Intra-host diversity of proviral FeLV and clinical outcomes

Feline leukemia virus (FeLV) is a retrovirus associated with a variety of clinical conditions, including either regenerative or non-regenerative anemia. The high mutation rate of retroviruses promotes the formation of quasispecies — populations composed of multiple genetically related haplotypes — which drives the evolution of the viral population within a host and can impact on disease development. This study investigated the intra-host diversity of proviral FeLV and evaluated their impact on the hematological parameters in naturally infected cats. Twenty-seven blood samples from FeLV-positive cats were analyzed. Total DNA was extracted, and the complete env gene was amplified by PCR, followed by next-generation sequencing (NGS). Raw sequence data were quality-checked, trimmed, and aligned to the FeLV SU reference sequence. Quasispecies with frequencies above 5% were inferred using CliqueSNV v2.0.3, phylogenetic reconstruction was performed with IQ-TREE v1.6.12 and recombinant sequences were identified using SimPlot v3.5.1. A remarkable variability in quasispecies composition was observed among infected cats. While 13 animals (48.1%) harbored infections driven by a single haplotype, others (n = 14) exhibited a much more complex viral landscape, with five or more distinct haplotypes detected. In samples with greater viral diversity, quasispecies frequencies were more evenly distributed. In the phylogenetic reconstruction, most quasispecies sequences isolated from the same host clustered together in well-supported clades within the FeLV-A lineage. Notably, one exception was identified as a FeLV-A/enFeLV recombinant. Additionally, five other recombinant quasispecies were detected. Analysis of hematological parameters showed no significant correlations between the number of haplotypes and the presence of anemia (regenerative or non-regenerative). However, classifying animals by their lymphocyte values’ adherence to normal reference ranges revealed a statistically significant difference in the number of viral haplotypes detected between groups (p = 0.04). Additionally, a negative binomial regression indicated that life stage had a positive trend in its relationship with the number of proviral quasispecies (p = 0.0593), indirectly suggesting that a higher number of quasispecies tends to be observed in more advanced disease stages. Although not statistically significant, the result suggests a potential relationship that warrants further investigation. This study, therefore, indicates that the number of intra-host quasispecies can be influenced by both the duration of infection and alterations in the immune response in naturally FeLV-infected cats.