H5N1 clade 2.3.4.4b dynamics in experimentally infected calves and cows.

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA1. Rapid dissemination to more than 190 farms in 13 states followed2. Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 isolates. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 108 TCID50/mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle.

Detection and characterization of H5N1 HPAIV in environmental samples from a dairy farm.

The recent expansion of HPAIV H5N1 infections in terrestrial mammals in the Americas, most recently including the outbreak in dairy cattle, emphasizes the critical need for better epidemiological monitoring of zoonotic diseases. In this work, we detected, isolated, and characterized the HPAIV H5N1 from environmental swab samples collected from a dairy farm in the state of Kansas, USA. Genomic sequencing of these samples uncovered two distinctive substitutions in the PB2 (E249G) and NS1 (R21Q) genes which are rare and absent in recent 2024 isolates of H5N1 circulating in the mammalian and avian species. Additionally, approximately 1.7% of the sequence reads indicated a PB2 (E627K) substitution, commonly associated with virus adaptation to mammalian hosts. Phylogenetic analyses of the PB2 and NS genes demonstrated more genetic identity between this environmental isolate and the 2024 human isolate (A/Texas/37/2024) of H5N1. Conversely, HA and NA gene analyses revealed a closer relationship between our isolate and those found in other dairy cattle with almost 100% identity, sharing a common phylogenetic subtree. These findings underscore the rapid evolutionary progression of HPAIV H5N1 among dairy cattle and reinforces the need for more epidemiological monitoring which can be done using environmental sampling.

Comparative evaluation of pathogenicity of three isolates of vesicular stomatitis virus (Indiana serotype) in pigs.

Vesicular stomatitis (VS) is a notifiable disease of livestock affecting cattle, horses, pigs and humans. Vesicular stomatitis virus (VSV) serotypes Indiana and New Jersey are endemic to Central America; however, they also cause sporadic and scattered outbreaks in various countries in South and North America, including the USA. In order to develop an effective experimental challenge model for VSV, we compared the pathogenicity of three VSV serotype Indiana isolates in 36 4-5 week-old pigs. Two bovine isolates of Central American origin and one equine isolate from the USA were used for the experimental infections. Each pig was inoculated with a single isolate by both the intradermal and intranasal routes. Clinical signs of VSV infection were recorded daily for 10 days post-inoculation (days p.i.). Nasal and tonsillar swab samples and blood were collected to monitor immune responses, virus replication and shedding. Post-challenge, characteristic signs of VS were observed, including vesicles on the nasal planum and coronary bands, lameness, loss of hoof walls and pyrexia. Pigs inoculated with the Central American isolates showed consistently more severe clinical signs in comparison to the pigs infected with the USA isolate. Genomic RNA was isolated from the original challenge virus stocks, sequenced and compared to VSV genomes available in GenBank. Comparative genome analysis demonstrated significant differences between the VSV isolate from the USA and the two Central American isolates. Our results indicate that the Central American isolates of VSV serotype Indiana used in this study are more virulent in swine than the USA VSV serotype Indiana isolate and represent good candidate challenge strains for future VSV studies.

Adjuvant Use of the Invariant-Natural-Killer-T-Cell Agonist α-Galactosylceramide Leads to Vaccine-Associated Enhanced Respiratory Disease in Influenza-Vaccinated Pigs.

Invariant natural killer T (iNKT) cells are glycolipid-reactive T cells with potent immunoregulatory properties. iNKT cells activated with the marine-sponge-derived glycolipid, α-galactosylceramide (αGC), provide a universal source of T-cell help that has shown considerable promise for a wide array of therapeutic applications. This includes harnessing iNKT-cell-mediated immune responses to adjuvant whole inactivated influenza virus (WIV) vaccines. An important concern with WIV vaccines is that under certain circumstances, they are capable of triggering vaccine-associated enhanced respiratory disease (VAERD). This immunopathological phenomenon can arise after immunization with an oil-in-water (OIW) adjuvanted WIV vaccine, followed by infection with a hemagglutinin and neuraminidase mismatched challenge virus. This elicits antibodies (Abs) that bind immunodominant epitopes in the HA2 region of the heterologous virus, which purportedly causes enhanced virus fusion activity to the host cell and increased infection. Here, we show that αGC can induce severe VAERD in pigs. However, instead of stimulating high concentrations of HA2 Abs, αGC elicits high concentrations of interferon (IFN)-γ-secreting cells both in the lungs and systemically. Additionally, we found that VAERD mediated by iNKT cells results in distinct cytokine profiles and altered adaptation of the challenge virus following infection compared to an OIW adjuvant. Overall, these results provide a cautionary note about considering the formulation of WIV vaccines with iNKT-cell agonists as a potential strategy to modulate antigen-specific immunity.

Pigs are highly susceptible to but do not transmit mink-derived highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b.

ABSTRACTRapid evolution of highly pathogenic avian influenza viruses (HPAIVs) is driven by antigenic drift but also by reassortment, which might result in robust replication in and transmission to mammals. Recently, spillover of clade 2.3.4.4b HPAIV to mammals including humans, and their transmission between mammalian species has been reported. This study aimed to evaluate the pathogenicity and transmissibility of a mink-derived clade 2.3.4.4b H5N1 HPAIV isolate from Spain in pigs. Experimental infection caused interstitial pneumonia with necrotizing bronchiolitis with high titers of virus present in the lower respiratory tract and 100% seroconversion. Infected pigs shed limited amount of virus, and importantly, there was no transmission to contact pigs. Notably, critical mammalian-like adaptations such as PB2-E627 K and HA-Q222L emerged at low frequencies in principal-infected pigs. It is concluded that pigs are highly susceptible to infection with the mink-derived clade 2.3.4.4b H5N1 HPAIV and provide a favorable environment for HPAIV to acquire mammalian-like adaptations.

Gene editing of pigs to control influenza A virus infections.

Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to WT pigs. Our findings could support the commercial use of GE pigs to minimize (i) the economic losses caused by IAV infection in pigs, and (ii) the emergence of novel IAVs with pandemic potential through genetic reassortment in the "mixing vessel", the pig.

Gene editing of pigs to control influenza A virus infections.

Proteolytic activation of the haemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in a significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to wild-type pigs. Our findings support the commercial use of GE pigs to mitigate influenza A virus infection in pigs, as an alternative approach to prevent zoonotic influenza A transmissions from pigs to humans.

Generation and Genetic Stability of a PolX and 5' MGF-Deficient African Swine Fever Virus Mutant for Vaccine Development.

The African swine fever virus (ASFV) causes fatal disease in pigs and is currently spreading globally. Commercially safe vaccines are urgently required. Aiming to generate a novel live attenuated vaccine (LAV), a recombinant ASFV was generated by deleting the viral O174L (PolX) gene. However, during in vitro generation, an additional spontaneous deletion of genes belonging to the multigene families (MGF) occurred, creating a mixture of two viruses, namely, Arm-ΔPolX and Arm-ΔPolX-ΔMGF. This mixture was used to inoculate pigs in a low and high dose to assess the viral dynamics of both populations in vivo. Although the Arm-ΔPolX population was a much lower proportion of the inoculum, in the high-dose immunized animals, it was the only resulting viral population, while Arm-ΔPolX-ΔMGF only appeared in low-dose immunized animals, revealing the role of deleted MGFs in ASFV fitness in vivo. Furthermore, animals in the low-dose group survived inoculation, whereas animals in the high-dose group died, suggesting that the lack of MGF and PolX genes, and not the PolX gene alone, led to attenuation. The two recombinant viruses were individually isolated and inoculated into piglets, confirming this hypothesis. However, immunization with the Arm-ΔPolX-ΔMGF virus did not induce protection against challenge with the virulent parental ASFV strain. This study demonstrates that deletion of the PolX gene alone neither leads to attenuation nor induces an increased mutation rate in vivo.

Experimental inoculation of pigs with monkeypox virus results in productive infection and transmission to sentinels.

Monkeypox virus (MPXV) is a re-emerging zoonotic poxvirus responsible for producing skin lesions in humans. Endemic in sub-Saharan Africa, the 2022 outbreak with a clade IIb strain has resulted in ongoing sustained transmission of the virus worldwide. MPXV has a relatively wide host range, with infections reported in rodent and non-human primate species. However, the susceptibility of many domestic livestock species remains unknown. Here, we report on a susceptibility/transmission study in domestic pigs that were experimentally inoculated with a 2022 MPXV clade IIb isolate or served as sentinel contact control animals. Several principal-infected and sentinel contact control pigs developed minor lesions near the lips and nose starting at 12 through 18 days post-challenge (DPC). No virus was isolated and no viral DNA was detected from the lesions; however, MPXV antigen was detected by IHC in tissue from a pustule of a principal infected pig. Viral DNA and infectious virus were detected in nasal and oral swabs up to 14 DPC, with peak titers observed at 7 DPC. Viral DNA was also detected in nasal tissues or skin collected from two principal-infected animals at 7 DPC post-mortem. Furthermore, all principal-infected and sentinel control animals enrolled in the study seroconverted. In conclusion, we provide the first evidence that domestic pigs are susceptible to experimental MPXV infection and can transmit the virus to contact animals.

Experimental co-infection of calves with SARS-CoV-2 Delta and Omicron variants of concern.

Since emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants.

Outcome of H5N1 clade 2.3.4.4b virus infection in calves and lactating cows.

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA. Rapid dissemination to more than 190 farms in 13 states followed. Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 genotypes/strains. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 108 TCID50/mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle.

Ancestral Lineage of SARS-CoV-2 Is More Stable in Human Biological Fluids than Alpha, Beta, and Omicron Variants of Concern.

SARS-CoV-2 is a zoonotic virus first identified in 2019, and has quickly spread worldwide. The virus is primarily transmitted through respiratory droplets from infected persons; however, the virus-laden excretions can contaminate surfaces which can serve as a potential source of infection. Since the beginning of the pandemic, SARS-CoV-2 has continued to evolve and accumulate mutations throughout its genome leading to the emergence of variants of concern (VOCs) which exhibit increased fitness, transmissibility, and/or virulence. However, the stability of SARS-CoV-2 VOCs in biological fluids has not been thoroughly investigated. The aim of this study was to determine and compare the stability of different SARS-CoV-2 strains in human biological fluids. Here, we demonstrate that the ancestral strain of the Wuhan-like lineage A was more stable than the Alpha VOC B.1.1.7, and the Beta VOC B.1.351 strains in human liquid nasal mucus and sputum. In contrast, there was no difference in stability among the three strains in dried biological fluids. Furthermore, we also show that the Omicron VOC B.1.1.529 strain was less stable than the ancestral Wuhan-like strain in liquid nasal mucus. These studies provide insight into the effect of the molecular evolution of SARS-CoV-2 on environmental virus stability, which is important information for the development of countermeasures against SARS-CoV-2. IMPORTANCE Genetic evolution of SARS-CoV-2 leads to the continuous emergence of novel virus variants, posing a significant concern to global public health. Five of these variants have been classified to date into variants of concern (VOCs); Alpha, Beta, Gamma, Delta, and Omicron. Previous studies investigated the stability of SARS-CoV-2 under various conditions, but there is a gap of knowledge on the survival of SARS-CoV-2 VOCs in human biological fluids which are clinically relevant. Here, we present evidence that Alpha, Beta, and Omicron VOCs were less stable than the ancestral Wuhan-like strain in human biological fluids. Our findings highlight the potential risk of contaminated human biological fluids in SARS-CoV-2 transmission and contribute to the development of countermeasures against SARS-CoV-2.

Stability of SARS-CoV-2 in Biological Fluids of Animals.

Since its first emergence in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve genetically, jump species barriers, and expand its host range. There is growing evidence of interspecies transmission including infection of domestic animals and widespread circulation in wildlife. However, knowledge of SARS-CoV-2 stability in animal biological fluids and their role in transmission is still limited as previous studies focused on human biological fluids. Therefore, this study aimed to determine the SARS-CoV-2 stability in biological fluids from three animal species, cats, sheep and white-tailed deer (WTD). Saliva, feces, 10% fecal suspensions, and urine of cats, sheep, and WTD were mixed with a known concentration of virus and incubated under indoor and three different climatic conditions. Our results show that the virus was stable for up to 1 day in the saliva of cats, sheep, and WTD regardless of the environmental conditions. The virus remained infectious for up to 6 days in feces and 15 days in fecal suspension of WTD, whereas the virus was rather unstable in cat and sheep feces and fecal suspensions. We found the longest survival of SARS-CoV-2 in the urine of cats, sheep, and WTD. Furthermore, side-by-side comparison with different SARS-CoV-2 strains showed that the Alpha, Delta, and Omicron variants of concern were less stable than the ancestral Wuhan-like strain in WTD fecal suspension. The results of our study provide valuable information for assessing the potential role of various animal biological fluids in SARS-CoV-2 transmission.

Rapid Identification of ASFV, CSFV and FMDV from Mongolian Outbreaks with MinION Short Amplicon Sequencing.

African swine fever virus (ASFV), classical swine fever virus (CSFV), and foot-and-mouth disease virus (FMDV) cause important transboundary animal diseases (TADs) that have a significant economic impact. The rapid and unequivocal identification of these pathogens and distinction from other animal diseases based on clinical symptoms in the field is difficult. Nevertheless, early pathogen detection is critical in limiting their spread and impact as is the availability of a reliable, rapid, and cost-effective diagnostic test. The purpose of this study was to evaluate the feasibility to identify ASFV, CSFV, and FMDV in field samples using next generation sequencing of short PCR products as a point-of-care diagnostic. We isolated nucleic acids from tissue samples of animals in Mongolia that were infected with ASFV (2019), CSFV (2015), or FMDV (2018), and performed conventional (RT-) PCR using primers recommended by the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH). The (RT-) PCR products were then sequenced in Mongolia using the MinION nanopore portable sequencer. The resulting sequencing reads successfully identified the respective pathogens that exhibited 91-100% nucleic acid similarity to the reference strains. Phylogenetic analyses suggest that the Mongolian virus isolates are closely related to other isolates circulating in the same geographic region. Based on our results, sequencing short fragments derived by conventional (RT-) PCR is a reliable approach for rapid point-of-care diagnostics for ASFV, CSFV, and FMDV even in low-resource countries.

Preliminary Study on the Efficacy of a Recombinant, Subunit SARS-CoV-2 Animal Vaccine against Virulent SARS-CoV-2 Challenge in Cats.

The objective of this work was to evaluate the safety and efficacy of a recombinant, subunit SARS-CoV-2 animal vaccine in cats against virulent SARS-CoV-2 challenge. Two groups of cats were immunized with two doses of either a recombinant SARS-CoV-2 spike protein vaccine or a placebo, administered three weeks apart. Seven weeks after the second vaccination, both groups of cats were challenged with SARS-CoV-2 via the intranasal and oral routes simultaneously. Animals were monitored for 14 days post-infection for clinical signs and viral shedding before being humanely euthanized and evaluated for macroscopic and microscopic lesions. The recombinant SARS-CoV-2 spike protein subunit vaccine induced strong serologic responses post-vaccination and significantly increased neutralizing antibody responses post-challenge. A significant difference in nasal and oral viral shedding, with significantly reduced virus load (detected using RT-qPCR) was observed in vaccinates compared to mock-vaccinated controls. Duration of nasal, oral, and rectal viral shedding was also significantly reduced in vaccinates compared to controls. No differences in histopathological lesion scores were noted between the two groups. Our findings support the safety and efficacy of the recombinant spike protein-based SARS-CoV-2 vaccine which induced high levels of neutralizing antibodies and reduced nasal, oral, and rectal viral shedding, indicating that this vaccine will be efficacious as a COVID-19 vaccine for domestic cats.

Ancestral lineage of SARS-CoV-2 is more stable in human biological fluids than Alpha, Beta and Omicron variants of concern.

SARS-CoV-2 is a zoonotic virus which was first identified in 2019, and has quickly spread worldwide. The virus is primarily transmitted through respiratory droplets from infected persons; however, the virus-laden excretions can contaminate surfaces which can serve as a potential source of infection. Since the beginning of the pandemic, SARS-CoV-2 has continued to evolve and accumulate mutations throughout its genome leading to the emergence of variants of concern (VOCs) which exhibit increased fitness, transmissibility, and/or virulence. However, the stability of SARS-CoV-2 VOCs in biological fluids has not been thoroughly investigated so far. The aim of this study was to determine and compare the stability of different SARS-CoV-2 strains in human biological fluids. Here, we demonstrate that the ancestral strain of Wuhan-like lineage A was more stable than the Alpha VOC B.1.1.7, and the Beta VOC B.1.351 strains in human liquid nasal mucus and sputum. In contrast, there was no difference in stability among the three strains in dried biological fluids. Furthermore, we also show that the Omicron VOC B.1.1.529 strain was less stable than the ancestral Wuhan-like strain in liquid nasal mucus. These studies provide insight into the effect of the molecular evolution of SARS-CoV-2 on environmental virus stability, which is important information for the development of countermeasures against SARS-CoV-2. Importance: Genetic evolution of SARS-CoV-2 leads to the continuous emergence of novel variants, posing a significant concern to global public health. Five of these variants have been classified so far into variants of concern (VOCs); Alpha, Beta, Gamma, Delta, and Omicron. Previous studies investigated the stability of SARS-CoV-2 under various conditions, but there is a gap of knowledge on the survival of SARS-CoV-2 VOCs in human biological fluids which are clinically relevant. Here, we present evidence that Alpha, Beta, and Omicron VOCs were less stable than the ancestral Wuhan-like strain in human biological fluids. Our findings highlight the potential risk of contaminated human biological fluids in SARS-CoV-2 transmission and contribute to the development of countermeasures against SARS-CoV-2.

Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic agent capable of infecting humans and a wide range of animal species. Over the duration of the pandemic, mutations in the SARS-CoV-2 spike (S) protein have arisen, culminating in the spread of several variants of concern (VOCs) with various degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we used pseudoviruses that express specific SARS-CoV-2 S protein substitutions and cell lines that express angiotensin-converting enzyme 2 (ACE2) from nine different animal species to gain insights into the effects of VOC mutations on viral entry and antibody neutralization capability. All animal ACE2 receptors tested, except mink, support viral cell entry for pseudoviruses expressing the ancestral prototype S at levels comparable to human ACE2. Most single S substitutions did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency. Conversely, combinatorial VOC substitutions in the S protein were associated with increased entry of pseudoviruses. Neutralizing titers in sera from various animal species were significantly reduced against pseudoviruses expressing the S proteins of Beta, Delta, or Omicron VOCs compared to the parental S protein. Especially, substitutions in the S protein of the Omicron variant significantly reduced the neutralizing titers of the sera. This study reveals important insights into the host range of SARS-CoV-2 and the effect of recently emergent S protein substitutions on viral entry, virus replication, and antibody-mediated viral neutralization. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to have devastating impacts on global health and socioeconomics. The recent emergence of SARS-CoV-2 variants of concern, which contain mutations that can affect the virulence, transmission, and effectiveness of licensed vaccines and therapeutic antibodies, are currently becoming the common strains circulating in humans worldwide. In addition, SARS-CoV-2 has been shown to infect a wide variety of animal species, which could result in additional mutations of the SARS-CoV-2 virus. In this study, we investigate the effect of mutations present in SARS-CoV-2 variants of concern and determine the effects of these mutations on cell entry, virulence, and antibody neutralization activity in humans and a variety of animals that might be susceptible to SARS-CoV-2 infection. This information is essential to understand the effects of important SARS-CoV-2 mutations and to inform public policy to create better strategies to control the COVID-19 pandemic.

Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 by experimental and/or natural infections. Sheep are a commonly farmed domestic ruminant that have not been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cells and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived kidney cells support SARS-CoV-2 replication. Furthermore, the experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs at 1 and 3-days post challenge (DPC); viral RNA was also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naïve sheep was not highly efficient; however, viral RNA was detected in respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used a challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern, to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection and that the alpha variant outcompeted the lineage A strain.

Infection and transmission of ancestral SARS-CoV-2 and its alpha variant in pregnant white-tailed deer.

ABSTRACTSARS-CoV-2 was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks have demonstrated the significant role of intermediate hosts in viral maintenance and transmission. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (WTD) are amongst the most abundant and geographically widespread wild ruminant species in the US. Recently, WTD fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult WTD. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the alpha variant of concern (VOC) B.1.1.7 through co-infection of WTD. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult WTD are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in WTD, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from the genome of virus present in tissues of principal infected deer, fetuses and contact animals.

Experimental Infection of Domestic Pigs with African Swine Fever Virus Isolated in 2019 in Mongolia.

African swine fever (ASF) is an infectious viral disease caused by African swine fever virus (ASFV), that causes high mortality in domestic swine and wild boar (Sus scrofa). Currently, outbreaks are mitigated through strict quarantine measures and the culling of affected herds, resulting in massive economic losses to the global pork industry. In 2019, an ASFV outbreak was reported in Mongolia, describing a rapidly progressing clinical disease and gross lesions consistent with the acute form of ASF; the virus was identified as a genotype II virus. Due to the limited information on clinical disease and viral dynamics within hosts available from field observations of the Mongolian isolates, we conducted the present study to further evaluate the progression of clinical disease, virulence, and pathology of an ASFV Mongolia/2019 field isolate (ASFV-MNG19), by experimental infection of domestic pigs. Intramuscular inoculation of domestic pigs with ASFV-MNG19 resulted in clinical signs and viremia at 3 days post challenge (DPC). Clinical disease rapidly progressed, resulting in the humane euthanasia of all pigs by 7 DPC. ASFV-MNG19 infected pigs had viremic titers of 108 TCID50/mL by 5 DPC and shed virus in oral secretions late in disease, as determined from oropharyngeal swabs. Whole-genome sequencing confirmed that the ASFV-MNG19 strain used in this study was a genotype II strain highly similar to other regional strains. In conclusion, we demonstrate that ASFV-MNG19 is a virulent genotype II ASFV strain that causes acute ASF in domestic swine.