Genetic drift and purifying selection shape within-host influenza A virus populations during natural swine infections.

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

Evolution of influenza A viruses in exhibition swine and transmission to humans, 2013-2015.

AIMS: Swine are a mixing vessel for the emergence of novel reassortant influenza A viruses (IAV). Interspecies transmission of swine-origin IAV poses a public health and pandemic risk. In the United States, the majority of zoonotic IAV transmission events have occurred in association with swine exposure at agricultural fairs. Accordingly, this human-animal interface necessitates mitigation strategies informed by understanding of interspecies transmission mechanisms in exhibition swine. Likewise, the diversity of IAV in swine can be a source for novel reassortant or mutated viruses that pose a risk to both swine and human health. METHODS AND RESULTS: In an effort to better understand those risks, here we investigated the epidemiology of IAV in exhibition swine and subsequent transmission to humans by performing phylogenetic analyses using full genome sequences from 272 IAV isolates collected from exhibition swine and 23 A(H3N2)v viruses from human hosts during 2013-2015. Sixty-seven fairs (24.2%) had at least one pig test positive for IAV with an overall estimated prevalence of 8.9% (95% CI: 8.3-9.6, Clopper-Pearson). Of the 19 genotypes found in swine, 5 were also identified in humans. There was a positive correlation between the number of human cases of a genotype and its prevalence in exhibition swine. Additionally, we demonstrated that A(H3N2)v viruses clustered tightly with exhibition swine viruses that were prevalent in the same year. CONCLUSIONS: These data indicate that multiple genotypes of swine-lineage IAV have infected humans, and highly prevalent IAV genotypes in exhibition swine during a given year are also the strains detected most frequently in human cases of variant IAV. Continued surveillance and rapid characterization of IAVs in exhibition swine can facilitate timely phenotypic evaluation and matching of candidate vaccine strains to those viruses present at the human-animal interface which are most likely to spillover into humans.

Genetic drift and purifying selection shape within-host influenza A virus populations during natural swine infections.

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intra-host single nucleotide variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

Lack of SARS-CoV-2 Viral RNA Detection among a Convenience Sampling of Ohio Wildlife, Companion, and Agricultural Animals, 2020-2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly, becoming a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter the virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g., shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g., pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n = 76 individuals), free-ranging mink (n = 57), multiple species of bats (n = 59), and other wildlife in addition to domestic cats (n = 275) and pigs (n = 184). In total, we tested 792 individual animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing the re-emergence of SARS-CoV-2 in humans or animals.

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

Shortening Duration of Swine Exhibitions to Reduce Risk for Zoonotic Transmission of Influenza A Virus.

Reducing zoonotic influenza A virus (IAV) risk in the United States necessitates mitigation of IAV in exhibition swine. We evaluated the effectiveness of shortening swine exhibitions to <72 hours to reduce IAV risk. We longitudinally sampled every pig daily for the full duration of 16 county fairs during 2014-2015 (39,768 nasal wipes from 6,768 pigs). In addition, we estimated IAV prevalence at 195 fairs during 2018-2019 to test the hypothesis that <72-hour swine exhibitions would have lower IAV prevalence. In both studies, we found that shortening duration drastically reduces IAV prevalence in exhibition swine at county fairs. Reduction of viral load in the barn within a county fair is critical to reduce the risk for interspecies IAV transmission and pandemic potential. Therefore, we encourage fair organizers to shorten swine shows to protect the health of both animals and humans.

SARS-CoV-2 infection in free-ranging white-tailed deer.

Humans have infected a wide range of animals with SARS-CoV-21-5, but the establishment of a new natural animal reservoir has not been observed. Here we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2, are exposed to multiple SARS-CoV-2 variants from humans and are capable of sustaining transmission in nature. Using real-time PCR with reverse transcription, we detected SARS-CoV-2 in more than one-third (129 out of 360, 35.8%) of nasal swabs obtained from O. virginianus in northeast Ohio in the USA during January to March 2021. Deer in six locations were infected with three SARS-CoV-2 lineages (B.1.2, B.1.582 and B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. We detected probable deer-to-deer transmission of B.1.2, B.1.582 and B.1.596 viruses, enabling the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are observed infrequently in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have been transmitted in wildlife in the USA, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive 'One Health' programmes to monitor the environment, deer and other wildlife hosts globally.

SARS-CoV-2 infection in free-ranging white-tailed deer ( Odocoileus virginianus ).

Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer ( Odocoileus virginianus ) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies. ONE-SENTENCE SUMMARY: A significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.

Influenza Vaccination of Swine Reduces Public Health Risk at the Swine-Human Interface.

Influenza A viruses (IAV) in swine (IAV-S) pose serious risk to public health through spillover at the human-animal interface. Continued zoonotic transmission increases the likelihood novel IAV-S capable of causing the next influenza pandemic will emerge from this animal reservoir. Because current mitigation strategies are insufficient to prevent IAV zoonosis, we investigated the ability of swine vaccination to decrease IAV-S zoonotic transmission risk. We assessed postchallenge viral shedding in market-age swine vaccinated with either live-attenuated influenza virus (LAIV), killed influenza virus (KV), or sham vaccine (NV). We also assessed postchallenge transmission by exposing naive ferrets to pigs with contact types reflective of those experienced by humans in a field setting. LAIV and KV swine groups exhibited a nearly 100-fold reduction in peak nasal titer (LAIV mean, 4.55 log 50% tissue culture infectious dose [TCID50]/ml; KV mean, 4.53 log TCID50/ml) compared to NV swine (mean, 6.40 log TCID50/ml). Air sampling during the postchallenge period revealed decreased cumulative IAV in LAIV and KV study room air (LAIV, area under the concentration-time curve [AUC] of 57.55; KV, AUC = 24.29) compared to the NV study room (AUC = 86.92). Pairwise survival analysis revealed a significant delay in onset of infection among ferrets exposed to LAIV pigs versus NV pigs (rate ratio, 0.66; P = 0.028). Ferrets exposed to vaccinated pigs had lower cumulative virus titers in nasal wash samples (LAIV versus NV, P < 0.0001; KV versus NV, P= 0.3490) and experienced reduced clinical signs during infection. Our findings support the implementation of preexhibition influenza vaccination of swine to reduce the public health risk posed by IAV-S at agricultural exhibitions. IMPORTANCE Swine exhibited at agricultural fairs in North America have been the source of repeated zoonotic influenza A virus transmission, which creates a pathway for influenza pandemic emergence. We investigated the effect of using either live-attenuated influenza virus or killed influenza virus vaccines as prefair influenza vaccination of swine on zoonotic influenza transmission risk. Ferrets were exposed to the pigs in order to simulate human exposure in a field setting. We observed reductions in influenza A virus shedding in both groups of vaccinated pigs as well as the corresponding ferret exposure groups, indicating vaccination improved outcomes on both sides of the interface. There was also significant delay in onset of infection among ferrets that were exposed to live-attenuated virus-vaccinated pigs, which might be beneficial during longer fairs. Our findings indicate that policies mandating influenza vaccination of swine before fairs, while not currently common, would reduce the public health risk posed by influenza zoonosis.

Tracing the Source of Influenza A Virus Zoonoses in Interconnected Circuits of Swine Exhibitions.

BACKGROUND: Since 2011, influenza A viruses circulating in US swine exhibited at county fairs are associated with >460 zoonotic infections, presenting an ongoing pandemic risk. Swine "jackpot shows" that occur before county fairs each summer intermix large numbers of exhibition swine from diverse geographic locations. We investigated the role of jackpot shows in influenza zoonoses. METHODS: We collected snout wipe or nasal swab samples from 17 009 pigs attending 350 national, state, and local swine exhibitions across 8 states during 2016-2018. RESULTS: Influenza was detected in 13.9% of swine sampled at jackpot shows, and 76.3% of jackpot shows had at least 1 pig test positive. Jackpot shows had 4.3-fold higher odds of detecting at least 1 influenza-positive pig compared to county fairs. When influenza was detected at a county fair, almost half of pigs tested positive, clarifying why zoonotic infections occur primarily at county fairs. CONCLUSIONS: The earlier timing of jackpot shows and long-distance travel for repeated showing of individual pigs provide a pathway for the introduction of influenza into county fairs. Mitigation strategies aimed at curtailing influenza at jackpot shows are likely to have downstream effects on disease transmission at county fairs and zoonoses.

Genomic Evidence for Sequestration of Influenza A Virus Lineages in Sea Duck Host Species.

Wild birds are considered the natural reservoir of influenza A viruses (IAVs) making them critical for IAV surveillance efforts. While sea ducks have played a role in novel IAV emergence events that threatened food security and public health, very few surveillance samples have been collected from sea duck hosts. From 2014-2018, we conducted surveillance focused in the Mississippi flyway, USA at locations where sea duck harvest has been relatively successful compared to our other sampling locations. Our surveillance yielded 1662 samples from sea ducks, from which we recovered 77 IAV isolates. Our analyses identified persistence of sea duck specific IAV lineages across multiple years. We also recovered sea duck origin IAVs containing an H4 gene highly divergent from the majority of North American H4-HA with clade node age of over 65 years. Identification of IAVs with long branch lengths is indicative of substantial genomic change consistent with persistence without detection by surveillance efforts. Sea ducks play a role in the movement and long-term persistence of IAVs and are likely harboring more undetected IAV diversity. Sea ducks should be a point of emphasis for future North American wild bird IAV surveillance efforts.

A Heterogeneous Swine Show Circuit Drives Zoonotic Transmission of Influenza A Viruses in the United States.

Influenza pandemics are associated with severe morbidity, mortality, and social and economic disruption. Every summer in the United States, youths attending agricultural fairs are exposed to genetically diverse influenza A viruses (IAVs) circulating in exhibition swine, resulting in over 450 lab-confirmed zoonotic infections since 2010. Exhibition swine represent a small, defined population (∼1.5% of the U.S. herd), presenting a realistic opportunity to mitigate a pandemic threat by reducing IAV transmission in the animals themselves. Through intensive surveillance and genetic sequencing of IAVs in exhibition swine in six U.S. states in 2018 (n = 212), we characterized how a heterogeneous circuit of swine shows, comprising fairs with different sizes and geographic coverage, facilitates IAV transmission among exhibition swine and into humans. Specifically, we identified the role of an early-season national show in the propagation and spatial dissemination of a specific virus (H1δ-2) that becomes dominant among exhibition swine and is associated with the majority of zoonotic infections in 2018. These findings suggest that a highly targeted mitigation strategy, such as postponing swine shows for 1 to 2 weeks following the early-season national show, could potentially reduce IAV transmission in exhibition swine and spillover into humans, and this merits further study.IMPORTANCE The varying influenza A virus (IAV) exposure and infection status of individual swine facilitates introduction, transmission, and dissemination of diverse IAVs. Since agricultural fairs bring people into intimate contact with swine, they provide a unique interface for zoonotic transmission of IAV. Understanding the dynamics of IAV transmission through exhibition swine is critical to mitigating the high incidence of variant IAV cases reported in association with agricultural fairs. We used genomic sequences from our exhibition swine surveillance to characterize the hemagglutinin and full genotypic diversity of IAV at early-season shows and the subsequent dissemination through later-season agricultural fairs. We were able to identify a critical time point with important implications for downstream IAV and zoonotic transmission. With improved understanding of evolutionary origins of zoonotic IAV, we can inform public health mitigation strategies to ultimately reduce zoonotic IAV transmission and risk of pandemic IAV emergence.

Year­Round Influenza A Virus Surveillance in Mallards (Anas platyrhynchos) Reveals Genetic Persistence During the Under­Sampled Spring Season.

Active influenza A virus (IAV) surveillance in wild waterfowl in the United States has revolved around convenience-based sampling methods, resulting in gaps in surveillance during the spring season. We conducted active IAV surveillance in mallards continuously from July 2017 to July 2019 in the coastal marshes of Lake Erie near Port Clinton, Ohio. We aimed to understand ecological and evolutionary dynamics of IAV across multiple seasons, including the under­sampled spring season. We collected 2096 cloacal swabs and estimated a 6.1% (95% confidence interval (CI): 0.050-0.071) prevalence during the study period. Prevalence was lowest during spring (1.0%, 95% CI: 0.004-0.015). Time­stamped phylogenetic analyses revealed local persistence of genetic lineages of multiple gene segments. The PA segment consists of a lineage detected in multiple seasons with a time to most recent common ancestor of 2.48 years (95% highest posterior density: 2.16-2.74). Analysis of the H3 and H6 segments showed close relation between IAVs detected in spring and the following autumn migration. Though the mechanisms behind viral persistence in a single location are not well understood, we provide evidence that viruses can persist across several seasons. Current surveillance methods should be evaluated to ensure they are capturing the breadth of genetic diversity of IAV in waterfowl and prepare for IAV outbreaks in both animals and humans.