Justin S. Lee

Gene flow and pathogen transmission among bobcats (Lynx rufus) in a fragmented urban landscape.

Urbanization can result in the fragmentation of once contiguous natural landscapes into a patchy habitat interspersed within a growing urban matrix. Animals living in fragmented landscapes often have reduced movement among habitat patches because of avoidance of intervening human development, which potentially leads to both reduced gene flow and pathogen transmission between patches. Mammalian carnivores with large home ranges, such as bobcats (Lynx rufus), may be particularly sensitive to habitat fragmentation. We performed genetic analyses on bobcats and their directly transmitted viral pathogen, feline immunodeficiency virus (FIV), to investigate the effects of urbanization on bobcat movement. We predicted that urban development, including major freeways, would limit bobcat movement and result in genetically structured host and pathogen populations. We analysed molecular markers from 106 bobcats and 19 FIV isolates from seropositive animals in urban southern California. Our findings indicate that reduced gene flow between two primary habitat patches has resulted in genetically distinct bobcat subpopulations separated by urban development including a major highway. However, the distribution of genetic diversity among FIV isolates determined through phylogenetic analyses indicates that pathogen genotypes are less spatially structured—exhibiting a more even distribution between habitat fragments. We conclude that the types of movement and contact sufficient for disease transmission occur with enough frequency to preclude structuring among the viral population, but that the bobcat population is structured owing to low levels of effective bobcat migration resulting in gene flow. We illustrate the utility in using multiple molecular markers that differentially detect movement and gene flow between subpopulations when assessing connectivity.

Novel gammaherpesviruses in North American domestic cats, bobcats and pumas: identification, prevalence and risk factors

ABSTRACT Gammaherpesviruses (GHVs) are a diverse and rapidly expanding group of viruses associated with a variety of disease conditions in humans and animals. To identify felid GHVs, we screened domestic cat (Felis catus), bobcat (Lynx rufus), and puma (Puma concolor) blood cell DNA samples from California, Colorado, and Florida using a degenerate pan-GHV PCR. Additional pan-GHV and long-distance PCRs were used to sequence a contiguous 3.4-kb region of each putative virus species, including partial glycoprotein B and DNA polymerase genes. We identified three novel GHVs, each present predominantly in one felid species: Felis catus GHV 1 (FcaGHV1) in domestic cats, Lynx rufus GHV 1 (LruGHV1) in bobcats, and Puma concolor GHV 1 (PcoGHV1) in pumas. To estimate infection prevalence, we developed real-time quantitative PCR assays for each virus and screened additional DNA samples from all three species (n = 282). FcaGHV1 was detected in 16% of domestic cats across all study sites. LruGHV1 was detected in 47% of bobcats and 13% of pumas across all study sites, suggesting relatively common interspecific transmission. PcoGHV1 was detected in 6% of pumas, all from a specific region of Southern California. The risk of infection for each host varied with geographic location. Age was a positive risk factor for bobcat LruGHV1 infection, and age and being male were risk factors for domestic cat FcaGHV1 infection. Further characterization of these viruses may have significant health implications for domestic cats and may aid studies of free-ranging felid ecology. IMPORTANCE Gammaherpesviruses (GHVs) establish lifelong infection in many animal species and can cause cancer and other diseases in humans and animals. In this study, we identified the DNA sequences of three GHVs present in the blood of domestic cats (Felis catus), bobcats (Lynx rufus), and pumas (Puma concolor; also known as mountain lions, cougars, and panthers). We found that these viruses were closely related to, but distinct from, other known GHVs of animals and represent the first GHVs identified to be native to these feline species. We developed techniques to rapidly and specifically detect the DNA of these viruses in feline blood and found that the domestic cat and bobcat viruses were widespread across the United States. In contrast, puma virus was found only in a specific region of Southern California. Surprisingly, the bobcat virus was also detected in some pumas, suggesting relatively common virus transmission between these species. Adult domestic cats and bobcats were at greater risk for infection than juveniles. Male domestic cats were at greater risk for infection than females. This study identifies three new viruses that are widespread in three feline species, indicates risk factors for infection that may relate to the route of infection, and demonstrates cross-species transmission between bobcats and pumas. These newly identified viruses may have important effects on feline health and ecology.

Evolution of Puma Lentivirus in Bobcats (Lynx rufus) and Mountain Lions (Puma concolor) in North America

ABSTRACT Mountain lions (Puma concolor) throughout North and South America are infected with puma lentivirus clade B (PLVB). A second, highly divergent lentiviral clade, PLVA, infects mountain lions in southern California and Florida. Bobcats (Lynx rufus) in these two geographic regions are also infected with PLVA, and to date, this is the only strain of lentivirus identified in bobcats. We sequenced full-length PLV genomes in order to characterize the molecular evolution of PLV in bobcats and mountain lions. Low sequence homology (88% average pairwise identity) and frequent recombination (1 recombination breakpoint per 3 isolates analyzed) were observed in both clades. Viral proteins have markedly different patterns of evolution; sequence homology and negative selection were highest in Gag and Pol and lowest in Vif and Env. A total of 1.7% of sites across the PLV genome evolve under positive selection, indicating that host-imposed selection pressure is an important force shaping PLV evolution. PLVA strains are highly spatially structured, reflecting the population dynamics of their primary host, the bobcat. In contrast, the phylogeography of PLVB reflects the highly mobile mountain lion, with diverse PLVB isolates cocirculating in some areas and genetically related viruses being present in populations separated by thousands of kilometers. We conclude that PLVA and PLVB are two different viral species with distinct feline hosts and evolutionary histories. IMPORTANCE An understanding of viral evolution in natural host populations is a fundamental goal of virology, molecular biology, and disease ecology. Here we provide a detailed analysis of puma lentivirus (PLV) evolution in two natural carnivore hosts, the bobcat and mountain lion. Our results illustrate that PLV evolution is a dynamic process that results from high rates of viral mutation/recombination and host-imposed selection pressure.

Characterization of Regionally Associated Feline Immunodeficiency Virus (FIV) in Bobcats (Lynx rufus)

Feline immunodeficiency virus (FIV) classically infects felid species with highly divergent species-specific FIVs. However, recent studies have detected an FIV strain infecting both bobcats (Lynx rufus) and pumas (Puma concolor) in California and Florida. To further investigate this observation, we evaluated FIV from bobcats in Florida (n=25) and Colorado (n=80) between 2008 and 2011. Partial viral sequences from five Florida bobcats cluster with previously published sequences from Florida panthers. We did not detect FIV in Colorado bobcats.

Feline immunodeficiency virus cross-species transmission: Implications for emergence of new lentiviral infections.

ABSTRACT Owing to a complex history of host-parasite coevolution, lentiviruses exhibit a high degree of species specificity. Given the well-documented viral archeology of human immunodeficiency virus (HIV) emergence following human exposures to simian immunodeficiency virus (SIV), an understanding of processes that promote successful cross-species lentiviral transmissions is highly relevant. We previously reported natural cross-species transmission of a subtype of feline immunodeficiency virus, puma lentivirus A (PLVA), between bobcats (Lynx rufus) and mountain lions (Puma concolor) for a small number of animals in California and Florida. In this study, we investigate host-specific selection pressures, within-host viral fitness, and inter- versus intraspecies transmission patterns among a larger collection of PLV isolates from free-ranging bobcats and mountain lions. Analyses of proviral and viral RNA levels demonstrate that PLVA fitness is severely restricted in mountain lions compared to that in bobcats. We document evidence of diversifying selection in three of six PLVA genomes from mountain lions, but we did not detect selection among 20 PLVA isolates from bobcats. These findings support the hypothesis that PLVA is a bobcat-adapted virus which is less fit in mountain lions and under intense selection pressure in the novel host. Ancestral reconstruction of transmission events reveals that intraspecific PLVA transmission has occurred among panthers (Puma concolor coryi) in Florida following the initial cross-species infection from bobcats. In contrast, interspecific transmission from bobcats to mountain lions predominates in California. These findings document outcomes of cross-species lentiviral transmission events among felids that compare to the emergence of HIV from nonhuman primates. IMPORTANCE Cross-species transmission episodes can be singular, dead-end events or can result in viral replication and spread in the new species. The factors that determine which outcome will occur are complex, and the risk of new virus emergence is therefore difficult to predict. We used molecular techniques to evaluate the transmission, fitness, and adaptation of puma lentivirus A (PLVA) between bobcats and mountain lions in two geographic regions. Our findings illustrate that mountain lion exposure to PLVA is relatively common but does not routinely result in communicable infections in the new host. This is attributed to efficient species barriers that largely prevent lentiviral adaptation. However, the evolutionary capacity for lentiviruses to adapt to novel environments may ultimately overcome host restriction mechanisms over time and under certain ecological circumstances. This phenomenon provides a unique opportunity to examine cross-species transmission events leading to new lentiviral emergence.

Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species

ABSTRACT Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.

Urban Landscapes can change virus gene flow and evolution in a fragmentation-sensitive carnivore

Urban expansion has widespread impacts on wildlife species globally, including the transmission and emergence of infectious diseases. However, there is almost no information about how urban landscapes shape transmission dynamics in wildlife. Using an innovative phylodynamic approach combining host and pathogen molecular data with landscape characteristics and host traits, we untangle the complex factors that drive transmission networks of feline immunodeficiency virus (FIV) in bobcats (Lynx rufus). We found that the urban landscape played a significant role in shaping FIV transmission. Even though bobcats were often trapped within the urban matrix, FIV transmission events were more likely to occur in areas with more natural habitat elements. Urban fragmentation also resulted in lower rates of pathogen evolution, possibly owing to a narrower range of host genotypes in the fragmented area. Combined, our findings show that urban landscapes can have impacts on a pathogen and its evolution in a carnivore living in one of the most fragmented and urban systems in North America. The analytical approach used here can be broadly applied to other host–pathogen systems, including humans.

Complete Genome Sequences of Two Novel Puma concolor Foamy Viruses from California

ABSTRACT We report two complete foamy retrovirus (FV) genomes isolated from Puma concolor, a large cat native to the Americas. Due to high overall genetic relatedness to known feline foamy viruses (FFVs), we propose the name Puma concolor FFV (FFVPc). The data confirm that felines are infected with distinct but closely related FVs.

A review of potential bluetongue virus vaccine strategies

Bluetongue (BT) is an economically important, non-zoonotic arboviral disease of certain wild and domestic species of cloven-hooved ungulates. Bluetongue virus (BTV) is the causative agent and the occurrence of BTV infection is distinctly seasonal in temperate regions of the world, and dependent on the presence of vector biting midges (e.g. Culicoides sonorensis in much of North America). In recent years, severe outbreaks have occurred throughout Europe and BTV is endemic in most tropical and temperate regions of the world. Several vaccines have been licensed for commercial use, including modified live (live-attenuated) and inactivated products, and this review summarizes recent strategies developed for BTV vaccines with emphasis on technologies suitable for differentiating naturally infected from vaccinated animals. The goal of this review is to evaluate realistic vaccine strategies that might be utilized to control or prevent future outbreaks of BT.

First Complete Genome Sequence of Felis catus Gammaherpesvirus 1.

ABSTRACT We sequenced the complete genome of Felis catus gammaherpesvirus 1 (FcaGHV1) from lymph node DNA of an infected cat. The genome includes a 121,556-nucleotide unique region with 87 predicted open reading frames (61 gammaherpesvirus conserved and 26 unique) flanked by multiple copies of a 966-nucleotide terminal repeat.