Experimental SARS-CoV-2 Infection of Elk and Mule Deer.

To assess the susceptibility of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) to SARS-CoV-2, we performed experimental infections in both species. Elk did not shed infectious virus but mounted low-level serologic responses. Mule deer shed and transmitted virus and mounted pronounced serologic responses and thus could play a role in SARS-CoV-2 epidemiology.

SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection.

SARS-CoV-2 spillback from humans into domestic and wild animals has been well documented, and an accumulating number of studies illustrate that human-to-animal transmission is widespread in cats, mink, deer, and other species. Experimental inoculations of cats, mink, and ferrets have perpetuated transmission cycles. We sequenced full genomes of Vero cell-expanded SARS-CoV-2 inoculum and viruses recovered from cats (n = 6), dogs (n = 3), hamsters (n = 3), and a ferret (n = 1) following experimental exposure. Five nonsynonymous changes relative to the USA-WA1/2020 prototype strain were near fixation in the stock used for inoculation but had reverted to wild-type sequences at these sites in dogs, cats, and hamsters within 1- to 3-d postexposure. A total of 14 emergent variants (six in nonstructural genes, six in spike, and one each in orf8 and nucleocapsid) were detected in viruses recovered from animals. This included substitutions in spike residues H69, N501, and D614, which also vary in human lineages of concern. Even though a live virus was not cultured from dogs, substitutions in replicase genes were detected in amplified sequences. The rapid selection of SARS-CoV-2 variants in vitro and in vivo reveals residues with functional significance during host switching. These observations also illustrate the potential for spillback from animal hosts to accelerate the evolution of new viral lineages, findings of particular concern for dogs and cats living in households with COVID-19 patients. More generally, this glimpse into viral host switching reveals the unrealized rapidity and plasticity of viral evolution in experimental animal model systems.

Role of Spillover and Spillback in SARS-CoV-2 Transmission and the Importance of One Health in Understanding the Dynamics of the COVID-19 Pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is notable both for its impact on global public health as well as its well-publicized transmissibility to other species of animals. Infection of incidental animal hosts is concerning because of possible emergence of novel variants due to viral mutation. Species that are susceptible to SARS-CoV-2 include domestic and nondomestic cats, domestic dogs, white-tailed deer, mink, and golden hamsters, among others. We detail possible origins of transmission of SARS-CoV-2 to humans, as well as the ecological and molecular mechanisms needed for the virus to establish infection in humans from animals. We highlight examples of SARS-CoV-2 spillover, spillback, and secondary spillover, demonstrating the breadth in the variability of hosts and current transmission events that have been documented in domestic, captive, and wild animals. Lastly, we turn our focus to the importance of animal hosts as potential reservoirs and sources of variant emergence that can have profound effects on the human population. We note that a One Health approach emphasizing surveillance of animals and humans in certain environments using interdisciplinary collaboration is encouraged to manage disease surveillance, regulation on animal trade and testing, and animal vaccine development that will mitigate further disease outbreaks. These efforts will minimize the spread of SARS-CoV-2 and advance knowledge to prevent the spread of future emerging infectious diseases.

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is probable that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to naive cats. We report that cats are highly susceptible to infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats developed a robust neutralizing antibody response that prevented reinfection following a second viral challenge. Conversely, we found that dogs do not shed virus following infection but do seroconvert and mount an antiviral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human infection; however, reverse zoonosis is possible if infected owners expose their domestic pets to the virus during acute infection. Resistance to reinfection holds promise that a vaccine strategy may protect cats and, by extension, humans.

Acute vaping in a golden Syrian hamster causes inflammatory response transcriptomic changes.

E-cigarette vaping is a major aspect of nicotine consumption, especially for children and young adults. Although it is branded as a safer alternative to cigarette smoking, murine and rat models of subacute and chronic e-cigarette vaping exposure have shown many proinflammatory changes in the respiratory tract. An acute vaping exposure paradigm has not been demonstrated in the golden Syrian hamster, and the hamster is a readily available small animal model that has the unique benefit of becoming infected with and transmitting respiratory viruses, including SARS-CoV-2, without genetic alteration of the animal or virus. Using a 2-day, whole body vaping exposure protocol in male golden Syrian hamsters, we evaluated serum cotinine, bronchoalveolar lavage cells, lung, and nasal histopathology, and gene expression in the nasopharynx and lung through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Depending on the presence of nonnormality or outliers, statistical analysis was performed by ANOVA or Kruskal-Wallis tests. For tests that were statistically significant (P < 0.05), post hoc Tukey-Kramer and Dunn's tests, respectively, were performed to make pairwise comparisons between groups. In nasal tissue, RT-qPCR analysis revealed nicotine-dependent increases in gene expression associated with type 1 inflammation (CCL-5 and CXCL-10), fibrosis [transforming growth factor-ß (TGF-ß)], nicotine-independent increase oxidative stress response (SOD-2), and a nicotine-independent decrease in vasculogenesis/angiogenesis (VEGF-A). In the lung, nicotine-dependent increases in the expression of genes involved in the renin-angiotensin pathway [angiotensin-converting enzyme (ACE), ACE2], coagulation (tissue factor, Serpine-1), extracellular matrix remodeling (MMP-2, MMP-9), type 1 inflammation (IL-1ß, TNF-α, and CXCL-10), fibrosis (TGF-ß and Serpine-1), oxidative stress response (SOD-2), neutrophil extracellular traps release (ELANE), and vasculogenesis and angiogenesis (VEGF-A) were identified. To our knowledge, this is the first demonstration that the Syrian hamster is a viable model of e-cigarette vaping. In addition, this is the first report that e-cigarette vaping with nicotine can increase tissue factor gene expression in the lung. Our results show that even an acute exposure to e-cigarette vaping causes significant upregulation of mRNAs in the respiratory tract from pathways involving the renin-angiotensin system, coagulation, extracellular matrix remodeling, type 1 inflammation, fibrosis, oxidative stress response, neutrophil extracellular trap release (NETosis), vasculogenesis, and angiogenesis.

Susceptibility of Wild Canids to SARS-CoV-2.

We assessed 2 wild canid species, red foxes (Vulpes vulpes) and coyotes (Canis latrans), for susceptibility to SARS-CoV-2. After experimental inoculation, red foxes became infected and shed infectious virus. Conversely, experimentally challenged coyotes did not become infected; therefore, coyotes are unlikely to be competent hosts for SARS-CoV-2.

Potential Use for Serosurveillance of Feral Swine to Map Risk for Anthrax Exposure, Texas, USA.

Anthrax is a disease of concern in many mammals, including humans. Management primarily consists of prevention through vaccination and tracking clinical-level observations because environmental isolation is laborious and bacterial distribution across large geographic areas difficult to confirm. Feral swine (Sus scrofa) are an invasive species with an extensive range in the southern United States that rarely succumbs to anthrax. We present evidence that feral swine might serve as biosentinels based on comparative seroprevalence in swine from historically defined anthrax-endemic and non-anthrax-endemic regions of Texas. Overall seropositivity was 43.7% (n = 478), and logistic regression revealed county endemicity status, age-class, sex, latitude, and longitude were informative for predicting antibody status. However, of these covariates, only latitude was statistically significant (ß = -0.153, p = 0.047). These results suggests anthrax exposure in swine, when paired with continuous location data, could serve as a proxy for bacterial presence in specific areas.

Peridomestic Mammal Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Wild animals have been implicated as the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but it is largely unknown how the virus affects most wildlife species and if wildlife could ultimately serve as a reservoir for maintaining the virus outside the human population. We show that several common peridomestic species, including deer mice, bushy-tailed woodrats, and striped skunks, are susceptible to infection and can shed the virus in respiratory secretions. In contrast, we demonstrate that cottontail rabbits, fox squirrels, Wyoming ground squirrels, black-tailed prairie dogs, house mice, and racoons are not susceptible to SARS-CoV-2 infection. Our results expand the knowledge base of susceptible species and provide evidence that human-wildlife interactions could result in continued transmission of SARS-CoV-2.

Quantifying Proximity, Confinement, and Interventions in Disease Outbreaks: A Decision Support Framework for Air-Transported Pathogens.

The inability to communicate how infectious diseases are transmitted in human environments has triggered avoidance of interactions during the COVID-19 pandemic. We define a metric, Effective ReBreathed Volume (ERBV), that encapsulates how infectious pathogens, including SARS-CoV-2, transport in air. ERBV separates environmental transport from other factors in the chain of infection, allowing quantitative comparisons among situations. Particle size affects transport, removal onto surfaces, and elimination by mitigation measures, so ERBV is presented for a range of exhaled particle diameters: 1, 10, and 100 µm. Pathogen transport depends on both proximity and confinement. If interpersonal distancing of 2 m is maintained, then confinement, not proximity, dominates rebreathing after 10-15 min in enclosed spaces for all but 100 µm particles. We analyze strategies to reduce this confinement effect. Ventilation and filtration reduce person-to-person transport of 1 µm particles (ERBV1) by 13-85% in residential and office situations. Deposition to surfaces competes with intentional removal for 10 and 100 µm particles, so the same interventions reduce ERBV10 by only 3-50%, and ERBV100 is unaffected. Prior knowledge of size-dependent ERBV would help identify transmission modes and effective interventions. This framework supports mitigation decisions in emerging situations, even before other infectious parameters are known.

Bourbon Virus in Wild and Domestic Animals, Missouri, USA, 2012-2013.

Since its recent discovery, Bourbon virus has been isolated from a human and ticks. To assess exposure of potential vertebrate reservoirs, we assayed banked serum and plasma samples from wildlife and domestic animals in Missouri, USA, for Bourbon virus-neutralizing antibodies. We detected high seroprevalence in raccoons (50%) and white-tailed deer (86%).

Cottontail rabbits shed clade 2.3.4.4 H5 highly pathogenic avian influenza A viruses.

During 2014-2015, clade 2.3.4.4 H5Nx highly pathogenic (HP) avian influenza A viruses (IAV) were first detected in North America and subsequently caused one of the largest agricultural emergencies in U.S. HISTORY: Recent evidence has suggested that cottontail rabbits can shed multiple IAV subtypes. We experimentally infected cottontail rabbits with three HP H5Nx IAVs. All rabbits tested shed virus on at least one day by at least one route. Cottontail rabbits appear to be an exception to the limited capacity for replication that has been previously reported for certain other mammalian species inoculated with clade 2.3.4.4 HP H5Nx avian influenza A viruses.

Experimental evolution of an RNA virus in wild birds: evidence for host-dependent impacts on population structure and competitive fitness.

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Characterization of non-lethal West Nile Virus (WNV) infection in horses: Subclinical pathology and innate immune response.

Most natural West Nile virus (WNV) infections in humans and horses are subclinical or sub-lethal and non-encephalitic. Yet, the main focus of WNV research remains on the pathogenesis of encephalitic disease, mainly conducted in mouse models. We characterized host responses during subclinical WNV infection in horses and compared outcomes with those obtained in a novel rabbit model of subclinical WNV infection (Suen et al. 2015. Pathogens, 4: 529). Experimental infection of 10 horses with the newly emerging WNV-strain, WNVNSW2011, did not result in neurological disease in any animal but transcriptional upregulation of both type I and II interferon (IFN) was seen in peripheral blood leukocytes prior to or at the time of viremia. Likewise, transcript upregulation for IFNs, TNFα, IL1ß, CXCL10, TLRs, and MyD88 was detected in lymphoid tissues, while IFNα, CXCL10, TLR3, ISG15 and IRF7 mRNA was upregulated in brains with histopathological evidence of mild encephalitis, but absence of detectable viral RNA or antigen. These responses were reproduced in the New Zealand White rabbits (Oryctolagus cuniculus) experimentally infected with WNVNSW2011, by intradermal footpad inoculation. Kinetics of the anti-WNV antibody response was similar in horses and rabbits, which for both species may be explained by the early IFN and cytokine responses evident in circulating leukocytes and lymphoid organs. Given the similarities to the majority of equine infection outcomes, immunocompetent rabbits appear to represent a valuable small-animal model for investigating aspects of non-lethal WNV infections, notably mechanisms involved in abrogating morbidity.

West Nile Virus Infection in Ruffed Grouse ( Bonasa umbellus): Experimental Infection and Protective Effects of Vaccination.

Ruffed grouse ( Bonasa umbellus) population numbers in Pennsylvania dramatically declined during the early 2000s and have subsequently remained depressed throughout much of the state. While this decline has been temporally associated with the presence of West Nile virus (WNV), lack of information on the WNV susceptibility of this popular game bird species has limited the ability to interpret the potential impacts of WNV. To address this knowledge gap, virologic, immunologic, pathologic, and clinical responses as well as protective effects of vaccination following experimental WNV inoculation in ruffed grouse were assessed. Four of 10 (40%) naive, WNV-inoculated grouse succumbed to infection within 8 days and had moderate mean peak viremia titers (107.0 plaque-forming units [PFU]/ml serum); severe necrotizing myocarditis with widespread, corresponding immunohistochemical labeling; and minimal encephalitis. Grouse that survived to the prescribed end point of 14 days postinoculation (6/10; 60%) had slightly lower mean peak viremia titers (106.8 PFU/ml serum), moderate myocardial lesions, and more widespread brain lesions with rare corresponding immunohistochemical labeling. Vaccinated, WNV-inoculated birds ( n = 5) had lower mean peak viremia titers (103.6 PFU/ml serum) and minimal lesions, and sham-inoculated, in-contact control birds ( n = 3) had no evidence of infection. All surviving, inoculated birds seroconverted, and WNV-specific antibodies were detectable in serum and Nobuto filter paper strip-eluted blood samples. These data suggest that WNV could serve as an additional population pressure on ruffed grouse in regions where transmission levels are high and WNV competent, ornithophilic vectors exist.

Vertebrate Host Susceptibility to Heartland Virus.

Heartland virus (HRTV) is a recently described phlebovirus initially isolated in 2009 from 2 humans who had leukopenia and thrombocytopenia. Serologic assessment of domestic and wild animal populations near the residence of 1 of these persons showed high exposure rates to raccoons, white-tailed deer, and horses. To our knowledge, no laboratory-based assessments of viremic potential of animals infected with HRTV have been performed. We experimentally inoculated several vertebrates (raccoons, goats, chickens, rabbits, hamsters, C57BL/6 mice, and interferon-α/ß/γ receptor-deficient [Ag129]) mice with this virus. All animals showed immune responses against HRTV after primary or secondary exposure. However, neutralizing antibody responses were limited. Only Ag129 mice showed detectable viremia and associated illness and death, which were dose dependent. Ag129 mice also showed development of mean peak viral antibody titers >8 log10 PFU/mL, hemorrhagic hepatic lesions, splenomegaly, and large amounts of HRTV antigen in mononuclear cells and hematopoietic cells in the spleen.

Programmed ribosomal frameshift alters expression of west nile virus genes and facilitates virus replication in birds and mosquitoes.

West Nile virus (WNV) is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed -1 ribosomal frameshift (PRF) resulting in production of an additional NS protein NS1'. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts.

Kinetics of the West Nile virus induced transcripts of selected cytokines and Toll-like receptors in equine peripheral blood mononuclear cells.

West Nile virus (WNV) is one of the most common causes of epidemic viral encephalitis in horses worldwide. Peripheral blood mononuclear cells (PBMCs) are amongst the first to encounter the virus following a mosquito bite. This study aimed to elucidate the transcription kinetics of cytokine, Toll-like receptor (TLRs) and TLRs-associated genes following WNV challenge of equine PBMCs. PBMCs were challenged with an Australian strain of WNV (WNVNSW2011) and transcriptomes were quantified at 2, 6, 12 and 24 h post-infection (pi) using qRT-PCR. Type I and II interferons (IFNα, ß and γ) mRNA transcription increased following WNV exposure, as did the transcripts for IL1α, IL1ß, IL6, IL8, and IL22, but with slightly varying kinetics. TLR1, 3, 5, 7-9 transcripts were also upregulated in equine PBMCsin response to WNV challenge, as were those for MyD88, NF-κB, TRAF3, STAT1 and 2, IRF3 and 7, ISG15, as well as SOCS1 and 3 compared to the control cells. Expression of selected genes in the draining lymph node, spleen and brain (medulla oblongata) of experimentally infected horses was also assessed and transcription of most of these genes was also upregulated here. Although qRT-PCR detected higher viral RNA at 24 h pi compared to 6 h pi, the virus did not replicate productively in equine PBMCs. The up-regulation of gene-transcription for selected cytokines, IFNs, TLRs and TLRs-associated molecules suggests their involvement in virus recognition and control of WNV infection in the horse.

Susceptibility of Carrion Crows to Experimental Infection with Lineage 1 and 2 West Nile Viruses.

West Nile virus (WNV) outbreaks in North America have been characterized by substantial die-offs of American crows (Corvus brachyrhynchos). In contrast, a low incidence of bird deaths has been observed during WNV epidemic activity in Europe. To examine the susceptibility of the western European counterpart of American crows, we inoculated carrion crows (Corvus corone) with WNV strains isolated in Greece (Gr-10), Italy (FIN and Ita09), and Hungary (578/10) and with the highly virulent North American genotype strain (NY99). We also inoculated American crows with a selection of these strains to examine the strains' virulence in a highly susceptible bird species. Infection with all strains, except WNV FIN, resulted in high rates of death and high-level viremia in both bird species and virus dissemination to several organs. These results suggest that carrion crows are highly susceptible to WNV and may potentially be useful as part of dead bird surveillance for early warning of WNV activity in Europe.

A novel bacterium-free method for generation of flavivirus infectious DNA by circular polymerase extension reaction allows accurate recapitulation of viral heterogeneity.

A novel bacterium-free approach for rapid assembly of flavivirus infectious cDNAs using circular polymerase extension reaction was applied to generate infectious cDNA for the virulent New South Wales isolate of the Kunjin strain of West Nile virus (KUNV) that recently emerged in Australia. Recovered virus recapitulated the genetic heterogeneity present in the original isolate. The approach was utilized to generate viral mutants with designed phenotypic properties and to identify E protein glycosylation as one of the virulence determinants.