| Herpes B virus belongs to alpha herpes virus, and it is a zoonotic pathogen which has serious harm to human health. In the natural host, the herpes B virus's infection rate range from 10%-60% and the virus is latent. The virus can lurk in the trigeminal and lumbosacral ganglion. Clinical symptoms include oral or genital ulcers, or no obvious symptoms, similar to people infected with the herpes simplex virus type 1 and type 2. But the herpes B virus is deadly to human and clinical symptoms include encephalitis, encephalomyelitis and even respiratory arrest to die. Mortality rate is more than 70 percent and neurologic sequelae will be left to survivors. At present, there is no relevant B virus vaccine. Rapid detection and early diagnosis are the major measures to prevent the B virus from infection. In medical clinic, people adopt the protein of herpes simplex virus (HSV), simian agent 8(SA8) to be diagnostic antigens. But the detection results appear to falsely positive and falsely negatives. The membrane protein of monkey B virus play an important role in the process of spreading the virus, such as adsorption, membrane fusion, entrancing the host cells, evading the immune monitoring. In the host, glycoprotein B, C, D appeared in the earliest time and disappeared in the latest time. They have good immunogenicity and reactogenicity. And they are the ideal targets to differential diagnosis and effectively prevent the monkey B virus.1. The epidemiological survey of monkey B virus's infection rate in four regionsFor surveying the epidemiology of monkey B virus, we used the immunoenzymatic technique to analyze 953 serums samples which were collected from Sichuan, Beijing, Hainan, and Guangxi, statis the infection rate in four regions, compared their difference in different regions.210 serums samples were positive. The seropositivity rate was 22.04%. Monkey B virus's infection rates were different in different macaque farms. Such as the infection was 10.37% in some macaque farm in Sichuan province, but the B virus's infection was 46.15% in some macaque in Beijing。There was no significant difference by statistical analysis.2. Comparison of detection results of monkey B virus between PCR and immunoenzymatic techniqueFor more effective understanding of the virus dormant state in animals, according to the UL1 gene and US6 gene of the genomic DNA of E2490 published on Genbank and 22 rhesus monkey fed in the Laboratory Animal Center of Military academy of medical sciences, We designed the specificity primer gL (UL1) and gD(US6), established the PCR detection methods for monkey B virus and compared the results of PCR and serologic detection.We detected 22 serums and only 13 serums were positive by immunoenzymatic technique. The seropositivity rate was 59.09%. Trough the PCR amplification,4 serums were positive. The positive rate was 18.18%. The positive coincidente rate was 30.77% between the both detection results. The serological detection of monkey B virus can determine the monkey B virus's carrier and don't determine the virus in the incubation or in active proliferation period.3. The expression and purification of glycoprotein C of monkey B virusAdopting the bioinformatics software DNAMAN, we compared the homology of glycoprotein B, glycoprotein C and glycoprotein D of the monkey B virus with the herpes simplex virus (HSV) type 1 or type 2, and determined the membrane protein C as the experiment research object. The B virus genome GC content is higher (73%) and contains a lot of rare codon, such as ccc, cgg, gga. That has the potentiality to impact the gene amplification and protein expression. We obtained the purpose gene by PCR amplification and overlap PCR. The constructed gene was cloned into pET-28b(+) and transformed into E.coli BL21. The culture was induced by IPTG. The recombinant protein was mainly located in the inclusion body. The western blot showed the protein could react specifically to the monkey B virus positive serum and anti-histidine monoclonal antibody.4. The establishment of gC-ELISA of monkey B virusThe recombinant protein gC of monkey B virus expressed in Escherichia coli BL21 (DE3) was used as antigen for developing an indirect-ELISA assay to detect monkey B virus specific antibody. Conditions of the gC-ELISA were optimized, and suggested that the optimistic concentration of recombinant protein was 1.5μg/ml,1:10 dilution of testing serums and 1:25000 dilution of HRP-labeled rabbit anti-monkey IgG. The gC-ELISA method has good specificity and sensitivity, and no cross reactivity with positive serum of herpes simplex virus type 1 and type 2. 187 serums of monkey B virus selected randomly were sent to the U.S. monkey B virus detection laboratory-China representative office. The coincidence rate between the detetion results of indirect ELISA and U.S. monkey B virus detection laboratory was 96.3%.
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