Live attenuated vaccines (LAVs) replicate in the respiratory/oral mucosa, mimic natural infection, and can induce mucosal and systemic immune responses to the full repertoire of SARS-CoV-2 structural/nonstructural proteins. Generally, LAVs produce broader and more durable protection than current COVID-19 vaccines. We generated a temperature-sensitive (TS) SARS-CoV-2 mutant TS11 via cold-adaptation of the WA1 strain in Vero E6 cells. TS11 replicated at >4 Log₁₀-higher titers at 32 °C than at 39 °C. TS11 has multiple mutations, including those in nsp3, a 12-amino acid-deletion spanning the furin cleavage site of the S protein and a 371-nucleotide-deletion spanning the ORF7b-ORF8 genes. We tested the pathogenicity and protective efficacy of TS11 against challenge with a heterologous virulent SARS-CoV-2 D614G strain 14B in Syrian hamsters. Hamsters were randomly assigned to mock immunization-challenge (Mock-C) and TS11 immunization-challenge (TS11-C) groups. Like the mock group, TS11-vaccinated hamsters did not show any clinical signs and continuously gained body weight. TS11 replicated well in the nasal cavity but poorly in the lungs and caused only mild lesions in the lungs. After challenge, hamsters in the Mock-C group lost weight. In contrast, the animals in the TS11-C group continued gaining weight. The virus titers in the nasal turbinates and lungs of the TS11-C group were significantly lower than those in the Mock-C group, confirming the protective effects of TS11 immunization of hamsters. Histopathological examination demonstrated that animals in the Mock-C group had severe pulmonary lesions and large amounts of viral antigens in the lungs post-challenge; however, the TS11-C group had minimal pathological changes and few viral antigen-positive cells. In summary, the TS11 mutant was attenuated and induced protection against disease after a heterologous SARS-CoV-2 challenge in Syrian hamsters.

Bovine coronavirus (BCoV) poses a threat to cattle health worldwide, contributing to both respiratory and enteric diseases. However, few contemporary strains have been isolated. In this study, 71 samples (10 nasal and 61 fecal) were collected from one farm in Ohio in 2021 and three farms in Georgia in 2023. They were screened by BCoV-specific real-time reverse transcription-PCR, and 15 BCoV-positive samples were identified. Among them, five BCoV strains from fecal samples were isolated using human rectal tumor-18 (HRT-18) cells. The genomic sequences of five strains were obtained. The phylogenetic analysis illustrated that these new strains clustered with US BCoVs that have been detected since the 1990s. Sequence analyses of the spike proteins of four pairs of BCoVs, with each pair originally collected from the respiratory and enteric sites of one animal, revealed the potential amino acid residue patterns, such as D1180 for all four enteric BCoVs and G1180 for three of four respiratory BCoVs. This project provides new BCoV isolates and sequences and underscores the genetic diversity of BcoVs, the unknown mechanisms of disease types, and the necessity of sustained surveillance and research for BCoVs.

Coronaviruses are known to cross species barriers, and spill over among animals, from animals to humans, and vice versa. SARS-CoV-2 emerged in humans in late 2019. It is now known to infect numerous animal species, including companion animals and captive wildlife species. Experimental infections in other animals have established that many species are susceptible to infection, with new ones still being identified. We have developed an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to SARS-CoV-2 nucleocapsid (N) and spike (S) proteins, that is both sensitive and specific. It can detect S antibodies in sera at dilutions greater than 1:10,000, and does not cross-react with antibodies to the other coronaviruses tested. We used the S antibody ELISA to test serum samples collected from 472 deer from ten sites in northeastern Ohio between November 2020 and March 2021, when the SARS-CoV-2 pandemic was first peaking in humans in Ohio, USA. Antibodies to SARS-CoV-2 were found in serum samples from every site, with an overall positivity rate of 17.2%; we further compared the viral neutralizing antibody titers to our ELISA results. These findings demonstrate the need to establish surveillance programs to monitor deer and other susceptible wildlife species globally.

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen in pigs and a newly recognized zoonotic coronavirus in humans. Genetic analyses suggest that PDCoV originated from avian deltacoronaviruses, with sparrow deltacoronaviruses (SpDCoVs) being the most closely related. House sparrows (Passer domesticus) frequently visit farms and interact directly with pigs in barns, raising the possibility of interspecies transmission. We hypothesized that PDCoV can be transmitted between pigs and house sparrows. To investigate this, 200 house sparrows near Ohio swine farms were sampled and screened for gammacoronaviruses and deltacoronaviruses using RT-PCR targeting the conserved RNA polymerase region. Deltacoronaviruses and gammacoronaviruses were detected in 18.0% (36/200) and 5.5% (11/200) of fecal samples, respectively. Genomic sequence analysis of representative samples revealed that SpDCoVs are closely related to, but not direct ancestors of, PDCoVs. These SpDCoVs appear to be widespread in the U.S. Midwest and may contribute to PDCoV evolution. Attempts to isolate SpDCoV from these samples in embryonated chicken eggs and four cell lines were unsuccessful. Because coronaviruses frequently cross species barriers to cause epidemics and/or pandemics in humans and livestock, these findings underscore the need for ongoing surveillance of deltacoronaviruses in diverse wild animals, livestock, and humans to safeguard public health.

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen in pigs and a newly recognized zoonotic coronavirus in humans. Genetic analyses suggest that PDCoV origi-nated from avian deltacoronaviruses, with sparrow deltacoronaviruses (SpDCoVs) being the most closely related. House sparrows (Passer domesticus) frequently visit farms and in-teract directly with pigs in barns, raising the possibility of interspecies transmission. We hypothesized that PDCoV can be transmitted between pigs and house sparrows. To inves-tigate this, 200 house sparrows near Ohio swine farms were sampled and screened for gamma- and deltacoronaviruses using RT-PCR targeting the conserved RNA polymerase region. Deltacoronaviruses and gammacoronaviruses were detected in 18.0% (36/200) and 5.5% (11/200) of fecal samples, respectively. Genomic sequence analysis of representative samples revealed that SpDCoVs are closely related to, but not the direct ancestors of, PDCoVs. These SpDCoVs appear to be widespread in the U.S. Midwest and may contrib-ute to PDCoV evolution. Attempts to isolate SpDCoV from these samples in embryonated chicken eggs and four cell lines were unsuccessful. Because coronaviruses frequently cross species barriers to cause epidemics and/or pandemics in humans and livestock, these findings underscore the need for ongoing surveillance of deltacoronaviruses in diverse wild animals, livestock, and humans to safeguard public health.