Epidemiologic Investigation, Envelop Protein Expression And Epitope Identification Of Simian B Virus

Herpes B virus belongs to alpha herpes virus, and it is a zoonotic pathogen which hasserious harm to human health. In the natural host, the herpes B virus’s infection rates rangefrom10%to60%, and the virus can lurk in the trigeminal and lumbosacral ganglion. Clinicalsymptoms include oral or genital ulcers, or no obvious symptoms, similar to people infectedwith the herpes simplex virus type1and type2. But the herpes B virus is deadly to humanand clinical symptoms include encephalitis, encephalomyelitis and even respiratory arrest todie. Mortality rate is more than70percent and neurologic sequelae will be left to survivors.At present, there is no relevant B virus vaccine. Rapid detection and early diagnosis are themajor measures to prevent the B virus from infection. In medical clinic, the protein of herpessimplex virus （HSV）, simian agent8（SA8） were used as diagnostic antigens. But somedetection results were falsely positive and falsely negatives. The membrane protein of simianB 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 late period. Andthey are ideal targets of differential diagnosis and effective vaccine, for they have goodimmunogenicity and reactogenicity.1. The epidemiological survey of herpes B virus’s infection rate in four regionsFor surveying the epidemiology of monkey B virus through immunoenzymatic method,953serum samples collected from Sichuan, Beijing, Hainan, and Guangxi, were used. Theseropositivity rate was36.1%（344positive samples）. Monkey B virus’s infection ratesranged from10.4%to48.2%in different regions and significant difference also appearedbeteen young monkeys （1-2age） and adult mokeys （18.4%VS53.2%）.Results of PCR showed that virus DNAs of4samples were present in trigeminal ganglia（TG） of the13seropositve monkeys （30.8%） tested, but no virus being detected in all serasamples. Sequencing products of PCR showed G+C content in gD gene was very high（74.2%） and middle in gL gene （64.1%）. Our results indicated that B virus would be latent in trigeminal ganglia only in part of infected moneys and gL gene of BV is a suitable target forspecific and rapid identification of viral infection by PCR technology.2. Preparation of herpes B virus recombinant glycoprotein C and D and evaluation ofits diagnostic potentialTo establish the foundation for rapid diagnosis tech and to prepare for vaccine develop，herpes B virus glycoprotein C and D were expressed and its diagnostic potential wasevaluated. In the study, the gC and gD gene was amplified by PCR and cloned into theprokaryotic expressive vector pET-28b（+） after sequencing. Recombinant plasmidpET-28b（+）-gC and pET-28b（+）-gD were transfected into E.coli BL21（DE3） and gC and gDproteins were expressed by IPTG induction. The purified protein was identified by Hisantibody and B virus positive serum, and its diagnostic value was evaluated by ELISA.Results showed that the recombinant protein gD had a MW of46kD, accounted for35％total proteins, and expressed in form of inclusion body but no gC protein was expressed. Andthen, coding optimizing fragments of gC was obtained by overlap PCR and expressed theprotein with a higher level, had a MW of50KD and accounting for30%total proteins, andexpressed in form of inclusion body. Western blot showed that the recombinant proteins gCand gD could react specifically to the herpes B virus positive sera and anti-histidinemonoclonal antibody.The recombinant proteins gC and gD of herpes B virus expressed in Escherichia coliBL21（DE3） was used as antigen for developing an indirect-ELISA assay to detect herpes Bvirus specific antibody. Conditions of the ELISA were optimized, and suggested that theoptimistic concentration of recombinant gC and gD protein were150ng/hole and100ng/holerespectively,1:8¹0dilution of testing serums and1:30000dilution of HRP-labeled rabbitanti-monkey IgG. The indirect ELISA of standard positive serum and negative serum showedthe sensitivity and specificity were90.0%（27/30）and100%（20/20）for gD and83.3%（25/30）and100%（20/20）for gC. When recombinant gC and gD was mixed and coated, thesensitivity and specificity were100%（30/30）and95.0%（19/20）. 3. Antibodie preparation and epitopes identification of herpes B virus gC and gDRecombinant gC and gD immunized mice to produce antiserum or used as ligands topurify antibodies from positive monkey sera. Only antibodies against envelop glycoprotein Dcould inhibite BV infection in Vero cell, but antibodies to gC is not.Using bioinformatics analysis the structure of monkey B virus gC and gD gene,hydrophilic, flexible, structure regions, antigenicindex and B-cell epitope. Then, potentialepitope peptides were synthetized and identified by standard positive sera. The resultsshowed that4peptides on gD (⁴⁶LPPLEQKTD⁵⁴,¹⁰⁶RGAPEATRSDA¹¹⁶,²⁹¹PELAPEERGTSRTPGD³⁰⁶ and³⁶¹AVYLVRRRGR³⁷⁰) and3peptides on gC(³²STPAGRPGASRPGGVKRANR⁵¹,⁵³AAPARGRGSSNGTGPGSTSAQFRCRRPD⁸¹ and²⁹⁸RDSVSFSRRNAT³⁰⁹) reacted with positive sera. The sensitivity ranged from40.0%⁸0.0%and specificity was100%.