Influenza poses a great threat to life and health in populations worldwide. Studies regarding the protein components of influenza viruses will facilitate the research and development of vaccines and diag nostic reagents. The influenza virus contains both structural and non-structural proteins. From the outset, it has been accepted that an influenza A virus possesses eight gene segments that encode eight corresponding viral proteins, respectively. Research has demonstrated that the M gene encodes the M2 ion channe! protein and the NS gene encodes the non-structural protein, NS2. In recent years, several novel viral proteins have been identified from influenza A viruses. This article will briefly describe the state of current research into PA-related proteins of influenza A viruses.
Animals ; Humans ; Influenza A virus ; enzymology ; genetics ; Influenza, Human ; virology ; RNA Replicase ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

To develop a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Norwalk GII. 4 primers which recognized 6 distinct regions on the RNA-dependent RNA polymerase gene of Norwalk GII were designed and used for LAMP assay. Norwalk GII RNA was amplified under isothermal conditions (65 degrees C) for 120 min, and LAMP results were then judged with naked eye, SYBR Green I staining, electrophoretic analysis and restriction digestion. To evaluate the specificity of the RT-LAMP, 48 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses were tested. To compare the sensitivity of the RT-LAMP with that of conventional RT-PCR, Norwalk GII RNA was serially diluted and amplified by RT-LAMP and RT-PCR, respectively. With 46 fecal specimens of Norwalk GII, observation with naked eyes, SYBR Green I staining and electrophoretic analysis were able to detect the PCR products in the RT-LAMP assay. The specificity of RT-LAMP products was also confirmed by digestion of the RT-LAMP products with restriction enzymes. No RNA amplification was observed in 2 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses. The specificity of the RT-LAMP assay with regard to RT-PCR were 100% for Norwalk GII. The detection limits of RT-LAMP was 15.6 pg/tube for Norwalk GII and similar to that of a RT-PCR assay. Compared to RT-PCR, the RT-LAMP assay has been proven to be a rapid, sensitive, specific and accurate method for detection of the Norwalk GII in fecal specimens, and that RT-LAMP assay is potentially useful for the rapid detection of Norwalk GII from fecal specimens in outbreaks of infectious diarrhea.
Caliciviridae Infections ; virology ; Feces ; virology ; Humans ; Norwalk virus ; genetics ; isolation & purification ; RNA Replicase ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Proteins ; genetics

OBJECTIVETo determine the molecular characterization of Polymerase complex (PA, PB1 and PB2) genes of H9N2 avian influenza viruses and the genetic relationship of Iranian H9N2 viruses and other Asian viruses.

METHODSThe Polymerase complex (PA, PB1 and PB2) genes from seven isolates of H9N2 viruses isolated from commercial chickens in Iran during 2008-2009 were amplified (by RT-PCR method) and sequenced. Nucleotide sequences (Open Reading Frame: orf) of the PA, PB1 and PB2 genes were used for phylogenetic tree construction.

RESULTSMost PB2 and PA genes of the H9N2 viruses isolated in 2008-2009 belonged to the unknown avian sublineage which grouped with the 2004 Pakistani H7N3 viruses. The PB1 genes of Iranian viruses indicated greater genetic diversity and shared a high level of similarity to PB1 genes from either H5 or H7 subtypes with compared to established H9N2 Eurasian sublineages.

CONCLUSIONSOur findings demonstrated that the H9N2 viruses in Iran exhibit striking reassortment which has led to the generation of new genotypes.

Animals ; Chickens ; virology ; Ducks ; virology ; Genotyping Techniques ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; Influenza in Birds ; virology ; Iran ; Pakistan ; RNA Replicase ; genetics ; Viral Proteins ; genetics

It has been suggested that non-structural protein 5A (NS5A) of hepatitis C virus (HCV) may have a regulatory role similar to other RNA viruses in RNA replication. In order to investigate the replication function of NS5A, we tried to purify recombinant His(6) tagged NS5A expressed in Escherichia coli by a denature-renaturing method. The native lysis buffer was used to remove most of the soluble non-specific proteins. His-NS5A protein was solublized with the denaturing lysis buffer containing 8 mol/L Urea, and then bound to Ni2+ -NTA resin. The protein bound resin was successively washed with buffer containing reducing concentrations of Urea in the presence of NaCl and DTT to renature the protein. The renatured His-NS5A protein was eluted from the resin and it was capable of interacting with glutathione S-transferase fused form NS5Bt (GST-NS5Bt). The purified His-NS5A exhibited an inhibitory effect on RNA-dependent RNA Polymerase (RdRP) activity of GST-NS5Bt in vitro. Conclusively, in this study, we have established a purification method of bacterial recombinant HCV NS5A, and the results support the notion that NS5A may involve in the regulation of HCV replication through direct interaction with NS5B.
DNA Primers ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; physiology ; Plasmids ; Protein Binding ; RNA Replicase ; metabolism ; Recombinant Proteins ; metabolism ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; Virus Replication

Sequence analysis of a new norovirus (NV) isolated from Lanzou city of China was performed based on partial sequence of RNA dependent RNA polymerase (RdRp) and complete capsid protein (VP1) gene. The isolated strain CHN02/LZ35666 shared high sequence homology with GII-4 NVs. Nucleotide homologies of RdRp region and encoded capsid protein region were 90.4% -- 98.6% and 89.8% -- 95.7% , respectively, while amino acid homology of capsid protein region was 94.4% -- 97.4%. The analysis of GDD motif in RdRp region indicated this GDD motif of Lanzhou strain differed from those of the GII-4 predominant epidemic strains. Lanzhou strain formed an independent branch in GII-4 cluster in the phylogenetic tree based on nucleotide sequence of RdRp region and amino acid sequence of capsid protein. Sequence alignment revealed a mutation at the fourth key site of the receptor-binding interface in the strains isolated after 2002 compared with those of previous strains suggesting a possible change of binding pattern to HBGAs receptors.
Amino Acid Sequence ; Base Sequence ; Capsid Proteins ; genetics ; China ; Gastroenteritis ; virology ; Humans ; Molecular Sequence Data ; Norovirus ; classification ; genetics ; Phylogeny ; RNA Replicase ; genetics ; Sequence Alignment ; Sequence Analysis, DNA

OBJECTIVETo investigate the effect of telomerase reverse transcriptase (TERT) to the proliferation of 5-HT induced pulmonary artery smooth muscle cells (PASMCs).

METHODSThe PASMCs proliferation experiment was performed to detect the effort on PASMCs of 5-HT or ASODN TERT (antisense oligoribonucleotides TERT designed according to the rat TERT mRNA sequence of gene bank). The immunohistochemistry staining experiment and the in situ hybridization experiment were to detect the TERT protein and mRNA expression with 5-HT or ASODN TERT. FITC marked ASODN TERT experiment was done to research the distribution of ASODN TERT in PASMCs.

RESULTS5-HT promoted PASMCs proliferation in a dose-dependent manner (10(-9) - 10(-5) mol/L). 5-HT also significantly increased TERT expression at protein and mRNA levels as shown by immunohistochemistry staining and the in situ hybridization studies. This effect could be blocked by ASODN TERT in a time and dose-dependent manner.

CONCLUSIONSOur experiments show TERT is one of the key factors in the procession of 5-HT induced PASMCs proliferation. ASODN TERT might be a potential therapy agent for pulmonary hypertension.

Animals ; Cell Proliferation ; Cells, Cultured ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Pulmonary Artery ; cytology ; RNA Replicase ; RNA, Messenger ; genetics ; Rats ; Serotonin ; pharmacology ; Telomerase ; pharmacology

OBJECTIVETo study the molecular characteristics of Noroviruses causing outbreaks of acute gastroenteritis in Huzhou.

METHODSFrom 2008 to 2010, total 119 fecal specimens collected from outbreaks of acute gastroenteritis were tested for Norovirus. Partial sequence of RNA dependent RNA polymerase (RdRp) of the positive samples were amplified by RT-PCR, then the PCR production were purified, sequenced and put into phylogenetic analysis.

RESULTS50 of 119 specimens were positive for Norovirus by real-time RT-PCR. Out of those 50 Norovirus positive specimens, 9 were Norovirus Genogroup I (GI) positive, 35 were Norovirus Genogroup II (GII) positive, 6 was both Norovirus GI and GII positive. 12 PCR products for RdRp were selected for further studies on sequencing. Phylogenetic analysis revealed that the 5 GI norovirus isolates were belonged to genotype GI/2 and GI/3. Of the 7 GII norovirus isolates, 6 were belonged to genotype GII/4, 1 was belonged to genotype Glib.

CONCLUSIONNorovirus is a major cause of outbreaks of acute gastroenteritis in Huzhou and the epidemic strains of norovirus isolated from Huzhou had a high degree of genetic diversity.

Acute Disease ; China ; epidemiology ; Disease Outbreaks ; Female ; Gastroenteritis ; epidemiology ; Genetic Variation ; Humans ; Male ; Norovirus ; classification ; genetics ; Phylogeny ; RNA Replicase ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate whether the nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus RNA genome could affect the expression of the survivin gene.

METHODSHCV NS5A expression plasmid (pCNS5A) was transfected into HepG2 cells using a lipofectin reagent and the HCV NS5A protein was detected using immuno-histochemistry staining. Survivin mRNA was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and the expression of survivin protein was detected using Western blot.

RESULTSHCV NS5A protein was detected in the cytoplasm of the HepG2 cells transfected with pCNS5A, and the expressions of survivin mRNA and survivin protein were both up-regulated in the presence of the HCV NS5A protein.

CONCLUSIONSHCV NS5A protein can stimulate survivin protein expression, and this might be the result of the strengthening of the survivin gene transcription.

Carcinoma, Hepatocellular ; pathology ; Humans ; Inhibitor of Apoptosis Proteins ; Liver Neoplasms ; pathology ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA Replicase ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Tumor Cells, Cultured ; Viral Nonstructural Proteins ; genetics

In order to study the proliferation inhibition effect of H5N1 subtype avian influenza virus (AIV) with small interfere RNA (siRNA), a total of 4 siRNAs were designed in accordance with the NP and PA genes of H5N1 subtype AIV, the siRNAs were then transfected to chicken embryo fibroblast(CEF), CEF was infected with H5N1 subtype AIV after 6 hrs. Virus titer of cell supernatant was tested at 16-56hrs post infection, and pathological changes of the cells was observed; mRNA levels of NP, PA, HA and p13-actin gene were tested at 36hrs post infection. The results showed that these 4 siRNAs could inhibit the prolif-eration of H5N1 subtype AIV in CEF in varying degrees, and one siRNA targeting PA was best per-formed. The experimental results also showed that the inhibition effect was decreased with the time prolonged. This research provides a basis for further studying RNAi on AIV prevention and control.
Actins ; genetics ; Animals ; Chick Embryo ; DNA Primers ; genetics ; Fibroblasts ; virology ; Hemagglutination ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Hemagglutinins ; genetics ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; growth & development ; physiology ; RNA Interference ; RNA Replicase ; genetics ; RNA, Small Interfering ; chemical synthesis ; genetics ; RNA-Binding Proteins ; genetics ; Real-Time Polymerase Chain Reaction ; Specific Pathogen-Free Organisms ; Transfection ; Viral Core Proteins ; genetics ; Viral Proteins ; genetics ; Virus Replication

The R (replicase) protein is the uniquely defined non-structural protein (NSP) responsible for RNA replication, mutation rate or fidelity, regulation of transcription in coronaviruses and many other ssRNA viruses. Based on our complete genome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China, we analyzed the structure and predicted functions of the R protein in comparison with 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF (open-reading frame) encodes for two major enzyme activities, RNA-dependent RNA polymerase (RdRp) and proteinase activities. The R polyprotein undergoes a complex proteolytic process to produce 15 function-related peptides. A hydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identified within NSP1. The substitution rate of the R protein is close to the average of the SARS-CoV genome. The functional domains in all NSPs of the R protein give different phylogenetic results that suggest their different mutation rate under selective pressure. Eleven highly conserved regions in RdRp and twelve cleavage sites by 3CLP (chymotrypsin-like protein) have been identified as potential drug targets. Findings suggest that it is possible to obtain information about the phylogeny of SARS-CoV, as well as potential tools for drug design, genotyping and diagnostics of SARS.
Amino Acid Sequence ; Base Composition ; Base Sequence ; Cluster Analysis ; Computational Biology ; Conserved Sequence ; genetics ; Evolution, Molecular ; Gene Components ; Genome, Viral ; Molecular Sequence Data ; Mutation ; genetics ; Phylogeny ; Protein Structure, Tertiary ; RNA Replicase ; genetics ; SARS Virus ; genetics ; Sequence Analysis, DNA