Genotyping atypical porcine pestivirus using NS5a

Atypical porcine pestivirus (APPV) is an emerging virus discovered in 2014 and it can cause congenital tremors in pigs. Molecular epidemiology serves as an essential tool in monitoring and controlling the disease. Virus epidemiology mainly relies on genome sequencing and phylogenetic characterization. Previous molecular epidemiology studies have been using different genes/regions for phylogeny, namely whole genome, Npro, and E2 coding sequences. However, with increasing number of APPV sequences available in GenBank, no systemic studies have been performed for detailed classification of APPV strains around the globe. The goal of this study is to propose a classification strategy or taxonomy of APPV strains at genotype, subgenotype, and isolate levels. A total of 76 whole genomes and 16 partial polyprotein coding sequences were analyzed for genetic variability and suitability of all individual genes for viral phylogenies. Our results revealed that, among all the viral genes, NS5a coding sequences were proved to be the most suitable alternative for tracing APPV strains supported by its capability of reproducing the same phylogenetic and evolutionary information as the whole viral genome did. Also, a reliable cutoff to accurately classify APPV at different levels is established. We propose a genotyping scheme with three well-defined genotypes (1–3) and 7 subgenotypes for genotype 1 (1.1–1.7). For whole genome analysis, a threshold value of 84%–91% pairwise identity allows separation of all APPV subgenotypes, whereas 80% identity clearly segregate the three major APPV genotypes. For NS5a gene analysis, 82%–91% identity allows subgenotype separation and 76% identity segregate APPV genotypes. Additionally, genetic distance of whole genome exhibits ≤8% in isolate level, 9%–14% in subgenotype level, and 17%–22% in genotype level, while for NS5a encoding sequences the genetic distance displays ≤9% in isolate level, 9.9%–19.1% in subgenotype level, and 21.6%–29.7% in genotype level. These allow a clear segregation among APPV genotypes, subgenotypes, and isolates. Therefore, the proposed strategy in this study provides a solid and improved basis for molecular phylogenetics to understand APPV genetic diversity, trace the origins and control the spread of new disease outbreaks.