Genetic Evolution Analysis Of CSFV E2 Gene In Guangxi And Construction And Rescue Of PRRSV Recombinant Clone Expressing CSFV E2

Classical swine fever(CSF)is a highly lethal infectious disease caused by classical swine fever virus(CSFV).Swine is the only natural host of this disease.The attenuated CSFV vaccine has good immune protection against CSFV yet it can’t differenate beween infected and vaccinated animals.Porcine reproductive and respiratory syndrome(PRRS)is also a highly contagious disease.The current use of two live attenuated vaccines for immunization will interfere with each other.Clinical co-infections of CSFV and PRRSV are commonly observed.In this study,clinical samples from different swine herds in Guangxi province from 2018 to2019 were detected for CSFV detection.The CSFV positive samples were used for virus isolation and sequencing.The genetic variation analysis of E2 gene of CSFV was also carried out.At the same time,PRRSV recombinant infectious clone expressing CSFV E2 gene was constructed and resued in MARC-145 cells.The results are shown as below:1.Genetic evolution analysis of CSFV E2 gene in Guangxi Province.14 of 115 samples from different swine herds in Guangxi province from 2018 to 2019 were positive for CSFV by RT-PCR,with a positive rate of 12.17%(14/115).The samples positive for CSFV were used to inoculate PK-15 cells for virus isolation.After serial passages in cell,four CSFV strains were isolated.The E2 gene was cloned and used for genetic evolution analysis.The results showed that all the 14 CSFV strains belong to 2.1 subtype of genotype II,8 of them belong to 2.1d subtype,3 of them belong to 2.1c subtype and 3 of them belong to 2.1b subtype.The identity of nucleotide and deduced amino acid with reference vaccine strains was 80.3%-83.7% and 87.9%-90.9%,respectively.Comparison of E2 protein domain showed that all the strains had mutations at the "D725G" "N725D" "K734R" and "V738T" sites,14 strains of CSFV have the same mutation site 986 "NYTK" 989 with HCLV.This study complements the epidemiological data of CSFV in Guangxi province,and is instructive for the prevention and control of CSFV.2.Construction and rescue of PRRSV recombinant clone expressing CSFV E2PRRSV infectious clone(pGXAM)and mutant clone(pGX12BS-TRS)were used as vectors.pGX12BS-TRS has the restriction sites(Bst BI and Sbf I)and transcriptional regulatory sequences(TRS)between ORF1 and ORF2 of genome.The recombinant plasmid pGXAM-CSFV-E2 was obtained by inserting CSFV E2 gene between ORF1 and ORF2 through the digestion sites of Bst B I and Sbf I.BHK-21 cells were transfected with the recombinant plasmid.At 48 hour post transfections,the supernatant of the transfected cells was inoculated into MARC-145 cells,and passaged continuously in MARC-145 cells.RT-PCR detection and IFA showed that the recombinant virus was successfully rescued.The recombinant virus could stably express exogenous CSFV E2 protein for at least10 passages in MARC-145 cells.The recombinant virus and its parental virus had similar plaque morphology and growth kinetics in MARC-145 cells.This study laid a foundation for the development of novel vaccine using recombinant PRRSV vector expressing CSFV E2.