Phagocyte-expressed glycosaminoglycans promote capture of alphaviruses from the blood circulation in a host species-specific manner.

The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya, eastern- (EEEV), and Venezuelan- (VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology.

Vaccination against Rabbit Hemorrhagic Disease Virus 2 (RHDV2) Using a Baculovirus Recombinant Vaccine Provides Durable Immunity in Rabbits.

Rabbit hemorrhagic disease virus 2 (RHDV2) emerged in the United States in 2018 and has spread in both domestic and wild rabbits nationwide. The virus has a high mortality rate and can spread rapidly once introduced in a rabbit population. Vaccination against RHDV2 provides the best protection against disease and should be considered by all rabbit owners. Here, we investigate the duration of immunity provided by vaccination with the Medgene Platform conditionally licensed commercial vaccine 6 months following the initial series. Rabbits received either the vaccination or a placebo and were challenged with RHDV2 6 months later. All vaccinated rabbits survived challenge whereas 18/19 non-vaccinated controls succumbed to infection within 10 or fewer days post-challenge. These results demonstrate lasting immunity following vaccination with the Medgene RHDV2 vaccine.

Seroprevalence of Anti-SARS-CoV-2 Antibodies in Cats during Five Waves of COVID-19 Epidemic in Thailand and Correlation with Human Outbreaks.

Human-to-animal SARS-CoV-2 transmission was observed, including a veterinarian contracting COVID-19 through close contact with an infected cat, suggesting an atypical zoonotic transmission. This study investigated the prevalence of SARS-CoV-2 antibodies in cats during human outbreaks and elucidated the correlation between cat infections and human epidemics. A total of 1107 cat serum samples were collected and screened for SARS-CoV-2 antibodies using a modified indirect ELISA human SARS-CoV-2 antibody detection kit. The samples were confirmed using a cPass™ neutralization test. The SARS-CoV-2 seropositivity rate was 22.67% (199/878), mirroring the trend observed in concomitant human case numbers. The waves of the epidemic and the provinces did not significantly impact ELISA-positive cats. Notably, Chon Buri exhibited a strong positive correlation (r = 0.99, p = 0.009) between positive cat sera and reported human case numbers. Additionally, the cPass™ neutralization test revealed a 3.99% (35/878) seropositivity rate. There were significant differences in numbers and proportions of positive cat sera between epidemic waves. In Samut Sakhon, a positive correlation (r = 1, p = 0.042) was noted between the proportion of positive cat sera and human prevalence. The findings emphasize the need for ongoing surveillance to comprehend SARS-CoV-2 dynamics in both human and feline populations.

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.

African Swine Fever: A Review of Current Disease Management Strategies and Risks Associated with Exhibition Swine in the United States.

African swine fever is a high-consequence foreign animal disease endemic to sub-Saharan Africa and the island of Sardinia. The U.S. is the world's third largest pork producer, and ASF introduction would severely disrupt the pork supply chain, emphasizing the need to protect market access for U.S. proteins. However, niche producers raising swine intended for exhibition may not follow stringent biosecurity protocols, and livestock show circuits may promote untracked animal movement across the country, potentially exacerbating virus' spread in the event of ASF incursion into the U.S. Youth membership in state or national swine organizations offers a route for outreach and educational activities to enhance foreign animal disease preparedness, and adult presence at swine exhibitions allows a wide variety of programming for all ages to better serve all levels of understanding.

Phagocyte-expressed glycosaminoglycans promote capture of alphaviruses from the blood circulation in a host species-specific manner.

The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya (CHIKV), eastern-(EEEV), and Venezuelan-(VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate (HS) impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology. Significance Statement: Previously, evidence of arbovirus-GAG interactions in vivo has been limited to associations between viral residues shown to promote enhanced GAG-binding phenotypes in vitro and in vivo phenotypes of viral dissemination and pathogenesis. By directly manipulating host GAG expression, we identified virion-GAG interactions in vivo and discovered a role for phagocyte-expressed GAGs in viral vascular clearance. Moreover, we observe species-specific differences in viral vascular clearance of enhanced GAG-binding virions between murine and avian hosts. These data suggest species-specific variation in GAG structure is a mechanism to distinguish amplifying from dead-end hosts for arbovirus transmission.

Intracellular Diversity of WNV within Circulating Avian Peripheral Blood Mononuclear Cells Reveals Host-Dependent Patterns of Polyinfection.

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.

Disease Progression and Serological Assay Performance in Heritage Breed Pigs following Brucella suis Experimental Challenge as a Model for Naturally Infected Feral Swine.

Invasive feral swine (Sus scrofa) are one of the most important wildlife species for disease surveillance in the United States, serving as a reservoir for various diseases of concern for the health of humans and domestic animals. Brucella suis, the causative agent of swine brucellosis, is one such pathogen carried and transmitted by feral swine. Serology assays are the preferred field diagnostic for B. suis infection, as whole blood can be readily collected and antibodies are highly stable. However, serological assays frequently have lower sensitivity and specificity, and few studies have validated serological assays for B. suis in feral swine. We conducted an experimental infection of Ossabaw Island Hogs (a breed re-domesticated from feral animals) as a disease-free proxy for feral swine to (1) improve understanding of bacterial dissemination and antibody response following B. suis infection and (2) evaluate potential changes in the performance of serological diagnostic assays over the course of infection. Animals were inoculated with B. suis and serially euthanized across a 16-week period, with samples collected at the time of euthanasia. The 8% card agglutination test performed best, whereas the fluorescence polarization assay demonstrated no capacity to differentiate true positive from true negative animals. From a disease surveillance perspective, using the 8% card agglutination test in parallel with either the buffered acidified plate antigen test or the Brucella abortus/suis complement fixation test provided the best performance with the highest probability of a positive assay result. Application of these combinations of diagnostic assays for B. suis surveillance among feral swine would improve understanding of spillover risks at the national level.

Feral Swine as Indirect Indicators of Environmental Anthrax Contamination and Potential Mechanical Vectors of Infectious Spores.

Anthrax is a disease that affects livestock, wildlife, and humans worldwide; however, its relative impacts on these populations remain underappreciated. Feral swine (Sus scrofa) are relatively resistant to developing anthrax, and past serosurveys have alluded to their utility as sentinels, yet empirical data to support this are lacking. Moreover, whether feral swine may assist in the dissemination of infectious spores is unknown. To address these knowledge gaps, we intranasally inoculated 15 feral swine with varying quantities of Bacillus anthracis Sterne 34F2 spores and measured the seroconversion and bacterial shedding over time. The animals also were inoculated either one or three times. The sera were evaluated by enzyme-linked immunosorbent assay (ELISA) for antibodies against B. anthracis, and nasal swabs were cultured to detect bacterial shedding from the nasal passages. We report that the feral swine developed antibody responses to B. anthracis and that the strength of the response correlated with the inoculum dose and the number of exposure events experienced. Isolation of viable bacteria from the nasal passages of the animals throughout the study period suggests that feral swine may assist in the spread of infectious spores on the landscape and have implications for the identification of environments contaminated with B. anthracis as well as the exposure risk to more susceptible hosts.

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.

H7N9 influenza A virus transmission in a multispecies barnyard model.

Influenza A viruses are a diverse group of pathogens that have been responsible for millions of human and avian deaths throughout history. Here, we illustrate the transmission potential of H7N9 influenza A virus between Coturnix quail (Coturnix sp.), domestic ducks (Anas platyrhynchos domesticus), chickens (Gallus gallus domesticus), and house sparrows (Passer domesticus) co-housed in an artificial barnyard setting. In each of four replicates, individuals from a single species were infected with the virus. Quail shed virus orally and were a source of infection for both chickens and ducks. Infected chickens transmitted the virus to quail but not to ducks or house sparrows. Infected ducks transmitted to chickens, resulting in seroconversion without viral shedding. House sparrows did not shed virus sufficiently to transmit to other species. These results demonstrate that onward transmission varies by index species, and that gallinaceous birds are more likely to maintain H7N9 than ducks or passerines.

Intracellular diversity of WNV within circulating avian peripheral blood mononuclear cells reveals host-dependent patterns of polyinfection.

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure. Author Summary: WNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.

Rapid evolution of SARS-CoV-2 in domestic cats.

SARS-CoV-2 (SARS2) infection of a novel permissive host species can result in rapid viral evolution. Data suggest that felids are highly susceptible to SARS2 infection, and species-specific adaptation following human-to-felid transmission may occur. We employed experimental infection and analysis of publicly available SARS2 sequences to observe variant emergence and selection in domestic cats. Three cohorts of cats (N = 23) were inoculated with SARS-CoV-2 USA-WA1/2020 or infected via cat-to-cat contact transmission. Full viral genomes were recovered from RNA obtained from nasal washes 1-3 days post-infection and analyzed for within-host viral variants. We detected 118 unique variants at ≥3 per cent allele frequency in two technical replicates. Seventy of these (59 per cent) were nonsynonymous single nucleotide variants (SNVs); the remainder were synonymous SNVs or structural variants. On average, we observed twelve variants per cat, nearly 10-fold higher than what is commonly reported in human patients. We observed signatures of positive selection in the spike protein and the emergence of eleven within-host variants located at the same genomic positions as mutations in SARS2 variant lineages that have emerged during the pandemic. Fewer variants were noted in cats infected from contact with other cats and in cats exposed to lower doses of cultured inoculum. An analysis of ninety-three publicly available SARS2 consensus genomes recovered from naturally infected domestic cats reflected variant lineages circulating in the local human population at the time of sampling, illustrating that cats are susceptible to SARS2 variants that have emerged in humans, and suggesting human-to-felid transmission occurring in domestic settings is typically unidirectional. These experimental results underscore the rapidity of SARS2 adaptation in felid hosts, representing a theoretical potential origin for variant lineages in human populations. Further, cats should be considered susceptible hosts capable of shedding virus during infections occurring within households.

A novel vaccine candidate against rabbit hemorrhagic disease virus 2 (RHDV2) confers protection in domestic rabbits.

OBJECTIVE: To evaluate efficacy of a novel vaccine against rabbit hemorrhagic disease virus 2 (RHDV2) in domestic rabbits. ANIMALS: 40 New Zealand White rabbits obtained from a commercial breeder. PROCEDURES: Rabbits were vaccinated and held at the production facility for the duration of the vaccination phase and transferred to Colorado State University for challenge with RHDV2. Rabbits were challenged with oral suspensions containing infectious virus and monitored for clinical disease for up to 10 days. Rabbits that died or were euthanized following infection were necropsied, and livers were evaluated for viral RNA via RT-PCR. RESULTS: None of the vaccinated animals (0/9) exhibited clinical disease or mortality following infection with RHDV2 while 9/13 (69%) of the control animals succumbed to lethal disease following infection. CLINICAL RELEVANCE: The novel vaccine described herein provided complete protection against lethal infection following RHDV2 challenge. Outside of emergency use, there are currently no licensed vaccines against RHDV2 on the market in the United States; as such, this vaccine candidate would provide an option for control of this disease now that RHDV2 has become established in North America.

Experimental Infection of Brazilian Free-Tailed Bats (Tadarida brasiliensis) with Two Strains of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presumed to have originated from wildlife and shares homology with other bat coronaviruses. Determining the susceptibility of North American bat species to SARS-CoV-2 is of utmost importance for making decisions regarding wildlife management, public health, and conservation. In this study, Brazilian free-tailed bats (Tadarida brasiliensis) were experimentally infected with two strains of SARS-CoV-2 (parental WA01 and Delta variant), evaluated for clinical disease, sampled for viral shedding and antibody production, and analyzed for pathology. None of the bats (n = 18) developed clinical disease associated with infection, shed infectious virus, or developed histopathological lesions associated with SARS-CoV-2 infection. All bats had low levels of viral RNA in oral swabs, six bats had low levels of viral RNA present in the lungs during acute infection, and one of the four bats that were maintained until 28 days post-infection developed a neutralizing antibody response. These findings suggest that Brazilian free-tailed bats are permissive to infection by SARS-CoV-2, but they are unlikely to contribute to environmental maintenance or transmission.

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.

American alligators are capable of West Nile virus amplification, mosquito infection and transmission.

West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle.

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.