Genomic Characterization and Molecular Epidemiology of Tusaviruses and Related Novel Protoparvoviruses (Family ) from Ruminant Species (Bovine, Ovine and Caprine) in Hungary.

Tusavirus 1 of species (family ) was first identified in humans and later in small ruminants (caprine and ovine). This study reports the full-length coding sequences (~4400-4600 nt) of three novel tusavirus-related protoparvoviruses from ovine ("misavirus", PV540792), for the first time bovine ("sisavirus", PV540793) and subsequently from caprine ("gisavirus" PV540850/51) fecal samples, using next-generation sequencing (NGS) and PCR techniques. Their NS1, VP1 and VP2 proteins shared 61-63% amino acid identities with each other and with tusaviruses, suggesting these three viruses belong to three novel species in the genus . Phylogenetic analyses placed them with tusaviruses on a separate main branch, implying a shared origin among these most likely ruminant protoparvoviruses. A small-scale epidemiological investigation on 318 ruminant enteric samples using novel generic NS1 primers found misavirus in 14/51 (27.5%) ovine and sisavirus in 19/203 (9.4%) bovine samples from multiple Hungarian farms. Tusavirus was present in 5/51 (9.8%) ovine and 15/62 (24.2%) caprine samples, all from one farm. The highest prevalences for all three viruses were found in animals aged 2-12 months, though sporadic cases were also found in other age groups. Partial NS and VP sequence-based phylogenetic trees showed virus-specific lineages for misa-, sisa-, gisa- and tusaviruses, with various strains forming sub-lineages. These findings suggest the presence of multiple genotypes and/or members of additional species, which was supported by a VP sequence-based hierarchical cluster analysis. The study's viruses were mostly phylogenetically separated by host; however, two bovine sisavirus strains with diverse phylogenetic localizations in the NS (belonging to bovine sisaviruses) and VP1 trees (distantly related to ovine misaviruses) could indicate previous (interspecies?) recombination events.