cFeline leukemia virus (FeLV) subgroups have emerged in infected cats via the mutation or recombination of the env gene of subgroup A FeLV (FeLV-A), the primary virus. We report the isolation and characterization of a novel env gene, TG35-2, and report that the TG35-2 pseudotype can be categorized as a novel FeLV subgroup. The TG35-2 envelope protein displays strong sequence identity to FeLV-A Env, suggesting that selection pressure in cats causes novel FeLV subgroups to emerge. Feline leukemia viruses (FeLVs) are pathogenic retroviruses of domestic cats (1, 2), which are classified into subgroups A (the parent virus), B, C, D, and T based on their interference and in vitro host range properties (3,4,5,6,7,8). Subgroups B and D arose from the recombination of FeLV-A env and the env genes of endogenous FeLV or endogenous retroviruses in the genomes of domestic cats (ERV-DCs) (7, 9, 10). Subgroups C and T possibly arose from mutations in FeLV-A env (11,12). The recombination or mutation of env often alters the interference and host ranges of FeLVs by affecting their receptor usage (5,6,13,14,15,16).FeLV env genes were isolated by PCR from the blood DNA of a 1-year-old castrated male cat, TG35, with a bite injury, stomatitis, loss of appetite, and FeLV infection, although he had been vaccinated with inactivated FeLV (genotype III) (16). Five clones (TG35-1 to -5) were isolated, and we focused on TG35-2, TG35-4, and TG35-5. The env sequences of these clones showed strong similarity ( Fig. 1), and the viruses clustered phylogenetically with those of genotype I/clade I FeLV, found mainly in Japan (16). The encompassing variable region A (VRA) of TG35-2 Env differs at eight amino acids from those of the TG35-4 and TG35-5 Env proteins. The proline-rich regions of TG35-2 and TG35-4, but not TG35-5, contain an inserted sequence of 25 amino acids (Fig. 1) not found in the cat genome database and of unknown origin.To identify the FeLV subgroup to which this viral strain belongs, we used an interference assay (16) and generated ␤-galactosidase (LacZ)-encoding pseudotype viruses expressing TG35-2, TG35-4, or TG35-5 envelope (Env) proteins in GPLac cells (7). Pseudotype viruses TG35-2, -4, and -5 infected uninfected HEK293T cells (Table 1). However, HEK293T cells preinfected with FeLV-A/clone 33 (293T/clone 33 cells) (17) or FeLV-A/Glasgow-1 (293T/Glasgow-1 cells) (9) were infected by pseudotype virus TG35-2, but not by TG35-4 or TG35-5. Neither cell type was infected by FeLV-A/clone 33 or FeLV-A/Glasgow-1. Therefore, only the TG35-4 and TG35-5 viruses interfered with FeLV-A. Neither the TG35-2, TG35-4, nor TG35-5 pseudotype interfered with other subgroups of FeLV, or with retroviruses such as ERV-DC10, a replication-competent feline ERV (7) ( Table 1). Therefore, FeLV TG35-4 and TG35-5 belong to the FeLV-A subgroup. However, TG35-2 could not be categorized.We next constructed a replication-competent virus (33TGE2) containing the TG35-2 env gene and the LTR, gag, and pol genes of When we examined AH927 feline cells, the TG35-2...

Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections of germ cells. Previous work identified one of the youngest feline ERV groups, ERV-DC, and reported that two ERV-DC loci, ERV-DC10 and ERV-DC18 (ERV-DC10/DC18), can replicate in cultured cells. Here, we identified another replication-competent provirus, ERV-DC14, on chromosome C1q32. ERV-DC14 differs from ERV-DC10/DC18 in its phylogeny, receptor usage, and, most notably, transcriptional activities; although ERV-DC14 can replicate in cultured cells, it cannot establish a persistent infection owing to its low transcriptional activity. Furthermore, we examined ERV-DC transcription and its regulation in feline tissues. Quantitative reverse transcription-PCR (RT-PCR) detected extremely low ERV-DC10 expression levels in feline tissues, and bisulfite sequencing showed that 5= long terminal repeats (LTRs) of ERV-DC10/DC18 are significantly hypermethylated in feline blood cells. Reporter assays found that the 5=-LTR promoter activities of ERV-DC10/DC18 are high, whereas that of ERV-DC14 is low. This difference in promoter activity is due to a single substitution from A to T in the LTR, and reverse mutation at this nucleotide in ERV-DC14 enhanced its replication and enabled it to persistently infect cultured cells. Therefore, ERV-DC LTRs can be divided into two types based on this nucleotide, the A type or T type, which have strong or attenuated promoter activity, respectively. Notably, ERV-DCs with T-type LTRs, such as ERV-DC14, have expanded in the cat genome significantly more than A-type ERV-DCs, despite their low promoter activities. Our results provide insights into how the host controls potentially infectious ERVs and, conversely, how ERVs adapt to and invade the host genome. IMPORTANCEThe domestic cat genome contains many endogenous retroviruses, including ERV-DCs. These ERV-DCs have been acquired through germ cell infections with exogenous retroviruses. Some of these ERV-DCs are still capable of producing infectious virions. Hosts must tightly control these ERVs because replication-competent viruses in the genome pose a risk to the host. Here, we investigated how ERV-DCs are adapted by their hosts. Replication-competent viruses with strong promoter activity, such as ERV-DC10 and ERV-DC18, were suppressed by promoter methylation in LTRs. On the other hand, replication-competent viruses with weak promoter activity, such as ERV-DC14, seemed to escape strict control via promoter methylation by the host. Interestingly, ERV-DCs with weak promoter activity, such as ERV-DC14, have expanded in the cat genome significantly more than ERV-DCs with strong promoter activity. Our results improve the understanding of the host-virus conflict and how ERVs adapt in their hosts over time. Endogenous retroviruses (ERVs) are resident DNA copies that abound in host chromosomal DNA and compromise ϳ8 to 10% of human and mouse genomes (1, 2). They have been found in all vertebrates, including mammals, fish, birds, reptiles, and amphibians (3). ERVs we...

Felis catus gammaherpesvirus 1 (FcaGHV1) is a widely endemic infection of domestic cats. Current epidemiological data identify domestic cats as the sole natural host for FcaGHV1. The Tsushima leopard cat (TLC; Prionailurus bengalensis euptilurus) is a critically endangered species that lives only on Tsushima Island, Nagasaki, Japan. Nested PCR was used to test the blood or spleen of 89 TLCs for FcaGHV1 DNA; three (3.37%; 95% CI, 0.70–9.54) were positive. For TLC management purposes, we also screened domestic cats and the virus was detected in 13.02% (95% CI, 8.83–18.27) of 215 cats. Regarding phylogeny, the partial sequences of FcaGHV1 from domestic cats and TLCs formed one cluster, indicating that similar strains circulate in both populations. In domestic cats, we found no significant difference in FcaGHV1 detection in feline immunodeficiency virus-infected (p = 0.080) or feline leukemia virus-infected (p = 0.163) cats, but males were significantly more likely to be FcaGHV1 positive (odds ratio, 5.86; 95% CI, 2.27–15.14) than females. The higher frequency of FcaGHV1 detection in domestic cats than TLCs, and the location of the viral DNA sequences from both cats within the same genetic cluster suggests that virus transmission from domestic cats to TLCs is likely.

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.