OBJECTIVETo clone, identify and phylogenetically characterize a clade B-Thai HIV isolate representing the most prevalent virus in Henan province.

METHODSPeripheral blood mononuclear cells (PBMCs) from an HIV-1 infected patient in Henan Province were separated, and co-cultivated with phytohemagglutinin-stimulated healthy donor PBMCs. Proviral DNA was extracted from productively infected PBMCs. The full-length HIV-1 genome was amplified by using the LA Tag long template PCR system. Primers were positioned in conserved regions within the HIV-1 long terminal repeats. Purified PCR products were T-A ligated into a pWSK29-T vector(CNHN 24 clone). Three recombinant clones containing virtually full-length HIV-1 genome were identified by PCR. The full-length genome was sequenced by using the primer-walking approach. Nucleotide sequence similarities were calculated by the local-homology algorithm. Phylogenetic trees of gag, pol and env reading frames were constructed using the Phylip software.

RESULTSHIV-1 C3V4 sequences indicate that the epidemic in this area was B-Thai subtype. V3 loop multiple amino acid sequence alignments showed amino acid alterations at nine positions. The 9,010 bp genomic sequence derived from isolate CNHN 24 contained all known structural and regulatory genes of an HIV-1 genome. No major deletions, insertions, or rearrangements were found. The highest homologies of the gag, pol, vpr, and vif reading frames to the corresponding clade B-Thai RL 42 sequences were 95.42%-97.08%. Phylogenetic trees showed the closest relationship of CNHN 24 and RL 42.

CONCLUSIONThe cloning and characterization of a virtually full-length HIV-1 B-Thai subtype in central China was completed in our laboratory. The data should be helpful to future studies on the genetic diversity of HIV-1.

Amino Acid Sequence ; Base Sequence ; Blood Donors ; China ; Cloning, Molecular ; DNA, Viral ; genetics ; Female ; Genome, Viral ; HIV Infections ; virology ; HIV-1 ; classification ; genetics ; Humans ; Leukocytes, Mononuclear ; virology ; Phylogeny ; Reading Frames ; Sequence Analysis, DNA ; Sequence Homology

OBJECTIVETo evaluate the first and second assay kits currently used in blood centers for screening HCV infected blood, and to provide basis for a better match of the two assay kits.

METHODSUsing the newly developed multi-recombinant-HCV-antigen supplementary assay kit, the authors evaluated concurrently the specificity and sensitivity of two domestic and one imported anti-HCV detection kits.

RESULTSDiscrepancy in specificity and sensitivity existed among the two domestic HCV kits, and overall quality was slightly below that of leading or main stream imported HCV kit.

CONCLUSIONThe newly developed multi-recombinant-HCV-antigen supplementary assay kit is useful in the evaluation of HCV antibody detection kit currently in use. It provides qualified assessing kit to capture antibodies against various HCV antigens. The present paper provided guidance for selecting a better match of the two screening kits and improved screening efficiency.

Blood Donors ; Evaluation Studies as Topic ; Hepacivirus ; genetics ; immunology ; Hepatitis C Antibodies ; blood ; immunology ; Hepatitis C Antigens ; genetics ; immunology ; Humans ; Reagent Kits, Diagnostic ; standards ; Sensitivity and Specificity

OBJECTIVETo study the relationship between late mRNA and the cytopathic effect(CPE) and ultrastructural features after human cytomegalovirus (HCMV) infection in vitro.

METHODSHuman embryo fibroblast cells(HEL) were infected with HCMV AD169 strain. The expression of the HCMV late mRNA was measured by semiquantitative RT-PCR, the cytopathic effect (CPE) and the cell ultrastructure were observed by means of light microscopy and electron microscopy, respectively.

RESULTSThe HCMV late mRNA could be detected 12 hours postinfection and increased gradually, but the CPE appeared 48 hours postinfection in HEL cells. The HCMV infected cells exhibited significant mitochondrial enlargement and the number of mitochondrial ridge deletion, the cisternae lumen of endoplasmic reticulum (ER) dilation and vacuolization (at the end age). The mature nucleocapsid could be observed 96 hours postinfection.

CONCLUSIONThe ultrastructural changes have an intimate correlation with the expression of HCMV late mRNA and play an important role in the life circle of the virus. HCMV late mRNA may serve as a indicator of the clinical effect of treatment in active HCMV infection.

Cytomegalovirus ; genetics ; physiology ; Cytopathogenic Effect, Viral ; Embryo, Mammalian ; Endoplasmic Reticulum ; pathology ; virology ; Fibroblasts ; metabolism ; ultrastructure ; virology ; Humans ; Inclusion Bodies ; pathology ; virology ; Mitochondrial Swelling ; RNA, Messenger ; metabolism

OBJECTIVETo study the biological features of the adapted rabies virus 3aG-V in Vero cell.

METHODSThe virus structure, culture condition, pathogenicity, immunocompetence, toxicity and protective efficiency were studied.

RESULTSThe rabies virus 3aG-V strain could well adapt to Vero cell, the pathogenicity was very weak and the immunogenicity was very good.

CONCLUSIONThe Vero cell-adapted rabies virus strain may possibly be used as a vaccine strain.

Animals ; Cercopithecus aethiops ; Guinea Pigs ; Mice ; Rabbits ; Rabies ; immunology ; virology ; Rabies virus ; growth & development ; immunology ; pathogenicity ; ultrastructure ; Vero Cells ; Virulence ; Virus Cultivation

OBJECTIVETo identify the location of major immunodominant antigenic region and study the relationship between the gene heterogeneity and immunoreactivity via detecting antigenic reactivity of synthetic peptides deriving from immunodominant region in different genotypes of hepatitis C virus (HCV) NS5a gene.

METHODSIn total, 305 non-identical 30-mer long and overlapping by 15 aa peptides derived from HCV NS5a region from codon 2,182 to 2,343 among 45 unique published HCV sequences in GenBank corresponding to different genotype were designed and synthesized. The amino acid sequences of all peptides were compared with DNA Star software. The antigenic reactivity of those peptides was detected with indirect ELISA with both anti-HCV and anti-NS5 positive serum.

RESULTSThe sequences showed highly conserved among HCV genotype in regions 2,272-2,301 and 2,302-2,331 as compared to regions 2,212-2,241 and 2,257-2,286. The peptides basing on amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331 showed stronger immunoreactivity than any other peptides. Eighteen peptides derived from this region showed a broad immunoreactivity, 3 of them could react with 96% of anti-HCV positive sera. Whereas the immunoreactivity of the peptides derived from the region showing highly variable among HCV genotype was found to react more strongly with homologous-genotype sera.

CONCLUSIONThe major linear antigenic region was located at amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331; the short synthetic peptide derived from NS5a region at position 2,212-2,241, 2,272-2,301 and 2,302-2,331 can be used for efficient detection of HCV antibody; some peptides showed genotype specific immuunoreactivity.

Amino Acid Sequence ; Antigenic Variation ; Antigens, Viral ; blood ; immunology ; Epitope Mapping ; Genotype ; Hepacivirus ; classification ; genetics ; immunology ; Hepatitis C ; immunology ; virology ; Hepatitis C Antibodies ; blood ; immunology ; Humans ; Immunodominant Epitopes ; Peptides ; chemical synthesis ; immunology ; Recombinant Proteins ; genetics ; immunology ; Sequence Homology, Amino Acid ; Viral Nonstructural Proteins ; genetics ; immunology

OBJECTIVETo study the mechanism of hepatitis C virus (HCV) gene regulation and the inhibitory effect of antisense RNA on HCV gene expression in vitro.

METHODSThe hepatoblastoma cell line (HepG2) was co-transfected by recombinant plasmid of antisense RNA complementary to HCV 5' untranslated region (5'UTR)and HCV 5' UTR Directed luciferase (luc) gene expression recombinant plasmid. Meanwhile a reversed HCV 5'UTR recombinant plasmid which can not transcribe as antisense RNA in the cell and a recombinant plasmid in which the luc was regulated by simian virus 40 (sv40) 5'UTR were used as controls respectively. The level of luc gene expression was determined by an enzymatic assay.

RESULTSThe antisense RNA which directed to HCV 5'UTRcould obviously knock down the level of luc gene expression and the close-dependent inhibition of antisense RNA was observed at the same time. However the above inhibition was not shown in the cells co-transfected by reversed HCV 5'UTR recombinant plasmid and HCV 5'UTR directed luc gene expression recombinant plasmid. No reduction was observed in luc gene expression level in the cell co-transfected by both antisense RNA recombinant plasmid and SV40 5'UTR directed luc gene expression recombinant plasmid.

CONCLUSIONHCV 5'UTR plays an important role in regulation of viral gene expression. The antisense RNA complementary to HCV 5'UTR could effectively inhibit the gene expression regulated by HCV 5'UTR in vitro.

5' Untranslated Regions ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Viral ; Genes, Viral ; Hepacivirus ; genetics ; Hepatoblastoma ; pathology ; Humans ; Liver Neoplasms ; pathology ; Luciferases ; genetics ; metabolism ; Plasmids ; RNA, Antisense ; pharmacology ; RNA, Viral ; genetics ; Recombinant Proteins ; genetics ; Transfection

OBJECTIVETo understand the hereditary and variant characteristics of rubella virus(RV), especially the strains isolated from China, investigating the epidemical trend and variation principle of RV.

METHODSThe envelope glycoprotein E1 gene was amplified from rubella virus by RT-PCR. After sequencing, the gene sequence was handled by the software DNASTAR and the phylogenetic tree was drawn to analyze the molecular epidemiological characteristics of RV.

RESULTSThe sequence of RV strain JR23 was sequenced, the phylogenetic tree was drawn taking 30 strains isolated at different times and locations in GenBank, including three strains from China as reference. The regions that encode the peptides which react with the HI antibody and the neutralization antibody were compared to show if there was any amino acid mutation in the sequence. (1) In general, the nucleotide and amino acid sequences of RV were highly conserved. The four strains isolated from China had relatively large variations. Strain 379 and strain BRD constituted genotype II, which is different from the other 29 strains. Further study is needed to understand their heritable resources and biological characteristics. (2) Strain JR23 showed little difference from the strains that were epidemic during 1960s in UK, USA and Japan, so maybe it is the derivative strain of that in epidemic 1960s. But the accurate epidemic time is not known.

CONCLUSIONThere are differences among areas and time in epidemics of rubella. The mobility and the region difference seem to be the key factors that affect the epidemic characteristics of RV.

Amino Acid Sequence ; Amino Acid Substitution ; Base Sequence ; China ; epidemiology ; Genotype ; Molecular Epidemiology ; Mutation ; Phylogeny ; Rubella ; epidemiology ; virology ; Rubella virus ; classification ; genetics ; Sequence Analysis, Protein ; Sequence Homology ; Viral Envelope Proteins ; genetics

OBJECTIVETo explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus.

METHODSThe recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus, which was produced from cloned PA317 cells screened by hygromycin B after "micro-pingpong" technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration.

RESULTSThe recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus.

CONCLUSIONHigh titer of retroviruses could be obtained in the culture medium of PA317 cell line through "micro-pingpong" technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.

Animals ; Breast Neoplasms ; enzymology ; pathology ; virology ; Cell Line ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; Humans ; Mice ; NIH 3T3 Cells ; Recombination, Genetic ; Retroviridae ; genetics ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Titrimetry ; Transfection

OBJECTIVETo investigate the expression dynamics and significance of matrix metalloproteinase-2 (MMP-2) membrane type-matrix metalloproteinase-2 (MT-MMP-2) in hepatic fibrosis and its reversal counterpart.

METHODSAn experimental CCl4 induced hepatic fibrosis rat model was established by intraperitoneal administration of carbon tetrachloride for 2, 4, 6, 8, 10 weeks, and normal rats were used as a control group. The immunohistochemical methods and in situ hybridization were used to detect MMP-2,MT-MMP-2 mRNA and related antigens in the liver.

RESULTSMMP-2,MT-MMP-2 mRNA and related antigens were expressed in mesenchymal cells and parts of hepatocytes besides active pathological changes, especially in the fibrous septum and portal area. Expression of MMP-2,MT-MMP-2 mRNA and related antigens were increased in hepatic fibrosis and decreased gradually in its reversal counterpart.

CONCLUSIONThis study suggested that mesenchymal cells are the main cellular origins of MMPs. The levels of MMP-2 and MT-MMP-2 antigens and gene expression were closely related to hepatic fibrosis. MMP-2 and MT-MMP-2 may play important roles in hepatic fibrosis and its reversal counterpart.

Animals ; Carbon Tetrachloride Poisoning ; Gene Expression Regulation, Enzymologic ; Hepatocytes ; enzymology ; Liver ; enzymology ; pathology ; Liver Cirrhosis, Experimental ; enzymology ; etiology ; pathology ; Male ; Matrix Metalloproteinase 2 ; biosynthesis ; genetics ; Matrix Metalloproteinases ; biosynthesis ; genetics ; Matrix Metalloproteinases, Membrane-Associated ; Mesenchymal Stromal Cells ; enzymology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo study the levels of SARS-associated coronavirus IgG antibody of SARS patients, people who closely contacted SARS patients and normal subjects in Gansu province.

METHODSThe levels of SARS-associated coronavirus IgG antibody were measured by ELISA. The material included acute and (or) recovery period sera of 9 SARS patients, sera from 1,109 doctors and nurses closely contacted with SARS patients, laboratory workers, personnel for disease control and prevention, persons who contacted SARS patients, and sera from 978 normal subjects.

RESULTSSARS coronavirus IgG antibody was detected positive in 6 of the 9 patients, it was still positive in the sera twelve months after recovery; 1 of the closely contacted persons and 3 normal subjects were found positive.

CONCLUSIONThe positive rate of SARS coronavirus IgG antibody of patients was consistent with the clinical diagnosis. The low positive rate of the persons who closely contacted SARS patients and normal subjects suggests that SARS probably had no subclinical infection.

Adolescent ; Adult ; Antibodies, Viral ; blood ; China ; epidemiology ; Health Personnel ; Humans ; Immunoglobulin G ; blood ; Infectious Disease Transmission, Patient-to-Professional ; analysis ; Middle Aged ; SARS Virus ; immunology ; Severe Acute Respiratory Syndrome ; epidemiology ; immunology ; transmission