Analysis of full-length gene sequence of rabies vaccine virus aG strain / 病毒学报

To sequence and analyze the full-length gene sequence of rabies vaccine virus aG strain. The full-length gene sequence of aG strain was amplified by RT-PCR by 8 fragments,each PCR product was cloned into vector pGEM-T respectively, sequenced and assemblied; The 5' leader sequence was sequenced with method of 5' RACE. The homology between aG and other rabies vaccine virus was analyzed by using DNAstar and Mega4. 0 software. aG strain was 11 925nt(GenBank accession number: JN234411) in length and belonged to the genotype I . The Bioinformatics revealed that the homology showed disparation form different rabies vaccine virus. the full-length gene sequence of rabies vaccine virus aG strain provided a support for perfecting the standard for quality control of virus strains for production of rabies vaccine for human use in China.

Analysis of full-length gene sequence of a rabies vaccine strain CTN-1 for human use in China / 病毒学报

CTN-1 is one of the rabies vaccine strains for human use in China, but there has been no report on the full-length gene sequence of CTN-1. In this study, the full-length gene of CTN-1 was amplified by RT-PCR, each PCR product was cloned into T vector and then sequenced, assemblied and compared with other vaccine strains as well as the wild Chinese rabies isolates. The phylogenetic tree of G gene was constructed and the genetic homology was analyzed. The results revealed that CTN-1 was 11 925nt (GenBank accession number: FJ959397)in length and belonged to the genotype I. The full-length nucleotide homologies among CTN-1 and other rabies virus strains were between 81.5%-93.4%, of which the lowest 81.5% was between CTN-1 strain and bat isolate SHBRV, and the highest 93.4% was between CTN-1 and Chinese isolate HN10. The phylogenetic analysis revealed that the majority of Chinese isolates could be grouped into the same clade with the CTN-1 strain, but aG and some vaccine strains from abroad such as Flury, PM, PV, ERA, RC-HL and a few Chinese strains were grouped in another clade. Comparsion of the G protein genes also showed that the homologies among CTN-1 and most of the Chinese isolates were higher than that of the other vaccine strains to those Chinese strains. Therefore, it suggests that the CTN-1 strain is more suitable and rational to be used for the production of rabies inactivated vaccine in China than the others.

Hantavirus mucosal vaccine through different mucosal with heat-labile enterotoxin B subunit as adjuvants / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluation the effect of different mucosal vaccination pathway on hantavirus with the recombinant E. coli heat-labile enterotoxin B subunit (rLTB) as adjuvant.</p><p><b>METHODS</b>The rLTB was expressed and purified. Take the inactivated hantavirus strain 84Fli as vaccine, and immunized C57 BL/6 mice through intranasal, oral and vaginal respectively. Specific IgG and sectory IgA were detected by ELISA in serum, and vaginal washing samples respectively.</p><p><b>RESULTS</b>The rLTB was efficiently expressed under the induction of lactose, identified by western blotting and GM-1 binding experiment. The vaccination through intranasal, oral and vaginal, can induce IgG and sectory IgA response.</p><p><b>CONCLUSION</b>Inactivated hantavirus can produce mucosal immune response with rLTB as adjuvants through intranasal, oral and vaginal vaccination respectively.</p>

Recombinant envelope glycoprotein domain III of dengue virus inhibit virus infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the ability of dengue virus recombinant envelope protein domain expressed in E. coli to inhibit virus infection and induce the neutralizing antibody.</p><p><b>METHODS</b>E III protein of Dengue virus serotypes 1-4 were expressed in E. coli BL21(DE3) then purified. Recombinant proteins were tested to inhibit DV2 from infecting BHK-21 cell. Rabbits were immunized with recombinant proteins to produce anti-E III serum. Antibody titers were determined by neutralizing assay.</p><p><b>RESULTS</b>The recombinant E III proteins of Dengue virus serotypes 1-4 were expressed in E. coli. They effectively protected BHK cells in culture against DV2 infection. All four type anti-E III sera can neutralize DV2 but their efficacies are different.</p><p><b>CONCLUSION</b>proteins of dengue virus expressed in E. coli can directly inhibit DV2 infection. Neutralizing antibodies were induced by E III proteins. Both E III protein of dengue virus and the neutralizing antibodies they induced are more efficient in inhibiting homologous dengue serotypes infection than heterologous serotypes.</p>

Prokaryotic expression and antigenic activity analysis on the matrix protein genes of two strains of human metapneumovirus recently identified in Beijing / 病毒学报

Human metapneumovirus (hMPV) is a recently identified respiratory virus more like human respiratory syncytial virus in clinical symptoms. Matrix protein (M) is one of the most important structural proteins. For further studying of hMPV, the full length of M genes from the recombinant plasmid pUCm-M1816 and pUCmM1817 were cloned by PCR and sub-cloned into the pET30a(+) vector, which is a prokaryotic expression vector, after dual-enzyme digestion with Bam HI and Xho I. The positive recombinated plasmids were transformed into E. coli BL21 (DE3) and expressed under the inducing of IPTG. Target proteins were characterized by SDS-PAGE and Western blotting. In this article, we' ve successfully constructed the recombinated plasmids pET30a-M1816 and pET30a-M1817 which have correct open reading frames confirmed by dual-enzyme digestion analysis and sequencing. The fusion proteins with 6 x His-N were highly produced after inducing by 1mmol/ L IPTG at 37 degrees C. A unique protein band with approximate 27.6 kD was characterized by SDS-PAGE. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the matrix protein of hMPV. So the M genes were highly expressed in the prokaryotic system and the expressed M proteins have specific antigenic activities. It can be used for further studying of hMPV infections in Beijing.

Rescue of minireplicon by using the cell line stably expressing the T7 RNA polymerase / 病毒学报

To improve the rescue efficiency of measles virus cDNA clone, the cell line that stably expressed the T7 RNA polymerase was established. Firstly, the T7 RNA polymerase gene was amplified by PCR and then the PCR product was inserted into pcDNA3 to obtain plasmid pcDNA3-T7. Vero cell was transfected with the plasmid and G418 was added to the cell 24h later to kill the cells without the plasmid. Western blotting analysis showed that the Vero/pcDNA3-T7 cell could express T7 RNA polymerase. To analyze the gene function of T7 RNA polymerase, the pT7IP-EGFP plasmid was transfected into the Vero/pcDNA3 T7 cell and EGFP was analized by fluorescence. The result suggested that T7 RNA polymerase expressed in the Vero/pcDNA3-T7 cell could transcribe the gene under control of the T7 promoter. Moreover, the minigenome PminiEGFP inserted reversely with report gene EGFP was established. After trans fection with the plasmid and infection with measles virus, EGFP was expressed, indicating the Vero/pcDNA3-T7 cell could rescue the minigenome.

Detection of IgM antibody against hantavirus by chemiluminescent enzyme-linked immunosorbent assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a chemiluminescent enzyme-linked immunosorbent assay (CLEIA) for the detection of HTNV IgM antibody.</p><p><b>METHODS</b>Black solid 96 well microplate was coated with anti-human IgM-microantibody, HRP labeled HTNV recombinant nucleotide antigen was used as detection antigen, luminol-H2O2 was used as substrate, a CLEIA was established for the detection of HFRS patient serum IgM antibody and comparison of detection sensitivity, specificity, and stability were made between CLEIA and MacELISA.</p><p><b>RESULTS</b>Correlate coefficient of CLEIA with MacELISA is 0.97; detection sensitivity of CLEIA is 100 percent while that of MacELISA is 92.1 percent; detection specificity of CLEIA and MacELISA are both 100 percent; coefficient of variance for intra-assay and inter-assay of CLEIA are both less than 15 percent, which are comparative with MacELISA.</p><p><b>CONCLUSION</b>The established method of CLEIA is a sensitive, selective, and stable method; it is suitable for the early detection of HFRS patient serum IgM antibody.</p>

Seroprevalence of antibody against human metapneumovirus in Beijing / 中华儿科杂志

<p><b>OBJECTIVE</b>To understand the seroprevalence of antibody against the newly identified human metapneumovirus (hMPV) in Beijing.</p><p><b>METHODS</b>The antigenic specificity of hMPV N protein cloned into vector pET30a and then expressed in E coli was verified by using SDS-PAGE and Western blotting in 116 serum specimens. The plasmid pET30a without insert was used as control. Totally 710 serum specimens collected from non-respiratory infection patients visited the Outpatient Departments of Children's Hospital affiliated to Capital Institute of Pediatrics and Xuanwu Hospital, Beijing from April 1996 to March 1997 were tested for specific IgG antibody against hMPV N protein.</p><p><b>RESULTS</b>The bands with expected molecular weight showed only on the membranes transferred by the expressed hMPV N protein and incubated with rabbit hyperimmune serum against hMPV N protein polypeptides as well as the collected human sera, indicating the specificity of the expressed hMPV N protein. Out of 710 specimens tested, 17.2% (122/710) were positive for antibody to N protein. Antibody positive rate was the lowest in 2 to 6 months old infants (3.1%); the rate declined from 13.2% in newborns to 6.1% in 1 to 2 months old infants, then to 3.1% in the 2 to 6 months group, and sustained at about 3.0% from 6 months group to 30 years of age, then increased to 28.1% in 30 to 39-year-old adults, 32.3% in 40 to 49-year-old adults and to 38.5% in the group over age of 50 years.</p><p><b>CONCLUSION</b>The expressed hMPV N protein is reliable when it was used as antigen for testing specific IgG antibody against hMPV in human sera. The high seroprevalence of antibody against hMPV N protein and early age antibody acquisition suggest that hMPV has been circulating in Beijing and the importance of the virus as pathogen should be further analyzed.</p>