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.

Antemortem cytologic diagnosis of pulmonary Langerhans cell histiocytosis in a cat.

Feline pulmonary Langerhans cell histiocytosis (FPLCH) is a rare histiocytic proliferative disease of middle-aged to older domestic cats. Langerhans cells in the terminal airways proliferate and infiltrate the interstitium and the airways to a lesser degree, widely effacing normal parenchyma. Historically, definitive diagnosis has required postmortem evaluation where pulmonary lesions have a classic gross and histologic morphology. Here, we present the first documented antemortem diagnosis of FPLCH using bronchoalveolar lavage (BAL) cytology and immunocytochemistry (ICC) in a 9-year-old British shorthair mix. The cat had a 3-month history of respiratory difficulty that was refractory to steroids and antimicrobials. Pulmonary radiographs had marked diffuse changes with a complex bronchointerstitial and micronodular pattern. BAL cytology revealed neutrophilic inflammation and markedly increased histiocytes with morphology distinct from typical pulmonary macrophages. ICC characterized histiocytes as CD1a+ /E-cadherin+ /CD11b- /PanCK- , consistent with a Langerhans cell phenotype. The cat was humanely euthanized due to poor prognosis and presented for necropsy. Gross, histopathologic, immunophenotypic, and ultrastructural findings confirmed a diagnosis of FPLCH. Proliferative cells were E-cadherin+ /Iba-1+ /CD18+ /CD1a+ /CD5+ /MHCII+ /CD204- /CD4- ; transmission electron microscopy identified the presence of Birbeck granules in the proliferating histiocytes, consistent with previous reports of FPLCH.

Apathogenic proxies for transmission dynamics of a fatal virus.

Identifying drivers of transmission-especially of emerging pathogens-is a formidable challenge for proactive disease management efforts. While close social interactions can be associated with microbial sharing between individuals, and thereby imply dynamics important for transmission, such associations can be obscured by the influences of factors such as shared diets or environments. Directly-transmitted viral agents, specifically those that are rapidly evolving such as many RNA viruses, can allow for high-resolution inference of transmission, and therefore hold promise for elucidating not only which individuals transmit to each other, but also drivers of those transmission events. Here, we tested a novel approach in the Florida panther, which is affected by several directly-transmitted feline retroviruses. We first inferred the transmission network for an apathogenic, directly-transmitted retrovirus, feline immunodeficiency virus (FIV), and then used exponential random graph models to determine drivers structuring this network. We then evaluated the utility of these drivers in predicting transmission of the analogously transmitted, pathogenic agent, feline leukemia virus (FeLV), and compared FIV-based predictions of outbreak dynamics against empirical FeLV outbreak data. FIV transmission was primarily driven by panther age class and distances between panther home range centroids. FIV-based modeling predicted FeLV dynamics similarly to common modeling approaches, but with evidence that FIV-based predictions captured the spatial structuring of the observed FeLV outbreak. While FIV-based predictions of FeLV transmission performed only marginally better than standard approaches, our results highlight the value of proactively identifying drivers of transmission-even based on analogously-transmitted, apathogenic agents-in order to predict transmission of emerging infectious agents. The identification of underlying drivers of transmission, such as through our workflow here, therefore holds promise for improving predictions of pathogen transmission in novel host populations, and could provide new strategies for proactive pathogen management in human and animal systems.

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.

Endogenous Feline Leukemia Virus (FeLV) siRNA Transcription May Interfere with Exogenous FeLV Infection.

Endogenous retroviruses (ERVs) are increasingly recognized for biological impacts on host cell function and susceptibility to infectious agents, particularly in relation to interactions with exogenous retroviral progenitors (XRVs). ERVs can simultaneously promote and restrict XRV infections using mechanisms that are virus and host specific. The majority of endogenous-exogenous retroviral interactions have been evaluated in experimental mouse or chicken systems, which are limited in their ability to extend findings to naturally infected outbred animals. Feline leukemia virus (FeLV) has a relatively well-characterized endogenous retrovirus with a coexisting virulent exogenous counterpart and is endemic worldwide in domestic cats. We have previously documented an association between endogenous FeLV (enFeLV) long terminal repeat (LTR) copy number and abrogated exogenous FeLV in naturally infected cats and experimental infections in tissue culture. Analyses described here examine limited FeLV replication in experimentally infected peripheral blood mononuclear cells, which correlates with higher enFeLV transcripts in these cells compared to fibroblasts. We further examine NCBI Sequence Read Archive RNA transcripts to evaluate enFeLV transcripts and RNA interference (RNAi) precursors. We find that lymphoid-derived tissues, which are experimentally less permissive to exogenous FeLV infection, transcribe higher levels of enFeLV under basal conditions. Transcription of enFeLV-LTR segments is significantly greater than that of other enFeLV genes. We documented transcription of a 21-nucleotide (nt) microRNA (miRNA) just 3' to the enFeLV 5'-LTR in the feline miRNAome of all data sets evaluated (n = 27). Our findings point to important biological functions of enFeLV transcription linked to solo LTRs distributed within the domestic cat genome, with potential impacts on domestic cat exogenous FeLV susceptibility and pathogenesis. IMPORTANCE Endogenous retroviruses (ERVs) are increasingly implicated in host cellular processes and susceptibility to infectious agents, specifically regarding interactions with exogenous retroviral progenitors (XRVs). Exogenous feline leukemia virus (FeLV) and its endogenous counterpart (enFeLV) represent a well-characterized, naturally occurring XRV-ERV dyad. We have previously documented an abrogated FeLV infection in both naturally infected cats and experimental fibroblast infections that harbor higher enFeLV proviral loads. Using an in silico approach, we provide evidence of miRNA transcription that is produced in tissues that are most important for FeLV infection, replication, and transmission. Our findings point to important biological functions of enFeLV transcription linked to solo-LTRs distributed within the feline genome, with potential impacts on domestic cat exogenous FeLV susceptibility and pathogenesis. This body of work provides additional evidence of RNA interference (RNAi) as a mechanism of viral interference and is a demonstration of ERV exaptation by the host to defend against related XRVs.

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.

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.

Endogenous Retroviruses Drive Resistance and Promotion of Exogenous Retroviral Homologs.

Endogenous retroviruses (ERVs) serve as markers of ancient viral infections and provide invaluable insight into host and viral evolution. ERVs have been exapted to assist in performing basic biological functions, including placentation, immune modulation, and oncogenesis. A subset of ERVs share high nucleotide similarity to circulating horizontally transmitted exogenous retrovirus (XRV) progenitors. In these cases, ERV-XRV interactions have been documented and include (a) recombination to result in ERV-XRV chimeras, (b) ERV induction of immune self-tolerance to XRV antigens, (c) ERV antigen interference with XRV receptor binding, and (d) interactions resulting in both enhancement and restriction of XRV infections. Whereas the mechanisms governing recombination and immune self-tolerance have been partially determined, enhancement and restriction of XRV infection are virus specific and only partially understood. This review summarizes interactions between six unique ERV-XRV pairs, highlighting important ERV biological functions and potential evolutionary histories in vertebrate hosts.

Presence of Endogenous Viral Elements Negatively Correlates with Feline Leukemia Virus Susceptibility in Puma and Domestic Cat Cells.

While feline leukemia virus (FeLV) has been shown to infect felid species other than the endemic domestic cat host, differences in FeLV susceptibility among species has not been evaluated. Previous reports have noted a negative correlation between endogenous FeLV (enFeLV) copy number and exogenous FeLV (exFeLV) infection outcomes in domestic cats. Since felids outside the genus Felis do not harbor enFeLV genomes, we hypothesized absence of enFeLV results in more severe disease consequences in felid species lacking these genomic elements. We infected primary fibroblasts isolated from domestic cats (Felis catus) and pumas (Puma concolor) with FeLV and quantitated proviral and viral antigen loads. Domestic cat enFeLV env and long terminal repeat (LTR) copy numbers were determined for each individual and compared to FeLV viral outcomes. FeLV proviral and antigen levels were also measured in 6 naturally infected domestic cats and 11 naturally infected Florida panthers (P. concolor coryi). We demonstrated that puma fibroblasts are more permissive to FeLV than domestic cat cells, and domestic cat FeLV restriction was highly related to enFeLV-LTR copy number. Terminal tissues from FeLV-infected Florida panthers and domestic cats had similar exFeLV proviral copy numbers, but Florida panther tissues have higher FeLV antigen loads. Our work indicates that enFeLV-LTR elements negatively correlate with exogenous FeLV replication. Further, Puma concolor samples lacking enFeLV are more permissive to FeLV infection than domestic cat samples, suggesting that endogenization can play a beneficial role in mitigating exogenous retroviral infections. Conversely, presence of endogenous retroelements may relate to new host susceptibility during viral spillover events.IMPORTANCE Feline leukemia virus (FeLV) can infect a variety of felid species. Only the primary domestic cat host and related small cat species harbor a related endogenous virus in their genomes. Previous studies noted a negative association between the endogenous virus copy number and exogenous virus infection in domestic cats. This report shows that puma cells, which lack endogenous FeLV, produce more virus more rapidly than domestic cat fibroblasts following cell culture challenge. We document a strong association between domestic cat cell susceptibility and FeLV long terminal repeat (LTR) copy number, similar to observations in natural FeLV infections. Viral replication does not, however, correlate with FeLV env copy number, suggesting that this effect is specific to FeLV-LTR elements. This discovery indicates a protective capacity of the endogenous virus against the exogenous form, either via direct interference or indirectly via gene regulation, and may suggest evolutionary outcomes of retroviral endogenization.

A Model Course to Enhance Veterinary Student Exposure to Research.

Despite many career opportunities available to veterinarians in research related fields and requirements for training in research methodologies by the American Veterinary Medical Association Council on Education (AVMA COE), formal approaches to development of veterinary curriculum related to research topics have not been widely reported. Colorado State University (CSU) offers a one-credit course that introduces first-year veterinary students to skills and career opportunities in research. Here we provide information about the course structure and content, and report outcomes of survey data that assesses the impact of the course on student appreciation and understanding of the research process. We found that most United States (US) veterinary colleges do not offer a didactic course on the research process. Student opinions of veterinary researchers were generally high, though a proportion of students (30%-40%) would have preferred a practice management class to a course on research principles. Nearly 25% of students reported that they were significantly influenced to consider research careers after taking the course. We document that this one-credit seminar course improved veterinary student perceptions of their understanding of the research process and resulted in self-reported influence of career choice.

Teaching Wildlife Disease Outbreak Response Through a Collaborative One Health Workshop.

Issues in the fields of wildlife disease and One Health are often difficult to address by single research groups because of the many disciplines and areas of expertise required to effectively solve complex problems. Although collaborations are becoming increasingly prevalent in the professional realm, many undergraduate, graduate, and professional students are merely introduced to the idea of collaboration without fully understanding how team-based approaches function. In this report, we describe the framework for a one-day workshop hosted by the Colorado State University student chapter of the Wildlife Disease Association (CSU WDA), where we gathered students and professionals to collectively investigate a simulated wildlife disease outbreak. CSU WDA student members designed the workshop and recruited professionals who are experts in their respective fields to run an outbreak simulation during the event. Based on pre- and post-event evaluation responses, this workshop was effective in increasing participants' knowledge of disease ecology, pathology, genetics, and microbiology, as well as the importance of collaboration among disciplines as it pertains to wildlife disease outbreaks.

Multiple Introductions of Domestic Cat Feline Leukemia Virus in Endangered Florida Panthers.

The endangered Florida panther (Puma concolor coryi) had an outbreak of infection with feline leukemia virus (FeLV) in the early 2000s that resulted in the deaths of 3 animals. A vaccination campaign was instituted during 2003-2007 and no additional cases were recorded until 2010. During 2010-2016, six additional FeLV cases were documented. We characterized FeLV genomes isolated from Florida panthers from both outbreaks and compared them with full-length genomes of FeLVs isolated from contemporary Florida domestic cats. Phylogenetic analyses identified at least 2 circulating FeLV strains in panthers, which represent separate introductions from domestic cats. The original FeLV virus outbreak strain is either still circulating or another domestic cat transmission event has occurred with a closely related variant. We also report a case of a cross-species transmission event of an oncogenic FeLV recombinant (FeLV-B). Evidence of multiple FeLV strains and detection of FeLV-B indicate Florida panthers are at high risk for FeLV infection.

Feline Leukemia Virus (FeLV) Disease Outcomes in a Domestic Cat Breeding Colony: Relationship to Endogenous FeLV and Other Chronic Viral Infections.

Exogenous feline leukemia virus (FeLV) is a feline gammaretrovirus that results in a variety of disease outcomes. Endogenous FeLV (enFeLV) is a replication-defective provirus found in species belonging to the Felis genus, which includes the domestic cat (Felis catus). There have been few studies examining interaction between enFeLV genotype and FeLV progression. We examined point-in-time enFeLV and FeLV viral loads, as well as occurrence of FeLV/enFeLV recombinants (FeLV-B), to determine factors relating to clinical disease in a closed breeding colony of cats during a natural infection of FeLV. Coinfections with feline foamy virus (FFV), feline gammaherpesvirus 1 (FcaGHV-1), and feline coronavirus (FCoV) were also documented and analyzed for impact on cat health and FeLV disease. Correlation analysis and structural equation modeling techniques were used to measure interactions among disease parameters. Progressive FeLV disease and FeLV-B presence were associated with higher FeLV proviral and plasma viral loads. Female cats were more likely to have progressive disease and FeLV-B. Conversely, enFeLV copy number was higher in male cats and negatively associated with progressive FeLV disease. Males were more likely to have abortive FeLV disease. FFV proviral load was found to correlate positively with higher FeLV proviral and plasma viral load, detection of FeLV-B, and FCoV status. Male cats were much more likely to be infected with FcaGHV-1 than female cats. This analysis provides insights into the interplay between endogenous and exogenous FeLV during naturally occurring disease and reveals striking variation in the infection patterns among four chronic viral infections of domestic cats.IMPORTANCE Endogenous retroviruses are harbored by many animals, and their interactions with exogenous retroviral infections have not been widely studied. Feline leukemia virus (FeLV) is a relevant model system to examine this question, as endogenous and exogenous forms of the virus exist. In this analysis of a large domestic cat breeding colony naturally infected with FeLV, we documented that enFeLV copy number was higher in males and inversely related to FeLV viral load and associated with better FeLV disease outcomes. Females had lower enFeLV copy numbers and were more likely to have progressive FeLV disease and FeLV-B subtypes. FFV viral load was correlated with FeLV progression. FFV, FcaGHV-1, and FeLV displayed markedly different patterns of infection with respect to host demographics. This investigation revealed complex coinfection outcomes and viral ecology of chronic infections in a closed population.

A Retrospective Examination of Feline Leukemia Subgroup Characterization: Viral Interference Assays to Deep Sequencing.

Feline leukemia virus (FeLV) was the first feline retrovirus discovered, and is associated with multiple fatal disease syndromes in cats, including lymphoma. The original research conducted on FeLV employed classical virological techniques. As methods have evolved to allow FeLV genetic characterization, investigators have continued to unravel the molecular pathology associated with this fascinating agent. In this review, we discuss how FeLV classification, transmission, and disease-inducing potential have been defined sequentially by viral interference assays, Sanger sequencing, PCR, and next-generation sequencing. In particular, we highlight the influences of endogenous FeLV and host genetics that represent FeLV research opportunities on the near horizon.