Emergence of a Candidate Novel Sub-Genotype 1.1d of Classical Swine Fever Virus in India: Insights from a Comprehensive NS5B Gene Analysis and Multi-Locus Phylogeny.

Abstract

Classical swine fever virus (CSFV) remains a major threat to the swine industry, causing severe economic losses in endemic regions. However, the limited use of conserved non-structural genes in molecular surveillance contains the understanding of viral evolution. This study aimed to evaluate the NS5B gene as a phylogenetic marker to elucidate the genetic diversity of circulating CSFV strains in India. A total of 870 clinical and tissue samples collected during a nationwide surveillance program (December 2023-2024) were screened using NS5B-specific RT-PCR, of which 43 (4.95%) samples tested positive for CSFV. Phylogenetic analysis involving 43 field-derived NS5B sequences and 66 reference sequences revealed a distinct lineage, PV820729, within genotype 1.1. Multi-locus comparison of NS5B, partial E2 (PX233330), and 5' UTR (PX237204) regions confirmed its genetic distinctness from known sub-genotypes, supported by pairwise nucleotide identities of 97.3% (recent Indian strains), 84-85% (older ones), 94.8-95.8% (E2), and 95.7-96.2% (5' UTR). Bayesian molecular clock analysis (GTR + G + I/UCLD/GMRF) estimated the mean tMRCA at 1837.9 (95% HPD: 1710.5-1926.9), aligning with previous E2- and whole-genome-based studies. SNP profiling across the NS5B region identified 133 substitutions, primarily C→T and A→G transitions, reflecting ongoing evolutionary activity within this conserved locus. Collectively, the results support the identification of a candidate novel sub-genotype, provisionally designated 1.1d, and emphasize the value of NS5B-based phylogenetic surveillance for monitoring CSFV evolution and informing control strategies in India.