Study an E protein epitopes and primary identification of main yellow virus / 中华流行病学杂志

Objective To analysis the E protein epitopes of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus and to distinguish the shared or specific epitopes among them. Methods Bioinformatic software DNAStar was used to analyze the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E prtein amino acid sequences. The influence of secondary structure was also considered. Based on the bio-informatic analysis of E protein epitopes, 6 specific epitopes were amplified and inserted into prokaryotic expression vector pMAL-c2x. The vectors was then transferred into E.coli BL21 (DE3) and Rosetta (DE3). Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of gene segments and SDS-PAGE were used identify the expression proteins. The antigenieity was tested, using Western blot. Results 15 shared epitopes and 47 specific epitopes were forecasted by bioinformatic analysis, and 6 specific epitopes from dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E protein were expressed in E.coli successfully. Two specific antigenic determinant from dengue virus type 1 and dengue virus type 2 were confirmed using Western blot, while the others epitopes shown no antigenic reaction property. Conclusion Two specific antigenic determinant were confirmed, under Western blot.

Detection and typing of dengue virus using polymerase chain reaction and microwell plate hybridization / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a specific, sensitive and practicable method for detection and typing of dengue virus.</p><p><b>METHODS</b>Based on the genomic sequence analysis of dengue virus types 1-4, 4 pairs of primers were designed. The specific capture probes of dengue virus types 1-4 were amplified using RT-PCR, cloned and sequenced before using them for precoating the microwell plate. The samples were amplified using biotin-labeled forward primer and reverse primer, and microwell plate hybridization was carried out for detection and typing of dengue virus types 1-4.</p><p><b>RESULTS</b>The absorbance of the positive samples were higher than 0.5, while the average absorbance of the negative samples was lower than 0.1, with the S/N higher than 10.</p><p><b>CONCLUSION</b>The method of PCR-ELISA we established for early detection and typing of all 4 dengue viruses seretypes.</p>

Development of multiplex reverse translation-polymerase chain reaction methods for detection of dengue virus type 1-4 and its application in clinical use / 中华流行病学杂志

<p><b>OBJECTIVE</b>To develop multiplex reverse translation-polymerase chain reaction (RT-PCR) method for detection of dengue virus type 1-4.</p><p><b>METHODS</b>Based on the genomes sequence analysis of dengue virus type 1-4, four-pair of primers were designed. The specificity of the primers was primarily tested by searching the GenBank DNA sequence database. The optimal reaction conditions of the multiplex RT-PCR were then established. The specificity of RT-PCR was tested using the homologous yellow fever virus and Japanese encephalitis virus. 30 serum samples of dengue virus from suspected sufferers in the prevalence of dengue virus in 2003 were detected using the methods we developed.</p><p><b>RESULTS</b>Positive segments about 295, 237, 118, 347 bp could be seen in the multiplex RT-PCR production of dengue virus type 1-4, respectively. There were no positive segments in the RT-PCR productions of Japanese encephalitis virus and yellow fever virus. 25 of the 30 serum samples showed dengue virus type 1 positive results, while the sequencing results suggesting the amplification sequence having a high homology with dengue virus type 1 strain Cambodia, GD14/97 and GD05/99 (97%, 97%, 98%, respective).</p><p><b>CONCLUSION</b>The method of multiplex RT-PCR we established could be used for early detection and identification of dengue virus type 1-4.</p>

Isolation, identification and sequence analyses of dengue virus type 2 strain GD19/2001 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To identify the virus isolated from Jiangmen, Guangdong province and to discuss the possible origin.</p><p><b>METHODS</b>Using characteristics of indirect fluorescent antibody tests (IFA), reverse transcription-polymerase chain reaction (RT-PCR), mouse neurovirulence and cell culture to identify the isolated virus. According to the nature of dengue virus type 2 NGC strain, two pairs of primers were designed. The structural protein gene of isolated dengue virus type 2 strain was then amplified by RT-PCR, cloned into pMD18-T vector and sequenced.</p><p><b>RESULTS</b>Twenty-two of 37 serum samples showed a positive reaction to dengue antibody IgG, and 36 of 37 with IgM with the highest antibody titer 1:640. Ten samples were resulted in a cytopathy on C6/36 cells and showed a neurovirulence in suckling mice when inoculated intracerebrally. The structural gene of new isolate GD19/2001 containing 2 325 nucleotides which encoded 774 amino acids. Data on nucleotide homology were 98%, 96%, 94%, 94%, 92%, 92%, 92% and 91% compared with TSV01, GD06/93, NGC and 44, ThNH81/93, 04 and GD08/98, and S1 respectively.</p><p><b>CONCLUSION</b>The isolated virus from Jiangmen, Guangdong province belonged to dengue virus type 2, which might come from Australia.</p>