Study On Biological Characteristics And Detection Methods Of Epidemic Strains Of Lumpy Skin Disease In Guangxi

Lumpy skin disease(LSD)is an acute or subacute infectious disease characterized by cattle fever,mucosal edema and extensive nodules on the skin surface caused by Lumpy skin disease virus(LSDV).Since it was first diagnosed in Xinjiang,China in August 2019,it has spread from northwest China to many provinces in southeast China within one year,causing great harm and threat to Chinese cattle breeding industry.Vaccination with attenuated vaccine is an effective means to prevent LSD.Attenuated Goatpox virus(GTPV)vaccine has been approved for use in LSD epidemic areas in China,but there are safety problems such as virulence reversion and strain recombination.For LSDV,it is urgent to monitor the newly discovered unknown epidemic strains.To grasp the sequence of viral genes and track the genetic evolution of viruses,and the virulence reversion of LSDV during transmission was preliminarily explored;A differential diagnosis method for LSDV and other members of the Capripoxvirus(Ca PV)was also developed to monitor the health status of cattle.In this study,the second-generation complete genome sequencing of LSDV isolated and identified from the skin nodules of diseased cattle in Guangxi was carried out by high-throughput sequencing technology,and its gene sequence,genetic evolution and gene recombination were analyzed;Aiming at the expression of virulence-related gene LSDV F3 L of LSDV,the recombinant transfer vectors were constructed with Vaccinia virus(VACV)Tiantan strain(VTT)as the research vector,and cotransfected into BHK-21 cells using VTT.The recombinant virus was successfully screened in BHK-21 cells;Based on the LSDV 010 gene and Ca PV 068 gene,a duplex real-time quantitative PCR detection method was established to simultaneously identify and diagnose LSDV and other members of Ca PV,and the detection method was applied to the epidemiological investigation of clinical samples of cattle and sheep collected from various regions of Guangxi.(1)The complete genome sequence of LSDV/LYGN/2020 strain was obtained by complete genome sequencing,the full length of LYGN/2020 strain was 150678 bp,and had 99.8～99.9 % homology with the strains isolated and identified in various provinces of China from 2019 to 2020.Phylogenetic tree analysis showed that the LYGN/2020 strain was in the same group as the strains reported in various provinces of China and southeast Asian countries after 2020,all belonging to the LSDV Recombinant group.Recombination analysis showed that LYGN/2020 strain had 13 potential recombination events and was a vaccinerecombinant strain;(2)A recombinant transfer vector plasmid p VTTd F3L-EGFP-gpt having a VACV p E/L early/late strong composite promoter,an EGFP green fluorescent protein gene,and gpt Escherichia coli xanthine-guanine phosphoribosyltransferase gene.The recombinant transfer vectors p VTT-d F3L-EGFP-gpt-LF3 Lr and p VTT-d F3L-EGFPgpt-v F3 L were obtained by inserting the LSDV F3 L and VTT F3 L gene expression cassettes into the vector plasmid p VTT-d F3L-EGFP-gpt.The recombinant virus r VTT-LF3 Lr and r VTT-v F3 L were finally obtained by co-transfection and screening purification with VTT;(3)The duplex realtime quantitative PCR detection method for identifying LSDV was established,which can simultaneously identify LSDV and other members of Ca PV.The limit of detection(LOD)was 15 copies/μL,and coefficients of variation of the intra-assay and inter-assay were all less than 2.0 %,thus showing high specificity,sensitivity and repeatability.A total of 2122 clinical samples of cattle and goats in Guangxi were detected by this method,including 1520 clinical samples of cattle and 602 clinical samples of goats.The positive rates of LSDV and Ca PV were 1.08 % and 1.41 %,respectively,and the agreements of the detection results by the developed and the reference q PCR method was more than 99.0 %.In conclusion,a vaccine-recombinant LSDV strain was isolated from the southwestern provinces of China and attracted attention.The recombinant virus was successfully constructed according to the virulencerelated genes of LSDV F3 L,which provided experimental conditions and preliminary preparation for the study of the biological characteristics of LSDV F3 L protein in the future.The differential LSDV detection method established for the specific sites of LSDV provides a fast and efficient technical means for accurate prevention and control of LSDV and epidemiological investigation.