Feline noroviruses: investigation of epidemic potential and genetic/structural insights of host-based separation and recombination.

Noroviruses (family Caliciviridae) are important enteric pathogens in humans and animals. The closely related GIV and GVI noroviruses in cats and dogs suggest interspecies circulation; however, their diversity and host restrictions have not been fully elucidated. In this study, we retrospectively investigated noroviruses in diarrheic cats in Hungary, determined the complete genomes of representative strains (n = 3), and performed comparative sequence and structural analyses of all available feline and canine GIV and GVI sequences. Fecal samples (n = 90; November 2024-January 2025) from stray and free-roaming cats were screened using RT-PCR. Capsid sequences and complete genomes were determined using 3'/5'RACE/RT-PCR, NovaSeq+ (Illumina), and Sanger sequencing techniques. Analyses included phylogenetic trees, p-distance comparison plots, recombination detection, VP1 P-domain dimer, and complete VP1-based capsid modeling. Eight (8.9%) samples were norovirus positive. All known feline genotypes (n = 4 GIV.2, n = 1 GVI.1, n = 1 GVI.2) were identified, each with the (GVI.P1) P-type. Nearly identical GIV.2 strains were found in the Budapest area (November 2024), representing the first non-shelter-associated case accumulation (a potential outbreak) of norovirus in cats. One complete genome of each genotype was determined. Multiple host-specific substitutions separate feline and canine noroviruses across viral proteins, including the surface-exposed VP1 P-domains and a feline-specific 10- or 12-amino-acid-long deletion in the GVI.2 P2 subdomain of currently unknown importance. Lower p-distance boundaries for inter-/intra-genogroups and inter-P-types were also established. Recombination at the ORF1/ORF2 junction was observed in two feline lineages, indicating that recombination and host adaptation jointly drive the evolution of carnivore noroviruses.IMPORTANCEDespite the major public health relevance of noroviruses, research has predominantly focused on human strains, while noroviruses of carnivores, particularly feline strains, remain poorly characterized. This study provides genetic, phylogenetic, and structural insights into feline noroviruses circulating in Hungary, including indications of the first reported large, non-shelter-associated potential outbreak of feline GVI.2 norovirus among free-living cats in Budapest and its surrounding areas, highlighting the natural epidemic potential of these viruses. Comparative genomic analyses demonstrated consistent host-based separation of closely related feline and canine GIV and GVI noroviruses across multiple genomic regions, supporting ongoing host-specific evolution. Structural analyses identified characteristic deletions within the highly variable surface-exposed P2 subdomain of feline GVI.2 capsid proteins, suggesting potential roles in interactions of receptors/attachment factors and/or immune evasion. Additionally, this study demonstrates the utility of pairwise p-distance analyses for recombinant strain recognition and for refining genetic demarcation criteria within feline and canine norovirus genogroups and P-types.