[Construction and recovery of chimeric rabies virus expressing envelop proteins E1E2 of hepatitis C].

OBJECTIVE: Construction and recovery of chimeric rabies virus expressing HCV envelop proteins E1E2. METHODS: On the basis of the previously established reverse genetic system CTN-GFP, HCV E1E2 genes were cloned to both replication competent and replication constrained viral vectors based on CTN181 strain and the chimeric viruses CTN-HCV E1E2 and CTNdeltaG-HCV E1E2 were recovered. RESULTS: The result demonstrated that both the chimeric viruses were rescued successfully, had the ability to re-infect normal sensitive cell lines and express HCV E1E2 genes detected in the level of mRNA. CONCLUSION: The establishment of chimeric RVs expressing HCV E1E2 genes provides the evidence that it is feasible to develop novel HCV vaccines based on viral vectors in theory and in practice.

[Expression and characterization of rabies virus nucleoprotein in baculovirus].

To construct a recombinant expression plasmid Bacmid-N containing the N gene of Rabies Virus, the N gene of RV CVS-11 strain was cloned into the baculovirus shuttle vector (Bacmid). Recombinant Baculovirus AcMNPV-N (P1 Viral stock) was obtained by transfecting the Bacmid-N into the insect cell line of Sf9. The expressed nucleoprotein was identified and analysised by ELISA, FA, SDS-PAGE and Western blot assays. The results showed that the NP protein was expressed intracellularly and had a good antigenicity, which would be potentially used for further study on the diagnostic reagent of rabies virus detection.

[Construction and analysis of full-length cDNA clone of rabies virus street strain].

To construct a expression plasmid containing the full-length cDNA of rabies virus, four overlapped fragments covering full length cDNA of rabies virus street stain HN10 were cloned into pVAX1 sequentially in the genome except for the G-L noncoding region which was replaced with GFP gene. The plasmid containing the full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequences and arranged under the control of the cytomegalovirus (CMV) promoter. The constructed plasmid could be directly used for the following procedure of producing the recombinant rabies virus street HN10.

[Characterization of rabies virus phosphoprotein in high prevalence provinces of China].

OBJECTIVE: Characterization of rabies virus phosphoprotein through analyzation of genetic variations about rabies virus phosphoproteins in high-incidence regions in China. METHODS: The nucleotide sequence of the P gene of Guangxi, Guizhou and Hunan provinces positive sample's were sequenced, and the P region's similarity and phylogenetic analyses were completed by using softer wares. RESULTS: The similarity of P region's nucleotide sequence is 82.1%-100%, while, the similarity of amino acid sequence is 87.5%-100%. A little variation in phosphoprotein cannot influence its biological functions. CONCLUSION: All rabies viruses isolated from Guangxi, Guizhou and Hunan provinces belong to genotype 1 and share same phylogenesis and same genome characteristic; Virus distribution presents unique Characterization; Some virus isolates from Hunan province and Thailand may come from the same virus.

[The primary application of direct rapid immunohistochemical test to rabies diagnosis in China].

OBJECTIVE: Evaluation of the direct rapid immumohistochemical test (DRIT) for laboratory surveillance of rabies. METHODS: 72 brain specimens of domestic dogs or patients collected from Guizhou, Guangxi, Hunan, Anhui, Jiangsu and Yunnan provinces were detected by conventional methods including Direct Fluorescent-antibody Assay (DFA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR), and by DRIT which was newly developed in the Rabies Section of the Centers for Disease Control and Prevention in the United States. The sensitivity and specificity of DRIT were evaluated by compare of the three results. By analysis of the index including cost of experiment, technique requirement and so on, the advancement and applicability of DRIT were discussed. RESULTS: Compared with DFA and RT-PCR, DRIT will be more applicable for laboratories with limited funds and weak techniques because of its lower cost needed and simpler techniques required while its sensitivity and specificity are equal to the other two methods. CONCLUSION: DRIT is more valuable in rabies diagnosis and more applicable for extension and popularization in rabies laboratory surveillance in local CDC.

[Characterization of RdRp gene of rabies virus in China].

OBJECTIVE: Characterization of RdRp gene (L) of rabies virus aG and CTN181 strain in China. METHODS: Overlapped fragments were amplified and assembled. Then characterization and phylogenetic analyses as well as prediction of functional regions were performed using biologic softwares. RESULTS: L gene of CTN181 and aG strains were composed with 6387nt and 6384nt respectively and there were two repeated ATG in the start of L gene in CTN181 strain. In addition, some mutations and new functional regions were discovered and presumed to be crucial to the role of these regions in replication of rabies virus. Analyses of L gene provide new insight into the functional regions, and phylogenetic analyses showed that human vaccine strain aG were independent to others recommended by WHO. CONCLUSIONS: The characterization of complete L genes will be helpful for research on new functional regions, antiviral drugs targeted at RdRp and phylogenetic analyses.