OBJECTIVETo construct the cloning vector of glycoprotein G2 gene of hantavirus (HV), to analyze the sequence of G2 gene by the phylogenetic tree, and to study the differences among glycoprotein G2 genes from the world around.

METHODSEnvelope glycoprotein G2 gene was amplified from four specimens of Shandong province by RT-PCR, and the product recombined into the PMD-18T vector. The clones that carry the G2 gene were identified. After sequencing, the gene sequence was handled with the software DNASTAR, compared with 24 strains worldwide and the phylogenetic tree was drawn.

RESULTSHV G2 gene was amplified by RT-PCR from 4 specimens, named GM04-38.G2, ZB8.G2, JUN5-14.G2, RCH5.G2, respectively. The map of the phylogenetic tree showed that all the 4 strains belonged to SEO-type hantavirus. The analysis of the sequence showed that all the four HV strains had the highest rates of homology with Z37 strain. The sequence homology of SEO-type HV strains was from 82.3% to 99.8%.

CONCLUSIONThe four cloning vectors containing the glycoprotein G2 genes were successfully constructed. Envelope glycoprotein G2 gene of four specimens from Shandong province had high homology rates.

Animals ; China ; Cloning, Molecular ; Hantavirus ; genetics ; Mice ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Viral Envelope Proteins ; genetics

Rubella virus (RV), one of pathogens in TORCH syndrome, can lead to anisotropy of the fetus, and therefore it is of great significance to screen RV infection among women at child-bearing age. At the present, the screening of RV infection is mainly based on the ELISA method, the specificity of RV antigens is very important for ELISA tests. The antigenicity of RV is closely associated with its structural proteins, including capsid protein C, and envelope glycoproteins E1 and E2, which are important surface antigens of RV. This paper reviews the advances in the studies on the structure features, immunogenicity and recombinant proteins of the three structural proteins.
Antibodies, Viral ; analysis ; Enzyme-Linked Immunosorbent Assay ; Recombinant Proteins ; immunology ; Rubella virus ; genetics ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; immunology

OBJECTIVEHepatitis B virus-associated glomerulonephritis (HBV-GN) is an immune complex-mediated glomerulonephritis. The present study was conducted to identify HBV S gene mutation in children with HBV-GN.

METHODSSerum HBV DNA was extracted in 53 children, including 30 with HBV-GN, 5 with HBV-carrying nephrosis (control group 1), and 18 HBV carriers (control group 2). HBV S gene sequence was amplified by polymerase chain reaction (PCR). The PCR products were sequenced directly and compared with AY167097.1, an epidemic HBV strain in China.

RESULTS(1) The adw serotype of HBV was found in all the 30 cases with HBV-GN, 5 cases with HBV-carrying nephrosis and 17 HBV carriers except for 1, in whom adr serotype was identified. (2) HBV genotype B was found in 29 children with HBV-GN, 5 cases with HBV-carrying nephrosis and 17 HBV carriers, genotype E was found in a child with HBV-GN, and genotype C in an HBV carrier. (3) A total of 17 kinds of different single nucleotide change in HBV S gene were identified in 21 of 30 (70%) HBV-GN patients. Among them, 16 of 21 (76.2%) nucleotide mutations resulted in amino acid substitution. It was interesting that most (11/16, 68.8%) amino acid substitutions involved threonine, serine and tyrosine, the potential phosphorylation sites of mitogen-activated protein kinase (MAPK) and protein tyrosine kinase (PTK) in HBV protein. Single nucleotide changes which didn not result in amino acid substitution were found in 2 HBV-carrying nephrosis patients, 2 HBV carriers and 5 cases with HBV-GN.

CONCLUSIONSingle nucleotide changes in HBV S gene were found in most children with HBV-GN. Most mutations in HBsAg resulted in amino acid substitutions involving threonine, serine and tyrosine, which may play a role in the pathogenesis of HBV-GN.

Amino Acid Substitution ; Carrier State ; Child ; DNA Mutational Analysis ; DNA, Viral ; genetics ; Genes ; Genotype ; Glomerulonephritis ; virology ; Hepatitis B virus ; genetics ; Humans ; Mutation ; Viral Envelope Proteins ; genetics

OBJECTIVEGene sequence data were clustered to explore evolution lineages of H3 antigen of influenza A virus.

METHODSAll data of H3 RNA sequence in NCBI Genbank and Influenza sequence database were downloaded and aligned in ClustalX while two step cluster method were applied to explore the data.

RESULTSAll sequences were aggregated into ten clusters, while seven of them mainly were human virus. Human virus and avian/other mammal virus were separated into different clusters distinctively, but coexisted into same clusters with swine virus. Time and host distribution were very distinctive in these clusters, but no geographic distribution features were found.

CONCLUSIONWith the interaction of human immunity system, H3 antigen mutated significantly every 5 - 7 years, and the speed of mutation had accelerated with the application of influenza vaccines in recent years. Mean while, human and swine influenza virus were not separated distinctly between clusters indicating that they had short inheritance distance. Result showed again that swine served as the mixer for antigenic recombination of different influenza virus.

Antigenic Variation ; genetics ; Antigens, Viral ; genetics ; Cluster Analysis ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Hemagglutinins, Viral ; genetics ; immunology ; Humans ; Influenza A virus ; genetics ; immunology ; Mutation ; Sequence Analysis, DNA ; Viral Envelope Proteins ; genetics

BACKGROUNDTo investigate sequence of the complete E region of genotype 2a hepatitis C virus genome and to set up the basis for the further study on biological functions of envelope proteins of HCV.

METHODSTwo cDNA fragments of 770bp, 1,100bp long, from serum of a patients infected HCV, were amplified by reverse transcription nested polymerase chain reaction (RT-nPCR). After being digested with two restriction endonucleases, the two fragments were cloned into JM105 respectively, then sequenced.

RESULTSThe two corresponding nucleotide sequences were obtained and ligated. The complete nucleotide sequences of envelope region and corresponding deduced amino acid sequences were compared with those of HCV-1, HC-C2, HCV-BK, HC-J6 and HC-J8. The homology of nucleotide sequence of the isolated strains was 60.5%, 60.1%, 59.7%, 87.8% and 67.5% respectively; the amino acid sequence homology to the isolated strains was found to be 67?3%, 66.4%, 65.0%, 87.8%, 79.0%, respectively.

CONCLUSIONSThe results indicate that the isolated strain (HCV-JS) belongs to genotype 2a, but there is heterogeneity between HCV-JS and HC-J6 strain.

Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; genetics ; Genotype ; Hepacivirus ; genetics ; Hepatitis C ; virology ; Humans ; Molecular Sequence Data ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Viral Envelope Proteins ; genetics ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo determine the sequence of S2 gene of SARS-associated coronavirus (SARS-CoV) GD322 and analyze the phyletic evolution of S2 gene.

METHODS2 gene fragment was amplified from SARS-CoV GD322 genome with RT-PCR and ligated to pGEM-T vector for sequence analysis after transformation of the plasmid into E. coli DH5a. The variability of S2 genes and S2 proteins from 12 strains isolated in the early, intermediate and advanced stages of the SARS outbreak were analyzed and the phylogenetic tree was constructed with Lasergene, Clustal X, DNAman and Treeview. T cell antigen epitopes of S2 protein were predicted on the basis of Internet database.

RESULTWith the epidemic spread of SARS-CoV, the S2 genes of the virus tended to become stable. Homology of S2 genes of SARS-CoV isolated in advanced stage of the outbreak reached 99.9%. Prediction of T cell antigen epitope showed that mutation at the 57th amino acid effected T cell antigen epitope.

CONCLUSIONS2 gene of GD322 SARS-CoV is relatively stable during the epidemic spread of the virus, and mutation at the 57th amino acids of S2 protein may affect the T cell antigen epitope.

Escherichia coli ; genetics ; Genetic Variation ; Humans ; Phylogeny ; Point Mutation ; SARS Virus ; genetics ; isolation & purification ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; virology ; Viral Envelope Proteins ; genetics

Our previous studies found that the Chinese attenuated EIAV vaccine was composed of a pool of quasispecies, which showed a complicated diversity called "multi-species". Further determining the viral composition of these species in the vaccine should improve the identification of predominant viruses in the vaccine and facilitate the analysis of in vivo evolution of EIAV and the vaccine. In this study, the comparison of fidelities in amplifying and sequencing the V3 to V5 fragment of EIAV envelope gp90 gene by either a single-genome amplification (SGA) approach or the traditional RT-PCR (bulk PCR) was performed. Results revealed that the diversities were 1.84% and 1.88% for SGA- and bulk PCR-derived sequences, respectively. Futher analysis revealed that beside the sequences highly homologous to those derived by the bulk PCR, nine of 73 sequences derived by SGA contained a deduced amino acid domain that was identical to the corresponding domain in the virulent strain LN40. In addition, sequences with deletion of one predicted amino acid residual was detected by using SGA The presence of these less populated sequences provided additional evidence for the "multi-species" hypothesis for the action mechanism of the EIAV vaccine. Furthermore, based on the analysis of sampling bias, Our results that the difference in copy number of each viral specie in the pool of quasispecies resulted in the inefficiency to amplify viral sequences that were in low population by bulk PCR. Therefore, the sequences amplified by bulk PCR could not correctly represent the composition of quasispecies. As an approach based on the amplification and sequencing single isolated genome, SGA significantly improved the weakness of bulk PCR and appeared its advantage in analysis of EIAV genome composition with high variety.
Calibration ; Cloning, Molecular ; DNA, Complementary ; genetics ; Genome, Viral ; genetics ; Infectious Anemia Virus, Equine ; immunology ; Nucleic Acid Amplification Techniques ; methods ; Polymerase Chain Reaction ; Sequence Analysis ; methods ; Vaccines, Attenuated ; genetics ; Viral Envelope Proteins ; genetics ; Viral Vaccines ; genetics

The biological and genetic characteristics of a highly neurovirulent JE virus strain SA4 were studied. Mice were inoculated intracerebrally with strain SA4 and SA14, and observed for 14 days, respectively. On different days, mice brains were harvested for titrations of the virus content in the brains. Full-length genome of SA4 was sequenced and compared with SA14 as well as other JE virus strains in the world. The results indicated that the mice inoculated by SA4 induced sickness and death more rapidly (24 hours faster) than those induced by the SA14. The virus titers in the brains of mice infected with SA4 were 0.5-1.0 lg PFU/mL higher than that infected with SA14. The sequence comparison indicated that the nucleotide and amino acid homology between SA4 and the other 21 JE strains were 84.6%-99.0% and 95.2%-99.7% respectively. Comparison with strain SA14 revealed that there were 17 amino acid differences between the two strains, of which 5 were in the E protein region. The results demonstrate that strain SA4 is a highly neurovirulent strain. The substitutions of the 17 amino acids in the SA4 strain can be the molecular basis for the biological characteristics of high neurovirulence.
Animals ; Brain ; virology ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; pathogenicity ; Encephalitis, Japanese ; mortality ; virology ; Genotype ; Humans ; Mice ; Sequence Analysis ; Viral Envelope Proteins ; genetics ; Virulence

BACKGROUNDTo study arboviruses carried by mosquitoes in Liaoning Province in 2002.

METHODSTotally 4927 mosquitoes were collected from Liaoning Province in July 2002. Virus strains were isolated by inoculating BHK-21, C6/36 and Vero cells. The newly isolated strains were identified by serological (ELISA and IFA) and molecular methods (Real-Time PCR and RT-PCR).

RESULTSTwo strains were isolated from mosquitoes causing cytopathogenic effect (CPE) on cells and were fatal to suckling mice. Serological tests showed that both were positive for the antibody to JEV. The PrM and E gene were cloned and sequenced. The phylogenetic analysis showed that the new isolates belonged to genotype I, JEV. Sequence analysis showed that the homology of nucleotide sequences and amino acid (AA) sequences between the two strains was 100%. Compared with the nucleotide sequences between the two strains and the standard JEV vaccine strain SA14-14-2, the difference was up to 4.11%, and the difference of AA was 0.6%.

CONCLUSIONTwo strains of JEV were isolated and identified in Liaoning province, both belonged to genotype I JEV.

Animals ; Cell Line ; Cercopithecus aethiops ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Analysis, DNA ; Vero Cells ; Viral Envelope Proteins ; genetics

This paper investigated the envelope protein E1/E2 quasispecies genetic characterization of 4 HCV positive sera (Genotype 1b: 274, 366, 383; Genotype 2a: 283) in China. Nucleotide acid was extracted and glycoprotein E1/E2 (191-764aa) coding genes were obtained by RT-PCR, positive clones were randomly selected for sequencing. The phylogenetic relationships and the homology of nucleotide and amino acid were analyzed based on E1/E2 coding genes, and some vital functional regions of E1/E2 were characterized. A total of 43 sequences (274: 10; 283: 12; 366: 13; 383: 8) were obtained showing high genetic heterogeneity in HVR1 and HVR2 regions, while sequences of the neutralizing epitopes, transmembrane domain I, II and N-terminal ectodomain were comparatively conservative. Single base (C) insertion mutation at nt1279 ( E1 region, aa313), resulting in a mutated E1 coding protein (beginning at aa 313) and interruption at N terminus (aa 398) of HVR1 region of E2, was dominant quasispecies sequence(11/12) found in serum 283 . This is the first report on E1/E2 quasispecies in Chinese HCV patients and this novel pattern of insertion mutation provides important information for further study on HCV pathogenesis and immune evasion.
Amino Acid Sequence ; Base Sequence ; DNA Mutational Analysis ; Hepacivirus ; genetics ; pathogenicity ; Hepatitis C ; virology ; Humans ; Molecular Sequence Data ; Mutagenesis, Insertional ; Viral Envelope Proteins ; chemistry ; genetics