Molecular Epidemiology Of Classical Swine Fever Virus In China Based On Virus Genome Analysis

Classical swine fever virus（CSFV）is a member of the Pestivirus genus within the Flaviviridae family and belongs to a single-stranded positive-sense RNA virus.Classical swine fever（CSF）caused by CSFV seriously harms the global pig industry and causes huge economic losses.CSFV is currently divided into 3 genotypes（genes1,2,and 3）and 11 subgenotypes（1.1 to 1.4,2.1 to 2.3,and 3.1 to 3.4）.Since 2000,the prevalence of the 2.1 subgenotype in China has become more widespread.Previous studies have shown that 2.1b is the dominant sub-subgenotype.Due to the mandatory vaccination of modified C-strain live vaccine,the CSF epidemic in China has been better controlled,but the outbreak still occurs from time to time.In-depth analysis based on the complete genome sequences of CSFV can reveal the most comprehensive information of the virus and predict its epidemic trend.In order to find the genetic derivation principles of CSFV,this study obtained 82 CSFV strain’s whole genome sequences of 3 genotypes which were collected between 1990and 2018 by high-throughput sequencing technology,and conducted multiple analysis with 119（as much as possible）CSFV whole genome sequences with clear information download from NCBI database.The results showed that the 14 2.1b strain’s complete genome measured by high-throughput technology shared 96.7%-99.6%and98.1%-99.8%sequence identity with each other at the nucleotide and amino acid level respectively.The genomic nucleotide and amino acid sequence identity of the 13 2.1c strain’s complete sequences were 95.5%-99.2%and 97.7%-99.4%,respectively.The homology of nucleotide and deduced amino acid sequence of the whole genome sequence between 2.1b epidemic strains and vaccine strain is 84%-85.1%and91.6%-92.6%respectively,and that of 2.1c strains and vaccine strain is 83.9%-84.8%and 91.7%-92.4%,respectively.The phylogenetic tree of the complete genome sequences of CSFV can reveal the most reliable evolutionary information compared to the 14 genes of CSFV.The evolution analysis of the full-length sequence of E^rns,NS2,and NS5A can all obtain results similar to those based on the E2 gene sequence.The 2.1b and 2.1c epidemic strains has 226 and 249 amino acid substitution sites compared to the vaccine strain,with substitution ratios of 5.8%and 6.4%,respectively.The 2.1b and 2.1c epidemic strains had the highest amino acid substitution ratio（10.6%）in the E^rnsprotein compared to the vaccine strain.Bayesian phylogenetic analysis of the whole genome sequence of CSFV showed that the evolution rate of2.1b strain is 1.604×10^-3substitutions per site per year,which is higher than that of the CSFV population and 2.1c strain.In order to clarify the current CSF epidemic situation and the genetic diversity of CSFV in Chin,epidemiological study of CSF in 12 provinces（municipalities/autonomous regions）was carried out in 2018-2019 including Jilin,Heilongjiang,Zhejiang,Guangdong,Yunnan,Beijing,Liaoning,Henan,Shandong,Hebei,Shanxi and Guangxi.A total of 99 CSFV positive pigs were detected in a total of 1031 samples collected from 873 pigs,with a total individual positive rate of11.34%（99/873）.Phylogenetic analysis showed that the epidemic strains include the2.1b,the 2.1c sub-subgenotype,and the 1.1 subgenotype.The dominant CSFV strain in China in the past two years has been the 2.1b sub-subgenotype.A case of persistently infected pigs found in Jilin was caused by the 2.1b strain.In order to conduct a more comprehensive and in-depth study of the evolutionary dynamics of CSFV,471（as much as possible）E2 full-length sequences with clear information including 15 full-length E2 amplified by epidemiological investigations and 82 E2 sequences cut from complete genome were analyzed.The results showed that the 2.1b sub-subgenotype is the dominant strain,which shared 81.2-82.8%and86.1-89.3%identity at the nucleotide and amino acid level with the full-length sequence of E2 gene of C strain.The 2.1b and 2.1c sub-subgenotype strains shared81.1%-83.1%and 86.1%-89.8%sequence identity with C strain at the nucleotide and amino acid level respectively.There are 27 and 36 amino acid substitution sites between the 2.1b/2.1c sub-subgenotype strains and the vaccine strain E2 gene,respectively,and the substitution ratios are 7.2%and 9.7%;Bayesian analysis based on the E2 full-length sequence showed that the evolution rate of CSFV population is1.299×10^-3substitutions per site per year,which is lower than the evolution rates of the 2.1b and 2.1c sub-subgenotype strains;The selective pressure measurement parametersω（dN/dS）of E2 gene of the 2.1b and 2.1c epidemic strains and all genotype CSFVs are all less than 1,indicating that the CSFVs are undergoing purification selection pressure.The above analysis explains to some extent the reasons why the 2.1b sub-subgenotype became the dominant genotype,and also implies the uncertainty of the 2.1c strain on the way of future evolution,reminding us to strengthen the monitoring of 2.1c epidemic strains.The detection of CSFV positive pigs in recovered piglets confirmed the clinical presence of persistently infected pigs.