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

OBJECTIVEEstablish the fluorescent quantitative RT-PCR detection method for rabies virus (RV) and construct RNase-resistant virus-like particles as positive controls.

METHODSAnalyze the database in GenBank, the probe and the primers were designed in the conservative region of N gene of rabies virus and the method of real-time fluorescent quantitative PCR was obtained; On the basis of MS2 phage, with the technology of gene recombination, prepare the RNase-resistant virus-like particles for RV positive controls;

RESULTSRNase-resistant virus-like particles were obtained after prokaryotic expression in E. coli. The designed primers and probe were confirmed to be very specific and conservative, and be sensitive to-concentration of 15 copies/microl.

CONCLUSIONEstablished the method of detecting rabies virus by reverse transcription real-time quantitative PCR,obtained the RNase-resistant and no infectivity virus-like particles as positive controls of rabies virus.

Animals ; DNA Probes ; DNA, Viral ; Dogs ; Humans ; RNA, Viral ; analysis ; Rabies ; virology ; Rabies virus ; chemistry ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Ribonuclease, Pancreatic ; metabolism ; Viral Load

RNA interference (RNAi) is a promising technology in development of specific antiviral therapy, but the quantitative detection of small interfering RNA (siRNA) expressed in vivo is the main challenge to assess its antiviral effect. In order to detect the siRNA molecules (siN1 and SiN2) particularly expressed in cells to inhibit the replication of classical swine fever virus (CSFV), serial specific stem-loop primers were designed and synthesized. Two of them (SLP-N1-6 and SLP-N2-8) were selected by screening in cross combination and successfully used in establishment of an optimal stem-loop RT-qPCR, which showed high specificity and sensitivity in detection of anti-CSFV siRNA expressed in PK-15 cells. The method was capable of detecting 10(2) to 10(8) copies of siRNA molecule with good parallel relationship (R(sq) = 0.999) and high amplification efficiency (Eff. = 98.2%). Therefore, the established stem-loop RT-qPCR can be used as an ideal tool in quantitative assessment of the anti-CSFV effects of RNAi in combination with detection of viral antigens using indirect immunofluorescent assay and TCID50, providing a novel technique for evaluating the antiviral effects of the siRNA expressed in anti-CSFV transgenic pigs to be established in future.
Animals ; Cell Line ; Classical swine fever virus ; genetics ; isolation & purification ; metabolism ; RNA Interference ; RNA, Small Interfering ; analysis ; genetics ; metabolism ; RNA, Viral ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Swine ; Transfection ; Viral Nonstructural Proteins ; genetics ; metabolism ; Virus Replication

Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.
Databases, Genetic ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Influenza A Virus, H1N1 Subtype/classification/*genetics/isolation & purification ; Influenza, Human/diagnosis/*virology ; Nasopharynx/*virology ; Nucleic Acid Amplification Techniques ; Phylogeny ; RNA, Viral/analysis/metabolism ; Sequence Analysis, RNA ; Viral Proteins/genetics

BACKGROUND/AIMS: It is essential to develop an in vitro culture model of primary hepatocytes for the study of hepatocellular function and the pathogenesis of hepatitis C virus (HCV) infection. In this study, we have established the immortalized primary human hepatocyte (IPHH) and performed in vitro culture of HCV derived from human patient. METHODS: Primary human hepatocytes were isolated from surgically resected liver tissue and then were immortalized by transfection with the SV40 large T antigen. The characterization of the IPHH during culture was analyzed by immunocytochemistry, RT-PCR, Western blot, ELISA, and soft agar assay. Next, sera and/or liver tissue homogenates from surgically resected liver tissues of patients with HCV infection were inoculated for the culture of HCV in IPHH. After HCV RNA extraction from IPHH and culture media, positive or negative stranded HCV RNA was examined by specific nest RT-PCR. RESULTS: IPHH expressed liver-associated proteins but did not express alpha-fetoprotein. Also IPHH showed ammonia removal activity. With regard to its malignant potential, colony formation in soft agar assay was not observed. Next, positive and negative stranded HCV RNAs in IPHH infected with patient's sera plus liver tissue homogenates were clearly detected whereas those in IPHH infected with only patient's sera were not detected. CONCLUSIONS: These results demonstrated the phenotypic characteristics of IPHH and the feasibility in vitro culture system of HCV infected human samples. This system might be useful for study of pathogenesis of HCV infection or hepatocyte-based applications.
Antigens, Viral, Tumor/genetics ; Base Sequence ; Carcinogenicity Tests ; Cell Culture Techniques ; Cells, Cultured ; Cells, Immobilized ; Hepacivirus/isolation & purification/*physiology ; Hepatocytes/metabolism/physiology/*virology ; Humans ; Liver Function Tests ; Models, Biological ; RNA Probes ; RNA, Viral/analysis ; Reverse Transcriptase Polymerase Chain Reaction

The transgenic mice that express Cre recombinase in a tissue specific manner is a powerful tool in generating the conditional gene knockout mice. The rat insulin promoter was cloned target the expression of Cre in pancreatic tissue. The Cre gene was modified by adding the nuclear localization signal and the sequence for initiation by eukaryotic ribosomes at 5' terminal of the Cre gene. Cre gene was linked to the intron of human growth factor gene. This construct was introduced into the mouse eggs using microinjection. Seven mice were identified as founders carrying the Cre gene by PCR. The results of RT-PCR showed that the transgenic mouse from one founder could transcribe the foreign gene in pancreas. The Southern blot analysis indicated that the Cre recombinase expressed in pancreas of the transgenic mouse was functional.
Animals ; Blotting, Southern ; Female ; Insulin ; genetics ; Integrases ; genetics ; Mice ; Mice, Transgenic ; Pancreas ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; analysis ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Proteins ; genetics

This study was purposed to constructe the three-plasmid system of the lentiviral vector carrying the green fluorescent protein (GFP) gene and to investigate the expression of GFP in T lymphocytes of the mouse. The polypurine tract (PPT) element, ubiquinone promoter (PUB) and GFP were ligated to plasmid pLO134 using subcloning technology to construct plasmid pTK153. Human kidney 293T cells were co-transfected with the three-plasmid system containing packaging plasmid DeltaNRF, plasmid pTK153 and envelope plasmid VSV-G by using calcium phosphate DNA precipation and the expression of GFP was observed under fluorescence microscope after 12 hours. The viral particles were collected after transfection 72 hours, were frozen at -80 degrees C and were used to infect mouse T lymphocytes at multiplicity of infection (m.o.i.) of 3. The expression of GFP in mouse T lymphocytes was observed by fluorescence microscopy and fluorescence-activated cell sorting (FACS). The results showed that the transfection efficacy was 63.04 +/- 7.24% in 293T cells analysed by FACS and the viral titer was (3.09 +/- 0.61) x 10(6) U/ml. The expression of GFP was also evident in mouse T lymphocytes and the transduction efficacy was (37.98 +/- 6.26)%. It is concluded that the three-plasmid system of lentiviral vector containing GFP gene is successfully constructed and the transduction efficacy is high in mouse T lymphocytes.
Animals ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; RNA, Viral ; analysis ; T-Lymphocytes ; metabolism ; Transduction, Genetic

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

BACKGROUND: During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. METHODS: PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively. RESULTS: The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition. CONCLUSIONS: The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.
Coronavirus Infections/diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/*genetics/isolation & purification ; Nasopharynx/virology ; Open Reading Frames/genetics ; RNA, Viral/*analysis/metabolism ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

OBJECTIVES: An outbreak of hepatitis A occurred at a residential facility for the disabled in July 10, 2011. This investigation was carried out to develop a response plan, and to find the infection source of the disease. METHODS: A field epidemiologist investigated the symptoms, vaccination histories, living environments, and probable infection sources with 51 residents and 31 teachers and staff members. In July 25, 81 subjects were tested for the hepatitis A virus antibody, and specimens of the initial 3 cases and the last case were genetically tested. RESULTS: Three cases occurred July 10 to 14, twelve cases August 3 to 9, and the last case on August 29. Among the teachers and staff, no one was IgM positive (on July 25). The base sequences of the initial 3 and of the last case were identical. The vehicle of the outbreak was believed to be a single person. The initial 3 patients were exposed at the same time and they might have disseminated the infection among the patients who developed symptoms in early August, and the last patient might have, in turn, been infected by the early August cases. CONCLUSIONS: The initial source of infection is not clear, but volunteers could freely come into contact with residents, and an infected volunteer might have been the common infection source of the initial patients. Volunteers' washing their hands only after their activity might be the cause of this outbreak. Although there may be other possible causes, it would be reasonable to ask volunteers to wash their hands both before and after their activities.
Adolescent ; Adult ; Antibodies, Viral/metabolism ; Assisted Living Facilities ; Child ; Disabled Persons ; *Disease Outbreaks ; Drinking Water/virology ; Female ; Hepatitis A/*epidemiology ; Hepatitis A Virus, Human/genetics ; Humans ; Interviews as Topic ; Male ; Middle Aged ; RNA, Viral/analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult