Genetic diversity, recombination, and codon-level selection of feline calicivirus ORF2 in Hokkaido (Japan)

To date, the genetic characteristics of feline caliciviruses (FCVs) in Hokkaido (Japan) have not been investigated. Here, 28 FCVs obtained in 2022-2023 from the Hidaka and Tokachi Districts were phylogenetically analyzed. Recombination breakpoints within open reading frame 2 (ORF2) were detected using the Genetic Algorithm for Recombination Detection (GARD) and the Recombination Detection Program v5.5 (RDP5). In addition, recombination was independently verified using permutation-based statistics in SimPlot++. Codon-level selection analysis using Hypothesis Testing using the Phylogenies framework were performed. Amino acid (aa) diversity at epitope-focused sites was quantified using entropy profiles. The ORF2 phylogeny grouped Hokkaido FCV strains into genogroups I and II. Their terminal branch lengths were comparable to other time-matched Japanese FCV strains, supporting a shared evolutionary background. Within region E of the capsid precursor protein encoded by ORF2, GARD delimited a recombination window at nucleotides 1284-1388 (aa 428-463), and RDP5 identified two events overlapping this region. SimPlot++ analyses also supported recombination signals. Among Hokkaido strains, aa diversity was detected at multiple epitope-focused sites. Codon-level analysis identified episodic positive selection at codon 447 within the linear neutralizing epitope (aa 445-451), supported across MEME, FEL, SLAC, and FUBAR. Two Hokkaido FCV strains carried an aa variation (T → D/S) at aa 447. Although entropy profiles indicated diversity across epitope-focused sites, aa 447 exhibited low entropy. The results support epitope-focused diversification in region E superimposed on a shared ORF2 backbone among Hokkaido FCV strains and highlight codon 447 as a candidate site warranting further structural and immunological validation.