Ecological divergence of wild birds drives avian influenza spillover and global spread.

The diversity of influenza A viruses (IAV) is primarily hosted by two highly divergent avian orders: Anseriformes (ducks, swans and geese) and Charadriiformes (gulls, terns and shorebirds). Studies of IAV have historically focused on Anseriformes, specifically dabbling ducks, overlooking the diversity of hosts in nature, including gull and goose species that have successfully adapted to human habitats. This study sought to address this imbalance by characterizing spillover dynamics and global transmission patterns of IAV over 10 years at greater taxonomic resolution than previously considered. Furthermore, the circulation of viral subtypes in birds that are either host-adapted (low pathogenic H13, H16) or host-generalist (highly pathogenic avian influenza-HPAI H5) provided a unique opportunity to test and extend models of viral evolution. Using Bayesian phylodynamic modelling we uncovered a complex transmission network that relied on ecologically divergent bird hosts. The generalist subtype, HPAI H5 was driven largely by wild geese and swans that acted as a source for wild ducks, gulls, land birds, and domestic geese. Gulls were responsible for moving HPAI H5 more rapidly than any other host, a finding that may reflect their long-distance, pelagic movements and their immuno-naïve status against this subtype. Wild ducks, long viewed as primary hosts for spillover, occupied an optimal space for viral transmission, contributing to geographic expansion and rapid dispersal of HPAI H5. Evidence of inter-hemispheric dispersal via both the Pacific and Atlantic Rims was detected, supporting surveillance at high latitudes along continental margins to achieve early detection. Both neutral (geographic expansion) and non-neutral (antigenic selection) evolutionary processes were found to shape subtype evolution which manifested as unique geographic hotspots for each subtype at the global scale. This study reveals how a diversity of avian hosts contribute to viral spread and spillover with the potential to improve surveillance in an era of rapid global change.

Host barriers to SARS-CoV-2 demonstrated by ferrets in a high-exposure domestic setting.

Ferrets (Mustela putorius furo) are mustelids of special relevance to laboratory studies of respiratory viruses and have been shown to be susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and onward transmission. Here, we report the results of a natural experiment where 29 ferrets in one home had prolonged, direct contact and constant environmental exposure to two humans with symptomatic disease, one of whom was confirmed positive for SARS-CoV-2. We observed no evidence of SARS-CoV-2 transmission from humans to ferrets based on viral and antibody assays. To better understand this discrepancy in experimental and natural infection in ferrets, we compared SARS-CoV-2 sequences from natural and experimental mustelid infections and identified two surface glycoprotein Spike (S) mutations associated with mustelids. While we found evidence that angiotensin-converting enzyme II provides a weak host barrier, one mutation only seen in ferrets is located in the novel S1/S2 cleavage site and is computationally predicted to decrease furin cleavage efficiency. These data support the idea that host factors interacting with the novel S1/S2 cleavage site may be a barrier in ferret SARS-CoV-2 susceptibility and that domestic ferrets are at low risk of natural infection from currently circulating SARS-CoV-2. We propose two mechanistically grounded hypotheses for mustelid host adaptation of SARS-CoV-2, with possible effects that require additional investigation.

Highly Pathogenic Avian Influenza A(H5N1) Virus Outbreak in New England Seals, United States.

We report the spillover of highly pathogenic avian influenza A(H5N1) into marine mammals in the northeastern United States, coincident with H5N1 in sympatric wild birds. Our data indicate monitoring both wild coastal birds and marine mammals will be critical to determine pandemic potential of influenza A viruses.

Global dissemination of influenza A virus is driven by wild bird migration through arctic and subarctic zones.

Influenza A viruses (IAV) circulate endemically among many wild aquatic bird populations that seasonally migrate between wintering grounds in southern latitudes to breeding ranges along the perimeter of the circumpolar arctic. Arctic and subarctic zones are hypothesized to serve as ecologic drivers of the intercontinental movement and reassortment of IAVs due to high densities of disparate populations of long distance migratory and native bird species present during breeding seasons. Iceland is a staging ground that connects the East Atlantic and North Atlantic American flyways, providing a unique study system for characterizing viral flow between eastern and western hemispheres. Using Bayesian phylodynamic analyses, we sought to evaluate the viral connectivity of Iceland to proximal regions and how inter-species transmission and reassortment dynamics in this region influence the geographic spread of low and highly pathogenic IAVs. Findings demonstrate that IAV movement in the arctic and subarctic reflects wild bird migration around the perimeter of the circumpolar north, favouring short-distance flights between proximal regions rather than long distance flights over the polar interior. Iceland connects virus movement between mainland Europe and North America, consistent with the westward migration of wild birds from mainland Europe to Northeastern Canada and Greenland. Though virus diffusion rates were similar among avian taxonomic groups in Iceland, gulls play an outsized role as sinks of IAVs from other avian hosts prior to onward migration. These data identify patterns of virus movement in northern latitudes and inform future surveillance strategies related to seasonal and emergent IAVs with potential public health concern.

Age and season predict influenza A virus dynamics in urban gulls: consequences for natural hosts in unnatural landscapes.

Gulls are ubiquitous in urban areas due to a growing reliance on anthropogenic feeding sites, which has led to changes in their abundance, distribution, and migration ecology, with implications for disease transmission. Gulls offer a valuable model for testing hypotheses regarding the dynamics of influenza A virus (IAV) - for which gulls are a natural reservoir in urban areas. We sampled sympatric populations of Ring-billed (Larus delawarensis), Herring (L. argentatus), and Great Black-backed Gulls (L. marinus) along the densely populated Atlantic rim of North America to understand how IAV transmission is influenced by drivers such as annual cycle, host species, age, habitat type, and their interplay. We found that horizontal transmission, rather than vertical transmission, played an outsized role in the amplification of IAV due to the convergence of gulls from different breeding grounds and age classes. We detected overlapping effects of age and season in our prevalence model, identifying juveniles during autumn as the primary drivers of the seasonal epidemic in gulls. Gulls accumulated immunity over their lifespan, however short-term fluctuations in seroprevalence were observed, suggesting that migration may impose limits on the immune system to maintain circulating antibodies. We found that gulls in coastal urban habitats had higher viral prevalence than gulls captured inland, correlating with higher richness of waterbird species along the coast, a mechanism supported by our movement data. The peak in viral prevalence in newly fledged gulls that are capable of long-distance movement has important implications for the spread of pathogens to novel hosts during the migratory season as well as for human health as gulls increasingly utilize urban habitats.

Longitudinal analysis of pinnipeds in the northwest Atlantic provides insights on endemic circulation of phocine distemper virus.

Phocine distemper virus (PDV) is a morbillivirus that circulates within pinnipeds in the North Atlantic. PDV has caused two known unusual mortality events (UMEs) in western Europe (1988, 2002), and two UMEs in the northwest Atlantic (2006, 2018). Infrequent cross-species transmission and waning immunity are believed to contribute to periodic outbreaks with high mortality in western Europe. The viral ecology of PDV in the northwest Atlantic is less well defined and outbreaks have exhibited lower mortality than those in western Europe. This study sought to understand the molecular and ecological processes underlying PDV infection in eastern North America. We provide phylogenetic evidence that PDV was introduced into northwest Atlantic pinnipeds by a single lineage and is now endemic in local populations. Serological and viral screening of pinniped surveillance samples from 2006 onward suggest there is continued circulation of PDV outside of UMEs among multiple species with and without clinical signs. We report six full genome sequences and nine partial sequences derived from harbour and grey seals in the northwest Atlantic from 2011 through 2018, including a possible regional variant. Work presented here provides a framework towards greater understanding of how recovering populations and shifting species may impact disease transmission.

Reticulate evolution is favored in influenza niche switching.

Reticulate evolution is thought to accelerate the process of evolution beyond simple genetic drift and selection, helping to rapidly generate novel hybrids with combinations of adaptive traits. However, the long-standing dogma that reticulate evolutionary processes are likewise advantageous for switching ecological niches, as in microbial pathogen host switch events, has not been explicitly tested. We use data from the influenza genome sequencing project and a phylogenetic heuristic approach to show that reassortment, a reticulate evolutionary mechanism, predominates over mutational drift in transmission between different host species. Moreover, as host evolutionary distance increases, reassortment is increasingly favored. We conclude that the greater the quantitative difference between ecological niches, the greater the importance of reticulate evolutionary processes in overcoming niche barriers.

Ecosystem Interactions Underlie the Spread of Avian Influenza A Viruses with Pandemic Potential.

Despite evidence for avian influenza A virus (AIV) transmission between wild and domestic ecosystems, the roles of bird migration and poultry trade in the spread of viruses remain enigmatic. In this study, we integrate ecosystem interactions into a phylogeographic model to assess the contribution of wild and domestic hosts to AIV distribution and persistence. Analysis of globally sampled AIV datasets shows frequent two-way transmission between wild and domestic ecosystems. In general, viral flow from domestic to wild bird populations was restricted to within a geographic region. In contrast, spillover from wild to domestic populations occurred both within and between regions. Wild birds mediated long-distance dispersal at intercontinental scales whereas viral spread among poultry populations was a major driver of regional spread. Viral spread between poultry flocks frequently originated from persistent lineages circulating in regions of intensive poultry production. Our analysis of long-term surveillance data demonstrates that meaningful insights can be inferred from integrating ecosystem into phylogeographic reconstructions that may be consequential for pandemic preparedness and livestock protection.

Reassortment of Influenza A Viruses in Wild Birds in Alaska before H5 Clade 2.3.4.4 Outbreaks.

Sampling of mallards in Alaska during September 2014-April 2015 identified low pathogenic avian influenza A virus (subtypes H5N2 and H1N1) that shared ancestry with highly pathogenic reassortant H5N2 and H5N1 viruses. Molecular dating indicated reassortment soon after interhemispheric movement of H5N8 clade 2.3.4.4, suggesting genetic exchange in Alaska or surrounds before outbreaks.

Transmission of influenza reflects seasonality of wild birds across the annual cycle.

Influenza A Viruses (IAV) in nature must overcome shifting transmission barriers caused by the mobility of their primary host, migratory wild birds, that change throughout the annual cycle. Using a phylogenetic network of viral sequences from North American wild birds (2008-2011) we demonstrate a shift from intraspecific to interspecific transmission that along with reassortment, allows IAV to achieve viral flow across successive seasons from summer to winter. Our study supports amplification of IAV during summer breeding seeded by overwintering virus persisting locally and virus introduced from a wide range of latitudes. As birds migrate from breeding sites to lower latitudes, they become involved in transmission networks with greater connectivity to other bird species, with interspecies transmission of reassortant viruses peaking during the winter. We propose that switching transmission dynamics may be a critical strategy for pathogens that infect mobile hosts inhabiting regions with strong seasonality.

Genetic characterization of H5N2 influenza viruses isolated from wild birds in Japan suggests multiple reassortment.

Low-pathogenic avian influenza viruses (LPAIVs) of the H5 subtype can mutate to highly pathogenic forms, potentially destabilizing the poultry industry. Wild migratory birds are considered a natural reservoir of LPAIVs capable of dispersing both high- and low-pathogenic forms of the virus. Therefore, surveillance and characterization of AIV in wild birds are essential. Here, we report on the isolation and genetic characterization of 10 AIVs of the H5N2 subtype obtained through surveillance in Hokkaido, Japan, during 2009 and 2011. Full-genome sequencing revealed that the H5 and N2 genes of these isolates are all closely related to each other, belonging to the Eurasian avian-like lineage, but they are unrelated to H5 highly pathogenic strains of clade 2.3.4.4. The internal genes of the isolates were found to be diverse, consistent with our hypothesis that these H5N2 strains have undergone multiple reassortment events. Even though all of the H5N2 isolates were characterized as LPAIV based on the amino acid sequences at the HA cleavage site, this analysis demonstrates a diverse pool of precursors that may seed future outbreaks in poultry and possible human transmissions, suggesting the need for high-quality surveillance.

Genetic characterization of a rare H12N3 avian influenza virus isolated from a green-winged teal in Japan.

This study reports on the genetic characterization of an avian influenza virus, subtype H12N3, isolated from an Eurasian green-winged teal (Anas crecca) in Japan in 2009. The entire genome sequence of the isolate was analyzed, and phylogenetic analyses were conducted to characterize the evolutionary history of the isolate. Phylogenetic analysis of the hemagglutinin and neuraminidase genes indicated that the virus belonged to the Eurasian-like avian lineage. Molecular dating indicated that this H12 virus is likely a multiple reassortant influenza A virus. This is the first reported characterization of influenza A virus subtype H12N3 isolated in Japan and these data contribute to the accumulation of knowledge on the genetic diversity and generation of novel influenza A viruses.

Stable Isotopes Suggest Low Site Fidelity in Bar-headed Geese (Anser indicus) in Mongolia: Implications for Disease Transmission.

Population connectivity is an important consideration in studies of disease transmission and biological conservation, especially with regard to migratory species. Determining how and when different subpopulations intermingle during different phases of the annual cycle can help identify important geographical regions or features as targets for conservation efforts and can help inform our understanding of continental-scale disease transmission. In this study, stable isotopes of hydrogen and carbon in contour feathers were used to assess the degree of molt-site fidelity among Bar-headed Geese (Anser indicus) captured in north-central Mongolia. Samples were collected from actively molting Bar-headed Geese (n = 61), and some individual samples included both a newly grown feather (still in sheath) and an old, worn feather from the bird's previous molt (n = 21). Although there was no difference in mean hydrogen isotope ratios for the old and new feathers, the isotopic variance in old feathers was approximately three times higher than that of the new feathers, which suggests that these birds use different and geographically distant molting locations from year to year. To further test this conclusion, online data and modeling tools from the isoMAP website were used to generate probability landscapes for the origin of each feather. Likely molting locations were much more widespread for old feathers than for new feathers, which supports the prospect of low molt-site fidelity. This finding indicates that population connectivity would be greater than expected based on data from a single annual cycle, and that disease spread can be rapid even in areas like Mongolia where Bar-headed Geese generally breed in small isolated groups.

Bird Migration and Avian Influenza: A Comparison of Hydrogen Stable Isotopes and Satellite Tracking Methods.

Satellite-based tracking of migratory waterfowl is an important tool for understanding the potential role of wild birds in the long-distance transmission of highly pathogenic avian influenza. However, employing this technique on a continental scale is prohibitively expensive. This study explores the utility of stable isotope ratios in feathers in examining both the distances traveled by migratory birds and variation in migration behavior. We compared the satellite-derived movement data of 22 ducks from 8 species captured at wintering areas in Bangladesh, Turkey, and Hong Kong with deuterium ratios (δD) of these and other individuals captured at the same locations. We derived likely molting locations from the satellite tracking data and generated expected isotope ratios based on an interpolated map of δD in rainwater. Although δD was correlated with the distance between wintering and molting locations, surprisingly, measured δD values were not correlated with either expected values or latitudes of molting sites. However, population-level parameters derived from the satellite-tracking data, such as mean distance between wintering and molting locations and variation in migration distance, were reflected by means and variation of the stable isotope values. Our findings call into question the relevance of the rainfall isotope map for Asia for linking feather isotopes to molting locations, and underscore the need for extensive ground truthing in the form of feather-based isoscapes. Nevertheless, stable isotopes from feathers could inform disease models by characterizing the degree to which regional breeding populations interact at common wintering locations. Feather isotopes also could aid in surveying wintering locations to determine where high-resolution tracking techniques (e.g. satellite tracking) could most effectively be employed. Moreover, intrinsic markers such as stable isotopes offer the only means of inferring movement information from birds that have died as a result of infection. In the absence of feather based-isoscapes, we recommend a combination of isotope analysis and satellite-tracking as the best means of generating aggregate movement data for informing disease models.

Host population structure and rare dispersal events drive leptospirosis transmission patterns among Rattus norvegicus in Boston, Massachusetts, US.

Leptospirosis (caused by pathogenic bacteria in the genus Leptospira ) is prevalent worldwide but more common in tropical and subtropical regions. Transmission can occur following direct exposure to infected urine from reservoir hosts, such as rats, or a urine-contaminated environment, which then can serve as an infection source for additional rats and other mammals, including humans. The brown rat, Rattus norvegicus , is an important reservoir of leptospirosis in urban settings. We investigated leptospirosis among brown rats in Boston, Massachusetts and hypothesized that rat dispersal in this urban setting influences the movement, persistence, and diversity of Leptospira . We analyzed DNA from 328 rat kidney samples collected from 17 sites in Boston over a seven-year period (2016-2022); 59 rats representing 12 of 17 sites were positive for Leptospira . We used 21 neutral microsatellite loci to genotype 311 rats and utilized the resulting data to investigate genetic connectivity among sampling sites. We generated whole genome sequences for 28 Leptospira isolates obtained from frozen and fresh tissue from some of the 59 Leptospira -positive rat kidneys. When isolates were not obtained, we attempted Leptospira genomic DNA capture and enrichment, which yielded 14 additional Leptospira genomes from rats. We also generated an enriched Leptospira genome from a 2018 human case in Boston. We found evidence of high genetic structure and limited dispersal among rat populations that is likely influenced by major roads and/or other unknown dispersal barriers, resulting in distinct rat population groups within the city; at certain sites these groups persisted for multiple years. We identified multiple distinct phylogenetic clades of L. interrogans among rats, with specific clades tightly linked to distinct rat populations. This pattern suggests L. interrogans persists in local rat populations and movement of leptospirosis in this urban rat community is driven by rat dispersal. Finally, our genomic analyses of the 2018 human leptospirosis case in Boston suggests a link to rats as the source. These findings will be useful for guiding rat control and human leptospirosis mitigation efforts in this and other urban settings.

Ecogeographic Drivers of the Spatial Spread of Highly Pathogenic Avian Influenza Outbreaks in Europe and the United States, 2016-Early 2022.

H5Nx highly pathogenic avian influenza (HPAI) viruses of clade 2.3.4.4 have caused outbreaks in Europe among wild and domestic birds since 2016 and were introduced to North America via wild migratory birds in December 2021. We examined the spatiotemporal extent of HPAI viruses across continents and characterized ecological and environmental predictors of virus spread between geographic regions by constructing a Bayesian phylodynamic generalized linear model (phylodynamic-GLM). The findings demonstrate localized epidemics of H5Nx throughout Europe in the first several years of the epizootic, followed by a singular branching point where H5N1 viruses were introduced to North America, likely via stopover locations throughout the North Atlantic. Once in the United States (US), H5Nx viruses spread at a greater rate between US-based regions as compared to prior spread in Europe. We established that geographic proximity is a predictor of virus spread between regions, implying that intercontinental transport across the Atlantic Ocean is relatively rare. An increase in mean ambient temperature over time was predictive of reduced H5Nx virus spread, which may reflect the effect of climate change on declines in host species abundance, decreased persistence of the virus in the environment, or changes in migratory patterns due to ecological alterations. Our data provide new knowledge about the spread and directionality of H5Nx virus dispersal in Europe and the US during an actively evolving intercontinental outbreak, including predictors of virus movement between regions, which will contribute to surveillance and mitigation strategies as the outbreak unfolds, and in future instances of uncontained avian spread of HPAI viruses.

Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in Shorebirds and Waterfowl in Southern Ukraine.

Emerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Health's capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia.

Migration strategy affects avian influenza dynamics in mallards (Anas platyrhynchos).

Studies of pathogen transmission typically overlook that wildlife hosts can include both migrant and resident populations when attempting to model circulation. Through the application of stable isotopes in flight feathers, we estimated the migration strategy of mallards (Anas platyrhynchos) occurring on California wintering grounds. Our study demonstrates that mallards- a principal host of avian influenza virus (AIV) in nature, contribute differently to virus gene flow depending on migration strategy. No difference in AIV prevalence was detected between resident (9.6%), intermediate-distance (9.6%) and long-distance migrants (7.4%). Viral diversity among the three groups was also comparable, possibly owing to viral pool mixing when birds converge at wetlands during winter. However, migrants and residents contributed differently to the virus gene pool at wintering wetlands. Migrants introduced virus from northern breeding grounds (Alaska and the NW Pacific Rim) into the wintering population, facilitating gene flow at continental scales, but circulation of imported virus appeared to be limited. In contrast, resident mallards acted as AIV reservoirs facilitating year-round circulation of limited subtypes (i.e. H5N2) at lower latitudes. This study supports a model of virus exchange in temperate regions driven by the convergence of wild birds with separate geographic origins and exposure histories.

Epidemiology and Ecology of Influenza A Viruses among Wildlife in the Arctic.

Arctic regions are ecologically significant for the environmental persistence and geographic dissemination of influenza A viruses (IAVs) by avian hosts and other wildlife species. Data describing the epidemiology and ecology of IAVs among wildlife in the arctic are less frequently published compared to southern temperate regions, where prevalence and subtype diversity are more routinely documented. Following PRISMA guidelines, this systematic review addresses this gap by describing the prevalence, spatiotemporal distribution, and ecological characteristics of IAVs detected among wildlife and the environment in this understudied region of the globe. The literature search was performed in PubMed and Google Scholar using a set of pre-defined search terms to identify publications reporting on IAVs in Arctic regions between 1978 and February 2022. A total of 2125 articles were initially screened, 267 were assessed for eligibility, and 71 articles met inclusion criteria. IAVs have been detected in multiple wildlife species in all Arctic regions, including seabirds, shorebirds, waterfowl, seals, sea lions, whales, and terrestrial mammals, and in the environment. Isolates from wild birds comprise the majority of documented viruses derived from wildlife; however, among all animals and environmental matrices, 26 unique low and highly pathogenic subtypes have been characterized in the scientific literature from Arctic regions. Pooled prevalence across studies indicates 4.23% for wild birds, 3.42% among tested environmental matrices, and seroprevalences of 9.29% and 1.69% among marine and terrestrial mammals, respectively. Surveillance data are geographically biased, with most data from the Alaskan Arctic and many fewer reports from the Russian, Canadian, North Atlantic, and Western European Arctic. We highlight multiple important aspects of wildlife host, pathogen, and environmental ecology of IAVs in Arctic regions, including the role of avian migration and breeding cycles for the global spread of IAVs, evidence of inter-species and inter-continental reassortment at high latitudes, and how climate change-driven ecosystem shifts, including changes in the seasonal availability and distribution of dietary resources, have the potential to alter host-pathogen-environment dynamics in Arctic regions. We conclude by identifying gaps in knowledge and propose priorities for future research.

A standardized instrument quantifying risk factors associated with bi-directional transmission of SARS-CoV-2 and other zoonotic pathogens: The COVID-19 human-animal interactions survey (CHAIS).

Similar to many zoonotic pathogens which transmit from animals to humans, SARS-CoV-2 (CoV-2), the virus responsible for the COVID-19 pandemic, most likely originated in Rhinolophus bats before spreading among humans globally. Early into the pandemic, reports of CoV-2 diagnoses in animals from various countries emerged. While most CoV-2 positive animals were confirmed to have been in close contact with CoV-2 positive humans, there has been a paucity of published evidence to-date describing risk factors associated with CoV-2 transmission among humans and animals. The COVID-19 Human-Animal Interactions Survey (CHAIS) was developed to provide a standardized instrument describing human-animal interactions during the pandemic and to evaluate behavioral, spatiotemporal, and biological risk factors associated with bi-directional zoonotic transmission of CoV-2 within shared environments, predominantly households with limited information about human-wildlife or human-livestock interactions. CHAIS measures four broad domains of transmission risk: 1) risk and intensity of infection in human hosts, 2) spatial characteristics of shared environments, 3) behaviors and human-animal interactions, and 4) susceptible animal subpopulations. Following the development of CHAIS, with a One Health approach, a multidisciplinary group of experts (n = 20) was invited to review and provide feedback on the survey for content validity. Expert feedback was incorporated into two final survey formats-an extended version and an abridged version for which specific core questions addressing zoonotic and reverse zoonotic transmission were identified. Both versions are modularized, with each section having the capacity to serve as independent instruments, allowing researchers to customize the survey based on context and research-specific needs. Further adaptations for studies seeking to investigate other zoonotic pathogens with similar routes of transmission (i.e. respiratory, direct contact) are also possible. The CHAIS instrument is a standardized human-animal interaction survey developed to provide important data on risk factors that guide transmission of CoV-2, and other similar pathogens, among humans and animals.