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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 (COVID-19) in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 infection by experimental and/or natural infections. Domestic and large cats, mink, ferrets, hamsters, deer mice, white-tailed deer, and non-human primates have been shown to be highly susceptible, whereas other species such as mice, dogs, pigs, and cattle appear to be refractory to infection or have very limited susceptibility. Sheep (Ovis aries) are a commonly farmed domestic ruminant that have not previously 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 cell cultures and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived cell cultures support SARS-CoV-2 replication. Furthermore, experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs primarily at 1-day post challenge (DPC), and also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was also 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 some of the respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used 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 (VOC), 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 VOC outcompeted the ancestral lineage A strain.

SARS-CoV-2, a novelBetacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, andin silicoandin vitrostudies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer 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 white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. 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 white-tailed deer 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 white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naïve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Histologic lesions that characterized primary SARS-CoV-2 infected cats at 4 DPC were absent in the re-challenged cats. Naïve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naïve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against reinfection.

The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.

Influenza A viruses (IAVs) circulate widely among different mammalian and avian hosts and sometimes give rise to zoonotic infections. Vaccination is a mainstay of IAV prevention and control. However, the efficacy of IAV vaccines is often suboptimal because of insufficient cross-protection among different IAV genotypes and subtypes as well as the inability to keep up with the rapid molecular evolution of IAV strains. Much attention is focused on improving IAV vaccine efficiency using adjuvants, which are substances that can modulate and enhance immune responses to co-administered antigens. The current review is focused on a non-traditional approach of adjuvanting IAV vaccines by therapeutically targeting the immunomodulatory functions of a rare population of innate-like T lymphocytes called invariant natural killer T (iNKT) cells. These cells bridge the innate and adaptive immune systems and are capable of stimulating a wide array of immune cells that enhance vaccine-mediated immune responses. Here we discuss the factors that influence the adjuvant effects of iNKT cells for influenza vaccines as well as the obstacles that must be overcome before this novel adjuvant approach can be considered for human or veterinary use.

Invariant natural killer T (iNKT) cells are an innate-like T cell subset expressed by some, but not all mammals. Unlike conventional T cells, iNKT cells express a semi-invariant T cell receptor (TCR) (Vα24-Jα18/Vβ11 in human and Vα14-Jα18/Vβ8.2, Vβ2, or Vβ7 chains in mice) that recognizes glycolipid antigens presented by the non-polymorphic molecule CD1d. Once activated, iNKT cells generate an extensive array of immune responses that may be useful for treating cancer, autoimmunity and infectious disease in humans. In addition, there may be potential to target iNKT cells for veterinary applications. Pigs express iNKT cells that can be detected using mouse or human CD1d-tetramer reagents loaded with the prototypic iNKT cell ligand, α-galactosylceramide (α-GalCer). We exploited this phenomenon to define the TCR repertoire of iNKT cells (CD3α+CD1d tetramer+) compared to conventional T cells (CD3α+CD1d tetramer−) sorted from the peripheral blood of commercial mixed-breed pigs. We found that iNKT cells are highly enriched for the α-chain and CDR3α loop from Vα87*-Jα60* that possess a high level of amino acid sequence homology with human Vα24 (85%) and Jα18 (86%) as well as mouse Vα14 (74%) and Jα18 (79%). Particularly striking was that the most abundant CDR3α sequence was a near perfect match for the human invariant CDR3α. Porcine iNKT cells were also biased for Vβ84* which is homologous to Vβ11 (74%) in humans, as well as Vβ8.2 (71%) and Vβ2 (80%) in mice. Our findings indicate that the porcine iNKT cell TCR is highly homologous to mouse and particularly human iNKT cell TCR, which suggests that iNKT cells among these species play a similar role in immunity and disease.

The continuing global burden of disease from influenza infections reveals an urgent need for new approaches that more efficiently protect and potentially avoid the emergence of drug-resistant strains over time. One strategy could be to therapeutically target natural killer T (NKT) cells, an innate-like lymphocyte subset capable of inducing potent antiviral immune responses. We previously reported that disease outcome is significantly improved when influenza-infected pigs are intranasally treated with the NKT cell superagonist α-galactosylceramide (αGC). Here, we compared the protective effects of αGC to 7DW8-5, an αGC derivative that promotes pro-inflammatory immune responses, as well as oseltamivir phosphate (Tamiflu®), a neuraminidase inhibitor used to treat and prevent influenza. A total of 20 pigs challenged with H1N1 A/California/04/2009 (CA04) (4 per group) were administered vehicle, 7DW8-5, or αGC and Tamiflu®, alone or in combination. Four other pigs were mock infected and vehicle treated. Symptoms, viral shedding and peripheral blood leukocytes were measured for 7 days post infection (dpi). Pigs were then euthanized to examine viral titers and immune responses in the airway and lymphoid organs. Pigs treated with αGC and 7DW8-5 but not Tamiflu restored weight gain of infected pigs to the level of un-infected animals. All treatments eliminated virus shedding by 2 dpi compared to infected and untreated pigs that continued to shed virus throughout the study. So far, our results suggest that NKT cell therapy is similar to Tamiflu® for reducing the severity and transmission of influenza infections in pigs. Thus, it may be possible to use NKT cell superagonists to limit the spread of influenza viruses amongst swine and humans.