| Rabies street virus targets the central nervous system (CNS) and causes fatal encephalitis in almost all species of mammals, the case fatality ratio (CFR) is100percent approximately. Rabies virus belongs to the genus Lyssavirus of the family Rhabdoviridae and has an unsegmented negative-sense RNA as the viral genome. The genome is about12kb in length and encodes five genes for the structural proteins:NP, PP, MP, GP, and LP. NP, PP, LP and the viral genomic RNA compose a ribonucleoprotein complex (RNP), only this one could initiate viral gene replication and expression.China is one of the most severe regions of rabies, and the number of rabies cases is increasing consecutively during recent years. The dead cases are about3000-4000every year in china in last several years.The data show that the93%of human rabies cases was caused by canine bite injure, so canine immunized rabies vaccine is most important to prevent from human rabies.To prepare live viral vector vaccines of rabies virus and convenient method for virus neutralization antibodies (VNA) detecting, based on the establishment of reverse genetic operation system of rabies virus Flury-LEP strain, this strain is widely used as inactivated vaccine of human rabies, and live-attenuated vaccine and inactivated one of animals in China, and its immunogenicity is very good. A recombinant rabies virus LEP-EGFP strain expressing enhanced green fluorescent protein (EGFP) was generated and analyzed. Our research on recombinant virus LEP-EGFP provided a useful platform for development of novel polyvalent vaccine and gene modified vaccine, and convenient, economical and reliable method for virus neutralization antibodies (VNA) detecting.1. To prepare rabies virus vector, based on establishment of reverse genetic system of rabies virus Flury-LEP strain, Flury-LEP cDNA with4096nt Pme I mutant was constructed by SOE-PCR, and the primers was devised according to acquired sequence. EGFP ORF as a reporter gene was modified with start point and end point of large transcription protein of rabies virus, and this one was inserted in Pme I of a full length cDNA clone, named PCI-LEP-eGFP.293T Cells were transfected with PCI-LEP-EGFP, PCAGG-N, PCAGG-P, PCAGG-L, the result showed that LEP-EGFP was successfully rescued.2. This study aimed to obtain the biological characteristics of LEP-EGFP vector. NA cells and BHK-21cells were infected with the wild type LEP (wtLEP) or LEP-EGFP virus at MOI=0.01. At24,72and120h after the infection, supernatants of the culture fluid were subjected with virus titration assay, the replication kinetics of LEP-EGFP did not differ significantly from one of wtLEP. Bal/BC mice were infected by intracerebral inoculation with wtLEP and LEP-EGFP, LEP-EGFP LD50=6.3FFU is similar in pathogenicity to wtLEP LD50=8.8FFU. The EGFP expression of LEP-EGFP was stable for at least nine passages in BHK-21cells.3. To obtain whether the immunogenicity of live viral vector of LEP-EGFP was influenced by EGFP expression, convenient method for virus neutralization antibodies (VNA) detecting was prepared. Seven-week-old Bal/BC was immunized with105FFU wtLEP and LEP-EGFP at medial vastus muscle, and the antibody titer of serum after three weeks showed good agreement (P>0.05). The LEP-EGFP strain could be used as challenge virus in the50%virus neutralization assay of canine serum from beagles immunized Flury-LEP live-attenuated vaccine and inactivated one after three weeks, the results obtained by the both LEP-EGFP method and IF AT showed good agreement (P>0.05)Taking above-mentioned data, this study can be concluded that LEP-EGFP is similar in pathogenicity, the replication kinetics and immunogenicity to that of the wild type. The EGFP expression of LEP-EGFP was stable for at least nine passages. The result between LEP-EGFP of VNA assay and IFAT showed good agreement. It might play a significant role in developing recoment rabies vaccine and VNA assay. |
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