BACKGROUND: Molecular detection of Middle East respiratory syndrome coronavirus (MERS-CoV) using real-time reverse transcription (rRT)-PCR assays is the method of choice for diagnosis of MERS. We evaluated the performance of the PowerChek MERS (upE & ORF1a) real-time PCR Kit (PowerChek MERS assay; Kogene Biotech, Korea) a one-step rRT-PCR assay for the qualitative detection of MERS-CoV. METHODS: We evaluated PowerChek MERS assay performance in comparison with nested RT-PCR and sequencing of the RNA-dependent RNA polymerase (RdRp) and N genes. To evaluate diagnostic sensitivity and specificity, 100 clinical specimens (50 positive and 50 negative for MERS-CoV) were simultaneously tested by using the PowerChek MERS and sequencing assays. Assay performance, including limit of detection and precision, was evaluated in vitro by using MERS-CoV RNA transcripts. Analytical specificity was evaluated with a diverse collection of 16 respiratory virus–positive clinical specimens and 14 respiratory bacterial isolates. RESULTS: The 95% limits of detection of the PowerChek MERS assay for the upE and the open rading frame (ORF)1a were 16.2 copies/µL and 8.2 copies/µL, respectively. No cross-reactivity was observed. The diagnostic sensitivity and specificity of the PowerChek MERS assay were both 100% (95% confidence interval, 91.1–100%). CONCLUSIONS: The PowerChek MERS assay is a straightforward and accurate assay for detecting MERS-CoV RNA. The assay will be a useful tool for the rapid diagnosis of MERS and could prove especially important for MERS outbreak control.
Coronavirus Infections* ; Diagnosis ; In Vitro Techniques ; Limit of Detection ; Methods ; Middle East Respiratory Syndrome Coronavirus* ; Middle East* ; Real-Time Polymerase Chain Reaction* ; Reverse Transcription ; RNA ; RNA Replicase ; Sensitivity and Specificity

A double-stranded RNA (dsRNA) mycovirus was detected in malformed fruiting bodies of Pleurotus ostreatus strain ASI2792, one of bottle cultivated commercial strains of the edible oyster mushroom. The partial RNA-dependent RNA polymerase (RdRp) gene of the P. ostreatus ASI2792 mycovirus (PoV-ASI2792) was cloned, and a cDNA sequences alignment revealed that the sequence was identical to the RdRp gene of a known PoSV found in the P. ostreatus strain. To investigate the symptoms of PoV-ASI2792 infection by comparing the isogenic virus-free P. ostreatus strains with a virus-infected strain, isogenic virus-cured P. ostreatus strains were obtained by the mycelial fragmentation method for virus curing. The absence of virus was verified with gel electrophoresis after dsRNA-specific virus purification and Northern blot analysis using a partial RdRp cDNA of PoV-ASI2792. The growth rate and mycelial dry weight of virus-infected P. ostreatus strain with PoV-ASI2792 mycovirus were compared to those of three virus-free isogenic strains on 10 different media. The virus-cured strains showed distinctly higher mycelial growth rates and dry weights on all kinds of experimental culture media, with at least a 2.2-fold higher mycelial growth rate on mushroom complete media (MCM) and Hamada media, and a 2.7-fold higher mycelial dry weight on MCM and yeastmalt-glucose agar media than those of the virus-infected strain. These results suggest that the infection of PoV mycovirus has a deleterious effect on the vegetative growth of P. ostreatus.
Agar ; Agaricales* ; Blotting, Northern ; Clone Cells ; Culture Media ; DNA, Complementary ; Electrophoresis ; Fruit ; Fungal Viruses* ; Methods ; Pleurotus* ; RNA Replicase ; RNA, Double-Stranded ; Weights and Measures

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

Hepatocellular carcinoma (HCC) related to hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is thought to account for more than 80% of primary liver cancers. Both HBV and HCV can establish chronic liver inflammatory infections, altering hepatocyte and liver physiology with potential liver disease progression and HCC development. Cyclophilin A (CypA) has been identified as an essential host factor for the HCV replication by physically interacting with the HCV non structural protein NS5A that in turn interacts with RNA-dependent RNA polymerase NS5B. CypA, a cytosolic binding protein of the immunosuppressive drug cyclosporine A, is overexpressed in many cancer types and often associated with malignant transformation. Therefore, CypA can be a good target for molecular cancer therapy. Because of antiviral activity, the CypA inhibitors have been tested for the treatment of chronic hepatitis C. Nonimmunosuppressive Cyp inhibitors such as NIM811, SCY-635, and Alisporivir have attracted more interests for appropriating CypA for antiviral chemotherapeutic target on HCV infection. This review describes CypA inhibitors as a potential HCC treatment tool that is contrived by their obstructing chronic HCV infection and summarizes roles of CypA in cancer development.
Carcinoma, Hepatocellular* ; Carrier Proteins ; Cyclophilin A* ; Cyclophilins ; Cyclosporine ; Cyclosporins ; Cytosol ; Hepacivirus* ; Hepatitis B virus ; Hepatitis C* ; Hepatitis C, Chronic ; Hepatitis ; Hepatocytes ; Liver ; Liver Diseases ; Liver Neoplasms ; RNA Replicase

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

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

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

Japanese encephalitis virus (JEV), a member of mosquito-borne flaviviruses, is the leading cause of viral encephalitis in a large geographic area of Southeast Asia and Australia. JEV contains a single-stranded positive-sense RNA genome, which encodes its own RNA-dependent RNA polymerase (NS5) that is required for genomic RNA replication. In this study, we have described a pair of mouse antisera specific to the N- or C-terminal region of the NS5. Initially, two hydrophilic regions corresponding to the N-terminus and C-terminus of the NS5 protein were individually amplified by reverse transcription-PCR from the genomic RNA of JEV K87P39 strain. The amplified DNA fragments were cloned into a prokaryotic expression vector, pGEX-4T-1; the resulting constructs were used for the expression of GST fusion proteins, designated GST/NS5N and GST/NS5C, in E. coli BL-21 strain. Following immunization of three BALB/c mice with each of the purified GST/NS5N and GST/NS5C, we obtained two pools of the antisera, specifically recognizing the ~103-kDa NS5 and several smaller NS5-related proteins in BHK-21 and Vero cells infected with JEV K87P39 strain. Overall, we have successfully expressed the N- and C-terminal regions of JEV NS5 fused to the C-terminus of GST and generated the mouse antisera capable of recognizing the NS5 and its related proteins in JEV-infected cells. This would provide a valuable reagent for the study of JEV NS5 in the viral life cycle.
Animals ; Antibody Formation ; Asia, Southeastern ; Asian Continental Ancestry Group ; Australia ; Clone Cells ; DNA ; Encephalitis Virus, Japanese ; Encephalitis, Japanese ; Encephalitis, Viral ; Flavivirus ; Genome ; Humans ; Immune Sera ; Immunization ; Mice ; Proteins ; RNA ; RNA Replicase ; Sprains and Strains ; Vero Cells