OBJECTIVETo study the mechanism of the cellular proteins involved in the process of replication of hepatitis C virus (HCV) negative-strand RNA.

METHODSUltraviolet (UV) cross-linking was used to identify the cellular proteins that would bind to the 3'-end of HCV negative-strand RNA. Competition experiment was used to confirm the specificity of this binding, in which excess nonhomologous protein and RNA transcripts were used as competitors. The required binding sequence was determined by mapping, then the binding site was predicted through secondary structure analysis.

RESULTSA cellular protein of 45 kD (p45) was found to bind specifically to the 3'-end of HCV negative-strand RNA by UV cross-linking. Nonhomologous proteins and RNA transcripts could not compete out this binding, whereas the unlabeled 3'-end of HCV negative-strand RNA could. Mapping of the protein-binding site suggested that the 3'-end 131-278nt of HCV negative-strand RNA was the possible protein-binding region. Analysis of RNA secondary structure presumed that the potential binding site was located at 194-GAAAGAAC-201.

CONCLUSIONThe cellular protein p45 could specifically bind to the secondary structure of the 3'-end of HCV intermediate negative-strand RNA, and may play an important role in HCV RNA replication.

Binding Sites ; Hepacivirus ; genetics ; Nucleic Acid Conformation ; RNA, Viral ; chemistry ; metabolism ; RNA-Binding Proteins ; analysis ; metabolism ; Virus Replication

BACKGROUND: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. METHODS: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). RESULTS: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). CONCLUSIONS: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.
Acetylcysteine/chemistry ; Citrates/chemistry ; Coronavirus Infections/diagnosis ; Deoxyribonuclease I/metabolism ; Endopeptidase K/metabolism ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/*isolation & purification ; RNA, Viral/analysis/*isolation & purification/metabolism ; Real-Time Polymerase Chain Reaction ; Sputum/*virology

During the 2015 outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea, 186 persons were infected, resulting in 38 fatalities. We isolated MERS-CoV from the oropharyngeal sample obtained from a patient of the outbreak. Cytopathic effects showing detachment and rounding of cells were observed in Vero cell cultures 3 days after inoculation of the sample. Spherical virus particles were observed by transmission electron microscopy. Full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade B of MERS-CoV.
Animals ; Cercopithecus aethiops ; Coronavirus Infections/*diagnosis/epidemiology/*virology ; Disease Outbreaks ; Humans ; Microscopy, Electron ; Middle East Respiratory Syndrome Coronavirus/classification/genetics/*isolation & purification/ultrastructure ; Phylogeny ; Polymerase Chain Reaction ; RNA, Viral/analysis/chemistry/metabolism ; Republic of Korea/epidemiology ; Sequence Analysis, RNA ; Vero Cells

Hepatitis C virus (HCV) is a major causative agent in liver disease. In order to investigate if Korean type HCV core protein and its related mutants, S99Q and S116I, are cytopathic to liver, three types of transgenic mice were established. The expression of transgenes was confirmed by HCV specific RT-PCR and Western immunoblotting. The livers of all wild type core and S116I transgenic lineages remained largely histologically normal. However, the livers of the S99Q transgenic mice showed significant high level of cell dysplasia associated with the transgene expression in hepatocytes largely located around the central veins by in situ hybridization analysis. In conclusion, the mutant HCV core protein at S99Q may contribute to the progress of HCV induced liver disease.
Amino Acid Sequence ; Animals ; Base Sequence ; Gene Expression ; Genetic Vectors/genetics ; Hepatitis C/*pathology/virology ; Hepatitis, Viral, Animal/*pathology/virology ; Hepatocytes/pathology/virology ; Liver/pathology/*virology ; Mice ; Mice, Transgenic ; Molecular Sequence Data ; Mutation/genetics ; RNA, Messenger/chemistry/metabolism ; Research Support, Non-U.S. Gov't ; Transgenes ; Viral Core Proteins/analysis/*genetics/metabolism

OBJECTIVETo investigate the antiviral effects of the aqueous extract of Spatholobus suberectus Dunn. (A.E.), a Chinese medicinal herb, against coxsackievirus B3 (CVB3).

METHODSThe antiviral effects of A.E. against CVB3 in vitro (primarily cultured myocardial cells) and in vivo (BALB/c mice) were determined. Serum pharmacological method was also adopted by in vitro experiments. The effects of A.E. inhibiting the CVB3 mRNA expression were compared by RT-PCR in mice in vivo.

RESULTSA.E. exhibited obvious antiviral: effects in vivo, and serum samples obtained from the rats with oral administration of A.E. (10 μg/mL, 5 μg/mL), reduced the virus titers in the infected myocardial cells (3.00±0.70, 3.55±0.52, P<0.01). Meanwhile, the viral myocarditis induced by CVB3 was inhibited significantly by A.E., and the 15-day mortality was reduced to 40% and 45% (P<0.01) in mice treated with A.E. at doses of 50 mg/kg and 100 mg/kg, respectively, while the 30-day mortality was decreased to 45% and 50%, respectively (P<0.01). Moreover, the mRNA expression of Coxsackie virus B3 was significantly inhibited by A.E.

CONCLUSIONAqueous extract of Spatholobus suberectus Dunn. (A.E.) has inhibitory effect on CVB3 both in vitro and in vivo.

Animals ; Antiviral Agents ; pharmacology ; therapeutic use ; Body Weight ; drug effects ; Cercopithecus aethiops ; Coxsackievirus Infections ; blood ; drug therapy ; pathology ; virology ; Enterovirus ; drug effects ; Fabaceae ; chemistry ; Gene Expression Regulation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Myocardium ; pathology ; Organ Size ; drug effects ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Survival Analysis ; Vero Cells ; Viral Load