The replicase genes of five isolates of Cucumber green mottle mosaic virus from Jiangsu, Zhejiang, Hunan and Beijing were amplificated, sequenced and analyzed. The similarities of nucleotide acid sequences indicated that 129 kD and 57 kD replicase genes of CGMMV-No. 1, CGMMV-No. 2, CGMMV-No. 3, CGMMV-No. 4 and CGMMV-No. 5 were 99.64% and 99.74%, respectively. The similarities of 129 kD and 57 kD replicase genes of CGMMV-No. 1, CGMMV-No. 3 and CGMMV-No. 4 were 99.95% and 99.94%, while they were lower between CGMMV-No. 2 and the rest of four reference sequences, just from 99.16% to 99.27% and from 99.04% to 99.18%. All reference sequences could be divided into six groups in neighbor-joining (NJ) phylogenetic trees based on the replicase gene sequences of 129 kD, 57 kD protein respectively. CGMMV-No. 1, CGMMV-No. 3 and CGMMV-No. 4 were clustered together with Shandong isolate (Accession No. KJ754195) in two NJ trees; CGMMV-No. 5 was clustered together with Liaoning isolate (Accession No. EF611826) in two NJ trees; CGMMV-No. 2 was clustered together with Korea watermelon isolate (Accession No. AF417242) in phylogenetic tree of 129 kD replicase gene of CGMMV; Interestingly, CGMMV-No. 2 was classified as a independent group in phylogenetic tree of 57 kD replicase gene of CGMMV. There were no significant hydrophobic and highly coiled coil regions on 129 kD and 57 kD proteins of tested CGMMV isolates. Except 129 kD protein of CGMMV-No. 4, the rest were unstable protein. The number of transmembrane helical segments (TMHs) of 129 kD protein of CGMMV-No. 1, CGMMV-No. 2, CGMMV-No. 3 and CGMMV-No. 5 were 6, 6, 2 and 4, respectively, which were 13, 13 and 5 on the 57 kD protein of CGMMV-No. 2, CGMMV-No. 4 and CGMMV-No. 5. The glycosylation site of 129 kD protein of tested CGMMV isolates were 2, 4, 4, 4 and 4, and that of 57 kD protein were 2, 5, 2, 5 and 2. There were difference between the disorders, globulins, phosphorylation sites and B cell antigen epitopes of 129 kD and 57 kD proteins of tested CGMMV isolates. The current results that there was no significant difference between the replicase gene sequences, it was stable and conservative for intra-species and clearly difference for inter-species. CGMMV-No. 1, CGMMV-No. 3, CGMMV-No. 4 and CGMMV-No. 5 had. a close genetic relationship with Shandong and Liangning isolates (Accession No. KJ754195 and EF611826), they are potentially originate from the same source. CGMMV-No. 2 was closer with Korea isolate. High sequence similarity of tested samples were gathered for a class in phylogenetic tree. It didn't show regularity of the bioinformatics analysis results of 129 kD and 57 kD proteins of tested CGMMV isolates. There was no corresponding relationship among the molecular phylogeny and the bioinformatics analysis of the tested CGMMV isolates.
Computational Biology ; Cucumis sativus ; chemistry ; classification ; enzymology ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Diseases ; virology ; RNA Replicase ; chemistry ; genetics ; metabolism ; Sequence Homology, Nucleic Acid ; Viral Proteins ; chemistry ; genetics ; metabolism

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

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

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

Influenza virus RNA-dependent RNA polymerase (RdRP) is essential for replication and expression of influenza virus genome. Viral genomic sequences encoding RdRP are highly conservative, thus making it a potential anti-influenza drug target. A cell-based influenza RdRP inhibitor screening assay was established by a luciferase reporter system to analyze the activity of RdRP. Specificity study and statistic analysis showed that the screening assay is sensitive and reproducible.
Amantadine ; pharmacology ; Antiviral Agents ; isolation & purification ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Genes, Reporter ; HEK293 Cells ; Humans ; Influenzavirus A ; enzymology ; Luciferases ; genetics ; metabolism ; Oseltamivir ; pharmacology ; Plasmids ; RNA Replicase ; antagonists & inhibitors ; metabolism ; Reproducibility of Results ; Ribavirin ; pharmacology ; Sensitivity and Specificity ; Transfection ; Zanamivir ; pharmacology

Enterovirus 71 (EV71), one of the major causative agents for hand-foot-and-mouth disease (HFMD), has caused more than 100 deaths among Chinese children since March 2008. The EV71 genome encodes an RNAdependent RNA polymerase (RdRp), denoted 3D(pol), which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics. Here we report the crystal structures of EV71 RdRp (3D(pol)) and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4 Å resolution. The structure of EV71 RdRp (3D(pol)) has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp. And the EV71 RdRp (3D(pol)) complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding. The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus (FMDV) 3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase. These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.
Amino Acid Sequence ; Child ; China ; epidemiology ; Crystallography, X-Ray ; Drug Discovery ; Enterovirus A, Human ; chemistry ; enzymology ; Hand, Foot and Mouth Disease ; drug therapy ; epidemiology ; virology ; Humans ; Models, Molecular ; Molecular Sequence Data ; Molecular Targeted Therapy ; Protein Conformation ; Protein Folding ; RNA Replicase ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Substrate Specificity

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

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

In this study, we describe Korean isolates of Trichomonas vaginalis infected with double-stranded (ds) RNA virus (TVV). One T. vaginalis isolate infected with TVV IH-2 evidenced weak pathogenicity in the mouse assay coupled with the persistent presence of a dsRNA, thereby indicating a hypovirulence effect of dsRNA in T. vaginalis. Cloning and sequence analysis results revealed that the genomic dsRNA of TVV IH-2 was 4,647 bp in length and evidenced a sequence identity of 80% with the previously-described TVV 1-1 and 1-5, but only a 42% identity with TVV 2-1 and 3 isolates. It harbored 2 overlapping open reading frames of the putative capsid protein and dsRNA-dependent RNA polymerase (RdRp). As previously observed in the TVV isolates 1-1 and 1-5, a conserved ribosomal slippage heptamer (CCUUUUU) and its surrounding sequence context within the consensus 14-nt overlap implied the gene expression of a capsid protein-RdRp fusion protein, occurring as the result of a potential ribosomal frameshift event. The phylogenetic analysis of RdRp showed that the Korean TVV IH-2 isolate formed a compact group with TVV 1-1 and 1-5 isolates, which was divergent from TVV 2-1, 3 and other viral isolates classified as members of the Giardiavirus genus.
Abscess/parasitology/pathology ; Animals ; Capsid Proteins/genetics ; Cloning, Molecular ; Disease Models, Animal ; Female ; Frameshifting, Ribosomal ; Giardiavirus/classification/genetics/*isolation & purification ; Humans ; Korea ; Mice ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; RNA Replicase/genetics ; RNA, Double-Stranded/*genetics ; RNA, Viral/*genetics ; Sequence Analysis, DNA ; Sequence Homology ; Trichomonas Infections/*virology ; Trichomonas vaginalis/genetics/isolation & purification/pathogenicity/*virology ; Virulence

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