Monkey B Virus Detection Method And Its Preliminary Applications

It is defined that there is no herpes B virus in SPF experimental monkeys.Herpes B virus is a pathogen of BSL 4,we primarily use HSVI to detect BV antibody to determine whether a monkey is infected by BV at present.The problem is that it can not distinguish HSVI antibody from BV antibody,and often mistakes the positive serum of BV antibody as negtive.We manage to detect BV by two assays: to detect the DNA of BV by PCR and to detect BV antibody with antigen expressed artificially.To detect the DNA of BV,the first problem is the specificity of the assay.We compare the genome of BV E2490 in NCBI with other herpes virus to find the sequence with diversity,and try to design primers in the sequences.We get two pairs of primers,there is a unique band by PCR with the molecular weight of 187 bp and 144 bp separately.We set up a few controls to verify the specificity of the primers: HSVI, HSVII, varicella zoster virus,simian parainfluenza virus,simian vacuolating virus, masern virus, epidemic parotitis virus, human parainfluenza viruses, sendai virus and normal Vero cells.We use these virus'DNA as template separately,there is a band with the predicted molecular weight when BV DNA is used.It shows the specificity of the assay.After the optimization of the PCR system,the minimum concentration of DNA we can detect is 1.292ng/ml and12.92ng/ml, respectively.We also design a pair of primers to discriminate BV and HSVI.There is the band with the same MW of 382bp afer PCR.The product of BV PCR shows two bands afer cut by SacII,but the product of HSVI cannot be cut into two bands.We expressed two kinds of polypeptide from BL21(DE3):the entire BV glycoprotein C and the segment near membrane of the BV glycoprotein G and BV glycoprotein D expressed by Miss Xiao. Afer purification and renaturation of the three kinds of polypeptide,we analysed their antigenicity by the means of Western Blot.The results show that they react with macaque sera which the BV antibody is positive,but don't react with rabbit sera which the HSVI antibody is positive.It shows that they are specific to detect BV antibody.We set up ELISA based on BVgC,BVgD and both of them.Based on the results of BV-ELISA,the sensitivity of BVgC-ELISA, BVgD-ELISA and BVgC+gD-ELISA is 84.6% ( 22/26 ) ,88.5%(23/26) and 96.2%(25/26),the specificity of them is 93%(13/14),85.7%(12/14) and 64.3% (9/14) ,respectively.Campared with the results of BV-ELISA,there is no significant deviation of the ratio of positive rusults.We compared the assay with HSVI-ELISA,BVgD polypeptide-ELISA(the linkage product of 9-amino acid polypeptide and protein vector CP39) and HPV2-DIA.The results show that the positive ratio of BVgC+BVgD-ELISA is higher than that of BVgD polypeptide-ELISA,and there is no significant deviation rest.We cannot set up ELISA using BVgG,the cause of it is not clear.The PCR assay is specific and sensitive and can be used to verify the presence of BV.It can also discriminate BV and HSVI.We expressed the three glycoprotein of BV by the means of prokaryotic expression first and analysed the character of them primarily,then set up corresponding ELISA. It can be a basic assay to detect BV antibody precisely.