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Since 1996, goose/Guangdong lineage H5 highly pathogenic influenza A viruses (HPIAV) have caused an unprecedented panzootic. In 2020, subclade 2.3.4.4b HPIAV H5N1 emerged and spread to multiple continents causing substantial [...]

The recent incursion of highly pathogenic influenza viruses into dairy cattle opens new insights for influenza virus ecology and its interspecies transmission and may have a significant impact on public health and agriculture. The aim of this study was to determine the stability of a bovine highly pathogenic avian influenza H5N1 virus isolate in the milk byproduct lactose and to evaluate two inactivation methods using industrial procedures. The bovine isolate of the highly pathogenic avian influenza H5N1 virus was stable for 14 days in a concentrated lactose solution under refrigerated conditions. Heat or citric acid treatments successfully inactivated the virus in lactose. This study highlights the persistence of HPAIV in lactose and its efficient inactivation under industrial standards. [ABSTRACT FROM AUTHOR]

Robust testing capacity was necessary for public health agencies to respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the coronavirus disease 19 (COVID-19) pandemic. As the nation faced the need for robust testing capacity, it became necessary to use all possible resources. In many cases, veterinary diagnostic laboratories rose to meet this demand because these facilities routinely perform high throughput diagnostic testing of large animal populations and are typically familiar with pathogens of high pandemic concern. In this study, we evaluated the impact of veterinary diagnostic laboratories in the United States on SARS-CoV-2 testing. Results of surveys, semi-structured interviews, and analysis of publicly available information showed that veterinary diagnostic laboratories had a substantial impact on human health through population-level testing in the COVID-19 response, supporting timely and informed public health interventions. This success was not without significant hurdles, as many participating veterinary diagnostic laboratories experienced restriction in their response due to difficulties obtaining the Clinical Laboratory Improvement Amendments (CLIA) certification required to conduct human diagnostic testing. Our results point out the importance of reducing hurdles before the next major public health emergency to enhance access to testing resources overall and to ultimately improve population health. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the seroprevalence and spatial and temporal clustering of SARS-CoV-2 antibodies in household cats within 63 counties in Illinois from October 2021 to May 2023. The analysis followed a stepwise approach. First, in a choropleth point map, we illustrated the distribution of county-level seroprevalence of SARS-CoV-2 antibodies. Next, spatial interpolation was used to predict the seroprevalence in counties without recorded data. Global and local clustering methods were used to identify the extent of clustering and the counties with high or low seroprevalence, respectively. Next, temporal, spatial, and space-time scan statistic was used to identify periods and counties with higher-than-expected seroprevalence. In the last step, to identify more distinct areas in counties with high seroprevalence, city-level analysis was conducted to identify temporal and space-time clusters. Among 1,715 samples tested by serological assays, 244 samples (14%) tested positive. Young cats had higher seropositivity than older cats, and the third quarter of the year had the highest odds of seropositivity. Three county-level space-time clusters with higher-than-expected seroprevalence were identified in the northeastern, central-east, and southwest regions of Illinois, occurring between June and October 2022. In the city-level analysis, 2 space-time clusters were identified in Chicago's downtown and the southwestern suburbs of Chicago between June and September 2022. Our results suggest that the high density of humans and cats in large cities such as Chicago, might play a role in the transmission and clustering of SARS-CoV-2. Our study provides an in-depth analysis of SARS-CoV-2 epidemiology in Illinois household cats, which will aid in COVID-19 control and prevention. [ABSTRACT FROM AUTHOR]

African Swine Fever Virus (ASFV) is a large dsDNA virus that encodes at least 150 proteins. The complexity of ASFV and lack of knowledge of effector immune functions and protective antigens have hindered the development of safe and effective ASF vaccines. In this study, we constructed four Orf virus recombinant vectors expressing individual ASFV genes B602L, -CP204L, E184L, and -I73R (ORFVΔ121-ASFV-B602L, -CP204L, -E184L, and -I73R). All recombinant viruses expressed the heterologous ASFV proteins in vitro. We then evaluated the immunogenicity of the recombinants by immunizing four-week-old piglets. In two independent animal studies, we observed high antibody titers against ASFV p30, encoded by CP204L gene. Using Pepscan ELISA, we identified a linear B-cell epitope of 12 amino acids in length (Peptide 15) located in an exposed loop region of p30 as an immunodominant ASFV epitope. Additionally, antibodies elicited against ASFV p30 presented antibody-dependent cellular cytotoxicity (ADCC) activity. These results underscore the role of p30 on antibody responses elicited against ASFV and highlight an important functional epitope that contributes to p30-specific antibody responses. [ABSTRACT FROM AUTHOR]

On April 4, 2021, a 5-year-old male Malayan tiger (Panthera tigris jacksoni) at the Virginia Zoo (Norfolk, VA, USA) began exhibiting lethargy, labored breathing, coughing, intermittent upper respiratory sounds, hyporexia, [...]

Orf virus (ORFV) is a large DNA virus that can harbor and efficiently deliver viral antigens in swine. Here we used ORFV as a vector platform to deliver chimeric hemagglutinins (HA) of Influenza A virus of swine (IAV-S). Vaccine development against IAV-S faces limitations posed by strain-specific immunity and the antigenic diversity of the IAV-S strains circulating in the field. A promising alternative aiming at re-directing immune responses on conserved epitopes of the stalk segment of the hemagglutinin (HA2) has recently emerged. Sequential immunization with chimeric HAs comprising the same stalk but distinct exotic head domains can potentially induce cross-reactive immune responses against conserved epitopes of the HA2 while breaking the immunodominance of the head domain (HA1). Here, we generated two recombinant ORFVs expressing chimeric HAs encoding the stalk region of a contemporary H1N1 IAV-S strain and exotic heads derived from either H6 or H8 subtypes, ORFVD121cH6/1 and ORFVD121cH8/1, respectively. The resulting recombinant viruses were able to express the heterologous protein in vitro. Further, the immunogenicity and cross-protection of these vaccine candidates were assessed in swine after sequential intramuscular immunization with OV-cH6/1 and OV-cH8/1, and subsequent challenge with divergent IAV-S strains. Humoral responses showed that vaccinated piglets presented increasing IgG responses in sera. Additionally, cross-reactive IgG and IgA antibody responses elicited by immunization were detected in sera and bronchoalveolar lavage (BAL), respectively, by ELISA against different viral clades and a diverse range of contemporary H1N1 IAV-S strains, indicating induction of humoral and mucosal immunity in vaccinated animals. Importantly, viral shedding was reduced in nasal swabs from vaccinated piglets after intranasal challenge with either Oh07 (gamma clade) or Ca09 (npdm clade) IAV-S strains. These results demonstrated the efficiency of ORFV-based vectors in delivering chimeric IAV-S HA-based vaccine candidates and underline the potential use of chimeric-HAs for prevention and control of influenza in swine. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is the most significant threat to pork production worldwide. Over the past year, the virus has emerged in new countries and continents, including Belgium (1), [...]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The unprecedented COVID-19 pandemic posed major challenges to local, regional, and global economies and health systems, and fast clinical diagnostic workflows were urgently needed to contain the spread of SARS-CoV-2. Here, we describe the platform and workflow established at the Cornell COVID-19 Testing Laboratory (CCTL) for the high-throughput testing of clinical samples from the university and the surrounding community. This workflow enabled efficient and rapid detection and the successful control of SARS-CoV-2 infection on campus and its surrounding communities. Our cost-effective and fully automated workflow enabled the testing of over 8000 pooled samples per day and provided results for over 2 million samples. The automation of time- and effort-intensive sample processing steps such as accessioning and pooling increased laboratory efficiency. Customized software applications were developed to track and store samples, deconvolute positive pools, track and report results, and for workflow integration from sample receipt to result reporting. Additionally, quality control dashboards and turnaround-time tracking applications were built to monitor assay and laboratory performance. As infectious disease outbreaks pose a constant threat to both human and animal health, the highly effective workflow implemented at CCTL could be modeled to establish regional high-capacity testing hubs for infectious disease preparedness and emergency response. [ABSTRACT FROM AUTHOR]

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs. [ABSTRACT FROM AUTHOR]

Newcastle disease (ND) is endemic in Bangladesh. Locally produced or imported live Newcastle disease virus (NDV) vaccines based on lentogenic virus strains, locally produced live vaccines of the mesogenic Mukteswar strain, as well as imported inactivated vaccines of lentogenic strains, are being used in Bangladesh under different vaccination regimens. Despite these vaccinations, frequent outbreaks of ND are being reported in Bangladesh. Here we compared the efficacy of booster immunization with three different vaccines in chickens that had been primed with two doses of live LaSota vaccine. A total of 30 birds (Group A) were primed with two doses of live LaSota virus (genotype II) vaccine at days 7 and 28, while 20 birds (Group B) remained unvaccinated. At day 60, birds of Group A were divided into three sub-groups, which received booster immunizations with three different vaccines; A1: live LaSota vaccine, A2: inactivated LaSota vaccine, and A3: inactivated genotype XIII.2 vaccine (BD-C161/2010 strain from Bangladesh). Two weeks after booster vaccination (at day 74), all vaccinated birds (A1–A3) and half of the unvaccinated birds (B1) were challenged with a genotype XIII.2 virulent NDV (BD-C161/2010). A moderate antibody response was observed after the primary vaccination, which substantially increased after the booster vaccination in all groups. The mean HI titers induced by the inactivated LaSota vaccine (8.0 log2/5.0 log2 with LaSota/BD-C161/2010 HI antigen) and the inactivated BD-C161/2010 vaccine (6.7 log2/6.2 log2 with LaSota/BD-C161/2010 HI antigen) were significantly higher than those induced by the LaSota live booster vaccine (3.6 log2/2.6 log2 with LaSota/BD-C161/2010 HI antigen). Despite the differences in the antibody titers, all chickens (A1–A3) survived the virulent NDV challenge, while all the unvaccinated challenged birds died. Among the vaccinated groups, however, 50% of the chickens in Group A1 (live LaSota booster immunization) shed virus at 5- and 7-days post challenge (dpc), while 20% and 10% of the chickens in Group A2 (inactivated LaSota booster immunization) shed virus at 3 and 5 dpc, respectively, and only one chicken (10%) in Group A3 shed virus at 5 dpc. In conclusion, the genotype-matched inactivated NDV booster vaccine offers complete clinical protection and a significant reduction in virus shedding. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAV-S) belonging to the H1 subtype are endemic in swine worldwide. Antigenic drift and antigenic shift lead to a substantial antigenic diversity in circulating IAV-S strains. As a result, the most commonly used vaccines based on whole inactivated viruses (WIVs) provide low protection against divergent H1 strains due to the mismatch between the vaccine virus strain and the circulating one. Here, a consensus coding sequence of the full-length of HA from H1 subtype was generated in silico after alignment of the sequences from IAV-S isolates obtained from public databases and was delivered to pigs using the Orf virus (ORFV) vector platform. The immunogenicity and protective efficacy of the resulting ORFVΔ121conH1 recombinant virus were evaluated against divergent IAV-S strains in piglets. Virus shedding after intranasal/intratracheal challenge with two IAV-S strains was assessed by real-time RT-PCR and virus titration. Viral genome copies and infectious virus load were reduced in nasal secretions of immunized animals. Flow cytometry analysis showed that the frequency of T helper/memory cells, as well as cytotoxic T lymphocytes (CTLs), were significantly higher in the peripheral blood mononuclear cells (PBMCs) of the vaccinated groups compared to unvaccinated animals when they were challenged with a pandemic strain of IAV H1N1 (CA/09). Interestingly, the percentage of T cells was higher in the bronchoalveolar lavage of vaccinated animals in relation to unvaccinated animals in the groups challenged with a H1N1 from the gamma clade (OH/07). In summary, delivery of the consensus HA from the H1 IAV-S subtype by the parapoxvirus ORFV vector decreased shedding of infectious virus and viral load of IAV-S in nasal secretions and induced cellular protective immunity against divergent influenza viruses in swine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Newcastle disease (ND) is endemic in poultry in Bangladesh. We performed genotypic and pathotypic characterization of four ND virus (NDV) isolates from recent outbreaks in broiler chickens in Bangladesh during the period of 2020–2021. Phylogenetic analysis based on the complete fusion protein gene coding sequences classified the viruses into NDV class II genotype VII.2 together with viruses from Indonesia isolated between 2014 and 2021 and a single 2020 Indian isolate. Pathogenicity testing using the intracerebral pathogenicity index in day-old chickens and mean embryo death time in embryonating chicken eggs revealed that the Bangladeshi isolates are velogenic. Inoculation of 35-day-old chickens with two NDV isolates (LT67 and N5) resulted in 100% morbidity by 3 days post inoculation (DPI), and all birds succumbed to infection by 7 DPI. Massive hemorrhages, congestion and necrotic lesions were observed in different visceral organs, which were typical for infection with a velogenic viscerotropic pathotype of NDV. At microscopic examination, tracheitis, severe pneumonia, focal proventriculitis, transmural enteritis, focal myocarditis, severe congestion and necrosis in kidneys, and lymphoid depletion in lymphoid tissues were found. Our study reports the first outbreak of the panzootic genotype VII.2 NDV in poultry in Bangladesh and documents a possible recent re-introduction of this NDV genotype from Southeast or East Asia. This study further provides viral distribution and epidemiological data that can facilitate the effective control of NDV. [ABSTRACT FROM AUTHOR]

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. Author summary: The high susceptibility of white-tailed deer (WTD) to SARS-CoV-2, their ability to transmit the virus to other deer, and the recent findings suggesting widespread SARS-CoV-2 infection in wild WTD populations in the U.S. underscore the need for a better understanding of the infection and transmission dynamics of SARS-CoV-2 in this potential reservoir species. Here we investigated the transmission dynamics of SARS-CoV-2 following intranasal inoculation of deer fawns by adding contact animals on days 3, 6, or 9 pi and defined the major sites of virus replication during the acute phase of infection. Additionally, we assessed the evolution of the virus as it replicated and transmitted between animals. The work provides important information on the infection dynamics of SARS-CoV-2 in WTD, an animal species that—if confirmed as a new reservoir of infection—may provide many opportunities for exposure and potential zoonotic transmission of the virus back to humans. [ABSTRACT FROM AUTHOR]

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination. [ABSTRACT FROM AUTHOR]

Transboundary movement of animal feed and feed ingredients has been identified as a route for pathogen incursions. While imports of animals and animal‐derived products are highly regulated for the purpose of infectious disease prevention, there has been less consideration of the viability of infectious agents in inanimate products, such as feed. This study investigated the ability of foot‐and‐mouth disease virus (FMDV) to remain infectious as a contaminant of commercial whole pig feed and select pig feed ingredients, and to establish the minimum infectious dose (MIDF) required to cause foot‐and‐mouth disease (FMD) in pigs that consumed contaminated feed. FMDV viability in vitro varied depending on virus strain, feed product, and storage temperature, with increased duration of infectivity in soybean meal compared to pelleted whole feed. Specifically, both strains of FMDV evaluated remained viable through to the end of the 37 day observation period in experimentally contaminated soybean meal stored at 4 or 20°C. The MIDF for pigs consuming contaminated feed varied across virus strains and exposure duration in the range of 106.2 to 107 TCID50. The ability of FMDV to cause infection in exposed pigs was mitigated by pre‐treatment of feed with two commercially available feed additives, based on either formaldehyde (SalCURB®) or lactic acid (Guardian™). Our findings demonstrate that FMDV may remain infectious in pig feed ingredients for durations compatible with transoceanic transport. Although the observed MIDF was relatively high, variations in feeding conditions and biophysical characteristics of different virus strains may alter the probability of infection. These findings may be used to parameterize modelling of the risk of FMDV incursions and to regulate feed importation to minimize the risk of inadvertent importation. [ABSTRACT FROM AUTHOR]

Animal feed and feed ingredients have recently been investigated as sources of pathogen introduction to farms and as a potential source of infection to animals post‐consumption of contaminated feed. Survival of several viruses for a prolonged period has been demonstrated in feed. Here, we determined the rate of decay of Senecavirus A (SVA) in swine feed ingredients as a function of time and temperature and established half‐life estimates for the virus. Select feed ingredients were spiked with a constant amount of SVA (105 median tissue culture infectious dose 50) and incubated at 4, 15 and 30°C for up to 91 days. Virus viability and the presence of viral RNA were assessed in samples collected over time. At the three different temperatures investigated, dried distillers' grains with solubles (DDGS) and soybean meal (SBM) provided the most stable matrices for SVA, resulting in half‐lives of 25.6 and 9.8 days, respectively. At 30°C, SVA was completely inactivated in all feed ingredients and in the control sample, which did not contain a feed matrix. Although virus infectivity was lost, viral RNA remained stable and at consistent levels throughout the experimental period. Additionally, the ability of SVA to infect swine via ingestion of contaminated feed was investigated in 3‐week‐old, weaned pigs. Animals were provided complete feed spiked with three concentrations of SVA (105, 106 and 107 per 200 g of feed) and allowed to naturally consume the contaminated feed. This procedure was repeated for three consecutive days. Infection of pigs through consumption of contaminated feed was confirmed by virus neutralization assay and the detection of SVA in serum, feces and in the tonsil of exposed animals by real‐time reverse transcriptase PCR. Our findings demonstrate that feed matrices are able to extend the survival of SVA, protecting the virus from decay. Additionally, we demonstrated that consumption of contaminated feed can lead to productive SVA infection. [ABSTRACT FROM AUTHOR]

Swine influenza is a highly contagious respiratory disease of pigs caused by influenza A viruses (IAV-S). IAV-S causes significant economic losses to the swine industry and poses challenges to public health given its zoonotic potential. Thus effective IAV-S vaccines are needed and highly desirable and would benefit both animal and human health. Here, we developed two recombinant orf viruses, expressing the hemagglutinin (HA) gene (OV-HA) or the HA and the nucleoprotein (NP) genes of IAV-S (OV-HA-NP). The immunogenicity and protective efficacy of these two recombinant viruses were evaluated in pigs. Both OV-HA and OV-HA-NP recombinants elicited robust virus neutralizing antibody response in pigs, with higher levels of neutralizing antibodies (NA) being detected in OV-HA-NP-immunized animals pre-challenge infection. Although both recombinant viruses elicited IAV-S-specific T-cell responses, the frequency of IAV-S-specific proliferating CD8+ T cells upon re-stimulation was higher in OV-HA-NP-immunized animals than in the OV-HA group. Importantly, IgG1/IgG2 isotype ELISAs revealed that immunization with OV-HA induced Th2-biased immune responses, whereas immunization with OV-HA-NP virus resulted in a Th1-biased immune response. While pigs immunized with either OV-HA or OV-HA-NP were protected when compared to non-immunized controls, immunization with OV-HA-NP resulted in incremental protection against challenge infection as evidenced by a reduced secondary antibody response (NA and HI antibodies) following IAV-S challenge and reduced virus shedding in nasal secretions (lower viral RNA loads and frequency of animals shedding viral RNA and infectious virus), when compared to animals in the OV-HA group. Interestingly, broader cross neutralization activity was also observed in serum of OV-HA-NP-immunized animals against a panel of contemporary IAV-S isolates representing the major genetic clades circulating in swine. This study demonstrates the potential of ORFV-based vector for control of swine influenza virus in swine. [ABSTRACT FROM AUTHOR]

Abstract Background Many viruses have evolved multiple strategies to prevent super infection of host cells by more than one virion. This phenomenon, known as super infection exclusion, may play an important role on virus evolution because it can affect the frequency of reassortment and/or recombination. Newcastle disease virus (NDV), a negative sense single-stranded RNA virus, is characterized by its continuous evolutionary dynamics and by a low frequency of recombination events. However, the mechanisms that contribute to the low recombination rates on NDV are still not completely understood. Methods In this study we assessed the ability of two NDV strains (LaSota and B1) to super infect host cells in vitro. We generated a recombinant NDV strain LaSota expressing the red fluorescent protein (RFP) and used it in co-infection assays with a related NDV strain B1 expressing the green fluorescent protein (GFP). DF-1 cells were inoculated with both viruses at the same time or at different intervals between primary infection and super infection. Results When both viruses were inoculated at the same time point, a 27% co-infection rate was observed, whereas when they were inoculated at different time points the super infection rates decreased to levels as low as 1.4%. Conclusions These results indicate that although different NDV strains can co-infect host cells in vitro, the super infection rates are low, specially as the time between the primary infection and super infection increases. These results confirm the occurrence of super infection exclusion between different strains of NDV.

African swine fever (ASF) is currently considered the most significant global threat to pork production worldwide. Disease caused by the ASF virus (ASFV) results in high case fatality of pigs. Importantly, ASF is a trade‐limiting disease with substantial implications on both global pork and agricultural feed commodities. ASFV is transmissible through natural consumption of contaminated swine feed and is broadly stable across a wide range of commonly imported feed ingredients and conditions. The objective of the current study was to investigate the efficacy of medium‐chain fatty acid and formaldehyde‐based feed additives in inactivating ASFV. Feed additives were tested in cell culture and in feed ingredients under a transoceanic shipment model. Both chemical additives reduced ASFV infectivity in a dose‐dependent manner. This study provides evidence that chemical feed additives may potentially serve as mitigants for reducing the risk of ASFV introduction and transmission through feed. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged as the cause of a global pandemic in 2019–2020. In March 2020, New York City became the epicenter in the United States for the pandemic. On 27 March 2020, a Malayan tiger (Panthera tigris jacksoni) at the Bronx Zoo in New York City developed a cough and wheezing with subsequent inappetence. Over the next week, an additional Malayan tiger and two Amur tigers (Panthera tigris altaica) in the same building and three lions (Panthera leo krugeri) in a separate building also became ill. The index case was anesthetized for diagnostic workup. Physical examination and bloodwork results were unremarkable. Thoracic radiography and ultrasonography revealed a bronchial pattern with peribronchial cuffing and mild lung consolidation with alveolar-interstitial syndrome, respectively. SARS-CoV-2 RNA was identified by real-time, reverse transcriptase PCR (rRT-PCR) on oropharyngeal and nasal swabs and tracheal wash fluid. Cytologic examination of tracheal wash fluid revealed necrosis, and viral RNA was detected in necrotic cells by in situ hybridization, confirming virus-associated tissue damage. SARS-CoV-2 was isolated from the tracheal wash fluid of the index case, as well as the feces from one Amur tiger and one lion. Fecal viral RNA shedding was confirmed in all seven clinical cases and an asymptomatic Amur tiger. Respiratory signs abated within 1–5 days for most animals, although they persisted intermittently for 16 days in the index case. Fecal RNA shedding persisted for as long as 35 days beyond cessation of respiratory signs. This case series describes the clinical presentation, diagnostic evaluation, and management of tigers and lions infected with SARS-CoV-2 and describes the duration of viral RNA fecal shedding in these cases. This report documents the first known natural transmission of SARS-CoV-2 from humans to nondomestic felids. [ABSTRACT FROM AUTHOR]

Classical swine fever virus (CSFV) and pseudorabies virus (PRV) are two of the most significant trade‐limiting pathogens affecting swine worldwide. Both viruses are endemic to China where millions of kilograms of feed ingredients are manufactured and subsequently imported into the United States. Although stability and oral transmission of both viruses through contaminated pork products has been demonstrated as a risk factor for transboundary spread, stability in animal feed ingredients had yet to be investigated. The objective of this study was to determine the survival of CSFV and variant PRV in 12 animal feeds and ingredients exposed to environmental conditions simulating a 37‐day transpacific shipment. Virus was detected by PCR, virus isolation and nursery pig bioassay. CSFV and PRV nucleic acids were stable throughout the 37‐day period in all feed matrices. Infectious CSFV was detected in two ingredients (conventional soybean meal and pork sausage casings) at 37 days post‐contamination, whereas infectious PRV was detected in nine ingredients (conventional and organic soybean meal, lysine, choline, vitamin D, moist cat and dog food, dry dog food and pork sausage casings). This study demonstrates the relative stability of CSFV and PRV in different feed ingredients under shipment conditions and provides evidence that feed ingredients may represent important risk factors for the transboundary spread of these viruses. [ABSTRACT FROM AUTHOR]

Senecavirus A (SVA) is an emerging picornavirus causing vesicular disease (VD) clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Currently there are no vaccines currently available for SVA. Here we developed a recombinant SVA strain (rSVAm SacII) using reverse genetics and assessed its immunogenicity and protective efficacy in pigs. In vivo characterization of the rSVAm SacII strain demonstrated that the virus is attenuated, as evidenced by absence of lesions, decreased viremia and virus shedding in inoculated animals. Notably, while attenuated, rSVA mSacII virus retained its immunogenicity as high neutralizing antibody (NA) responses were detected in inoculated animals. To assess the immunogenicity and protective efficacy of rSVA mSacII, 4-week-old piglets were sham-immunized or immunized with inactivated or live rSVA mSacII virus-based formulations. A single immunization with live rSVA mSacII virus via the intramuscular (IM) and intranasal (IN) routes resulted in robust NA responses with antibodies being detected between days 3–7 pi. Neutralizing antibody responses in animals immunized with the inactivated virus via the IM route were delayed and only detected after a booster on day 21 pi. Immunization with live virus resulted in recall T cell proliferation (CD4+, CD8+, and CD4+/CD8+ T cells), demonstrating efficient stimulation of cellular immunity. Notably, a single dose of the live attenuated vaccine candidate resulted in protection against heterologous SVA challenge, as demonstrated by absence of overt disease and reduced viremia, virus shedding and viral load in tissues. The live attenuated vaccine candidate developed here represents a promising alternative to prevent and control SVA in swine. [ABSTRACT FROM AUTHOR]