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

Abstract

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.

Keywords: feline leukemia virus; genetic recombination; retroviruses.

FIG 1

FeLV-B phylogeny reveals a high degree of genetic dissimilarity within the closed cat colony and supports de novo recombination as a primary source of FeLV-B emergence. (A to D) Four potential scenarios for FeLV-B emergence were considered, which could be distinguished by phylogenetic analysis. All members of the Felis genus harbor enFeLV (yellow circles as shown for each scenario). (A) No recombination. Active FeLV-A infection (red circles) does not develop into FeLV-B, despite the cat harboring enFeLV (yellow circles). Ten of 32 cats evaluated were in this category. (B) A cat has an active FeLV-A infection (red circles) that recombines with enFeLV (yellow circles) to develop an FeLV-B (orange) infection. If all FeLV-B infections developed de novo and horizontal transmission was not possible or rare, genomic analysis would cluster viral sequences from the same individual together or show recombination events that are not identical between individuals. (C) A cat horizontally transmits both FeLV-A (red circles) and FeLV-B (orange circles) to another cat via social contact. In this case, phylogenies indicate branches with shared FeLV-B ancestry between infected individuals. (D) FeLV-B infection (orange circles) emerges via both de novo recombination and horizontal transmission. Resulting phylogeny would demonstrate both independent FeLV-B branches related to each cat as well as mixed branches indicating FeLV-B infections shared between individuals. It is important to note that different FeLV-B recombination break points that occur within an individual could be shared across individuals and would generate a phylogenic structure similar to that of direct transmission of FeLV-B and could confound interpretations. (E) Neighbor-joining phylogenetic tree illustrates the relationship between FeLV sequences (FeLV-A and -B and enFeLV) recovered in this study (red) and sequences reported previously (black). Two primary clades and several minor clades were identified. Several FeLV-B clades are seen here, which are indicative of the differing recombination patterns between FeLV-A and enFeLV. This results in FeLV-B isolates that are more closely related to FeLV-A, isolates that are more enFeLV-like, or variants that are intermediate between these two identities. Our data suggest scenario D, although the majority of FeLV-B variants arise de novo.

FIG 2

Several recombination sites in env identified among FeLV-A, enFeLV, and FeLV-B isolates. The schematic illustrates recombination events in FeLV-B inferred from parental FeLV-A and enFeLV sequences. The majority of sites were concentrated in the 5′ end of gp70. Recombination sites for each event are represented by arrows of different colors. Information below schematic the shows recombination event information, including event, cat ID harboring recombinant sequence, cat ID harboring parental sequence, detection methods, with best method (boldface), and the corresponding highest E value. For cat IDs, see Table 1.

FIG 3

Comparison of FeLV-A and FeLV-B sequences from individual cats illustrates both de novo recombination and horizontal transmission. Neighbor-joining phylogeny on the left illustrates FeLV-A env recovered from a single individual. (Right) The tanglegram illustrates the relationship with FeLV-B env recovered from the same individual. Colored lines highlight env of two subtypes recovered from the same individual. This figure demonstrates that env variants from an individual are usually closely related; however, in half the cats with multiple variants detected, FeLV-B variants were distinct enough to group into separate clades.