Genotype of hantavirus in Zhejiang province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To identify the genotype and clades of hantavirus (HV) in Zhejiang province.</p><p><b>METHODS</b>The partial S and M segment of the HV in Zhejiang province were amplified with RT-PCR using genotype-specific primers, and then were sequenced and compared with other known hantaviruses.</p><p><b>RESULTS</b>The genotype of 11 strains were HTNV and other 7 strains were SEOV by homology and phylogenesis analysis, yet the clade distribution was significantly different among foci of Zhejiang with 5 clades of HTNV and 3 clades of SEOV. There also existed special clade of HTNV named ZNB-1, ZNB-2, A3 and of SEOV named Gou3, ZJ5. The homology of M segments of ZNB-1 and ZNB-2 with other HTNV clades were 69.7%-74.0% except Nc167, A3 with other HTNV clades were 73.6%-76.3% except B78.</p><p><b>CONCLUSION</b>Zhejiang province is co-circulating with HTN and SEO. Say the least of the clades are 5 of HTNV and 3 of SEOV and there also existed special clade of HTNV and SEOV.</p>

The expression and bioactivity of S gene in Vero-E6 cells using baculovirus vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The S gene of a Hanta Virus (HV) Z10 strain was cloned into a baculovirus shuttle bacmid pDual-CMV contained a CMV promoter to generated a recombinant baculovirus BAC-pDual-CMV-HVS, then the recombinant baculovirus was transfected into Vero-E6 cell. The cells with recombinant baculovirus were applied to the detection of HV antiserum.</p><p><b>METHODS</b>To generate the recombinant baculovirus BAC-pDual-CMV-HVS, the sequence of CMV promoter was obtained from the plasmid pEGFP-N1 by PCR, and subsequently cloned to the baculovirus shuttle bacmid pFastBacDUAL resulting the recombinant plasmid pDual-CMV. Then the sequence of HV-S gene was inserted to the plasmid pDual-CMV, to generate the plasmid pDual-CMV-HVS. Plasmid pDual-CMV-HVS was transformed into the DH10BAC competent cells to get the recombinant baculovirus BAC-pDual-CMV-HVS. The antigen substrate slides were made by transfecting the recombinant virus into Vero-E6 cells.</p><p><b>RESULTS</b>The plasmid pDual-CMV-HVS was verified by sequencing. The recombinant virus BAC-pDual-CMV-HVS was generated according to the protocol of the baculovirus and transfected into Vero-E6 cells. The expression of the HV-S gene was verified by positive HV antiserum.</p><p><b>CONCLUSION</b>[corrected] The recombinant virus were successfully generated and applied to prepare the antigen substrate slides. The antigen substrate slides was conveniently prepared without special equipments, and can be used to detect the antiserum of HV virus.</p>

Molecular evolution analysis of hantaviruses in Zhejiang Province / 病毒学报

In order to analyze the molecular epidemiology of Hantavirus (HV) in Zhejiang Province, the complete M and S genome sequences of 12 HV strains from different hosts and locations in Zhejiang Province of China during the period of 1981-2007 were analyzed on genetic evolution by DNAstar and MEGA 4.0 software in this research. Phylogenetic analyses revealed that HTN and SEO strains were co-circulating in Zhejiang Province, and the difference in sequence similarity and the phylogeny was closely related to the isolated regions, but had no distinct relationship with the isolate year and the host, indicating a relationship between epidemiology of HFRS and the distribution region, especially in HTNV. The isolates in the same region could be assigned in same or near phylogenetic clade sharing high sequence similarity. Interestingly, the Gou3 strain and ZJ5 strain isolated from Jiande region in Zhejiang Province formed a distinct phylogenetic lineage in SEOV clade, and different from the other SEOV variants outside China. We believed that the special SEOV variants were distributed in Jiande region.

The distributions of mosquito vectors carrying Japanese encephalitis virus in Zhejiang province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the natural infection and genotype of Japanese encephalitis virus in mosquitoes and swine in some areas of Zhejiang province.</p><p><b>METHODS</b>Samples of mosquitoes and sera of swine were collected in three counties (Xianju, Longquan and Cixi) of Zhejiang province from May to October in 2007. 10 662 mosquitoes were collected during 2007, of which, C.pipiens pallens and C.quinquefasciatus were the most dominant species and 204 pig serum samples were detected. Japanese encephalitis virus in mosquitoes were detected by virus isolation and real time RT-PCR. The antibody against Japanese encephalitis virus in swine was detected by ELISA. The isolated strain were identified by real time RT-PCR. The PrM gene of the isolated strain was amplified by RT-PCR. Three strains were typed by the gene of PrM.</p><p><b>RESULTS</b>Seven positive mosquitoe samples were identified by real time RT-PCR. Three strains were isolated and identified by real time RT-PCR. The PrM gene was cloned and sequenced. The phylogenetic analysis showed that three isolates belong to genotype I of Japanese encephalitis virus. Of 204 swine serum samples, 121 positive samples were identified positives. Above 50% sera samples from swine were positive in June.</p><p><b>CONCLUSION</b>The vector of Japanese encephalitis virus existed and carried the Japanese encephalitis virus in these areas of Zhejiang province. Three strains of Japanese encephalitis virus were isolated from mosquito pools collected in Zhejiang province. It should be the first isolation of genotype I Japanese encephalitis virus in Zhejiang province in recent years.</p>

Study on the difference of genes and the type identification of hantavirus from Lishui,Zhejiang province / 中华流行病学杂志

Objective To isolate hantavirus from Lishui county-one of the epidemic regions for hemorrhagic fever with renal syndrome(HFRS),in Zhejiang province,and to identify the serotype and molecular/biological characteristics of a new HTN subtype hantavirus(HV)strains,hopefully to provide evidence for HFRS prevention and therapy.Methods Data on the host animals was collected from Lishui,Zhejiang province in 2007.Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues from HV infeeted Vero-E6 cells for HV isolation,then total RNA was extracted from Hantavirus Lishui strains and amplified by RT-PCR M,S segments of strains genome were also clened and sequenced and compared with those of other strains of HV Results 2 strains virus(ZLS6-11 and ZLS-12)Were successfully isolated from 7 positive lung samples of mice and were identified as HTNV by anti-McAb and phylogenetic analysis.With sequence compation.We found that 2 strains with complete M and S segment had higher homology with HTN-type strains than with other types of HV,but 13.4%-20.7%and 10.3%-16.1%of the genes were found which were difierent from HTNV.The phylogenetic trees constructed by complete S and M segment showed that ZLS6-11 and ZLS-12 strains were located in HTNV group,and structured independent embranchment.Conclusion ZLS6-11 and ZLS-12 Strains were believed to belong to HTN-type and phylogenetically different from the HTNV.

Complete genome sequence analysis of Japanese encephalitis virus newly isolated in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the complete genome sequence of Japanese encephalitis virus (JEV) strain XJ69 isolated in ZheJiang province and explore its evolution.</p><p><b>METHODS</b>Overlapping primers were designed according to the full-length genomes from GenBank. RT-PCR was used to amplify the fragments and RT-PCR products were cloned T vector, sequenced and analyzed.</p><p><b>RESULTS</b>The genome of strain XJ69 and XJP613 were 10 964 nucleotides in length with a single open reading frame encoding 3432 amino acids. Comparison of the complete genome sequences of different JEV isolates showed XJ69 and XJP613 were 83.5%-99.2% and 83.4%-99.4% nucleotide sequence homology among them respectively, which resulted in 94.8%-99.7% amino acid sequence homology. Phylogenetic analysis through PrM/C,E and full-length genome showed that the XJ69 and XJP613 strain belonged to genotype I.</p><p><b>CONCLUSION</b>The nucleotitede sequence and deduced amino acid sequence of XJ69 and XJP613 strain were similar to that of those of genotype I of Japanese encephalitis virus. It belonged to genotype I and were close to the isolates SH17M-07.</p>

Expression of partial S gene of Hantavirus Z10 in Pichia pastoris / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The purpose of this study is to express partial S gene of Hantavirus Z10.</p><p><b>METHODS</b>The 300 bp S gene of Z10 strain was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris and subcloned into pMD19-T. The SP300 gene was constructed into pPICZaA and sequenced. The recombinant pPICZaA-SP300 and pPICZaA-S300 was transformed into Pichia with LiCI.</p><p><b>RESULTS</b>The recombination Pichia were cultivate, and expressed the SP300 or S300 gene induced in Pichia by methanol.</p><p><b>CONCLUSION</b>The nucleocapsid secreted from the Pichia can be detected by ELISA and WesternBlot.</p>

Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus / 中华流行病学杂志

Objective To establish a TaqMan based real-time reverse transeription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus. Methods The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. Results Mg2+, primer and probe were optimized at 5 mmol/L, 0.2 μmol/L and 0.1 μmol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0.1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. Conclusion This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.

Isolation and identification of Japanese encephalitis virus from mosquitoes in Zhejiang province / 中华流行病学杂志

Objective To study the situation of arboviruses carried by mosquitoes in Zhejiang province.Methods Mosquitoes were collected from Zhejiang province in 2007.Virus strains were isolated by the inoculation of homogenates of the mosquitoes onto BHK-21 cell line.The isolated strains were identified by serological(IFA)and molecular methods(RT-PCR).Results Two strains were isolated from mosquitoes causing cytopathogenic effect(CPE)in BHK-21 cells.Results from serological tests showed that both of the two strains were positive for the antibody to Japanese encephalitis virus(JEV).PrM and E gene were then cloned and sequenced.Results from the phylogenetic analysis showed that the isolates belonged to genotype Ⅰ JEV while through sequence analysis it showed that the homology of nucleotide sequences and amino acid sequences between the two strains were 99.0% and 98.8% respectively.Compared with the JEV vaccine strain SA14-14-2 and two strains,the homology of nucleotide sequences Was up to 87.7% and homology of amino acid sequences was up to 96.4%.When comparing with the vaccine strain SA14-14-2,there were 14 common amino acid variations in all the two strains.Conclusion Two strains of JEV were isolated from mosquitoes collected in Zhejiang province whiCh was the first isolation of genotype Ⅰ JEV in the province in recent years.

Molecular characterization of hantavirus Zhejiang isolate ZT10 strain from M. fartis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To learn about the complete genomic sequence of the Seoul virus strain ZT10 isolated from M. fartis.</p><p><b>METHODS</b>The total RNA was extracted from the infected Vero E6 cells and amplified by RT-PCR. The purified PCR products were cloned into T-vector and sequenced.</p><p><b>RESULTS</b>The results demonstrated that the complete genome of ZT10 was comprised of L(6530), M(3651) and S(1753) segments which encoded 2151-1133 and 429 amino acids respectively.</p><p><b>CONCLUSION</b>Analysis of sequence revealed that the ZT10 belonged to Seoul virus. The nucleotide sequence identity of the M gene with Seoul virus was 84.0%-96.3%. The identity with Hantan vrisu (Prospect Hill virus, Tula virus) isolated from M. fartis was 57.5%-60.9%. The sequence identity of the S gene with Seoul virus was 87.9%-96.0% at nucleotide level and 96.9%-97.9% at amino acid level.</p>

Molecular characterization and sequence comparison of the M and S segments of Hantavirus ZJ5 strain / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to understand the molecular characters of Hantavirus ZJ5 strain, its complete M and S genome were sequenced and compared with that of other hantavirus strains.</p><p><b>METHODS</b>We prepared the total RNA from ZJ5. Infected cells and the raw or purified RT-PCR product was cloned and sequenced.</p><p><b>RESULTS</b>With sequence compation, we found ZJ5 strain complete M and S segment had higher homology with SEO-type strains than other type of HV, but differential genes were 11.7%-19.2% and 6.7%-14.5% from SEOV. The phylogenetic trees constructed by complete M ind S segment showed that ZJ5 strain was located in SEOV group, and structured alone embranchment.</p><p><b>CONCLUSION</b>The ZJ5 strain was believed to belong to SEO-type virus,and suggest that ZJ5 strain is a new subtype S SEOV group,and structured alone embranchment.</p><p><b>CONCLUSION</b>The ZJ5 strain was believed to belong to SEO-type virus, and suggest that ZJ5 strain is a new subtype from other SEO viruses.</p>

Study on the recombinant expression of Hantaan virus protein N and the establishment and application of rNP-IgM direct capture ELISA / 中华流行病学杂志

<p><b>OBJECTIVE</b>To clone the gene encoding nucleocapsid protein (NP) of hantavirus strain Z10 (HV-Z10), to construct its prokaryotic expression system as well as to establish a rNP-IgM direct capture ELISA based on HRP-labeled recombinant NP (rNP), in order to detect serum samples of patients suffering from hemorrhagic fever with renal syndrome (HFRS) and to evaluate the effects of detection.</p><p><b>METHODS</b>Gene encoding NP of strain HV-Z10 was amplified by PCR and then its prokaryotic expression system pET28a-Z10N-E. coli BL21DE3 was constructed, using routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Western blot assay was used to determine the specific immuno-reactivity of rNP while HRP-labeled rNP-IgM direct capture ELISA was established to detect the serum samples from 95 cases of confirmed HFRS patients. The detection effect was compared with that by routine HV-IgM indirect capture ELISA method.</p><p><b>RESULTS</b>pET28a-Z10N-E. coli BL21DE3 was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV-IgG could efficiently recognize rNP and hybridize with the recombinant protein. 94.73% (90/95) of HFRS patients' serum samples were positively confirmed by rNP-IgM direct capture ELISA, while a positive rate of 92.63% (88/95) in the same samples was confirmed by HV-IgM indirect capture ELISA. The distributions of A450 values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the A450 mean values from several serum samples with different dilutions were similar.</p><p><b>CONCLUSION</b>We successfully constructed a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10. The rNP-IgM direct capture ELISA that established in this study could be used as a new serological test for HFRS diagnosis because of its simplicity, safety, with high sensitivity and specificity.</p>