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Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 11/2012; 33(11):1198-9.
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ABSTRACT: The study was performed to examine the enterovirus 71(EV71) VP1 genetic feature and the epidemiology of hand-foot-mouth disease (HFMD) in Xinxiang in 2011. Real-time RT-PCR was used for the detection of Pan-enterovirus, Coxsackievirus A 16(CA16) and EV71 from stool specimens of HFMD. The VP1 region was amplified from 10 EV71 positive samples and the products were sequenced. EV71 genotypes were characterized by homology and phylogenetic tree analyses. Additionally, epidemic data of Xinxiang HFMD in 2011 was analyzed. The results revealed that 73% of the specimens from severe cases were determined as EV71 positive, which was significantly higher than CA16-positive ones (19%) (P < 0.01). Ten EV71 strains isolated in Xinxiang belonged to C4a cluster of sub-genotype C4, with 2.8% nucleotide and 0.9% amino acid sequences divergence among them. At position 170 in VP1 gene, an alanine(A) was predominant in 9 isolates, while a valine(V) residue was observed in one isolate. Compared to the representative C4a strains which were closely related to Xinxiang isolates, the amino acid variations of the pre-dominant Xinxiang strains generally occurred at position 292, threonine --> alanine (T --> A). A total of 1118 HFMD cases were reported in Xinxiang in 2011, and 92% of them were younger than 3 years old; the incidence rate peaked in April and December, suggesting that it is very necessary to strengthen HFMD prevention and control even in cold weather.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 11/2012; 28(6):675-80.
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ABSTRACT: Molecular detection of enterovirus (EV)71 RNA based on PCR methods is a quick and sensitive approach. At present, different PCR-based methods for EV71 RNA detection are available, but comparisons of results obtained using different approaches are limited. This study is to compare the analytical sensitivity and specificity of different real-time reverse transcription-polymerase chain reaction (rRT-PCR) and conventional reverse transcription-polymerase chain reaction (cRT-PCR) assays for enterovirus and EV71 detection, Altogether, three rRT-PCR assays and one cRT-PCR assay targeting the 5'UTR gene for universal detection of enterovirus; two rRT-PCR assays andone cRT-PCR assay targeting the VP1 gene for specific detection of EV 71 were examined. All assays showed good specificity. The detection sensitivity ranged from 8.19 x 10 to 8.19 x 10(5) copy equivalents. In general, rRT-PCR assays were more sensitive than cRT-PCR assays. All rRT-PCR assays showed 100% sensitivity for clinical specimens.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 11/2012; 28(6):670-4.
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ABSTRACT: To understand etiological types and distribution features of hand-foot-mouth disease (HFMD) in Henan province between 2008 and 2011.
A total of 30 486 specimens of feces, rectal swabs or throat swabs from HFMD patients were collected by each Municipal CDC in Henan from 2008 to 2011. The enterovirus 71 (EV71), coxsackie virus A16 (CA16) and other enterovirus (EV) were detected by RT-PCR or real time RT-PCR. The VP1 gene of EV71 was amplified and the sequences were analyzed by bioinformatics software. A genetic evolution tree of the sequence was constructed as well.
The positive rates of EV71, CA16 and other EV were 62.70% (11 209/17 876), 12.03% (2150/17 876), 25.27% (4517/17 876) in 17 876 laboratory diagnosed cases, respectively. The differences were statistically significant (χ(2) = 157.17, P < 0.05). The positive rates of EV71, CA16 and other EV were 63.40% (7370/11 624), 11.58% (1346/11 624) and 25.02% (2908/11 624) in male patients and 61.40% (3839/6252), 12.86% (804/6252) and 25.74% (1609/6252) in female patients, respectively. The differences were statistically significant (χ(2) = 4.06, P < 0.05). The children under 5 years old were high-risk population of HFMD, accounting to 97.67% (17 459/17 876) of the laboratory-diagnosed patients.86.92% (15 537/17 876) cases were children between 1 to 3 years old. Constituent ratio of EV71 changed seasonally during a year, there was a high infection ratio of EV71 between April and June, especially in May, the infection ratio reached 69.34% (2384/3438). The positive rates of EV71, CA16 and other EV were 82.48% (5715/6929), 1.76% (122/6929) and 15.76% (1092/6929) among the 6929 laboratory-diagnosed severe cases, respectively. The positive rates of EV71 was higher than CA16 and other EV (χ(2) = 9259.17, 6170.81, P < 0.05, respectively). There were 117 deaths because of severe HFMD, 55 (47.01%) of which were laboratory confirmed.50 death cases were infected by EV71, and according to the genetic evolution analysis, the VP1 gene of EV71 strain was belonged to subtype C4 of gene C.
The EV71 and CA16 were the main pathogens which caused HFMD in Henan province, and EV71 virus was the dominant strain, belonging to C4 subtype of gene C.
Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine] 10/2012; 46(10):883-7.
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ABSTRACT: This report presents an overview of human enterovirus B species in Henan Province. A total of 14 isolates of HEV-B species isolated under HFMD surveillance network during the six months in 2010 were examined. Based on molecular typing results targeting VP1 region, 14 isolates were classified into 6 serotypes of HEV-B. Furthermore, comparison of these 14 isolates with reference strains and strains in mainland China was conducted. The phylogenetic analysis revealed that E25, E11 and E6 showed homology with those from Shandong Province which adjoins Henan Province. E1 and E13 showed homology with those from Yunnan Province, and E30 showed homology with Henan strain isolated in 2008. Cocirculation of two lineages of echovirus 6 was observed.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 03/2012; 28(2):114-7.
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ABSTRACT: To reveal the genomic sequence characteristics of coxsackievirus A16 (CoxA16) strain isolated from patients with hand-foot-mouth disease (HFMD) in Henan province. A total of 406 samples were detected by reverse-transcription polymerase chain reaction (RT-PCR) and cell-culture-based isolation of coxsackievirus A16. The whole genome of CoxA16 isolate was amplified using 10 pairs of primers, the sequences were analyzed and phylogenetic tree was generated by bioinformatics software. The full length of HN1162/HN/CHN/2010 genome was 7411bp. Compared with the other CoxA16 strains released in GenBank, the nucleotide similarities were 87.0-97.9%, 77.0%-95.4%, 80.3%-96.9%, 77.9% 96.2%, 80.5-100% in 5'UTR, P1, P2, P3, 3'UTR region, respectively; The similarities of nucleotide and amino acid sequences in VP1 region were 91.4%-96.4% and 99.3%-99.7%, respectively. Phylogenetic tree analysis showed that CoxA16 strains isolated from Henan, Shenzhen, Guangzhou and Fujian belonged to the same cluster. The newly isolated CoxA16 from Henan province belonged to subgenotype C2/B-2. These results will have great significance in monitoring CoxA16 and for prevention and control of hand-foot-mouth disease.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 03/2012; 28(2):118-23.
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ABSTRACT: To reveal the genetic features and recombination of enterovirus 71 isolates between 2008 and 2010. A total of 5 enterovirus 71 isolates were sequenced completely and phylogenetic analysis and recombination were performed. Phylogenetic analysis based on VP1 regions revealed that the Henan enterovirus 71 between 2008 and 2010 belonged to C4a in subgenotype C4. Bootscan analyses and phylogenetic analysis based on the 5'UTR, P1, P2, P3 genomic regions revealed the recombinations between EV71 genotypes B and C at the 2A-2B junction, and between EV71 genotype B and CA16 strain G-10 at the 3B-3C junction. Henan enterovirus 71 isolates between 2008 and 2010 belonged to C4a in subgenotype C4 which was the predominant virus genotype circulating in mainland China since 2004, a combination of intratypic and intertypic recombination were found in EV71 subgenotype C4.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 09/2011; 27(5):433-7.
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ABSTRACT: To sequence the whole-genome of enterovirus 71 (EV71) srtain isolated from patient with hand, foot and mouth in Henan province in 2008.
Eight overlapping clones covering the whole viral genome were obtained by RT-PCR and the sequences were determined by Sanger dideoxg-mediated chain termination method.
Data it showed that the full length of enterovirus 71 (EV71) HENAN08 genome (not including Poly A tail) is 7405 bp. No deletion or insertion was detected in the coding region. There were several deletions and insertions in 5'UTR and 3'UTR regions. In P1 region, HENAN08 strain shared high homology with AnhuiFY08 strain, Zhejiang08 strain and SHZH strains (SHZH98, SHZH03) but low homology with Cox. A16. In P2 and P3 regions, HENAN08 strain shared higher nucleotide homology with Cox. A16 (81.7% and 83.7%) than that with BrCr and TW2086 strains. The phylogenetic analysis based on P1 region demonstrates that HENAN08 strain had the nearest genetic relationship with AnhuiFY and Zhejiang strains (isolated in 2008).
The HENAN08 strain might belong to the same genogroup with AnhuiFY08 and Zhejiang08 strains as C4 gene subtypes.
Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 09/2009; 30(9):938-41.
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Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 05/2006; 27(4):366-7.
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ABSTRACT: To investigate the dynamics of amyloid fiber formation of yeast (Saccharomyces cerevisiae) prion protein Sup35NM under the native condition to provide materials and clues for the elucidation of amyloid fiber formation.
The Sup35NM gene was cloned and expressed in E. coli. The recombinant Sup35NM protein was purified under denaturing conditions through Nickel-Sepharose chromatography. Aliquots were removed at designated time points for transmission electron microscopy (TEM), circular dichroism (CD) spectra, protease K resistance assay, as well as thioflavin T (ThT) binding assay.
The Sup35NM expressed and purified under denaturing conditions. The morphological alteration of the Sup35NM in PBS (pH7.4) during the protein aggregation and amyloid fiber formation was visualized by TEM. The CD assay showed that the course of amyloid fiber formation underwent a conformational shift from alpha-helix to beta-sheet. The fibers had higher capacity of resistance to protease K digestion compared to the monomers. ThT fluorescence assay displayed a rapid growth phase before reaching a final equilibrium phase during the fiber formation, and the higher concentration of Sup35NM could greatly accelerate the fiber formation in vitro.
Yeast prion protein Sup35NM forms amyloid readily under native conditions in vitro. The dynamics of Sup35NM amyloid formation may provide supporting evidences for the nucleating polymerization models of amyloid fiber formation.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 04/2006; 20(1):39-42.
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ABSTRACT: To investigate the interaction between the host cell and the truncated S fragments to identify the receptor-binding domain of the spike (S) protein of SARS-associated coronavirus (SARS-CoV).
Two different fragments S260-600 and S397-796 of the SARS-CoV S protein were expressed in Escherichia coli (E.coli) using a pET expression vector, respectively. The two recombinant proteins were separately verified by Western blot, purified by nickel-affinity chromatography, and incubated with Vero cells, a susceptible cell line of SARS-CoV infection, for cell binding assay. After the sequential probing with sera from convalescent SARS-patients and FITC-labeled anti-human IgG, the cells were analyzed by flow cytometry. The NIH 3T3 cell, a non-permissive cell line of SARS-CoV infection, was used as controls.
The recombinant proteins S260-600 and S397-796 were efficiently expressed in an insoluble form in E.coli. The appropriate expression of the proteins was confirmed by Western blotting using both SARS patients' sera and anti-6 x histidine antibody. The flow cytometry results showed that the both proteins were able to bind Vero cells, but the binding ability of S260-600 was somewhat stronger than that of S397-796. In contrast, the S260-600 protein did not bind NIH3T3 cells.
Both S260-600 and S397-796 exhibited different receptor binding activity. The S260-600 fragment probably contains the important receptor binding domain and could be a potential candidate for the development of SARS vaccine and anti-SARS therapeutics.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 01/2006; 19(4):353-7.
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ABSTRACT: To observe the in vitro expression of DNA vaccine (recombinant eukaryotic expression plasmid pcDNA3-TspE1) encoding a Mr 31,000 antigen of Trichinella spiralis in Chinese hamster ovary (CHO) cells and analyze the antigenicity of the products expressed.
The recombinant plasmid pcDNA3-TspE1 was transfected into CHO cells by using cationic lipids (Lipofectamine 2000). The positive cell clones were screened by the selective antibiotic G418. The expressed products were identified by RT-PCR, IFAT, SDS-PAGE and Western blotting.
The results of RT-PCR amplification showed that there was one band with 876 bp in CHO cells transfected with pcDNA3-TspE1 and no any bands in CHO cells transfected with the empty plasmid pcDNA3. The IFAT demonstrated that the pcDNA3-TspE1 transfected CHO cells reacted with sera from mice immunized with the recombinant fusion protein and from mice infected with T. spiralis, the bright yellow green fluorescence staining appeared in the transfected CHO cells. The pcDNA3 transfected and un-transfected CHO cells exhibited as orange color. The results of SDS-PAGE showed that there was one band with Mr 31,000 in culture supernatant of CHO cells transfected with pcDNA3-TspE1. Western blotting confirmed that the band with Mr 31,000 could be recognized by sera from mice immunized with the recombinant fusion protein, from rabbits immunized with T. spiralis muscle larval soluble antigens, from mice infected with T. spiralis and from patients with trichinellosis. Conclusion The mammalian CHO cells were transfected by the recombinant plasmid pcDNA3-TspE1, and the TspE1 gene of T. spiralis was expressed in the transfected CHO cells. The proteins expressed are secreted into cell culture supernatants and show the antigenicity of T. spiralis.
Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases 11/2004; 22(5):266-70.
