Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats.

Endogenous retroviruses (ERV) are indicators of vertebrate evolutionary history and play important roles as homeostatic regulators. ERV long terminal repeat (LTR) elements may act as cis-activating promoters or trans-activating enhancer elements modifying gene transcription distant from LTR insertion sites. We previously documented that endogenous feline leukemia virus (FeLV)-LTR copy number variation in individual cats tracks inversely with susceptibility to virulent FeLV disease. To evaluate FeLV-LTR insertion characteristics, we assessed enFeLV-LTR integration site diversity in 20 cats from three genetically distinct populations using a baited linker-mediated PCR approach. We documented 765 individual integration sites unequally represented among individuals. Only three LTR integration sites were shared among all individuals, while 412 sites were unique to a single individual. When primary fibroblast cultures were challenged with exogenous FeLV, we found significantly increased expression of both exogenous and endogenous FeLV orthologs, supporting previous findings of potential exFeLV-enFeLV interactions; however, viral challenge did not elicit transcriptional changes in genes associated with the vast majority of integration sites. This study assesses FeLV-LTR integration sites in individual animals, providing unique transposome genotypes. Further, we document substantial individual variation in LTR integration site locations, even in a highly inbred population, and provide a framework for understanding potential endogenous retroviral element position influence on host gene transcription.

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.

Feline Leukemia Virus Frequently Spills Over from Domestic Cats to North American Pumas.

Feline leukemia virus (FeLV) is a gammaretrovirus with horizontally transmitted and endogenous forms. Domestic cats are the primary reservoir species, but FeLV outbreaks in endangered Florida panthers and Iberian lynxes have resulted in mortalities. To assess prevalence and interspecific/intraspecific transmission, we conducted an extensive survey and phylogenetic analysis of FeLV infection in free-ranging pumas (n = 641) and bobcats (n = 212) and shelter domestic cats (n = 304). Samples were collected from coincident habitats across the United States between 1985 and 2018. FeLV infection was detected in 3.12% of the puma samples, 0.47% of the bobcat samples, and 6.25% of the domestic cat samples analyzed. Puma prevalence varied by location, with Florida having the highest rate of infection. FeLV env sequences revealed variation among isolates, and we identified two distinct clades. Both progressive and regressive infections were identified in cats and pumas. Based on the time and location of sampling and phylogenetic analysis, we inferred 3 spillover events between domestic cats and pumas; 3 puma-to-puma transmissions in Florida were inferred. An additional 14 infections in pumas likely represented spillover events following contact with reservoir host domestic cat populations. Our data provide evidence that FeLV transmission from domestic cats to pumas occurs widely across the United States, and puma-to-puma transmission may occur in genetically and geographically constrained populations. IMPORTANCE Feline leukemia virus (FeLV) is a retrovirus that primarily affects domestic cats. Close interactions with domestic cats, including predation, can lead to the interspecific transmission of the virus to pumas, bobcats, or other feline species. Some infected individuals develop progressive infections, which are associated with clinical signs of disease and can result in mortality. Therefore, outbreaks of FeLV in wildlife, including the North American puma and the endangered Florida panther, are of high conservation concern. This work provides a greater understanding of the dynamics of the transmission of FeLV between domestic cats and wild felids and presents evidence of multiple spillover events and infections in all sampled populations. These findings highlight the concern for pathogen spillover from domestic animals to wildlife but also identify an opportunity to understand viral evolution following cross-species transmissions more broadly.

Feline Leukemia Virus (FeLV) Endogenous and Exogenous Recombination Events Result in Multiple FeLV-B Subtypes during Natural Infection.

Feline leukemia virus (FeLV) is associated with a range of clinical signs in felid species. Differences in disease processes are closely related to genetic variation in the envelope (env) region of the genome of six defined subgroups. The primary hosts of FeLV are domestic cats of the Felis genus that also harbor endogenous FeLV (enFeLV) elements stably integrated in their genomes. EnFeLV elements display 86% nucleotide identity to exogenous, horizontally transmitted FeLV (FeLV-A). Variation between enFeLV and FeLV-A is primarily in the long terminal repeat (LTR) and env regions, which potentiates generation of the FeLV-B recombinant subgroup during natural infection. The aim of this study was to examine recombination behavior of exogenous FeLV (exFeLV) and enFeLV in a natural FeLV epizootic. We previously described that of 65 individuals in a closed colony, 32 had productive FeLV-A infection, and 22 of these individuals had detectable circulating FeLV-B. We cloned and sequenced the env gene of FeLV-B, FeLV-A, and enFeLV spanning known recombination breakpoints and examined between 1 and 13 clones in 22 animals with FeLV-B to assess sequence diversity and recombination breakpoints. Our analysis revealed that FeLV-A sequences circulating in the population, as well as enFeLV env sequences, are highly conserved. We documented many recombination breakpoints resulting in the production of unique FeLV-B genotypes. More than half of the cats harbored more than one FeLV-B variant, suggesting multiple recombination events between enFeLV and FeLV-A. We concluded that FeLV-B was predominantly generated de novo within each host, although we could not definitively rule out horizontal transmission, as nearly all cats harbored FeLV-B sequences that were genetically highly similar to those identified in other individuals. This work represents a comprehensive analysis of endogenous-exogenous retroviral interactions with important insights into host-virus interactions that underlie disease pathogenesis in a natural setting. IMPORTANCE Feline leukemia virus (FeLV) is a felid retrovirus with a variety of disease outcomes. Exogenous FeLV-A is the virus subgroup almost exclusively transmitted between cats. Recombination between FeLV-A and endogenous FeLV analogues in the cat genome may result in emergence of largely replication-defective but highly virulent subgroups. FeLV-B is formed when the 3' envelope (env) region of endogenous FeLV (enFeLV) recombines with that of the exogenous FeLV (exFeLV) during viral reverse transcription and integration. Both domestic cats and wild relatives of the Felis genus harbor enFeLV, which has been shown to limit FeLV-A disease outcome. However, enFeLV also contributes genetic material to the recombinant FeLV-B subgroup. This study evaluates endogenous-exogenous recombination outcomes in a naturally infected closed colony of cats to determine mechanisms and risk of endogenous retroviral recombination during exogenous virus exposure that leads to enhanced virulence. While FeLV-A and enFeLV env regions were highly conserved from cat to cat, nearly all individuals with emergent FeLV-B had unique combinations of genotypes, representative of a wide range of recombination sites within env. The findings provide insight into unique recombination patterns for emergence of new pathogens and can be related to similar viruses across species.

Modelling the role of predation on disease burdens of prey.

Research Highlight: Brandell, E. E., Cross, P. C., Smith, D. W., Rogers, W., Galloway, N. L., MacNulty, D. R., Stahler, D. R., Treanor, J. & Hudson, P. J. (2022). Examination of the interaction between age-specific predation and chronic disease in the Greater Yellowstone Ecosystem. Journal of Animal Ecology, 00, 1-12. https://doi.org/10.1111/1365-2656.13661. Predation can alter disease dynamics in prey. If predators select for infected individuals, they can reduce disease burdens. In other cases, predators can increase disease burdens via various mechanisms such as altered prey behaviour. The influence of predation on disease dynamics is a result of interactions among various traits of the predators, prey and the pathogen itself. For example, pathogens tend to vary with age and predators typically select for certain age classes. Thus, the overlap between ages selected by predators and those infected will likely contribute to any effects of predation on reducing disease burdens. In this paper, Brandell et al. (2022) develop a model to evaluate the predator cleansing effect given age-based variation in pathogens and predation. The model was developed for Chronic Wasting Disease (CWD) infections in deer and elk facing predation by cougars and grey wolves in the Greater Yellowstone Ecosystem. The results indicate that predators can reduce CWD outbreak size, especially if selecting for infected individuals. CWD is an always fatal disease and this work suggests that predators could reduce disease burdens in cervids. The model is also applicable to other systems and promises to further our understanding of the role of predation on disease in prey, as well as drive future empirical studies.

Parasites as conservation tools.

Parasite success typically depends on a close relationship with one or more hosts; therefore, attributes of parasitic infection have the potential to provide indirect details of host natural history and are biologically relevant to animal conservation. Characterization of parasite infections has been useful in delineating host populations and has served as a proxy for assessment of environmental quality. In other cases, the utility of parasites is just being explored, for example, as indicators of host connectivity. Innovative studies of parasite biology can provide information to manage major conservation threats by using parasite assemblage, prevalence, or genetic data to provide insights into the host. Overexploitation, habitat loss and fragmentation, invasive species, and climate change are major threats to animal conservation, and all of these can be informed by parasites.

Los Parásitos como Herramienta de Conservación Resumen El éxito de los parásitos depende típicamente de la relación cercana con uno o más hospederos; por lo tanto, las características de la infección parasitaria tienen potencial para proporcionar detalles indirectos de la historia natural del hospedero y son biológicamente relevantes para la conservación animal. La caracterización de las infecciones parasitarias ha sido útil para definir a las poblaciones hospederas y ha servido como sustituto para la evaluación de la calidad ambiental. Los estudios innovadores de la biología de parásitos pueden proporcionar información para manejar las principales amenazas a la conservación mediante la información proporcionada por el conjunto de parásitos, su prevalencia o genética que proporciona conocimiento sobre el hospedero. La sobreexplotación, la pérdida del hábitat y la fragmentación, las especies invasoras y el cambio climático son las principales amenazas para la conservación animal y a todas pueden ser informadas mediante los parásitos.

A natural laboratory to elucidate the evolution of endogenous-exogenous retroviral interactions.

The advent of whole genome sequencing has revealed much about the genomes of animals including the relatively large percentage of the genome consisting of endogenous retroviruses (ERV; International Human Genome Sequencing Consortium, 2001). An ERV arises when a retrovirus integrates into a host germ cell genome through normal infection processes. Germline infections can be transmitted to offspring through Mendelian inheritance and at times become fixed elements of the host genome (Weiss, 2006). At their inception, these endogenized retroviruses maintain all the functions of their exogenous progenitors and can produce infectious virus (Figure 1). Mutations in the ERV randomly accumulate over time and can lead to the loss of the deleterious effects. In addition, the ERV can provide benefits to the host often via limiting exogenous viral infections (Chiu and VandeWoude, 2020a). However, most of these endogenous viruses are evolutionary relics representing historical infections and have no contemporary exogenous virus. This limits the opportunities to understand the evolution of ERVs and their interactions with exogenous viruses. In this issue of Molecular Ecology, Quigley et al. (2020) use a novel approach to show that koala retrovirus (KoRV) is undergoing endogenization along a geographic gradient with a variety of exogenous variants dispersed across the landscape. This system provides an opportunity to further elucidate the complex mechanisms in which endogenous and exogenous viruses interact and follow the evolution of an ERV in real time.

Variation in Intra-individual Lentiviral Evolution Rates: a Systematic Review of Human, Nonhuman Primate, and Felid Species.

Lentiviral replication mediated by reverse transcriptase is considered to be highly error prone, leading to a high intra-individual evolution rate that promotes evasion of neutralization and persistent infection. Understanding lentiviral intra-individual evolutionary dynamics on a comparative basis can therefore inform research strategies to aid in studies of pathogenesis, vaccine design, and therapeutic intervention. We conducted a systematic review of intra-individual evolution rates for three species groups of lentiviruses-feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), and human immunodeficiency virus (HIV). Overall, intra-individual rate estimates differed by virus but not by host, gene, or viral strain. Lentiviral infections in spillover (nonadapted) hosts approximated infections in primary (adapted) hosts. Our review consistently documents that FIV evolution rates within individuals are significantly lower than the rates recorded for HIV and SIV. FIV intra-individual evolution rates were noted to be equivalent to FIV interindividual rates. These findings document inherent differences in the evolution of FIV relative to that of primate lentiviruses, which may signal intrinsic difference of reverse transcriptase between these viral species or different host-viral interactions. Analysis of lentiviral evolutionary selection pressures at the individual versus population level is valuable for understanding transmission dynamics and the emergence of virulent and avirulent strains and provides novel insight for approaches to interrupt lentiviral infections.IMPORTANCE To the best of our knowledge, this is the first study that compares intra-individual evolution rates for FIV, SIV, and HIV following systematic review of the literature. Our findings have important implications for informing research strategies in the field of intra-individual virus dynamics for lentiviruses. We observed that FIV evolves more slowly than HIV and SIV at the intra-individual level and found that mutation rates may differ by gene sequence length but not by host, gene, strain, an experimental setting relative to a natural setting, or spillover host infection relative to primary host infection.

Altered lentiviral infection dynamics follow genetic rescue of the Florida panther.

Wildlife translocations are a commonly used strategy in endangered species recovery programmes. Although translocations require detailed assessment of risk, their impact on parasite distribution has not been thoroughly assessed. This is despite the observation that actions that alter host-parasite distributions can drive evolution or introduce new parasites to previously sequestered populations. Here, we use a contemporary approach to amplify viral sequences from archived biological samples to characterize a previously undocumented impact of the successful genetic rescue of the Florida panther (Puma concolor coryi). Our efforts reveal transmission of feline immunodeficiency virus (FIV) during translocation of pumas from Texas to Florida, resulting in extirpation of a historic Florida panther FIV subtype and expansion of a genetically stable subtype that is highly conserved in Texas and Florida. We used coalescent theory to estimate viral demography across time and show an exponential increase in the effective population size of FIV coincident with expansion of the panther population. Additionally, we show that FIV isolates from Texas are basal to isolates from Florida. Interestingly, FIV genomes recovered from Florida and Texas demonstrate exceptionally low interhost divergence. Low host genomic diversity and lack of additional introgressions may underlie the surprising lack of FIV evolution over 2 decades. We conclude that modern FIV in the Florida panther disseminated following genetic rescue and rapid population expansion, and that infectious disease risks should be carefully considered during conservation efforts involving translocations. Further, viral evolutionary dynamics may be significantly altered by ecological niche, host diversity and connectivity between host populations.

Urbanization reduces genetic connectivity in bobcats (Lynx rufus) at both intra- and interpopulation spatial scales.

Urbanization is a major factor driving habitat fragmentation and connectivity loss in wildlife. However, the impacts of urbanization on connectivity can vary among species and even populations due to differences in local landscape characteristics, and our ability to detect these relationships may depend on the spatial scale at which they are measured. Bobcats (Lynx rufus) are relatively sensitive to urbanization and the status of bobcat populations is an important indicator of connectivity in urban coastal southern California. We genotyped 271 bobcats at 13,520 SNP loci to conduct a replicated landscape resistance analysis in five genetically distinct populations. We tested urban and natural factors potentially influencing individual connectivity in each population separately, as well as study-wide. Overall, landscape genomic effects were most frequently detected at the study-wide spatial scale, with urban land cover (measured as impervious surface) having negative effects and topographic roughness having positive effects on gene flow. The negative effect of urban land cover on connectivity was also evident when populations were analyzed separately despite varying substantially in spatial area and the proportion of urban development, confirming a pervasive impact of urbanization largely independent of spatial scale. The effect of urban development was strongest in one population where stream habitat had been lost to development, suggesting that riparian corridors may help mitigate reduced connectivity in urbanizing areas. Our results demonstrate the importance of replicating landscape genetic analyses across populations and considering how landscape genetic effects may vary with spatial scale and local landscape structure.

Urbanization impacts apex predator gene flow but not genetic diversity across an urban-rural divide.

Apex predators are important indicators of intact natural ecosystems. They are also sensitive to urbanization because they require broad home ranges and extensive contiguous habitat to support their prey base. Pumas (Puma concolor) can persist near human developed areas, but urbanization may be detrimental to their movement ecology, population structure, and genetic diversity. To investigate potential effects of urbanization in population connectivity of pumas, we performed a landscape genomics study of 130 pumas on the rural Western Slope and more urbanized Front Range of Colorado, USA. Over 12,000 single nucleotide polymorphisms (SNPs) were genotyped using double-digest, restriction site-associated DNA sequencing (ddRADseq). We investigated patterns of gene flow and genetic diversity, and tested for correlations between key landscape variables and genetic distance to assess the effects of urbanization and other landscape factors on gene flow. Levels of genetic diversity were similar for the Western Slope and Front Range, but effective population sizes were smaller, genetic distances were higher, and there was more admixture in the more urbanized Front Range. Forest cover was strongly positively associated with puma gene flow on the Western Slope, while impervious surfaces restricted gene flow and more open, natural habitats enhanced gene flow on the Front Range. Landscape genomic analyses revealed differences in puma movement and gene flow patterns in rural versus urban settings. Our results highlight the utility of dense, genome-scale markers to document subtle impacts of urbanization on a wide-ranging carnivore living near a large urban center.

The Expectations and Challenges of Wildlife Disease Research in the Era of Genomics: Forecasting with a Horizon Scan-like Exercise.

The outbreak and transmission of disease-causing pathogens are contributing to the unprecedented rate of biodiversity decline. Recent advances in genomics have coalesced into powerful tools to monitor, detect, and reconstruct the role of pathogens impacting wildlife populations. Wildlife researchers are thus uniquely positioned to merge ecological and evolutionary studies with genomic technologies to exploit unprecedented "Big Data" tools in disease research; however, many researchers lack the training and expertise required to use these computationally intensive methodologies. To address this disparity, the inaugural "Genomics of Disease in Wildlife" workshop assembled early to mid-career professionals with expertise across scientific disciplines (e.g., genomics, wildlife biology, veterinary sciences, and conservation management) for training in the application of genomic tools to wildlife disease research. A horizon scanning-like exercise, an activity to identify forthcoming trends and challenges, performed by the workshop participants identified and discussed 5 themes considered to be the most pressing to the application of genomics in wildlife disease research: 1) "Improving communication," 2) "Methodological and analytical advancements," 3) "Translation into practice," 4) "Integrating landscape ecology and genomics," and 5) "Emerging new questions." Wide-ranging solutions from the horizon scan were international in scope, itemized both deficiencies and strengths in wildlife genomic initiatives, promoted the use of genomic technologies to unite wildlife and human disease research, and advocated best practices for optimal use of genomic tools in wildlife disease projects. The results offer a glimpse of the potential revolution in human and wildlife disease research possible through multi-disciplinary collaborations at local, regional, and global scales.

Phylogeography of the widespread creek chub Semotilus atromaculatus (Cypriniformes: Leuciscidae).

The extent and nature of genetic differentiation in Semotilus atromaculatus, one of the most abundant and widespread leuciscids in North America, were evaluated based on mitochondrial (mt) and nuclear DNA sequence variation. Phylogenetic relationships were first inferred based on a fragment of the cytochrome b (cytb) region and the nuclear intron s7 gene for S. atromaculatus and all other congeners as well as representative species from all other genera in the creek chub-plagopterin clade. The phylogeography of major haplogroups of S. atromaculatus was also assessed according to variation in a fragment of the mitochondrial cytb region from 567 individuals across its range. All analyses identified S. thoreauianus, S. lumbee and S. corporalis as reciprocally monophyletic groups. Analyses of nuclear sequence variation resolved S. atromaculatus as a single clade, where S. thoreauianus and S. lumbee were recovered as the sister group to S. atromaculatus, and S. corporalis was resolved as sister to all other species in the genus. Analyses of mtDNA sequence variation recovered S. atromaculatus as three well supported and differentiated monophyletic groups, with a widespread genetically homogeneous lineage extending across most of the current range of the species; a more geographically restricted and geographically structured lineage in the southern Appalachians, sister group to S. lumbee; and a geographically restricted lineage was identified from two Gulf Slope basins. Evidence of complex mito-nuclear discordance and phylogeographic differentiation within S. atromaculatus illustrates that further analysis of widespread species is warranted to understand North American freshwater fish diversity and distributions.

Mitogenomes and relatedness do not predict frequency of tool-use by sea otters.

Many ecological aspects of tool-use in sea otters are similar to those in Indo-Pacific bottlenose dolphins. Within an area, most tool-using dolphins share a single mitochondrial haplotype and are more related to each other than to the population as a whole. We asked whether sea otters in California showed similar genetic patterns by sequencing mitogenomes of 43 otters and genotyping 154 otters at 38 microsatellite loci. There were six variable sites in the mitogenome that yielded three haplotypes, one found in only a single individual. The other two haplotypes contained similar percentages (33 and 36%) of frequent tool-users and a variety of diet types. Microsatellite analyses showed that snail specialists, the diet specialist group that most frequently used tools, were no more related to each other than to the population as a whole. The lack of genetic association among tool-using sea otters compared with dolphins may result from the length of time each species has been using tools. Tool-use in dolphins appears to be a relatively recent innovation (less than 200 years) but sea otters have probably been using tools for many thousands or even millions of years.

Mutual dilution of infection by an introduced parasite in native and invasive stream fishes across Hawaii.

The presence of introduced hosts can increase or decrease infections of co-introduced parasites in native species of conservation concern. In this study, we compared parasite abundance, intensity, and prevalence between native Awaous stamineus and introduced poeciliid fishes by a co-introduced nematode parasite (Camallanus cotti) in 42 watersheds across the Hawaiian Islands. We found that parasite abundance, intensity and prevalence were greater in native than introduced hosts. Parasite abundance, intensity and prevalence within A. stamineus varied between years, which largely reflected a transient spike in infection in three remote watersheds on Molokai. At each site we measured host factors (length, density of native host, density of introduced host) and environmental factors (per cent agricultural and urban land use, water chemistry, watershed area and precipitation) hypothesized to influence C. cotti abundance, intensity and prevalence. Factors associated with parasitism differed between native and introduced hosts. Notably, parasitism of native hosts was higher in streams with lower water quality, whereas parasitism of introduced hosts was lower in streams with lower water quality. We also found that parasite burdens were lower in both native and introduced hosts when coincident. Evidence of a mutual dilution effect indicates that introduced hosts can ameliorate parasitism of native fishes by co-introduced parasites, which raises questions about the value of remediation actions, such as the removal of introduced hosts, in stemming the rise of infectious disease in species of conservation concern.

Metagenomics for Pathogen Detection During a Mass Mortality Event in Songbirds.

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria forming the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes, including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events.

Genetic Characterization of Microsporum canis Clinical Isolates in the United States.

Microsporum canis is the primary agent causing dermatophytosis in cats, and also infects humans, dogs, and other species. Assessment of genetic variation among M. canis isolates in the United States has not been conducted. Further, M. canis mating type and assessment of disease severity associated with genotypic characteristics have not been rigorously evaluated. We therefore isolated M. canis from 191 domestic cats across the US and characterized genotypes by evaluation of ITS sequence, MAT locus, and microsatellite loci analysis. The genes SSU1 and SUB3, which are associated with keratin adhesion and digestion, were sequenced from a subset of isolates to evaluate potential genetic associations with virulence. Analysis of microsatellite makers revealed three M. canis genetic clusters. Both clinic location and disease severity were significant predictors of microsatellite variants. 100% of the M. canis isolates were MAT1-1 mating gene type, indicating that MAT1-2 is very rare or extinct in the US and that asexual reproduction is the dominant form of replication. No genetic variation at SSU1 and SUB3 was observed. These findings pave the way for novel testing modalities for M. canis and provide insights about transmission and ecology of this ubiquitous and relatively uncharacterized agent.

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.

Prevalence and Genomic Sequence Analysis of Domestic Cat Hepadnavirus in the United States.

Hepadnaviruses are partially double-stranded DNA viruses that infect a variety of species. The prototypical virus in this family is the human hepatitis B virus, which chronically infects approximately 400 million people worldwide and is a risk factor for progressive liver disease and liver cancer. The first hepadnavirus isolated from carnivores was a domestic cat hepadnavirus (DCH), initially identified in Australia and subsequently detected in cats in Europe and Asia. As with all characterized hepadnaviruses so far, DCH infection has been associated with hepatic disease in its host. Prevalence of this infection in the United States has not been explored broadly. Thus, we utilized conventional and quantitative PCR to screen several populations of domestic cats to estimate DCH prevalence in the United States. We detected DCH DNA in 1 out of 496 animals (0.2%) in the U.S. cohort. In contrast, we detected circulating DCH DNA in 7 positive animals from a cohort of 67 domestic cats from Australia (10.4%), consistent with previous studies. The complete consensus genome of the U.S. DCH isolate was sequenced by Sanger sequencing with overlapping PCR products. An in-frame deletion of 157 bp was identified in the N-terminus of the core open reading frame. The deletion begins at the direct repeat 1 sequence (i.e., the 5' end of the expected double-stranded linear DNA form), consistent with covalently closed circular DNA resultant from illegitimate recombination described in other hepadnaviruses. Comparative genome sequence analysis indicated that the closest described relatives of the U.S. DCH isolate are those previously isolated in Italy. Motif analysis supports DCH using NTCP as an entry receptor, similar to human HBV. Our work indicates that chronic DCH prevalence in the U.S. is likely low compared to other countries.