml-1) was observed in Omicron-inoculated animals. In addition, tissue distribution of the Omicron variant was markedly reduced in comparison to the D614G and Delta variants, as evidenced by lower in situ viral RNA detection, in situ viral immunofluorescence staining, and viral loads in tissues on days 3, 5, and 14 pi. Nasal turbinate, trachea, and lung were the main-but not the only-sites of replication for all three viral variants. However, only scarce virus staining and lower viral titers suggest lower levels of viral replication in tissues from Omicron-infected animals. Notably, while D614G- and Delta-inoculated cats presented pneumonia, histologic examination of the lungs from Omicron-infected cats revealed mild to modest inflammation. Together, these results demonstrate that the Omicron variant BA.1.1 is less pathogenic than D614G and Delta variants in a highly susceptible feline model. IMPORTANCE The SARS-CoV-2 Omicron (B.1.1.529) variant of concern emerged in South Africa late in 2021 and rapidly spread across the world causing a significant increase in the number of infections. Importantly, this variant was also associated with an increased risk of reinfections. However, the number of hospitalizations and deaths due to COVID-19 did not follow the same trends. These early observations suggested effective protection conferred by immunizations and/or overall lower virulence of the highly mutated variant virus. In this study we present novel evidence demonstrating that the Omicron BA.1.1 variant of concern presents a lower pathogenicity when compared to D614G- or Delta variants in cats. Clinical, virological, and pathological evaluations revealed lower disease severity, viral replication, and lung pathology in Omicron-infected cats when compared with D614G and Delta variant inoculated animals, confirming that Omicron BA.1.1 is less pathogenic in a highly susceptible feline model of infection.

Comparative pathogenicity of a genotype XXI.1.2 pigeon Newcastle disease virus isolate in pigeons and chickens.

Here, we performed molecular and pathogenic characterization of a Newcastle disease virus (NDV) isolate from pigeons in Bangladesh. Molecular phylogenetic analysis based on the complete fusion gene sequences classified the three study isolates into genotype XXI (sub-genotype XXI.1.2) together with recent NDV isolates obtained from pigeons in Pakistan (2014-2018). The Bayesian Markov Chain Monte Carlo analysis revealed that the ancestor of Bangladeshi pigeon NDVs and the viruses from sub-genotype XXI.1.2 existed in the late 1990s. Pathogenicity testing using mean embryo death time pathotyped the viruses as mesogenic, while all isolates carried multiple basic amino acid residues at the fusion protein cleavage site. Experimental infection of chickens and pigeons revealed no or minimum clinical signs in chickens, while a relatively high morbidity (70%) and mortality (60%) were observed in pigeons. The infected pigeons showed extensive and systemic lesions including hemorrhagic and/or vascular changes in the conjunctiva, respiratory and digestive system and brain, and atrophy in the spleen, while only mild congestion in the lungs was noticed in the inoculated chickens. Histologically, consolidation in the lungs with collapsed alveoli and edema around the blood vessels, hemorrhages in the trachea, severe hemorrhages and congestion, focal aggregation of mononuclear cells, and single hepatocellular necrosis in the liver, severe congestion, multifocal tubular degeneration, and necrosis, as well as mononuclear cell infiltration in the renal parenchyma, encephalomalacia with severe neuronal necrosis with neuronophagia were noticed in the brain in infected pigeons. In contrast, only slight congestion was found in lungs of the infected chickens. qRT-PCR revealed the replication of the virus in both pigeons and chickens; however, higher viral RNA loads were observed in oropharyngeal and cloacal swabs, respiratory tissues, and spleen of infected pigeons than the chickens. In conclusion, genotype XXI.1.2 NDVs are circulating in the pigeon population of Bangladesh since 1990s, produce high mortality in pigeons with pneumonia, hepatocellular necrosis, renal tubular degeneration, and neuronal necrosis in pigeons, and may infect chickens without overt signs of clinical disease and are likely to shed viruses via the oral or cloacal routes.

A linear SARS-CoV-2 DNA vaccine candidate reduces virus shedding in ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 760 million cases and over 6.8 million deaths as of March 2023. Vaccination has been the main strategy used to contain the spread of the virus and to prevent hospitalizations and deaths. Currently, two mRNA-based vaccines and one adenovirus-vectored vaccine have been approved and are available for use in the U.S. population. The versatility, low cost, and rapid production of DNA vaccines provide important advantages over other platforms. Additionally, DNA vaccines efficiently induce both B- and T-cell responses by expressing the antigen within transfected host cells, and the antigen, after being processed into peptides, can associate with MHC class I or II of antigen-presenting cells (APCs) to stimulate different T cell responses. However, the efficiency of DNA vaccination needs to be improved for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been used successfully in the field of veterinary oncology, resulting in high rates of response after electrochemotherapy. Here, we evaluate the safety, immunogenicity, and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate delivered by intramuscular injection followed by electroporation (Vet-ePorator™) in ferrets. The linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects. Additionally, the vaccine elicited neutralizing antibodies and T cell responses on day 42 post-immunization using a low dose of the linear DNA construct in a prime-boost regimen. Most importantly, vaccination significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.

Granulomatous mural folliculitis in 16 domestic goats: Infection with malignant catarrhal fever viruses and colocalization with ovine herpesvirus-2 using in situ hybridization.

Granulomatous mural folliculitis (GMF) is an uncommon reaction pattern occasionally observed in nonadapted ruminant hosts infected with malignant catarrhal fever viruses. This report characterizes GMF and concurrent cutaneous lesions in 16 goats with crusting dermatitis using histochemistry including hematoxylin and eosin, periodic acid-Schiff, and Grocott's methenamine silver, and immunohistochemistry for CD3, CD20, ionized calcium binding adaptor molecule 1, and cytokeratin AE1/3. Infiltrates in all 16 GMF cases consisted of macrophages and fewer T lymphocytes, and variably included eosinophils, multinucleated histiocytic giant cells, and/or neutrophils. Formalin-fixed paraffin-embedded skin and fresh skin samples from caprine GMF cases were tested using pan-herpesvirus nested conventional polymerase chain reaction (PCR) and partial sequencing, ovine herpesvirus-2 (OvHV-2) real-time PCR, and OvHV-2 colorimetric in situ hybridization (ISH). Five of 16 goats with GMF (31%) were PCR positive for malignant catarrhal fever viruses, including caprine herpesvirus 3 in 1 goat and OvHV-2 in 4 goats. Three goats also had positive intranuclear OvHV-2 hybridization signal in follicular keratinocytes, among other cell types, localized to areas of GMF. Herpesviruses were not detected in the formalin-fixed paraffin-embedded skin of 9 goats without GMF. This case series describes relatively frequent detections of malignant catarrhal fever viruses in the skin of goats with GMF, including the first report of caprine herpesvirus 3, and localizes OvHV-2 infected follicular keratinocytes within areas of GMF.

Histopathologic and molecular characterization of Erethizon dorsatum papillomavirus 1 and Erethizon dorsatum papillomavirus 2 infection in North American porcupines (Erethizon dorsatum).

Erethizon dorsatum papillomavirus 1 (EdPV1) and Erethizon dorsatum papillomavirus 2 (EdPV2) are associated with cutaneous papillomas in North American porcupines (Erethizon dorsatum). This study defined gross, histopathologic, and molecular characteristics of viral papillomas in 10 North American porcupines submitted to the New York State Animal Health Diagnostic Center. Investigation for the presence of EdPV1 and EdPV2 DNA via polymerase chain reaction (PCR) was performed in 9 of the 10 (90.0%) porcupines, and all porcupines were investigated for the detection and localization of EdPV1 and EdPV2 E6 and E7 nucleic acid via chromogenic in situ hybridization (CISH). Next-generation sequencing (NGS) was performed in 2 porcupines. Papillomas were diagnosed on the muzzle (n = 4), caudal dorsum (n = 1), upper lip (n = 1), chin (n = 1), gingiva (n = 2), and nasal planum (n = 1). Histologically, the lesions consisted of hyperplastic epidermis or epithelium with orthokeratotic keratin, prominent keratohyalin granules, and intranuclear inclusion bodies. PCR identified EdPV1 in 6 of 9 samples and EdPV2 in the remaining 3 samples. NGS resulted in 100% genome coverage of EdPV1 and 76.20% genome coverage of EdPV2 compared with GenBank reference sequences, with 99.8% sequence identity to the complete EdPV2 L1 gene of a novel subtype recently identified in France. Hybridization patterns in 9 of the 10 (90.0%) porcupines were characterized by strong nuclear signals in the superficial epidermis, with strong nuclear and punctate cytoplasmic signals in the stratum spinosum and basale. In one animal, CISH suggested dual EdPV1 and EdPV2 infection.

Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2.

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

Genome sequence and experimental infection of calves with bovine gammaherpesvirus 4 (BoHV-4).

Bovine gammaherpesvirus 4 (BoHV-4) is ubiquitous in cattle worldwide, and it has been detected in animals exhibiting broad clinical presentations. The virus has been detected in the United States since the 1970s; however, its clinical relevance remains unknown. Here, we determined the complete genome sequences of two contemporary BoHV-4 isolates obtained from respiratory (SD16-38) or reproductive (SD16-49) tract specimens and assessed clinical, virological, and pathological outcomes upon intranasal (IN) inoculation of calves with the respiratory BoHV-4 isolate SD16-38. A slight and transient increase in body temperature was observed in BoHV-4-inoculated calves. Additionally, transient viremia and virus shedding in nasal secretions were observed in all inoculated calves. BoHV-4 DNA was detected by nested PCR in the tonsil and regional lymph nodes (LNs) of calves euthanized on day 5 post-inoculation (pi) and in the lungs of calves euthanized on day 10 pi. Calves euthanized on day 35 pi harbored BoHV-4 DNA in the respiratory tract (turbinates, trachea, lungs), regional lymphoid tissues, and trigeminal ganglia. Interestingly, in situ hybridization revealed the presence of BoHV-4 DNA in nerve bundles surrounding the trigeminal ganglia and retropharyngeal lymph nodes (day 35 pi). No histological changes were observed in the respiratory tract (turbinate, trachea, and lung), lymphoid tissues (tonsil, LNs, thymus, and spleen), or central nervous tissues (olfactory bulb and trigeminal ganglia) sampled throughout the animal studies (days 5, 10, and 35 pi). This study contributes to the understanding of the infection dynamics and tissue distribution of BoHV-4 following IN infection in calves. These results suggest that BoHV-4 SD16-38 used in our study has low pathogenicity in calves upon intranasal inoculation.

SARS-CoV-2 B.1.1.7 Variant Infection in Malayan Tigers, Virginia, USA.

We report infection of 3 Malayan tigers with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 (Alpha) variant at a zoologic park in Virginia, USA. All tigers exhibited respiratory signs consistent with SARS-CoV-2 infection. These findings show that tigers are susceptible to infection with the SARS-CoV-2 B.1.1.7 variant.

A virulent and pathogenic infectious clone of Senecavirus A.

Senecavirus A (SVA) is a picornavirus that circulates in swine populations worldwide causing vesicular disease (VD) in affected animals. Here we developed a reverse genetics system for SVA based on the well-characterized wild-type SVA strain SD15-26 (wt SVA SD15-26). The full-length cDNA genome of SVA was cloned into a plasmid under a T7 RNA polymerase promoter. Following in vitro transcription, the genomic viral RNA was transfected into BHK-21 cells and rescue of infectious virus (rSVA SD15-26) was shown by inoculation of highly susceptible H1299 cells. In vitro characterization of the rSVA SD15-26 showed similar replication properties and protein expression levels as the wt SVA SD15-26. A pathogenesis study was conducted in 15-week-old finishing pigs to evaluate the pathogenicity and infection dynamics of the rSVA SD15-26 virus in comparison to the wt SVA SD15-26. Animals from both rSVA- and wt SVA SD15-26-inoculated groups presented characteristic SVA clinical signs (lethargy and lameness) followed by the development of vesicular lesions on the snout and/or feet. The clinical outcome of infection, including disease onset, severity and duration was similar in rSVA- and the wt SVA SD15-26-inoculated animals. All animals inoculated with rSVA or with wt SVA SD15-26 presented a short-term viremia, and animals from both groups shed similar amounts of virus in oral and nasal secretion, and faeces. Our data demonstrates that the rSVA SD5-26 clone is fully virulent and pathogenic in pigs, presenting comparable pathogenesis and infection dynamics to the wt SVA SD15-26 strain. The infectious clone generated here is a useful platform to study virulence determinants of SVA, and to dissect other aspects of SVA infection biology, pathogenesis and persistence.

Identification and genetic characterization of an isolate of bovine adenovirus 7 from the United States, a putative member of a new species in the genus Atadenovirus.

The bovine adenovirus 7 (BAdV-7) isolate SD18-74 was recovered from lung tissue of calves in South Dakota. The 30,043-nucleotide (nt) genome has the typical organization of Atadenovirus genus members. The sequence shares over 99% nt sequence identity with two Japanese BAdV-7 sequences, followed by 74.9% nt sequence identity with the ovine adenovirus 7 strain OAV287, a member of the species Ovine atadenovirus D. SD18-74 was amplified in both bovine and ovine primary nasal turbinate cells, demonstrating greater fitness in bovine cells. The genomic and biological characteristics of BAdV-7 SD18-74 support the inclusion of the members of the BAdV-7 group in a new species in the genus Atadenovirus.

Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples.

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.