Two Lineages of Papillomaviruses Identified from Caracals (Caracal caracal) in South Africa.

Papillomaviruses (PV) infect epithelial cells and can cause hyperplastic or neoplastic lesions. In felids, most described PVs are from domestic cats (Felis catus; n = 7 types), with one type identified in each of the five wild felid species studied to date (Panthera uncia, Puma concolor, Leopardus wiedii, Panthera leo persica and Lynx rufus). PVs from domestic cats are highly diverse and are currently classified into three genera (Lambdapapillomavirus, Dyothetapapillomavirus, and Taupapillomavirus), whereas those from wild felids, although diverse, are all classified into the Lambdapapillomavirus genus. In this study, we used a metagenomic approach to identify ten novel PV genomes from rectal swabs of five deceased caracals (Caracal caracal) living in the greater Cape Town area, South Africa. These are the first PVs to be described from caracals, and represent six new PV types, i.e., Caracal caracal papillomavirus (CcarPV) 1-6. These CcarPV fall into two phylogenetically distinct genera: Lambdapapillomavirus, and Treisetapapillomavirus. Two or more PV types were identified in a single individual for three of the five caracals, and four caracals shared at least one of the same PV types with another caracal. This study broadens our understanding of wild felid PVs and provides evidence that there may be several wild felid PV lineages.

Leopardus wiedii Papillomavirus type 1, a novel papillomavirus species in the tree ocelot, suggests Felidae Lambdapapillomavirus polyphyletic origin and host-independent evolution.

The limited knowledge on Papillomavirus diversity (particularly in wild animal species) influences the accuracy of PVs phylogeny and their evolutionary history, and hinders the comprehension of PVs pathogenicity, especially the mechanism of virus - related cancer progression. This study reports the identification of Leopardus wiedii Papillomavirus type 1 (LwiePV1), the first PV type within Lambdapapillomavirus in a Leopardus host. LwiePV1 full genome sequencing allowed the investigation of its taxonomic position and phylogeny. Based on results, LwiePV1 should be assigned to a novel PV species providing evidence for a polyphyletic origin of feline lambda PVs, and representing an exception to codivergence between feline lambda PVs and their hosts. Results improve our knowledge on PV diversity and pave the way to future studies investigating biological and evolutionary features of animal PVs.

Epidemiologic survey of feline leukemia virus in domestic cats on Tsushima Island, Japan: management strategy for Tsushima leopard cats.

The Tsushima leopard cat (TLC) Prionailurus bengalensis euptilurus, a subspecies of P. bengalensis, is designated a National Natural Monument of Japan, and lives only on Tsushima Island, Nagasaki Prefecture, Japan. TLCs are threatened by various infectious diseases. Feline leukemia virus (FeLV) causes a serious infectious disease with a poor prognosis in cats. Therefore, the transmission of FeLV from Tsushima domestic cats (TDCs) to TLCs may threaten the TLC population. We investigated the FeLV infection status of both TDCs and TLCs on Tsushima Island by screening blood samples for FeLV p27 antigen and using PCR to amplify the full-length FeLV env gene. The prevalence of FeLV was 6.4% in TDCs and 0% in TLCs. We also demonstrated that the virus can replicate in the cells of TLCs, suggesting its potential cross-species transmission. The viruses in TDCs were classified as genotype I/clade 3, which is prevalent on a nearby island, based on previous studies of FeLV genotypes and FeLV epidemiology. The FeLV viruses identified on Tsushima Island can be further divided into 2 lineages within genotype I/clade 3, which are geographically separated in Kamijima and Shimojima, indicating that FeLV may have been transmitted to Tsushima Island at least twice. Monitoring FeLV infection in the TDC and TLC populations is highly recommended as part of the TLC surveillance and management strategy.

Molecular detection of viral agents in free-ranging and captive neotropical felids in Brazil.

We describe molecular testing for felid alphaherpesvirus 1 (FHV-1), carnivore protoparvovirus 1 (CPPV-1), feline calicivirus (FCV), alphacoronavirus 1 (feline coronavirus [FCoV]), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and canine distemper virus (CDV) in whole blood samples of 109 free-ranging and 68 captive neotropical felids from Brazil. Samples from 2 jaguars ( Panthera onca) and 1 oncilla ( Leopardus tigrinus) were positive for FHV-1; 2 jaguars, 1 puma ( Puma concolor), and 1 jaguarundi ( Herpairulus yagouaroundi) tested positive for CPPV-1; and 1 puma was positive for FIV. Based on comparison of 103 nucleotides of the UL24-UL25 gene, the FHV-1 sequences were 99-100% similar to the FHV-1 strain of domestic cats. Nucleotide sequences of CPPV-1 were closely related to sequences detected in other wild carnivores, comparing 294 nucleotides of the VP1 gene. The FIV nucleotide sequence detected in the free-ranging puma, based on comparison of 444 nucleotides of the pol gene, grouped with other lentiviruses described in pumas, and had 82.4% identity with a free-ranging puma from Yellowstone Park and 79.5% with a captive puma from Brazil. Our data document the circulation of FHV-1, CPPV-1, and FIV in neotropical felids in Brazil.

Identification and characterization of diverse groups of endogenous retroviruses in felids.

BACKGROUND: Endogenous retroviruses (ERVs) are genetic elements with a retroviral origin that are integrated into vertebrate genomes. In felids (Mammalia, Carnivora, Felidae), ERVs have been described mostly in the domestic cat, and only rarely in wild species. To gain insight into the origins and evolutionary dynamics of endogenous retroviruses in felids, we have identified and characterized partial pro/pol ERV sequences from eight Neotropical wild cat species, belonging to three distinct lineages of Felidae. We also compared them with publicly available genomic sequences of Felis catus and Panthera tigris, as well as with representatives of other vertebrate groups, and performed phylogenetic and molecular dating analyses to investigate the pattern and timing of diversification of these retroviral elements. RESULTS: We identified a high diversity of ERVs in the sampled felids, with a predominance of Gammaretrovirus-related sequences, including class I ERVs. Our data indicate that the identified ERVs arose from at least eleven horizontal interordinal transmissions from other mammals. Furthermore, we estimated that the majority of the Gamma-like integrations took place during the diversification of modern felids. Finally, our phylogenetic analyses indicate the presence of a genetically divergent group of sequences whose position in our phylogenetic tree was difficult to establish confidently relative to known retroviruses, and another lineage identified as ERVs belonging to class II. CONCLUSIONS: Retroviruses have circulated in felids along with their evolution. The majority of the deep clades of ERVs exist since the primary divergence of felids' base and cluster with retroviruses of divergent mammalian lineages, suggesting horizontal interordinal transmission. Our findings highlight the importance of additional studies on the role of ERVs in the genome landscaping of other carnivore species.

Feline immunodeficiency virus and feline leukemia virus infection in free-ranging guignas (Leopardus guigna) and sympatric domestic cats in human perturbed landscapes on Chiloé Island, Chile.

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are two of the most common viruses affecting domestic cats (Felis catus). During the last two decades, reports show that both viruses also infect or affect other species of the family Felidae. Human landscape perturbation is one of the main causes of emerging diseases in wild animals, facilitating contact and transmission of pathogens between domestic and wild animals. We investigated FIV and FeLV infection in free-ranging guignas (Leopardus guigna) and sympatric domestic cats in human perturbed landscapes on Chiloé Island, Chile. Samples from 78 domestic cats and 15 guignas were collected from 2008 to 2010 and analyzed by PCR amplification and sequencing. Two guignas and two domestic cats were positive for FIV; three guignas and 26 domestic cats were positive for FeLV. The high percentage of nucleotide identity of FIV and FeLV sequences from both species suggests possible interspecies transmission of viruses, facilitated by increased contact probability through human invasion into natural habitats, fragmentation of guigna habitat, and poultry attacks by guignas. This study enhances our knowledge on the transmission of pathogens from domestic to wild animals in the global scenario of human landscape perturbation and emerging diseases.

Surveillance using serological and molecular methods for the detection of infectious agents in captive Brazilian neotropic and exotic felids.

The aim of the current study was to investigate the exposure of captive wild felids to various infectious pathogens using serological and molecular methods. One hundred and fifty-nine neotropic felids and 51 exotic felids from 28 captive settings in Brazil were tested. While antibodies against Feline parvovirus and Feline coronavirus (FCoV), Feline calicivirus and Bartonella spp. were frequently detected by serologic tests, antibodies against Felid herpesvirus 1 or infection with hemotropic mycoplasmas were less prevalent. Serologic evidence of exposure to Ehrlichia spp., Feline immunodeficiency virus, and Feline leukemia virus (FeLV) was detected rarely, and infections with FeLV, Ehrlichia spp., and Cytauxzoon spp. were found infrequently. The detected Bartonella sequence was molecularly similar to B. koehlerae and B. henselae; for Cytauxzoon, the sequence resembled those from domestic cats. No Anaplasma phagocytophilum and Theileria spp. infections were detected. The positive test results varied significantly among different facilities and species. Additionally, FCoV seropositivity was more prevalent in captivity than in free-ranging populations. Results suggest that testing is appropriate prior to relocation of felids.

Quantification and molecular characterization of the feline leukemia virus A receptor.

Virus receptors and their expression patterns on the cell surface determine the cell tropism of the virus, host susceptibility and the pathogenesis of the infection. Feline thiamine transport protein 1 (fTHTR1) has been identified as the receptor for feline leukemia virus (FeLV) A. The goal of the present study was to develop a quantitative, TaqMan real-time PCR assay to investigate fTHTR1 mRNA expression in tissues of uninfected and FeLV-infected cats, cats of different ages, in tumor tissues and leukocyte subsets. Moreover, the receptor was molecularly characterized in different feline species. fTHTR1 mRNA expression was detected in all 30 feline tissues investigated, oral mucosa scrapings and blood. Importantly, identification of significant differences in fTHTR1 expression relied on normalization with an appropriate reference gene. The lowest levels were found in the blood, whereas high levels were measured in the oral mucosa, salivary glands and the musculature. In the blood, T lymphocytes showed significantly higher fTHTR1 mRNA expression levels than neutrophil granulocytes. In vitro activation of peripheral blood mononuclear cells with concanavalin A alone or followed by interleukin-2 led to a transient increase of fTHTR1 mRNA expression. In the blood, but not in the examined tissues, FeLV-infected cats tended to have lower fTHTR1 mRNA levels than uninfected cats. The fTHTR1 mRNA levels were not significantly different between tissues with lymphomas and the corresponding non-neoplastic tissues. fTHTR1 was highly conserved among different feline species (Iberian lynx, Asiatic and Indian lion, European wildcat, jaguarundi, domestic cat). In conclusion, while ubiquitous fTHTR1 mRNA expression corresponded to the broad target tissue range of FeLV, particularly high fTHTR1 levels were found at sites of virus entry and shedding. The differential susceptibility of different species to FeLV could not be attributed to variations in the fTHTR1 sequence.

Development of a PCR assay to detect papillomavirus infection in the snow leopard.

BACKGROUND: Papillomaviruses (PVs) are a group of small, non-encapsulated, species-specific DNA viruses that have been detected in a variety of mammalian and avian species including humans, canines and felines. PVs cause lesions in the skin and mucous membranes of the host and after persistent infection, a subset of PVs can cause tumors such as cervical malignancies and head and neck squamous cell carcinoma in humans. PVs from several species have been isolated and their genomes have been sequenced, thereby increasing our understanding of the mechanism of viral oncogenesis and allowing for the development of molecular assays for the detection of PV infection. In humans, molecular testing for PV DNA is used to identify patients with persistent infections at risk for developing cervical cancer. In felids, PVs have been isolated and sequenced from oral papillomatous lesions of several wild species including bobcats, Asian lions and snow leopards. Since a number of wild felids are endangered, PV associated disease is a concern and there is a need for molecular tools that can be used to further study papillomavirus in these species. RESULTS: We used the sequence of the snow leopard papillomavirus UuPV1 to develop a PCR strategy to amplify viral DNA from samples obtained from captive animals. We designed primer pairs that flank the E6 and E7 viral oncogenes and amplify two DNA fragments encompassing these genes. We detected viral DNA for E6 and E7 in genomic DNA isolated from saliva, but not in paired blood samples from snow leopards. We verified the identity of these PCR products by restriction digest and DNA sequencing. The sequences of the PCR products were 100% identical to the published UuPV1 genome sequence. CONCLUSIONS: We developed a PCR assay to detect papillomavirus in snow leopards and amplified viral DNA encompassing the E6 and E7 oncogenes specifically in the saliva of animals. This assay could be utilized for the molecular investigation of papillomavirus in snow leopards using saliva, thereby allowing the detection of the virus in the anatomical site where oral papillomatous lesions develop during later stages of infection and disease development.

Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

Ocelots on Barro Colorado Island are infected with feline immunodeficiency virus but not other common feline and canine viruses.

Transmission of pathogens from domestic animals to wildlife populations (spill-over) has precipitated local wildlife extinctions in multiple geographic locations. Identifying such events before they cause population declines requires differentiating spillover from endemic disease, a challenge complicated by a lack of baseline data from wildlife populations that are isolated from domestic animals. We tested sera collected from 12 ocelots (Leopardus pardalis) native to Barro Colorado Island, Panama, which is free of domestic animals, for antibodies to feline herpes virus, feline calicivirus, feline corona virus, feline panleukopenia virus, canine distemper virus, and feline immunodeficiency virus (FIV), typically a species-specific infection. Samples also were tested for feline leukemia virus antigens. Positive tests results were only observed for FIV; 50% of the ocelots were positive. We hypothesize that isolation of this population has prevented introduction of pathogens typically attributed to contact with domestic animals. The high density of ocelots on Barro Colorado Island may contribute to a high prevalence of FIV infection, as would be expected with increased contact rates among conspecifics in a geographically restricted population.

Ancient papillomavirus-host co-speciation in Felidae.

BACKGROUND: Estimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1. RESULTS: The feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 x 10(-8) (95% confidence interval 1.32 x 10(-8) to 2.47 x 10(-8)) nucleotide substitutions per site per year for the viral coding genome. CONCLUSION: Our work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence.