Molecular Analysis of Classical Swine Fever Virus Associated Field Infections Evidence Novel CSFV Sub Genotype in Tamil Nadu, Southern India

Classical swine fever (CSF) is an endemic and major viral infection of Indian swine husbandry, contributing to great economic losses with multiple genotypes associated with vast clinical and subclinical outcomes. Molecular detection and genotyping of CSF virus directly from field samples has great application in disease monitoring and control measures hence this study aimed to isolate and characterize CSFV genotypes circulating in southern states of India. Fifty-seven porcine post-mortem tissues (lymph nodes, spleens, livers, lungs, and kidneys) collected from pigs suspected of systemic infections and sudden death with the history of live attenuated CSF vaccination from different regions of Tamil Nadu were used in this study. An NS5B gene based CSFV specific RT-PCR screening confirmed CSFV positivity in 7% (4/57) of samples with a specific amplicon of 449 bp. Further molecular screening for other viral co-infections such as PCV2, PPV and PRRSV done by specific individual PCR assays to all the samples. Non-involvement of above screened three viral pathogens in all four field samples which showed positivity for CSFV confirming CSFV as primary pathogen. Two RT-PCR positive samples (TNI-4 and CHNL-2) selected randomly and sequenced. Aligned contig sequences of both samples were subjected to BLAST homology search and phylogentic characterization. BLAST study of TNI-4 sequence revealed 99% sequence identity with Indian CSFV sequences of genotype 1 and CHNL-2 showed 98% sequence identity with Indian CSFV sequences of genotype 2. Phylogenetic analysis of the TNI-4 and CHNL-2 sequences obtained in this study along with 38 published CSFV sequences consisting of all 5 new genotypes and 14 sub genotypes through the Maximum Likelihood tree method in MEGA 11 revealed that TNI-4 clustering together with 1.7 sub genotypes and CHNL-2 clustering together with 2.2 sub genotypes. TNI-4 and CHNL-2 partial NS5B gene sequences obtained in this study deposited in the GenBank database under accession numbers of MW822568 and MW822569 respectively. The study is the first to report CSF infections associated with the newer 1.7 sub genotype in Tamil Nadu, southern India. It is possible that vaccination could affect the genetic diversity of the CSFV through recombination and point mutations for immune evasion.