Research Of Molecular Methods For The Detection Of Lumpy Skin Disease And The Expression Of ORF132 Gene In Baculovirus System And Identification

Lumpy skin disease（LSD）is an acute,subacute and chronic highly contact infectious disease caused by Lumpy skin disease virus（LSDV）.LSDV of cattle belong to the Capripoxvirus genus of the Poxviridae family.It can infect cattle of any breed and age,which mainly causes the infected animals to abortion,temporary or permanent infertility of bulls,decrease of milk yield,emaciation of infected animals,and low leather utilization rate due to nodules on the skin,and the morbidity rate is 2%～45%,and the mortality rate is 10%,or even higher,which brings greater economics loss.The World Organization for Animal Health（OIE）lists it as one of the required animal diseases,and China also lists it as a Class I animal disease.Therefore,in the countries and regions where the disease is endemic,the export of susceptible animals and animal-derived products will be strictly restricted by trading countries in the world,which will greatly affect the development of animal husbandry and international trade.For this disease,vaccines are mainly used for prevention,and no drugs to treat,and the existing detection methods for the pathogen are single or not good for differential detection.In this study,describes a species-specific,CaPV-LAMP based on ORF001 gene of LSDV,a real-time Taq Man MGB PCR method based on ORF101 for LSDV,and a wild type LSDV strain Taq Man MGB real-time PCR with vaccine strains based on ORF126.Using the method established by this study,Capripoxvirus were detected from anticoagulated,mosquitoes and flies samples of cattle and sheep in Chongqing,Xinjiang,Heilongjiang,and Yunnan,respectively.And Lumpy skin disease virus were successfully isolated from LSDV nucleic acid-positive vector organisms.According to the ORF132 sequences of the genomes of LSDV registered in Gen Bank,Using LSDV LW-1959 strain as template,the target fragment was amplified and linked to p Fast Bac HTB vector.The pfastbac HTB/ORF005 and pfastbac HTB/ORF132 were transformed into E.coli DH10Bac,respectively,and screened by blue white spot to obtain Bacmid/ORF005 and Bacmid/ORF132.r Bacmid-ORF005 and r Bacmid-ORF132 were transfected into Sf9 cells with cellfectin recombinant transfection reagent to obtain ORF005 and ORF132 recombinant baculovirus,respectively.This research laid the foundation for the development of new vaccines and the establishment of antibody diagnostic methods.Results of the current study as follows:（1）The CaPV-LAMP method was established to detect the LSDV,GTPV and SPPV viruses of the genus Capripoxvirus with a sensitivity of 14.7 TCID₅₀,which is100 times higher than the ordinary PCR recommended by OIE,and developed with CaPV.The LAMP detection method matches the standardized detection reagents.The stability and repeatability test results show that the detection reagent has good stability and repeatability.The observation of the results is visually applicable and is suitable for clinical on-site rapid screening of CaPV.（2）The universal Taq Man MGB real-time PCR for the detection of LSDV,it has a minimum detectable amount of 21.6 copies/μL,which can specifically and rapidly identify LSDV,GTPV and SPPV.Amplification efficiency of this method is 95.3%.The diagnostic kit developed has good stability.It can be stored at 4℃for 6 months and at-20℃for at least 14 months.The sensitivity of clinical samples shows that the minimum detectable amount of the method is 3.8 pg/m L,which can specifically detect LSDV in the blood,skin,lymph nodes and other tissue samples of cattle and sheep.Diagnosis and epidemiological investigation of nodular skin diseases.（3）dual Taq Man MGB real-time PCR method for the detection of LSDV wild strains and vaccine strains was established,which can specifically and sensitively identify LSDV wild strains and vaccine strains,as well as GTPV and SPPV.The yields were 22.6copies/μL and 19.7 copies/μL,respectively,and the amplification efficiency was greater than 99.5%,and developed a corresponding standardized diagnostic kit.The detection of bovine EDTA anticoagulated blood of experimental LSDV wild strains was detected with a minimum detection limit of 9 DPI.The method is specific,sensitive,simple and rapid,and can provide identification techniques for epidemic surveillance of LSD and immune monitoring of attenuated vaccines.（4）The ORF005and ORF132 gene of LSDV were systematically analyzed,optimization of codons,design of primers,amplification of target genes,recovery of target genes.The pfastbac HTB/ORF005 and pfastbac HTB/ORF132 were transformed into E.coli DH10Bac,respectively,and screened by blue white spot to obtain r Bacmid/ORF005 and r Bacmid/ORF132.r Bacmid-ORF005 and r Bacmid-ORF132 were transfected into Sf9 cells with Cellfectin recombinant transfection reagent,to obtain ORF005 and ORF132 recombinant baculovirus,respectively.Through Western blot analysis and IFA identification,ORF005 and ORF132 proteins were successfully expressed in Sf9 insect cells,which laid the foundation for the development of new vaccines and the establishment of antibody diagnostic methods.（5）The epidemiological investigation on the prevalence of Capripoxvirus in cattle and sheep in Xinjiang,Yunnan,Heilongjiang and Chongqing was carried out by using the quantitative PCR method,CaPV-LAMP method and common PCR method established in this study.The lumpy skin disease virus was isolated from the vector organisms positive for the detection of LSDV DNA in the XINJIANG Yili border area,the MDBK cells were inoculated.After 3 generations of blind transmission,a lumpy skin disease virus,XJ1917 strain,was successfully isolated.By sequencing analysis,the isolated lumpy skin disease virus has 99%homology with the LSDV Neethling vaccine strain,and there are 27 base deletions and 3 mutations in the ORF126 region.The virus was measured by TCID₅₀,and the titer of XJ1917 strain fifth generation cell virus solution was 3.65×10⁶TCID₅₀/m L.