2009 evolution for VP4 of enterovirus 71 strains in Shenzhen / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the genetic evolution for the common causative agent of hand, foot and mouth disease(HFMD) that VP4 of human enterovirus 71 in Shenzhen district.</p><p><b>METHOD</b>491 sttol specimen were collected from, children with hand, foot and mouth diease in Shenzhen Children's Hospital 2009. After cell culture, VP4 gene of eight EV71 strains were amplified by reverse-transcriptase PCR( RT-PCR), phylogenetic analysis of the VP4 gene was constructed by using MEGA 4. 0.</p><p><b>RESULT</b>The VP4 gene of eight EV71 strains encoding 69 amino acids with full length 207 bp. The nucleotide homology of VP4 gene among eight EV71 strains was 94. 2% -98. 1%, compared with VP4 gene of EV71 strains retrieved from Shenzhen 2001 to 2004 and GenBank was 89. 1%-98. 1% and 79.2%-100% respectively. Asian epidemic strain Fuyang had the highest nucleotide homology, representative strain C4 and Shenzhen strain (AY895144) with 94. 2% -98. 1% secondly. Except for the 54th amino acid of VP4 gene of India reported strain and one of the eight EV71 strains, the homology of the rest amino acids between the eight EV71 strains and those in GenBank was 100%. Compared with representative strain C4,there were seventeen differences in nucleotide sequences of VP4 of the eight EV71 strains. All of the different nucleotides were located at the degenerate password sites except one. There was no significant difference in VP4 gene between the severe and the mild cases of strainS. The eight Shenzhen EV71 strains were classified as sub-genotype C4 in the phylogenetic tree.</p><p><b>CONCLUSIONS</b>The epidemic of EV71 in Shenzhen 2009 was sub-genotype C4. VP4 gene of EV71 was very conservative which dose not belong to the variation section. The variation of most of nucleotide was invalid variation. The amino acids encoded by VP4 gene which variation was almost zero.</p>

Full sequence analysis and characterization of the Shenzhen Norovirus strain SZ2010422 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain information on viral molecular structural and evolutionary characteristics, we conducted the SZ2010422 full-length genomic analysis.</p><p><b>METHODS</b>Primers were designed by New Orleans full sequence, SZ2010422 full genome was amplified by RT-PCR, the whole genome sequence and the capsid domain amino acid sites was analysised after cloned and sequenced.</p><p><b>RESULTS</b>The genome of G II-4 Norovirus SZ2010422 strain was consist of 7559 bp, it revealed three ORFs composites of the whole genome, ORF1 (5100 bp), ORF2 (1623 bp), ORF3 (807 bp) respectively, ORF1 and ORF2 had 19 nucleotide overlap. By evolutionary comparative analysis found SZ2010422 genomic nucleotide sequences with reference strains of G II-4 New Orleans1805 strains the highest homology with a total length of homology was 99.3%, of ORF1 (99.5%), ORF2 (99.2%), ORF3 (98.6%). Phylogenetic analyses showed SZ2010422 belonging to G II-4 New Orleans variant. Date of 541 amino acid analyses showed: New Orleans variant strains of popular sites: aa310N or K, --> S aa341D --> of N, aa359T--> S, aa396H --> P, aa460H --> Y.</p><p><b>CONCLUSION</b>Norovirus SZ2010422 belonged to the G II-4 New Orleans variant. In This study, SZ2010422 full sequence can be used not only as a full-length NoV variant sequence standard for future comparison studies, but also as useful material for the public health field by enabling the diagnosis, vaccine development, and prediction of new emerging variants. Noroviruses; Genes; Sequence analysis</p>

The genotype of norovirus in Shenzhen, 2010 / 病毒学报

To describe epidemiologic characteristics of norovirus infection and its genotype in Shenzhen area of 2010. Stool specimens were collected from four monitoring point and detected by reverse transcription polymerase chain reaction (PT-PCR). Positive PCR products were purified and sequenced, and the sequences were performed by Clustal W and MEGA 4.0 programs, then genotype was identified and phylogenetic tree was constructed. Nucleotide sequence analysis revealed that 79 strains of NV belonged to norovirus genogroup II and 6 belonged to genogroup 1 of all 85 positive products. While 55 strains belonged to G II/4(2006b), 16 strains belonged to G II/4(2008 variant), 2 strains belonged to G II /1, 4 strains belonged to G II/5, 2 strains belonged to G II/11, 1 strain belonged to GII/4, 2 strains belonged to GII/5, 3 strains belonged to GI/6. The main genogroups of norovirus in Shenzhen ware GI and G II. G II /4 was one of the most major genotype of norovirus , while G II /4(2006b) variant was identified as the predominant strain in Shenzhen area.

Genetic evolution of VP1 of enterovirus type 71 in Shenzhen / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Genetic evolution of VP1 of enterovirus type 71 in Shenzhen were analyzed.</p><p><b>METHODS</b>All samples were tested by RT-PCR using EV71 specific primer. The VP1 of EV71 were amplified and sequenced. A phylogenetic tree was constructed by comparison of the sequences with subgenotype A, B and C using DNAStar, BioEdit and Mega 3.1 software.</p><p><b>RESULTS</b>Among 35 strains, the homogeneity of the VP1 nucleotide sequence was between 92.1%-100%. The homogeneity of the VP1 nucleotide sequence with subgenotype A and B was between 81.4% -91.1%. The VP1 nucleotide sequence of 35 strains of Shenzhen shared between 93% -97.4% identity with cluster C4. The prevalence strains of EV71 were cluster C4b from 1998 to 2004, and gradually moved to C4a since 2003. All of EV71 were C4b from 2006 to 2008. Also, the homogeneity of the VP1 nucleotide sequence with Anhui FY23 EV71 strain were 94.5% -94.7%, 95.7% -95.8%, 96.2%, 95.4% -97.5%, 96.3% -99.2% from 2003 to 2008. It shows that the homogeneity was increased year by year. There was a mutation (A --> C) at No. 66 nucleotide of VP1 of EV71 that two strains were isolated in 2003 and 8 strains in 2008, that caused amino acid mutation (Q --> H) at No. 22 of VP1.</p><p><b>CONCLUSION</b>EV71 C4b was gradually moved to C4a from 1998 to 2008. There was a missense mutation at No. 66 nucleotide of VP1.</p>

Detection and genotype analysis of sapovirus associated with sporadic diarrhea in Shenzhen in 2009 / 南方医科大学学报

<p><b>OBJECTIVE</b>To conduct an epidemiological and genotype analysis of sapovirus (SaV) associated with sporadic diarrhea in Shenzhen in the year 2009.</p><p><b>METHODS</b>A total of 852 fecal samples were collected from sporadic cases of diarrhea in Shenzhen in 2009 and detected by reverse-transcription polymerase chain reaction (RT-PCR) using the primers of SLV5317/5749. The PCR products were analyzed with 1.5% agarose gel electrophoresis and sequenced to construct the phylogenetic tree.</p><p><b>RESULTS</b>Sixteen samples were found positive for SaV, with a positivity rate of 1.88%. Sequence analysis identified 8 isolates as SaV GI genotype (including 3 SaV GI.1 and 5 SaV GI.2), 7 as SaV GIV genotype, and 1 as GII genotype.</p><p><b>CONCLUSIONS</b>SaV infection is present in Shenzhen with GI as the predominant genotype. This is the first report of SaV GIV strains in China, which differs from the strains of Anhui-A141 and Beijing-CHN99/BJ360, suggesting the genotypic variety of SaV infection in China.</p>

The preparation of P particle of the norovirus strain SZ9711 from China and its affinity analysis with human histo-blood group antigens in saliva / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the binding profile of NV strain SZ9711 (GII-4) with human histo-blood group antigens (HBGAs).</p><p><b>METHODS</b>The P domain-encoding fragment was amplified by RT-PCR from the stain SZ9711 and cloned into the pGEX-4T-1 vector. The recombinant fusion protein was expressed in E. coli and purified using the column Sepharose 4B. The P protein was released by thrombin cleavage. The binding of P particles of SZ9711 and VA387 with the HBGAs were measured by saliva-based EIA method.</p><p><b>RESULTS</b>The expression of the recombinant fusion protein was shown by the SDS-PAGE, in which a 38 x 10(3)-P protein was obtained. Saliva-based EIA revealed that the P particle of SZ9711 bound to HBGAs in saliva similar to that of the strain VA387 reported previously. It bound strongly to saliva of type A, B and O(secretor) but did not interact with saliva of type O(non-secretor). Noteworthy, binding ability of SZ9711 P particle to type A saliva was lower than that of the VA387 P particle.</p><p><b>CONCLUSION</b>This is the first time that a P particle was prepared from a norovirus strain isolated in China and the binding ability of the P particle with HBGAs was analyzed. The result indicated the binding profile of the SZ9711 P particle was similar to that of VA387 reported previously. These data may be valuable in studying the relationship between noroviruses and their bindings to HGBA receptors.</p>

A survey of Japanese encephalitis antibody migrant workers in Shenzhen 2009 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To understand the immunological status of Japanese encephalitis (JE) antibodies amongst migrant workers and to provide epidemiological basis for public health strategies on JE prevention and control in Shenzhen.</p><p><b>METHODS</b>A multi-stage random sampling method was used, and 1003 migrant workers aged 18 to 60 from 44 factories were investigated and their serum specimens were collected. The enzyme-linked immunosorbent assay (ELISA) was used to detect JE antibodies qualitatively.</p><p><b>RESULTS</b>The gross IgG seroprevalence rate for JE was 20.2% (203/1003). Sex-specified seroprevalence was 21.2% (103/485) for male and 19.3% (100/518) for female, respectively (χ(2) = 579, P > 0.05). Age-specific seropositive rates were 22.6% (12/53) for those below 20 years old, 18.7% (120/642) for those between 20-years old, 26.0% (58/223) for those between 30-years old and 15.3% (13/85) for those on or above 40 years old (χ(2) = 7.96, P > 0.05). Proportions for self-reported positive immunization, non-immunization and unclear immunization history were 22.1% (30/136), 22.1% (51/231) and 19.2% (122/636), respectively (χ(2) = 501, P > 0.05). Seroprevalence by region of origins showed that workers from Guangdong province was the highest (30.5%, 50/164), followed by workers from Guangxi (29.7%, 22/74) whilst workers from Shan(3)xi (5.4%, 2/37) had the lowest rate. Seroprevalence rate for managers (29.0%, 31/107) was higher than that of technicians (7.1%, 1/14) (χ(2) = 21.78, P < 0.05). Serological positive rate of workers with university or above educational background was the highest (32.7%, 16/49), followed by that for individuals with college degree (10.3%, 10/97) (χ(2) = 13.02, P < 0.05).</p><p><b>CONCLUSION</b>No associations are detected between JE seroprevalence and age, or sex, or self-reported immunization histories amongst migrant labor workers in Shenzhen. However, correlations between JE serological positive rate and region of origins, occupation and educational attainment are found to be significant. The gross seroprevalence of JE antibodies suggests that the level of JE antibodies amongst Shenzhen migrant workers is low and the population immunity barrier has yet to be established. It is necessary to strengthen prevention and control strategies of JE among labor workers of Shenzhen.</p>

Identification and sequence analysis of E gene of Dengue virus type 2 strain isolated from patient serum in Shenzhen / 中华预防医学杂志

<p><b>OBJECTIVE</b>To isolate and identify the pathogen of Dengue fever from Shenzhen city in 2005 - 2006, and to analyze the molecular characteristics of the isolated Dengue virus strain as well as to explore its possible origin.</p><p><b>METHODS</b>IgM and IgG of serum samples taken from 60 suspected Dengue fever patients were detected by ELISA and immunochromatography, and 9 specimens were positive. Nine samples from patients with early stage Dengue fever were used to isolate virus with C6/36 cell line and the positive cell cultures were identified by MGB fluorescent PCR. The type of isolated virus strain was determined by RT-semi-nested-PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of Shenzhen Dengue virus with the strains isolated from other areas were constructed.</p><p><b>RESULTS</b>Of nine antibody-positive serum samples, one strain of Dengue virus was successfully isolated. The isolated virus strain was confirmed as Dengue virus type 2 and designated as DEN2-SZ0521. The homology of nucleotide sequence and the deduced amino acid sequence of E gene of SZ0521 with standard type 2 Dengue virus NGC strain was 94.2% and 98.2%, but the homology with standard Dengue virus 1, 3, 4 in the same fragment were 59.1%, 57.2%, 58.5% and 68.1%, 66.7%, 63.2%, respectively. The phylogenetic tree indicated that SZ0521 had the greatest similarity with the Malay0412a/Tw strain and they lied in the same branch of the phylogenetic tree. The corresponding homology of nucleotide sequence and amino acid sequence was 99.8% and 100%, respectively. The isolated Dengue virus type 2 belonged to genotype IV with Indonesia-76, Somalia-84 and Sri Lanka-90.</p><p><b>CONCLUSION</b>Dengue virus was isolated from Shenzhen for the first time, and it was classified as type 2. It was confirmed that the type 2 Dengue virus may come from the epidemic area in Malaysia.</p>

Genetype characteristics of enterovirus type 71 in Shenzhen from 2005 to 2008 / 病毒学报

Genetic characteristics of enterovirus type 71 in Shenzhen from 2005 to 2008 were analyzed. All samples were detected by RT-PCR using EV71-specific primers. The VP1s of EV71 strains were amplified and sequenced. A phylogenetic tree was constructed by comparison of the sequences with those of subgenotype A, B and C using DNASTAR, BioEdit and Mega 3.1 softwares. The VP1 nucleotide sequences of 17 strains of Shenzhen shared 95.3%-99.4% identities with cluster C4a, and one strain shared 93.0%-95.6% identities with cluster C4b. The homogeneity of the VP1 nucleotide sequence with subgenotype A and B was 81.8%-86.0%. Among 18 strains, the homogeneity of the VP1 nucleotide sequence was between 92.5%-100%. All EV71 strains circulating in Shenzhen were of subgenotype C4. The predominant strain of EV71 belonged to cluster C4a, also there was cluster C4b.

Genotype analysis of enterovirus type 71 detected from patients with hand-foot-mouth disease in Shenzhen / 中华流行病学杂志

<p><b>OBJECTIVE</b>To carry out genotype analysis of enterovirus type 71, detected from hand-foot-mouth disease patients in Shenzhen in 2004.</p><p><b>METHODS</b>All samples were tested by reverse transcription-polymerase chain reaction (RT-PCR) using EV71 specific primer. The VP1 and VP4 of EV71 were cloned and sequenced. A phylogenetic tree was constructed by comparing the sequences with genogroups A, B and C genotypes using TreeView and PHYLIP software (3.6b).</p><p><b>RESULTS</b>The VP1 nucleotide sequence of 4 strains isolated from Shenzhen shared 87.8% - 92.0% identity with genogroup C, while the homogeneity of the VP4 nucleotide sequence was between 85.9% - 87.4%. The homogeneity of the VP1 nucleotide sequence with genotypes A and B was between 81.9% - 84.2% and was 80.6% - 85.0% with VP4. Among the 4 strains, the homogeneity of the VP1 nucleotide sequence was between 94.1% - 99.8% and was 100% with VP4 which formed a small group and could denominate EV71 genetype C4.</p><p><b>CONCLUSION</b>Similar results were obtained from phylogenetic analysis of EV71 based on VP1 and VP4 nucleotide sequence. The four EV71 strains causing epidemic in Shenzhen could be named as C4 subgroups.</p>

Surveillance on natural infection of rodents with hantavirus in Shenzhen city and identification of a hantavirus strain SZ2083 / 中华流行病学杂志

<p><b>OBJECTIVE</b>For clarifying the situation of the natural infection of rodents having hemorrhagic fever with renal syndrome (HFRS) virus and to type Hantavirus (HV) using molecular technique in Shenzhen city in 2005, and offering guidance for prevention and control of HFRS.</p><p><b>METHODS</b>Data on the host animals was collected from the city of Shenzhen. ELISA and indirect immunofluorscent antibody(IFA) test were applied to the specific antibodies against HV in the sera of captured rats. Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues of the HV infected rats were inoculated into Meriones unguiculata to isolate HV. The whole viral RNA was extracted from the lung tissues of the HV infected rats and amplified the partial M fragments with RT-nested-PCR, using the HV genotype specific primers. The amplified genes were then sequenced, and subjected to genotyping and homology analysis.</p><p><b>RESULTS</b>472 rodents were captured from Shenzhen in 2005. Surveillance on rats demonstrated 9.96% rats carrying HV (with a density of 8.25%) and the main host was Rattus norvegicus. In the blood samples of rats, anti-HV IgG antibodies were detectable in 56 cases by IFA, and proved to be positive in 76 cases by ELISA. We successfully isolated a HV strain designated as SZ2083 from Rattus norvegicus for the first time in Shenzhen and was identified to SEO type by RT-nested-PCR. Compared with the coding region of the M gene of HV L99 virus strain, the homologies of nucleotide among them were 97%, but the homology was 76% of the SZ2083 with HTN 76-118 virus strain.</p><p><b>CONCLUSION</b>Results showed the existence of natural epidemic areas of HFRS in Shenzhen city. Based on the results of sequencing, it is possible that the Seoul strain of HV might be the predominant serotype of virus harbored.</p>

Nucleotides sequence analysis of EV71 Chinese strain SHZH98 3C segment / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the EV71 Chinese strain SHZH98 and analyze its genetic evolution using 3c gene as index.</p><p><b>METHODS</b>The 3C gene cDNA of EV71 Chinese strain SHZH98 was amplified by PCR, the PCR product was sequenced.</p><p><b>RESULTS</b>The EV71 Chinese mainland strain SHZH98 3C segment was 549 bps in length. Comparison of nucleotide sequences from other enteroviruses which have been published, revealed a higher homology to strain MS, 78.7% at nucleotide level and 93.45% at deduced amino acid level. The homology to strain BrCr was 76.7% at nucleotide level and 89.1% at deduced amino acid level. Taiwan strains POLY,NCKU,TW2086,TW2272 shared a lower homology with Chinese mainland strain SHZH98, 74.0%, 73.8%, 71.9%, 69.8% at nucleotide level and 90.7%, 90.2%, 84.2%, 82.5% at deduced amino acid level. The genetic progress analysis revealed that EV71 Chinese mainland strain SHZH98 3C segment shares more homology with European and American strains than Taiwan strains.</p><p><b>CONCLUSION</b>The non-structural protein of EV71 Chinese strains may have different evolutionary process from Taiwan strains.</p>