Genetic Evolution And Molecular Characterization Of Classical Swine Fever Virus Recently Isolated In China

Classical swine fever（CSF）is an OIE-listed,contagious and fatal disease of pigs caused by classical swine fever virus（CSFV）.China has the biggest swine industry in the world,while CSF is an important disease seriously threatening pig farming.At present,CSF has been effectively controlled and successfully eradicated in North America,Brazil,Cuba,Australia,and New Zealand and many European countries after years of continuous efforts.However,CSF situation is not satisfactory for some countries including,Russia,China and Southeast Asia.For decades,the disease is controlled by a modified live vaccine C-strain in China.Despite the use of mass and intense vaccination regimens,the disease is still endemic in China.In the past,genotype 2 strains that genetically distant from the vaccine strain（genotype 1）have being predominant in the field.The recent epidemic strains and epidemiological status of CSF in China are still unclear.Thus,the aim of our study was to analyze the genetic and molecular characteristics of the recent field strains of CSFV to ascertain the field status of CSF in China.CSFV is a single-stranded,positive-sense RNA of around 12.3 kb containing one open reading frame（ORF）that is surrounded by 5?and 3?un-translated regions（UTRs）.The ORF is capable to translate 3,898-AA of polyprotein that contains four structural(C,E^rns,E1,and E2)and eight non-structural(N^pro,NS2,NS3,NS4A,NS4B,NS5A and NS5B)proteins.E1,E2 and E^rnsare the major structural protein and important virulent determinants,and protective immunogen.E2 is the most important glycoprotein of the virus and has been shown to induce strong neutralizing antibodies.E^rnsis unique which possesses intrinsic ribonuclease（RNase）activity that can inhibit the production of type-I interferon.E1 is involved in the formation of hetrodimer with E2 and essential for viral entry and infectivity.The non-structural protein NS5B is an important RNA-dependent RNA polymerase（Rd Rp）.At present,E2 and NS5B genes are mainly used for genotyping of CSFV strains.Here,we identified and characterized the 24 new isolates of CSFV in different areas of China during2016-2018.Phylogenetic analysis of the NS5B,E2 and full-length genome of the new strains revealed that the new strains were clustered into subgenotype 2.1b or 2.1d.We observed that most new isolates belong to 2.1d,while strains HL18-490,HL18-462,SD18-461 and NM16-333 belong to 2.1b branch.Further,we found some genomic variations in 5?UTR and 3?UTR.Moreover,molecular characterization of E1,E2 and E^rnsshowed unique variations.These alterations were observed in antigenic regions of E^rns（AR1,AR2 and AR3）and E2（B/C and A/D）.The sequence distance of nucleotide and amino acid of the 8 newly isolated strains was compared with 8 representative CSFV strains,including Shimen（1.1）,Paderborn（2.1a）,HEBZ（2.1b）,HNSD-20（2.1c）,Zj0801（2.1d）,CSFV39（2.2）,Alfort（2.3）and TWN（3.4）.The average range of similarity percentage was observed between 83%-100%.Site-by-site analysis of positive selection pressure clarified specific codons that underwent evolutionary selection.The positive selection was identified on eight sites in E2 and five sites in E^rns（posterior probabilities≥0.7）.Among them,three sites of E2（57,105,and 207）and five sites of E^rns（63,107,153,178,and 217）are located in the antigenic regions of the two proteins,respectively.Genome-wide scanning predicted recombination events between the vaccine strain and newly isolated strains（except isolate HL18-494）.We identified frequent recombination events occurring in the CSFV-encoded genes among the strains and most of them occurred between the strains of genotype 2.The putative recombination events were found in C,E^rns,E1,E2,N^pro,NS5A and p7 proteins.The isolated virus was confirmed by indirect fluorescence assay（IFA）and viral titration(TCID₅₀)was determined.We utilized four antibodies（MAbs 5B8-2,3F-1,5A12-1 and rabbit anti-E2 sera）for virus detection and end point titration.Our results demonstrated that MAb 5B8-2 showed better reaction with all CSFV strains than other monoclonal antibodies.Serum-virus-cross-neutralization test was performed to investigate antigenicity of the new isolates against C-strain vaccinated porcine sera and we found that the recent strains had tendency to escape from antibody neutralization,which might be due to the variations in antigenic regions,especially neutralizing epitopes in B/C domain of E2.The growth properties of two new representative strains（HL18-494 and NM16-232）of CSFV were also found distinguishable from Shimen strain.In summary,the new isolates of CSFV were found with molecular variations that might lead to neutralization escape of the antibodies derived from C-strains-vaccinated pigs.The study also predicted recombination events occurred among the vaccine strain and wild-type strains and traces of vaccination pressure were also observed.It exposed the myth that evolutionary changes in CSFV might be due to disproportionate usage of the C-strain vaccines in endemic areas.This underscores the need to develop mitigation vaccination strategies to minimize the substantial risk associated with the emergence of vaccine-escaping mutants.Taken together,this study uncovered the genetic diversity and molecular features of CSFV currently circulating in China,which will provide insight of epidemiology and designing effective vaccines and diagnostic purposes for CSF.