The genetical evolution of the full length genes of 5 EV 71 strains from 5 Shenzhen patients with hand-food-mouth disease associated with EV71 infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the characteristics of molecular epidemiology and molecular evolution of 5 EV 71 (enterovirus 71, EV71) strains from 5 Shenzhen patients with hand-food-mouth disease associated with EV 71 infection.</p><p><b>METHODS</b>5 EV 71 strains were isolated, and sequenced to analyzed the full length gene sequences in order to compare nucleotide and amino acid homology with other EV71 strains from other regions and countries as well as previous strains across the world through bioinformatics software.</p><p><b>RESULTS</b>5 strains of EV 71 belonged to sub-genotype C4 by analysis of nucleotide sequences of VP1 and VP4 of EV 71. The differences of nucleotide and amino acid sequences were much small with nucleotide homology of 93% and amino acid homology of 98% among these 5 strains. A phylogenetic tree analysis indicated that 2008 Shenzhen epidemic strains were the most close to 2004 Shenzhen circulating strains, and also much close to 1998 Shenzhen epidemic strains and 2008 Fuyang Anhui strains. The dead strain was very close to 2008 Fuyang Anhui epidemic strains.</p><p><b>CONCLUSION</b>It can be speculated that this epidemic strains of EV 71 probably originate from the same ancient strain in the history, may from 1998 Shenzhen strain.</p>

Integration sites in HCC biopsy / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To compare the performance of Inverse-PCR, Alu-PCR and Cassette-ligation-mediated PCR (CLM-PCR) in HBV DNA integration sites identification.</p><p><b>METHODS</b>One HCC biopsy was obtained from surgically resected sample. The patient was positive for serum hepatitis B surface antigen (HBsAg). The genomic DNA was purified by the standard phenol/chloroform extraction and ethanol precipitation method. Seperated set of primers were designed to amplify the HBV DNA integration region by means of 3 different PCR methods respectively. The PCR products were analyzed by electrophoresis, then cloned to PMD18-T vector for DNA sequencing. The sequence alignment was performed under Blast software.</p><p><b>RESULTS</b>7 bands and 22 sequencing results was obtained from IPCR and 3 integration sites was identified. Alu-PCR provided 12 bands and 32 sequencing results, and CLM-PCR showed 12 bands and 4 sequencing results. No integration site was identified from the latter two.</p><p><b>CONCLUSION</b>IPCR compared with another two methods showed a reliable capacity in HBV DNA integration site identification.</p>

Study on characteristics of cellular-mediated immune responses of novel H1N1 influenza A patients with pneumonia / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the phenotype, frequency and function of CD4+ T cell subsets and the relevant cytokines, as well as the relationship between these cells and appearance of pneumonia of novel (H1N1) influenza A patients.</p><p><b>METHODS</b>68 healthy people, 53 confirmed novel A(H1N1) influenza patients without pneumonia and 16 confirmed severe novel A (H1N1) influenza patients with pneumonia were enrolled in this study. Viral load in nasopharyngeal swabs specimens was measured by real time PCR assay. The phenotype and percentage of CD4+ T cell subsets including Th1, Th2, Th17, and Treg cells were measured by Flow cytometry analysis. The relevant cytokines in plasma including TGF-beta, IL-6 and IFN-gamma were measured by ELISA. Data was analyzed by one way ANOVA.</p><p><b>RESULTS</b>It was found that peak viral load and viral shedding period of severe patients with pneumonia was significantly increased compared with mild patients without pneumonia (P < 0.05). The percentage of Th17 cells of severe patients with pneumonia was significantly diminished compared to that of healthy subjects and mild patients without pneumonia (P < 0.05). However, Th1, Th2, Treg cells frequencies had no significant differences (P > 0.05) among these three groups. The level of TGF-beta in plasma for the severe patients with pneumonia was also significantly decreased compared to that of healthy subject and mild patients without pneumonia (P < 0.05). The viral shedding period inversely correlated with the frequency of Th17 cells (r = - 0.38, P < 0.05).</p><p><b>CONCLUSION</b>H1N1 influenza A virus can inhibit Th17 cells to differentiate, particularly more extent in patients with pneumonia. Impaired Th17 cells may correlate with viral clearance and pneumonia of novel H1N1 influenza A patients.</p>

To investigate pathogen of hand, foot and mouth disease in Shenzhen in 2008 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate EV71 and CA16 pathogen of HFMD in Shenzhen in 2008, and to provide the evidence for the prevention and treatment HFMD.</p><p><b>METHOD</b>Using RT-PCR technology to detect the EV71 and CoxA16 genes of 307 samples HFMD; sequencing the purified PCR products from 14 samples. Using ClustalW2 online analysis software for sequence and phylogenetic analysis of enterovirus 71.</p><p><b>RESULT</b>Percentage of positive EV71 from different samples is shown as follows respectively: positive EV71 from stool samples is 24.4% (75/307), from throat swab--7.8% (24/307), from peripheral blood--12.5% (1/8). Percentage of positive CoxA16 is shown as follows respectively: positive EV71 from stool samples is 13.8% (28/203), from throat swab-11.0% (20/181). Among all the 307 samples, three are positive for both EV71 and CoxA16. EV71 and CoxA16 are not detected in the samples of cerebrospinal fluid.Comparative analysis of nucleotide sequences of EV71 with those of strains BrCr and 11 deposited in GenBank demonstrated numerous disparities from 8 samples, but residue 595 from 2 samples and residue 658 from 1 sample are variable. The phylogenetic analysis based on VP1 region demonstrates that strains from 2 samples has the nearest genetic relationship with anhui strains, the farthest with BrCr and SHH02-6, SHZH02-40, SHZH03-58 strains, also strains from other 12 samples have the farthest genetic relationship with them. The genotypes A, B and C were classified as proposed by Brown et al. (1999). The EV71 from 14 samples were the member of genotype C.</p><p><b>CONCLUSION</b>EV71 among the pathogen of HFMD in Shenzhen in 2008 was majority. These EV71 may belong to the same genegroup with Anhui predominant strains.</p>

Expression of a single-chain trimer of MHC restricted HBsAg CTL epitope using adenovirus vector containing GFP-report gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To generate a recombinant Adenovirus encoding a GFP (green fluorescent protein)-report gene and a single-chain trimer of MHC restricted HBsAg CTL epitope.</p><p><b>METHODS</b>An oligonucleotide encoding H-2L(d) restricted HBsAg CTL epitope was synthesized and fused with H-2L(d) DNA molecule to construct the eukaryotic expression vector carrying the HBsAg-SCT gene. The HBsAg-SCT gene was subcloned into a GFP adenovirus expression vector,which was transfected into Ad293 cells for packaging and amplification of recombinant adenovirus encoding HBsAg-SCT.</p><p><b>RESULTS</b>HBsAg-SCT has been cloned into an adenovirus vector encoding GFP report gene successfully as confirmed by double enzyme digestion and direct sequencing. HBsAg-SCT was expressed by infected Ad293 cells demonstrated by western blot assay.</p><p><b>CONCLUSION</b>A recombinant adenovirus expressing HBsAg-SCT and green fluorescent protein report gene has been generated.</p>

Study of the clinical and laboratory features of hand-foot-mouth disease / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the clinical and laboratory features of the mild and severe hand-foot-mouth diseases (HFMD) in Shenzhen in 2008.</p><p><b>METHODS</b>145 cases were observed in East-Lake Hospital and Shenzhen Children's Hospital. Of the 145 cases, 124 mild cases and 21 severe cases were involved.All the clinical data and laboratory findings were collected and summarized. After collection of the acute and convalescent consecutive stools and peripheral bloods from the patients with HFMDI, EV71 genes were amplified from these samples by RT-PCR. Enterovirus 71 were cultured and isolated using Vero cell line and R&D cell line.</p><p><b>RESULTS</b>The WBC counts and blood glucose levels of the severe cases were significantly elevated, but the ages of the severe ones significantly decreased compared with those of the mild cases (P < 0.05). EV71 genes could be detected by RT-PCR with 35% positive rate in mild cases and 67% in severe cases. The EV71 gene detection rate of the severe cases was significantly increased in contrast to that of the mild ones. The EV71 were isolated and cultured from the stools of 9 patients, one specimens from the dead's stool. Two severe cases died of neurogenic pulmonary edema and brain-stem encephalitis.</p><p><b>CONCLUSIONS</b>EV71 mainly contributes to HFMD and is responsible for death of some severe cases. High fever, less rash, elevated white blood cell counts and blood glucose concentrations as well as age less than 4 years old should be used for prediction of severe cases.</p>

Expression of recombinant VP1 protein of enterovirus 71 and development of serological assay for detection of EV71 infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain a recombinant purified Enterovirus 71 VPI protein and establishment of an early, rapid and accurate serological ELISA (enzyme-linked immunosorbent assay) for detection of EV71 infection.</p><p><b>METHODS</b>VP1 gene was amplified by PCR and clonel into pET-21b (+) vector, the positive recombinant plasmid were transformed into E. coli BI21(DE3), and was induced with IPTG, the recombinant protein by SDS-PAGE and Western Blot assays. Finally, the recombinant purified VP1 protein was used as a coated antigen for detection of serum anti-IgM and IgG against EV71 by ELISA.</p><p><b>RESULTS</b>The purified VP1 was obtained, and it can be recognized by sera of patients with EV71 infection associated with hand-foot-mouth disease. The A values of anti-EV71 IgM and IgG were significantly elevated as compared to healthy objects and HFMD patients without EV71 infection (P < 0.05). The sensitivity and specificity of IgM to EV71 were 73% and 77% compared with the RT-PCR results, respectively;and those of IgG being 82% and 83%, respectively.</p><p><b>CONCLUSIONS</b>The recombinant protein VP1 was produced and purified, and it was proved to have a good antigenicity and could be used to develop a serological diagnosis kit for EV71 infection in the future.</p>