ABSTRACT
<p><b>OBJECTIVE</b>To evaluate anti-HEV diagnostic kits by experimental infecting rhesus monkeys with HEV.</p><p><b>METHODS</b>Eight rhesus monkeys were infected with genotype 1 and 4 HEV separately. The alanine aminotransferase (ALT) level of all monkeys were detected before and after the process of infection. HEV RNA in stool specimens was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Anti-HEV IgG in serum was detected by GL-IgG and WT-IgG.</p><p><b>RESULTS</b>HEV RNA presented in the stool of all the 8 monkeys after infection. The ALT level of 1 monkey infected with genotype 1 HEV and 2 monkeys infected with genotype 4 HEV appeared abnormally after infection. Tested by GL-IgG, 2 of the 4 monkeys infected with genotype 1 HEV and 1 of 4 monkeys infected with genotype 4 HEV seroconverted to anti-HEV IgG. However, when tested by WT-IgG, all the infected monkeys seroconverted to anti-HEV IgG. The anti-HEV IgG tested by WT-IgG was positive during the whole observation period,and the anti-HEV IgG measured by GL-IgG only remained 12 weeks after infection. Detected by GL-IgG and WT-IgG, seropositive conversion of the anti-HEV IgG happened almost at the same time.</p><p><b>CONCLUSION</b>Both GL-IgG and WT-IgG could detect the anti-HEV IgG of experimentally infected rhesus monkeys but the WT-IgG had a higher sensitivity for detection of anti-HEV IgG than</p>
Subject(s)
Animals , Alanine Transaminase , Blood , Disease Models, Animal , Genotype , Hepatitis E , Allergy and Immunology , Virology , Hepatitis E virus , Genetics , Allergy and Immunology , Immunoglobulin G , Allergy and Immunology , Macaca mulatta , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>BACKGROUND</b>To clone and express the ss1 recombinant gene containing S gene and preS1 (10-50 AA) gene in P. pastoris expression system.</p><p><b>METHODS</b>The fusion gene ss1 containing the S (1-222 AA) gene and preS1 (10-50 AA) gene was constructed with PCR method. The fusion ss1 gene was cloned into the expression vector of pPIC3.5k. The linear vector DNA was transformed into the host cell of GS115 with electroporation method. After screening with G418, the product was induced to express with methanol and its antigenicity was analyzed.</p><p><b>RESULTS</b>The molecular weight of expressed ss1 protein was about 30,000 dalton. The product was reactive to anti-HBs and anti-preS1 mAb.</p><p><b>CONCLUSION</b>The fusion gene was efficiently expressed in P. pastoris expression system.The expressed products have the antigenicity of both S and preS1 protein.</p>
Subject(s)
Blotting, Western , Enzyme-Linked Immunosorbent Assay , Gene Expression , Hepatitis B Surface Antigens , Genetics , Metabolism , Pichia , Genetics , Plasmids , Genetics , Protein Precursors , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , Metabolism , Transformation, GeneticABSTRACT
<p><b>OBJECTIVE</b>To study the genome sequence of hepatitis A virus L-A-1 strain which has been applied for live attenuated vaccine production in China, to compare with other HAV strains, to understand some characteristics of L-A-1 strain, and to find the mechanism of attenuation and cell adaptation.</p><p><b>METHODS</b>Genome fragments were prepared by antigen-capture PCR from infected cell (2BS), PCR products were cloned into T vector, sequenced and analyzed by using bioinformatics program.</p><p><b>RESULTS</b>Analysis of the genomic sequences(nt 25-7,418) showed that the open reading frame contains 6,675 nucleotides in length encoding 2,225 amino acids. Sequence homology comparison showed 98.00% and 94.00% homology at nucleotide level, and 98.51% and 98.65% homology at amino acid level with international strains MBB and HM 175, respectively. Through comparison with other attenuated, cell adapted and cytopathic effect (CPE) strains, L-A-1 strain had mutation at nt 152, 591, 646, 687 and insertion at nt 180-181 in 5?NTR and had mutation at nt 3,889 (aa 1 052-Val) in 2B region, these mutations and insertion are molecular basis for cell adaptation; mutation at nt 4,185 (aa 1 152-Lys) in 2C region should be attenuated marker; deletion in 3A region (nt 5,020-5,025) that caused two amino acids deletion is virus fast growth basis.</p><p><b>CONCLUSION</b>Through analyzing L-A-1 strain genomic sequence, certain sites related to cell adaptation and attenuation were found.</p>
Subject(s)
Adaptation, Biological , Genetics , Amino Acid Sequence , Base Sequence , Gene Deletion , Genome, Viral , Hepatitis A Vaccines , Genetics , Hepatitis A virus , Genetics , Mutation , Open Reading Frames , Genetics , Sequence Homology , Vaccines, Attenuated , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To study the safety and immunogenicity of the Bilive combined hepatitis A and B vaccine produced by Sinovac Biotech Co., Ltd.</p><p><b>METHODS</b>Samples were selected from first year students of a senior high school (adults group) and first to fifth grade 1-5 students of 3 primary schools (children group). Those who were susceptible to both hepatitis A virus (HAV) and hepatitis B virus (HBV), HAV only or HBV only were assigned to group AB, A and B respectively and were vaccinated with three doses (0, 1 and 6 month schedule) of Bilive combined hepatitis A and B vaccine, inactivated hepatitis A vaccine and recombined hepatitis B vaccine respectively. The dosage for adult group was 500 U hepatitis A antigen and/or 10 micro g hepatitis B surface antigen and the dosage for children group was half the dosage of adult group. The potential adverse effects were observed within 72 hours after vaccination. Serum samples were collected for testing anti-HAV and anti-HBs at month 2 and 7 after the initial dose.</p><p><b>RESULTS</b>The rates of local adverse effects were 0.58% and 2.56% in children AB group and adults AB group and the general adverse effects rates were 9.88% and 5.45% respectively. Both local and general adverse effect rates were not significantly different to the control group. The sero-conversion rate of anti-HAV in children and adults AB group reached 100%, one month after 3 doses. The geometric mean titer (GMTs) reached 33,910 mIU/ml and 23,435 mIU/ml respectively, significant higher than that in control group (group A). The sero-conversion rates of anti-HBs were 97.30% and 96.63%, and GMTs were 103 mIU/ml and 102 mIU/ml in children and adults AB group respectively. No significant difference on sero-conversion and GMT was observed when compared with control group.</p><p><b>CONCLUSION</b>The Bilive combined hepatitis A and B vaccine had good safety profile, and the immunogenicity both on anti-HAV and anti-HBs was similar to that of separated components.</p>
Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Hepatitis A , Hepatitis A Antibodies , Blood , Hepatitis A Vaccines , Allergy and Immunology , Hepatitis Antibodies , Blood , Hepatitis B , Hepatitis B Antibodies , Blood , Hepatitis B Vaccines , Allergy and Immunology , Safety , Vaccines, Combined , Allergy and Immunology , Vaccines, Synthetic , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVES</b>To calibrate the national hepatitis B virus (HBV) DNA standard according to world health organization's standard material and prepare the national reference panel for HBV DNA reagents.</p><p><b>METHODS</b>Sera from blood donors and HBV patients were collected and detected by home-made HBV DNA PCR kits, HBsAg kits, and anti-HBc kits, and then confirmed by HBV DNA PCR kits produced by Roche in German, which was recognized by the world health organization. The stability of the panel was detected by acceleration method.</p><p><b>RESULTS</b>The convinced copies of the sensitivity samples were gotten by seven independent experiments, the coefficients of variation of logarithm of the copies of L0-L5 were all less than 15%. Regarding the national reference panel as the standard, the quality of most domestic HBV DNA PCR kits was improved, while part of the kits should be further qualified.</p><p><b>CONCLUSION</b>The national reference panel for HBV DNA reagents is developed. It contains eight negative, nine positive sera and seven samples for sensitivity test</p>
Subject(s)
DNA, Viral , Reference Standards , Hepatitis B virus , Genetics , Polymerase Chain Reaction , Reference Standards , Sensitivity and SpecificityABSTRACT
<p><b>OBJECTIVE</b>To examine sensitivity of the tree shrews and Macaca assamensis to human hepatitis B virus (HHBV) by serologic methods.</p><p><b>METHODS</b>Totally 233 tree shrews and 28 Macaca assamensis were inoculated with human sera containing HBV. After inoculation, the sera were collected weekly from them and HBV markers were detected with HBV ditecting ELISA kits.</p><p><b>RESULTS</b>Ninety percent of the tree shrews developed acute infection, among them, 44.4 % persisted for over one year, 33.3% of them developed chronic infection persisted for 2 years and one month; the persistence of HBV in Macaca assamensis was much shorter.</p><p><b>CONCLUSION</b>These data clearly indicated that tree shrew may be used as an animal model for study of chronic HBV infection, whereas, Macaca assamensis, showed only a transient sensitivity to HHBV.</p>
Subject(s)
Animals , Female , Humans , Male , Disease Models, Animal , Hepatitis B , Blood , Allergy and Immunology , Virology , Hepatitis B Surface Antigens , Blood , Hepatitis B e Antigens , Blood , Hepatitis B virus , Macaca , TupaiidaeABSTRACT
ORF3 and partial ORF1 regions were amplified with RT-PCR f rom two patients (T1 and T11)infected with new genotype of hepatitis E Virus. Th e PCR products were cloned and sequenced. The results showed that G-C rich regi on in ORF3 was deleted when amplified with normal PCR reaction. However, PCR rea ction containing G-C melt solution can overcome this problem. The sequence anal ysis showed that T1 and T11 belong to a new genotype of HEV which differs from g enotype I,II and III reported.T1 and T11 have 79%～82%, 80%～81% and 83%～85% id entical to genotype I,II and III respectively.