BACKGROUNDTo determine the long-term efficacy and persistence of Chinese infants after receiving only active plasma-derived hepatitis B vaccine, and to evaluate if providing booster vaccination after basic hepatitis B immunization is necessary.

METHODSInfants who were born in 1986-1988 in four demonstrative hepatitis B immunization trial areas of Hunan, Guangxi, Hebei and Shanghai after receiving only active plasma-derived hepatitis B vaccination, had been randomly followed up for 15 years. HBsAg,anti-HBs and anti-HBc in 21 680 person-times were tested using commercial SPRIA kits.

RESULTSPrevalence of HBV carriers was less than 1.66% among all children vaccinated with only active plasma-derived hepatitis B vaccine in 4 clinical trial areas. Prevalence of HBsAg did not increase with years after vaccination,90%(95% Cl:83.1%-97.2%) effectiveness of hepatitis B vaccine persisted for 15 years in preventing chronic HBV infection. Carriage, HBV infection and efficacy were not different among all age groups (P>0.05). Seroprotection rate (anti-HBs?10 mIU/ml) and quantity of anti-HBs were significantly decreased with years after vaccination. Seroprotection rates of anti-HBs were 40%-50% and 30%-42% during the 9th-10th year and the 13th-14th ear of vaccination, respectively. Titer of anti-HBs declined?by 90% after 14 years.

CONCLUSIONSThese results showed that long-term efficacy of only active plasma-derived hepatitis B vaccination, which was not affected by decline in seroprotection rate and titer of anti-HBs. For children and adults whose immune status is normal, booster doses of vaccine are not recommended.

Adolescent ; Child ; Child, Preschool ; Follow-Up Studies ; Hepatitis B ; prevention & control ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B Vaccines ; immunology ; Humans ; Infant ; Infant, Newborn ; Vaccination

BACKGROUNDTo understand the characterization of genome of a strain of avian influenza A H9N2 virus repeatedly isolated from a child with influenza illness. Thereafter to reveal the origin of this H9N2 virus.

METHODSViruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares.

RESULTSGenome of A/Guangzhou/333/99 (H9N2) virus was closely related to avian influenza A H9N2 virus, but obvious difference from that of A/Duck/Hong Kong/Y439/97(H9N2) virus, as well as its genome did not include any RNA segment derived from human influenza A virus. However, the genes encoding the HA,NA,NP and NS proteins of A/Guangzhou/333/99 virus were derived from those of G9 lineage virus, the rest genes encoding the M and three polymerase (PB2,PB1 and PA) proteins were derived from G1 lineage strain.

CONCLUSIONSA/Guangzhou/333/99 virus was a reassortant derived from reassortment betweenG9 and G1 lineages of avian influenzaA(H9N2) viruses. Therefore, the most possibility is that it is derived from avian influenza A virus directly. The results do not only demonstrate that avian influenza A (H9N2) virus could infect men, but also firstly prove that the genetic reassortment could be occurred between different genetic lineages of avian influenza A (H9N2) viruses in the nature.

Animals ; Base Sequence ; Chick Embryo ; Child ; Genome, Viral ; Humans ; Influenza A Virus, H9N2 Subtype ; Influenza A virus ; genetics ; Influenza, Human ; virology ; Phylogeny

BACKGROUNDTo investigate expression of IL-6 in non?replicating vaccinia virus and its immune effects on recombinant virus.

METHODSThe recombinant non replicating vaccinia virus RVJ123 delta CK11 beta 75IL6 was constructed with non?replicating vaccinia virus vector pNEOCK11beta75IL6 and replicating vaccinia virus RVJ123. In animal model, immunization with the recombinant virus was carried out and its immune response was studied.

RESULTSThe recombinant virus could express IL-nd HBsAg simultaneously. Southern blot analysis demonstrated that the genes between vaccinia virus Hind? C and K fragments were deleted and IL-6 gene was integrated stably. Given intranasal inocula of the virus to immunize BALB/c mouse and New Zealand Rabbit, the elevated anti-HBsAg IgA and IgG antibody secreting cells in mouse lung lymphoid to vectors expressing IL-6 was at about two?fold higher level than those elicited by control virus at day 14 after immunization. Authors also could detect elevated anti-HBsAg IgA and IgG antibody conversion in mouse serum and lung fluid, rabbits serum, lung fluid, saliva, vagina and nasal washing samples.

CONCLUSIONSIL-6 expressed by non-replicating recombinant vaccinia virus could enhance the induced?immune effects, it could serve as the effective adjuvant for recombinant vector vaccine.

Adjuvants, Immunologic ; Animals ; Cells, Cultured ; Chick Embryo ; Female ; Genetic Vectors ; Humans ; Immunoglobulin A ; analysis ; Immunoglobulin G ; analysis ; Interleukin-6 ; biosynthesis ; genetics ; immunology ; Lung ; immunology ; Mice ; Mice, Inbred BALB C ; Rabbits ; Recombination, Genetic ; Vaccinia virus ; immunology ; metabolism ; physiology ; Virus Replication

BACKGROUNDBased on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity.

METHODSTwo unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB. Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project.

RESULTSThe mutated residues M31?D,D32?P of LoopAB in parent IFN were produced. The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed. The recombinant protein has been expressed in E.coli JM103. The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT. 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference.

CONCLUSIONSThe authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.

Amino Acid Substitution ; Antibodies, Viral ; Antiviral Agents ; pharmacology ; Cells, Cultured ; DNA Mutational Analysis ; Humans ; Interferon Type I ; genetics ; pharmacology ; Interferon-alpha ; Mutagenesis, Site-Directed ; Mutation ; Peptide Fragments ; genetics ; pharmacology ; Plasmids ; genetics ; Receptors, Interferon ; metabolism ; Recombinant Proteins

BACKGROUNDTo determine the relationship between diversity of hepatitis C virus quasispecies and viremia, activity of liver disease and response to interferon therapy.

METHODSHCV quasispecies heterogeneity in 68 patients with chronic hepatitis C were detected by single stranded conformational polymorphism (SSCP) analysis of the HCV E2 hypervaribale region 1 (HVR1); of these, 48 were subsequently treated with interferon-alpha for 6 months.

RESULTSHVR1 was amplified in 61 patients. The average number of SSCP bands was 6.2+/-2.4. Quasispecies heterogeneity significantly correlated with serum HCV RNA levels (P<0.01), but not with serum ALT, AST levels and histological activity index (P>0.05). Of the patients who received interferon therapy, 43 were HVR1 positive. Patients who gained sustained response (n=11) had lower pre-treatement quasispecies heterogeneity (3.3+/-1.2) compared to those who had complete end-of-treatment response (ETR) with relapse (6.3+/-2.2, n=12, P<0.5) or no response (8.0+/-3.3, n=20, P<0.01). At the end of treatment, HVR1 could still be detected in 16 patients. The number of quasispecies heterogeneity in these patients decreased to 3.4+/-1.2, which was significantly lower than that in the patients who didn't receive interferon therapy (6.8+/-2.5, P<0.01). Of these 16 patients, 10 had change in quasispecies patterns.

CONCLUSIONSIncreased quasispecies heterogeneity can cause high HCV viremia, but it is not related to severity of liver disease. Quasispecies heterogeneity is another marker to predict the response to interferon-alpha in patients with chronic hepatitis C.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; Female ; Genetic Heterogeneity ; Hepacivirus ; drug effects ; genetics ; Hepatitis C, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; therapeutic use ; Male ; Middle Aged ; Polymorphism, Single-Stranded Conformational ; RNA, Viral ; blood ; Viremia ; virology

BACKGROUNDTo select the mutants of HPV type 16 E6 and E7 genes suitable for construction of vaccine for treatment of cervical cancer.

METHODSE6 and E7 genes were modified by site-directed mutagenesis. Several recombinant vaccina viruses were constructed by inserting the E6 or E7 mutants into the genome of vaccina virus Tiantan strain and employed to study their antigenicity.

RESULTSWestern blot assay showed that the E6 ?mutant? with substitution of Gly for Leu at amino acid site 50 and E7 mutant with substitution of Gly for Cys-24 and Glu-26 had no effect on their stability and antigenicity, but change of the Cys at position 91 of E7 dramatically reduced its stability and antigencity. Conclusion The results confirmed that the Zinc-finger structure at the E7 C-terminal? plays an important role in the integrity and stability of E7 protein.

Animals ; Antibodies, Viral ; biosynthesis ; Female ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Insertional ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins ; Vaccinia virus ; immunology ; Zinc Fingers

BACKGROUNDTo establish an ELISA method for detecting the activity of HIV-1 integrase and screening of inhibitors.

METHODSHIV recombinant plasmid F185K/C280S IN1-288 was transformed into the E.coli strain BL21(DE3) integrase with a histag was induced by adding isopropyl-?-D?thiogalactopyranoside (IPTG)and purified by nickel affinity chromatography. The synthesized donor substrate oligonucleotide representing the HIV-1 U5LTR was immobilized onto covalink polystyrene microtiter plates, and a synthesized biotinlated 20 bp oligonucleotide was used as the target substrate. The products were detected and quantified using a colorimetic avidin?linked alkaline phosphatase reporter system,and identified by 32? autoradiography. Some natural products and chemically synthesized compounds were screened for HIV-1 integrase inhibitors.

RESULTSThe purified integrase was identified by SDS?PAGE and showed integration activity by ELISA and?32P radioisotopic assay.?Coefficients of variation (CV)of ELISA in a lot and among the lots were 4.63% and 5.89% respectively, the mean of P/N was 2.836 0.161 under the optimal experimental condition. Some plant extracts were found as potent integrase inhibitors. The IC50s for CEH and CEHL were (20.41 5.68)?g/ml and (7.56 1.86)?g/ml respectively.

CONCLUSIONSThe authors have established a simple and rapid ELISA method with stable repeatability for detecting integrase activity, which can be used for screening and studying of specific inhibitors of HIV-1 integrase.

Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; Enzyme-Linked Immunosorbent Assay ; methods ; HIV Integrase ; isolation & purification ; metabolism ; HIV Integrase Inhibitors ; pharmacology ; HIV-1 ; enzymology ; Recombinant Fusion Proteins ; isolation & purification ; metabolism

BACKGROUNDTo elucidate relationship between amino acid sequence of non-structural protein 5A (NS5A) and outcome of HCV (1 b) patients after interferon (IFNa) therapy.

METHODSSera of 24 patients were collected before, during and after IFNa therapy. Pretreatment RNA levels and the sequences of HCV NS5A interferon sensitivity determining region (ISDR) were determined. NS5A full-length sequences of 5 HCV isolates from 3 patients with different response types were also analyzed. Phylogenetic tree analysis and protein secondary structure prediction were undertaken.

RESULTSPretreatment RNA levels of sustained response group were significantly lower than that of non-response group and relapse group (4.50X104 copies/ml versus 1.82X107 copies/ml, P < 0.01).ISDR sequences of NS5A from pretreatment sera were compared with HCV-J strain (prototype). Thirteen of 24 isolates were wild type,11 of 24 were intermediate type and none of them was mutant type. 3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. Phylogenetic tree based on NS5A full-length sequences classified 5 isolates with 3 different response types into 3 groups. Non-response isolates belonged to the same group as HCV-J. Secondary structure prediction of 5 isolates revealed significant differences existing in 2 255- 2 289. This region was partly overlapped with PKR-binding domain.

CONCLUSIONSLow HCV RNA levels in serum are associated with favorable outcome of IFNa therapy. ISDR sequence alone could not predict outcome of IFN treatment. Combination of determination of HCV RNA levels in serum with sequence analysis of PKR-binding domain may be helpful in predicting the efficacy of IFN therapy.

Amino Acid Sequence ; Antiviral Agents ; therapeutic use ; Hepacivirus ; drug effects ; genetics ; Hepatitis C, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; therapeutic use ; RNA, Viral ; blood ; Viral Nonstructural Proteins ; genetics

BACKGROUNDConstructing replication defective recombinant adenovirus vector expressing the group specific antigen VP6 of human rotavirus and studying the immune responses induced in vivo.

METHODSThe cDNA of full length VP6 was inserted into the adenovirus vector pShuttle-CMV, and recombinant adenovirus genome DNA was obtained through homological recombination in E.coli,then the recombinant adenovirus was gained after transfecting 293 cell line with the genome DNA. Gene integration of VP6 in resultant adenovirus was confirmed by PCR and Southern blot, respectively gene expression was confirmed in 293 cells by Western blot. BALB/c mice were immunized intranasally(inl)and orally(ora), respectively, to test the immunization effects of the adenovirus.

RESULTSRecombinant adenovirus named rvAd-VP6 was obtained. The cDNA of VP6 was integrated in the adenovirus and was able to be expressed in 293 cells stably. The systemic immune responses to rotavirus VP6 could be induced effectively in both oral and intranasal group, the titer of serum IgG antibody in the two group of mice were 1?1 000 and 1?10 000-1?100 000, respectively. In addition to IgG, the serum IgA specific to VP6 could also be detected at a titer of 1?10-1?100. Secretory IgA(sIgA) was detected in both lung lavage fluid and intestinal homogenate when administered intranasally to BALB/c mice, whereas only found in intestinal homogenate in the oral group. The results indicated that the immunization efficacy of intranasal inoculation was superior to that of oral inoculation.

CONCLUSIONSThe recombinant adenovirus vector expressing human rotavirus VP6 was successfully constructed, its ability to induce immune responses has laid a solid foundation for the development of rotavirus genetically engineering vaccine against rotavirus infection.

Adenoviridae ; genetics ; Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; Capsid Proteins ; biosynthesis ; immunology ; Genetic Vectors ; Mice ; Mice, Inbred BALB C ; Recombination, Genetic ; Rotavirus ; immunology

BACKGROUNDAn amino acid polymorphism for Met to Val has been identified at PrP codon 129 from different human races. In this study,the characteristics of polymorphism of PRNP 129th amino acid in Han and Uighur Chinese have been investigated.

METHODSHuman DNAs were extracted from peripheral lymphocytes and PrP gene fragments were amplified with a specific PCR protocol. The distribution of 129th amino acid in PRNP was determined by a PCR-RFLP and the results were analyzed with software SAS for Windows 6.12.

RESULTSThe frequencies of the allele 129 Met and 129 Val were 97.0% and 3.0% in Han Chinese, whereas 91.4% and 8.6% in Uighur Chinese. The frequency of 129 M/M phenotypes in Han Chinese was significantly higher than that in Uighur Chinese (P=0.0490). Comparing the phenotype distributions of codon 129 of Han Chinese with that of Japanese and Caucasian, there was significant difference with Caucasian (P=0.0005),but there was no difference with Japanese (P=0.5040).

CONCLUSIONSThe polymorphism of 129th amino acid in PRNP of Han Chinese is similar to Japanese, but different from Uighur Chinese.

Asian Continental Ancestry Group ; genetics ; China ; Codon ; genetics ; European Continental Ancestry Group ; genetics ; Gene Frequency ; Genotype ; Humans ; Polymorphism, Genetic ; Prion Diseases ; genetics ; Prions ; genetics