High-throughout detection and analysis of drug-resistance gene mutation for 2465 cases with chronic hepatitis B in Shenzhen / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the character of drug-resistance gene mutation for patients with chronic hepatitis B in Shenzhen.</p><p><b>METHODS</b>2465 clinical cases with chronic hepatitis B were analyzed for gene mutation with MALDI-TOF-MS in order to know about the epidemiology of HBV drug resistance and its clinical significance.</p><p><b>RESULTS</b>763 cases were detected mutation among the 2465 cases. The frequency of Lamivudine related mutation was the highest (42.96%), especially on rtL180M (14. 72%), rtL204I (18. 50%), rtL204V (9. 74%). The frequency of Adefovir related mutation was about 8. 19% , among of which rtN236T was 4. 15%. The frequency of Entecavir related mutation was about 0. 49%. Among all samples, rtS202I mutation couldn't be detected. The existence of drug resistance could be detected earlier with MALDI-TOF-MS from the results of dynamic follow-up.</p><p><b>CONCLUSION</b>In Shenzhen, the main HBV mutation was associated with lamivudine and adefovir,and with lower frequency of mutation for entecavir,so the optimized treatment for HBV was entecavir. It could detect the existence of drug resistance effectively with MALDI-TOF-MS and guide clinical treatment.</p>

Efficacy of Peg-interferon alpha-2a combinated with entecavir on HBeAg positive chronic hepatitis B patients with high serum hepatitis B viral loads / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>This study aimed at evaluating the efficacy and safety of a combination treatment of entecavir and Peginterferon alpha-2a for HBeAg positive chronic hepatitis B patients with high serum hepatitis B viral loads.</p><p><b>METHODS</b>60 treatment-naive HBeAg-positive CHB patients with high serum hepatitis B viral loads were enrolled and randomly divided into three groups: group A received Peginterferon alpha-2a monotherapy for 48 weeks (n = 20); group B received entecavir monotherapy for more than 48 weeks (n = 20); group C received Peginterferona alpha-2a combined with entecavir for 12 weeks, then Peginterferon alpha-2a monotherapy for 36 weeks (n = 20). Virological response, ALT normalization, HBeAg and HBsAg seroclearance rate were analysed at the end of 4, 12 and 24 weeks after the treatment.</p><p><b>RESULTS</b>The ratio of undetectable hepatitis B virus (HBV) DNA were 50% and 10%, 95% and 25% and 100% and 30% in group C and group A respectively, 50% and 20%, 95% and 75% and 100% and 90% in group C and group B respectively at the end of 4, 12 and 24 weeks of treatment. The differences were significant between group C and A (Z = -4.6, P < 0.001), group C and B (Z = -2.53, P = 0.0114). ALT normalization rate was significantly lower in group A than that of group C (Z = -2.63, P = 0.0086). HBeAg levels declined more in group C than the other two groups after 24 weeks of treatment.</p><p><b>CONCLUSIONS</b>For HBeAg positive chronic hepatitis B patients with high serum hepatitis B viral loads, combination treament of Peginterferon alpha-2a with entecavir is more effective than Peginterferon alpha-2a monotherapy in virologic response and ALT normalization after 24 weeks of treatment.</p>

Evaluation of the effect of chronic virus infection on laboratory tests results in patients with osteoarticular tuberculosis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of chronic virus infection on laboratory tests results in patients with osteoarticular tuberculosis.</p><p><b>METHODS</b>A total of 121 patients with osteoarticular tuberculosis, who were hospitalized in Shenzhen Third People's Hospital during June 2008 to June 2012, were recruited for analysis. Clinical laboratory tests results were collected for comparison between patients with or without chronic co-infection with virus.</p><p><b>RESULTS</b>Among the 121 patients, thirty patients were co-infected with hepatitis B virus (HBV), two were with Human immunodeficiency virus (HIV), and one was co-infected with HBV, HIV and hepatitis C virus (HCV). Compared to patients with osteoarticular tuberculosis without HBV/HCV/HIV infection, patients with chronic HBV/HCV/HIV virus infection had similar positive rate of laboratory tests including tissue smear acid-fast bacilli (AFB) staining, tissue Mycobacterium tuberculosis (Mtb) culture, tissue Mtb DNA detection, serological test of antibodies against Mtb, and Mtb. antigen-specific interferon-gamma release assay. Similar results were also found for erythrocyte sedimentation rate, C-reative protein level and liver function including Alanine aminotransferase and Aspartate Aminotransferase.</p><p><b>CONCLUSION</b>Chronic infection with HBV/HCV in patients with have no obvious effect on clinical laboratory tests related to tuberculosis.</p>

Identification of HBx-related integration sites in HBsAg-positive hepatocellular carcinoma biopsy / 中华肝脏病杂志

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.

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>

The association between HBV genotyping and clinical characteristics and expression of TH1/TH2 cytokines / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the association between HBV genotyping and clinical characteristics and expression of TH1/TH2 cytokines.</p><p><b>METHODS</b>The expression of IL-4 and IFN-gamma was detected with flow cytometry for 102 HBV infections and 48 healthy controls. 50 CHB patients were randomly selected for HBV genotyping with real-time fluorescence PCR assay.</p><p><b>RESULTS</b>Higher expression of IL-4 in peripheral blood was detected in patients with HBV infection than healthy controls (P < 0.001); No significant differences on expression of Th1/Th2 cytokines were observed in CHB patients with different HBV DNA levels or HBeAg status (P > 0.05). There were 34 (68%) patients with genotype B infection and 16 (32%) with genotype C infection. Compared to patients with genotype B infection, the patients with genotype C infection showed higher levels of IL-4 (P = 0.018), and Th1/Th2 ratio decreased,but the difference was not statistically significant (P = 0.2262).</p><p><b>CONCLUSION</b>The different expression of TH1/TH2 cytokines may elucidate cellular immune response and clinical outcome difference between patients with genotype B infection and genotype C infection.</p>

Loss of the balance between Th17 and Th1 populations in HIV/AIDS patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the Th17/Th1 response in HIV infected patients and the mutual relationship between the response of Th17 and Th1.</p><p><b>METHODS</b>38 chronic HIV infected patients as well as 24 healthy volunteers were performed in this study. The patients were divided into two groups, one group before treatment, the other after therapy. The whole blood intracellular cytokine staining was used, samples detected by BD FACSCanto, after that, the expression of CD4+ IL-17+ T cell and CD4 IFN-gamma+ T cell were analyzed by FACSDiva software and lastly compared the differences among different groups.</p><p><b>RESULTS</b>The expression of CD4+ IL-17+ T cell in naive-therapy patients were significantly lower than that of the healthy controls (1.14 +/- 0.7)9% vs (3.98 +/- 1.14)%, P = 0.000, but increased remarkably after HARRT(highly antiretroviral treatment) (2.22 +/- 1.00)%, P = 0.001; however there were no significant differences in the expression of CD4+ IFN-gamma+ T cell before and after therapy (34.35 +/- 24.38)% vs (42.10 +/- 15.57%), also with the healthy control (P = 0.383). The frequency of CD4 IL-17+ T cell was positively correlated with CD4+ T counts (R = 0.345, P = 0.034), but no significant correlations was observed between the expression of CD4+ IFN-gamma T cell and CD4+ T counts (R = -0.247, P = 0.136).</p><p><b>CONCLUSION</b>The infection of HIV virus down-regulated Th17 immune response and disturbed the balances between Th17 and Th1 evidently in human. Th17 response may play an important role in the pathogenesis of HIV infection.</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>

Financial burden of hepatitis B-related diseases and factors influencing the costs in Shenzhen,China / 中华流行病学杂志

Objective To investigate the direct, indirect and intangible costs due to hepatitis B-related diseases and to explore main factors associated with the costs in Shenzhen. Methods Cluster sampling for cases collected consecutively during the study period was administrated. Subjects were selected fiom eligible hepatitis B-related patients. By pre-trained professional investigators,health economics-related information was collected, using a structured questionnaire. Hospitalization expenses were obtained through hospital records after the patients were discharged from hospital.Total economic burden of hepatitis B-related patients would involve direct, indirect and intangible costs. Direct costs were further divided into direct medical costs and direct nonmedical costs. Human Capital Approach was employed to measure the indirect costs both on patients and the caregivers in 1-year time span. Willing to pay method was used to estimate the intangible costs. Multiple linear stepwise regression models were conducted to determine the factors linked to the economic burden.Results On average, the total annual cost of per patient with hepatitis B-related diseases was 81 590.23 RMB Yuan. Among which, direct, indirect and intangible costs were 30 914.79 Yuan (account for 37.9% ), 15 258.01 Yuan (18.7% ), 35 417.43 Yuan (43.4%), respectively. The total annual costs per patient for hepatocellular carcinoma, severe hepatitis B, decompensated cirrhosis,compensated cirrhosis, chronic hepatitis B and acute hepatitis B were 194 858.40 Yuan, 144 549.20 Yuan, 120 333.60 Yuan, 79 528.81 Yuan, 66 282.46 Yuan and 39 286.81 Yuan, respectively. The ratio of direct to indirect costs based on the base-case estimation foot add to 2.0∶1, increased from hepato-eellular carcinoma (0.7∶1)to compensated cirrhosis (3.5∶ 1 ), followed by acute hepatitis B (3.3∶1 ), severe hepatitis B (2.8∶1 ), decompensate cirrhosis (2.3:1)and chronic hepatitis B(2.2∶1 ).Direct medical costs were more than direct nonmedical. Ratio between the sum total was 16∶1. The proportions of total annual cost per patient with hepatitis B-related diseases accounted for annual patient income were 285.3%, and 75.4% for annual household income. Furthermore, proportions of direct costs accounted for annual patient income and annual household income were 108.1% and 28.6%. The total annual indirect cost per person was 8123.38 Yuan for patients of all hepatitis B-related diseases, while 7134.63 Yuan for caregivers. Corresponding work-loss days were 55.74 days for patients and 19.83 days for caregivers. Based on multiple linear stepwise regression analysis, age of patients was a common influencing factor to all kinds of costs. Other factors were as follows:complicated with other diseases, antiviral medication, monthly household income and selfmedications. Conclusion The economic burden of hepatitis B-related diseases was substantial for patients and their families. All costs tended to increase with the severity of disease. The direct costs were larger than the indirect costs. And the direct medical costs were more than the direct ones.Indirect costs based on patients were larger than the ones of caregivers.

Association between CD4+CD25+Foxp3+ regulatory T cells and serum transforming growth factor beta 1 in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether the CD4+CD25+Foxp3+ regulatory T cells are associated with serum TGF beta 1 in patients with hepatitis B.</p><p><b>METHODS</b>Patients with chronic hepatitis B (CHB), chronic asymptomatic carriers (AsC), normal subjects (NS) and the resolved from HBV infection (Resolved) were recruited in this study. Flow cytometric analysis was used to detect the frequency and phenotype of peripheral CD4+CD25+Foxp3+ T cells, and Foxp3 gene expression were examined by real time PCR. Serum TGF beta 1 levels were measured by ELISA (enzyme-linked immunosorbent assay).</p><p><b>RESULTS</b>Patients with CHB or AsC exhibited significantly higher frequency of CD4+CD25+Foxp3+ T cells compared to healthy controls. CD4+CD25+ T cells derived from patients with CHB and AsC expressed higher level of Foxp3-mRNA. Furthermore, the frequency of CD4+CD25+Foxp3+ regulatory T cells was correlated with serum HBV DNA copy numbers in patients with CHB and AsC. Our results indicated that the serum TGF beta was increased in CHB and AsC patients compared to control patients, and that serum TGF beta was correlated with the expression of Foxp3-mRNA and the frequency of CD4+CD25+Foxp3+ regulatory T cells in patients with CHB and AsC.</p><p><b>CONCLUSIONS</b>The findings have important implication in the understanding of the role and mechanism of aberrant CD4+CD25+Foxp3+ regulatory T cells in the maintenance of chronicity in hepatitis B patients.</p>

The expression of some homing and co-receptor molecules on CD4+ T cells in AIDS patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the expression of CD49d, CCR9, CD62L and CCR5 on CD4+ T cells in AIDS patients before and after HAART.</p><p><b>METHODS</b>The study was performed in 42 cases of AIDS patients and 18 cases of healthy controls. The expression of CD49d, CCR9, CD62L and CCR5 on CD4+ T cells, and CD45RO on CD4+ CCCR9+ and CD4+ CCR5+ T cells in AIDS patients and healthy controls were analysed by Flow cytometry. Software BD FACSDiva was used to calculate the percentage of expression.</p><p><b>RESULTS</b>The number of peripheral CD4+ T cells in group pre-HAART was decreased compared with group HAART (P < 0.01); the frequency of CD3+ CD4+, CD4+ CCR9+, CD4+ CCR5+ T cells in group pre-HAART were decreased compared with group HAART (P < 0.01), the frequency of CD4+ CD49d+, CD4+ CD62L+, CD4+ CCR9+ CD45RO , CD4+ CCR9+ CD45RO- ,CD4+ CCR5+ CD45RO+, CD4+ CCR5+ CD45RO- T cells in group pre-HAART were significantly decreased compared with group HAART (P < 0.001); the frequency of CD3+ CD4, CD4+ CD62L+, CD4+ CCR5+ T cells in group HAART were significantly decreased compared with group HIV-neg (P < 0.05).</p><p><b>CONCLUSIONS</b>Not only the number but the function of CD4' T cells was impaired in AIDS patients: the lower expression frequency of gut homing receptor molecule CD49d and CCR9,1ymph node homing molecule CD62L, coreceptor molecule CCR5. HAART can partially reverse this pathological phenomena. CD49d, CCR9 and CD62L may be suggested to indicate the progression of AIDS and immunologic reconstitution after HAART.</p>

Construction and application of a genechip method for detection of hepatitis B virus lamivudine-resistant mutants and basal core promotor/Pre-C mutants / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The objective of this research is to construct a clinic-usable genechip method for detection of hepatitis B virus lamivudine-resistant mutants and basal core promotor/Pre-C mutants, compare this method with DNA sequencing to investigate this genechip's character (sensity, specificity, stability and practicability in clinic) and apply it in clinic.</p><p><b>METHODS</b>This genechip detection method can detect the DNA and 8 mutative site of HBV, include 3 lamivudine-resistant mutation site(No. 180, 204, 207 site in DNA polymerase gene), 5 HBeAg escape-related mutation site (nt 1896, 1899, 1862, 1764,1762 site in BCP/Pre-C region).The results of genechip method was verified by DNA sequencing.</p><p><b>RESULTS</b>In detecting HBV DNA, the results of genechip were agree with 100% of the results of DNA sequencing. In detecting HBV mutants, 251 sites (in 32 samples, 256 sites) showed the same results using both methods, and only 5 sites were not completely match (P > 0.05). In these 5 sites, genechip methods got multi-infection results, but sequencing got single-infection results.</p><p><b>CONCLUSION</b>These results suggest that genechip method has the same positive rate and almost these same specificity with DNA sequencing method, and is better than DNA sequencing method in detecting multi-infected HBV strains. [Key words]</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>

Evaluation of the accuracy of domestic commercial HBV DNA real-time polymerase chain reaction kits using COBAS TaqMan HBV Test as reference / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate of the accuracy of domestic commercial HBV DNA real-time polymerase chain reaction kits.</p><p><b>METHODS</b>Using COBAS TaqMan HBV Test as reference, we evaluate the accuracy of a domestic commercial HBV DNA real-time polymerase chain reaction kit (PG).</p><p><b>RESULTS</b>Among the samples with viral load at the range of 10(1), 10(2), 10(3), 10(4), 10(5), 10(6), 10(7) (IU/mL), the Coefficient of Correlation(r) between the result determined by domestic kit (PG) and those of Roche COBAS TaqMan HBV Test were: -0.08011, -0.05056, 0.105642, 0.312181, 0.908046, 0.866175, -0.23295, respectively; the percentage of false negative results were 60%, 30%, 33.3%, 8.3%, 0, 0, 0, respectively. Among the samples with viral load over than 10(7) (IU/ml), the result determined by PG is significantly lower.</p><p><b>CONCLUSION</b>The accuracy of PG is not satisfied, especially in those samples with viral load less than 10(4) (IU/ml). A implication from these observation is that samples from patients received antiviral treatment should be tested by Roche COBAS TaqMan HBV Test.</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>

Effect of hepatitis B virus C protein on function of natural killer cell in NK-92 cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the influence of hepatitis B virus C protein on the function of natural killer cell.</p><p><b>METHODS</b>Recombinant eukaryotic expression plasmid pHBI-CMV-HBC was constructed and confirmed by double restrictive enzyme digestion and DNA sequencing analysis. Then the recombinant plasmid was transfected into NK-92 cells with lipofectamine encapsuled. The transfected NK-92 cells containing expressive HBV C protein was confirmed by Western Blot analysis. ELISA was employed to determine the IFN-gamma level secreted by NK-92 cells. And finally the cytotoxicities of NK cells were analysed by MTT colorimetry, with the hepatoblastoma cell line (HepG2) as target cell.</p><p><b>RESULTS</b>Western blotting confirmed the expression of HBV C protein in the NK-92 cells transfected with pHBI-CMV-HBC. NK cytotoxicities and IFN-gamma secretion level of NK-92 cells transfected with recombinant plasmid significantly increased compared to control NK-92 cells transfected with blank plasmid (P < 0.01) and untransfected NK-92 cells(P < 0.01).</p><p><b>CONCLUSION</b>Transient expression of HBC can increase IFN-gamma secretion and cytotoxicities of NK-92 cells.</p>

Detection of cryptosporidium infection among AIDS patients in Guangdong and Yunnan / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the infection of Cryptosporidium and its epidemiological characteristics in AIDS patients of Southern China.</p><p><b>METHODS</b>Stool samples colleted from AIDS confirmed patients. The samples were detected for oocyst of Cryptosporidium by acid fast bacteria stain and indirect fluorescent antibody stain respectively, CD4 count was detected by Flow Cytometry.</p><p><b>RESULTS</b>212 samples of fresh stool obtained from the AIDS patients who live in Guangdong and Yunnan province. The total infection rate of Cryptosporidium in AIDS patients was 4.25% (9/212), the infectious rate of oocyst in the group of 50- 59-years-old was significantly higher than those in 30-39 (P < 0.01); the infectious rate of oocyst in patients with antiretroviral therapy (ART) was also significantly lower (P = 0.0000); we found the patients coinfected with Cryptosporidium with CD4 count all below 100 cells/microl. However, there were no any difference between the infectious rate to the patient's gender, areas and stool shape.</p><p><b>CONCLUSION</b>AIDS patients infected by Cryptosporidium are not rare in southern China, and the infectious rate was lower than western country. Patients received ART could decrease the infectious rate of Cryptosporidium, Cryptosporidium always happen in patient whose CD4 count was very low (< 100 cells/microl).</p>