Adult ; Antibodies, Viral/isolation & purification* ; COVID-19/virology* ; Feces/chemistry* ; Female ; Humans ; Intestines/virology* ; Male ; RNA, Ribosomal, 16S/genetics* ; RNA, Viral/isolation & purification* ; SARS-CoV-2/pathogenicity*

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: 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

In the present study, the complete genomes of four common (4/EV71/Wenzhou/CHN/2014, 15/ EV71/Wenzhou/CHN/2014, 116/EV71/Wenzhou/ CHN/2014, and 120/EV71/Wenzhou/CHN/2014) and two virulent (11/EV71/Wenzhou/CHN/2014 and 109/EV71/Wenzhou/CHN/2014) enterovirus 71 (EV71) isolates were sequenced and described. They are 7405 bp in length and belong to EV71 sub-genotype C4 (C4a cluster). Nucleotide sequence alignment revealed six nucleotide variations (GP151→TP151, GP199→AP199, GP261→TP261, AP328→CP328, GP422→AP422, and GP437→TP437) in the two virulent isolates within the 5'UTR of the IRES element. RNA secondary structure predictions of IRES and FCE indicated that the common isolates shared similar structures, which were different from those of the virulent isolates. Moreover, the GP114→CP114 and GP151→TP151 mutations in the virulent isolates contributed to the formation of the unique RNA secondary structures in SL II. Furthermore, nucleotide/amino acid sequence alignments of 82 EV71 isolates indicated that six sites (TP488 and CP577 in the 5'UTR; AsnP57 in 2A; IleP56 in 3C; CP10 and AP47 in the 3'UTR) are potentially associated with the neurovirulence of EV71. Finally, the 3D structures of 2A were analogous, whereas the structures of VP1 and 3C were variable.
Base Sequence ; Central Nervous System ; virology ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; pathogenicity ; Enterovirus Infections ; virology ; Genome, Viral ; Genomics ; Genotype ; Humans ; Molecular Sequence Data ; Nucleic Acid Conformation ; Phylogeny ; RNA, Viral ; chemistry ; genetics ; Virulence

OBJECTIVES: An outbreak of acute febrile illness occurred in the Republic of Korea Air Force boot camp from May to July 2011. An epidemiological investigation of the causative agent, which was of a highly infective nature, was conducted. METHODS: Throat swabs were carried out and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed to identify possible causative factors. RESULTS: The mean age of patients who had febrile illness during the study period was 20.24 years. The multiplex RT-PCR assay identified respiratory syncytial virus (RSV) as the causative agent. The main symptoms were sore throat (76.0%), sputum (72.8%), cough (72.1%), tonsillar hypertrophy (67.9%), and rhinorrhea (55.9%). The mean temperature was 38.75degreesC and the attack rate among the recruits was 15.7% (588 out of 3750 recruits), while the mean duration of fever was 2.3 days. The prognosis was generally favorable with supportive care but recurrent fever occurred in 10.1% of the patients within a month. CONCLUSIONS: This is the first epidemiological study of an RSV outbreak that developed in a healthy young adult group. In the event of an outbreak of an acute febrile illness of a highly infective nature in facilities used by a young adult group, RSV should be considered among the possible causative agents.
Adolescent ; Adult ; Antiviral Agents/therapeutic use ; Body Temperature ; Disease Outbreaks ; Humans ; Male ; Military Personnel ; Multiplex Polymerase Chain Reaction ; Oseltamivir/therapeutic use ; Pharynx/virology ; RNA, Viral/chemistry/genetics/metabolism ; Republic of Korea/epidemiology ; Respiratory Syncytial Virus Infections/drug therapy/*epidemiology/virology ; Respiratory Syncytial Viruses/*genetics/isolation & purification ; Sputum/virology ; Young Adult

To gain more insights into epidemiologic characteristics and genotype of hantavirus in Apodemus agrarius in Changbai Area. Complete hantavirus S segment sequences were amplified by RT-PCR and sequenced. The phylogenetic trees were constructed for analysis of genetic characters of hantavirus. A total of 58 Apodemus agrarius were trapped in the epidemic areas, and complete hantavirus S segment sequences were obtained from 4 lung samples of these rodents (6. 90%0). Phylogenetic analysis of the four S segment sequences indicated that all viruses isolated from Apodemu sagrarius were closely related to genotype 6 of Hantaan virus (95. 8%-96. 3%, nucleotide identity; 98. 6%-99. 5%, amino acid identity), all of them had a specific S387 different from other genotypes of Hantaan virus.
Animals ; Base Sequence ; China ; epidemiology ; DNA, Complementary ; chemistry ; genetics ; Disease Reservoirs ; virology ; Genotype ; Hantavirus ; classification ; genetics ; isolation & purification ; Hantavirus Infections ; epidemiology ; veterinary ; virology ; Lung ; virology ; Murinae ; virology ; Phylogeny ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Rodent Diseases ; virology ; Sequence Analysis, DNA ; Viral Proteins ; genetics

The purpose of this study was to determine whether manually plucked hairs might serve as an alternative sample for a quantitative real time polymerase chain reaction (qRT-PCR) testing. Twenty three, 1~3 week old, non-bovine viral diarrhea virus (BVDV) vaccinated calves, found to be positive for BVDV by immunohistochemical staining, were selected and hairs were manually plucked from the ear. qRT-PCR was performed on samples consisting of more than 30 hairs (30~100) and whole blood. All 23 animals were positive for the virus by qRT-PCR performed on the whole blood and when samples of more than 30 hairs were assayed. Additionally, qRT-PCR was performed on groups of 10 and 20 hairs harvested from 7 out of 23 immunohistochemical staining-positive calves. When groups of 20 and 10 hairs were tested, 6 and 4 animals, respectively, were positive for the virus.
Animals ; Antibodies, Viral/analysis/diagnostic use ; Bovine Virus Diarrhea-Mucosal Disease/blood/*diagnosis/virology ; Cattle ; Diarrhea Virus 1, Bovine Viral/genetics/*isolation & purification ; Diarrhea Virus 2, Bovine Viral/genetics/*isolation & purification ; Hair/virology ; RNA, Viral/chemistry/genetics ; Real-Time Polymerase Chain Reaction/methods/*veterinary

Human enterovirus 71 (EV71) is one of the major etiological agents for the hand, foot, and month disease (HFMD) and is causing frequent, widespread occurrence in the mainland of China. The single positive-stranded RNA genome of EV71 is translated into a single polyprotein which is autocleavaged into structural and nonstructural proteins. The functions of many nonstructural proteins characterized in the life cycle of virus are potential targets for blocking viral replication. This article reviews the studies of the structures and functions of nonstructural proteins of EV71 and the anti-enterovirus 71 drugs targeting on these nonstructural proteins.
Antiviral Agents ; pharmacology ; Enterovirus A, Human ; enzymology ; genetics ; isolation & purification ; Hand, Foot and Mouth Disease ; drug therapy ; virology ; Humans ; Molecular Targeted Therapy ; Peptide Hydrolases ; chemistry ; metabolism ; physiology ; Protein Kinase Inhibitors ; pharmacology ; RNA, Viral ; genetics ; Viral Nonstructural Proteins ; chemistry ; metabolism ; physiology ; Virus Replication ; drug effects

To efficiently express nucleoprotein (NP) of influenza A virus A/Jingke/30/95 (H3N2) in E. coli for further immunogenicity study, three forms of NP gene, NP(His) (NP fused with 6 x His tag), NPwt (wild type NP, non-fused NP with native codon) and NP(O) (codon optimized, non-fused NP) were cloned by the technologies of restriction enzyme digestion, PCR, codon optimization and gene synthesis. Three recombinant plasmids were subsequently constructed based on the prokaryotic vector pET-30a, respectively. The comparative studies with these plasmids were carried out on the gene expression efficiency, induction temperature and time, purification process and immune reactivity. It was confirmed by restriction enzyme digestion and sequencing analysis that the three NP genes were inserted into the expression plasmid pET-30a correctly. SDS-PAGE showed that all three forms of NP gene could be efficiently ex pressed in E. coli, among which NP(O) was expressed with the highest expression level. The lower temperature fermentation (T=25 degrees C) and longer time induction (t=10 h) were necessary for high-level expression of protein in soluble form. The purity of tag-free NP was up to 90% through the two-step purification process with anion-exchange and gel filtration chromatography. It was indicated by Western blot that purified NP reacted well with the serum from mice immunized with PR8 virus. These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli and the expressed NP protein with specific immune reactivity could be purified from the supernatant of bacterial lysate.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; RNA-Binding Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Solubility ; Viral Core Proteins ; chemistry ; genetics ; isolation & purification ; metabolism

Avian metapneumovirus (aMPV) causes upper respiratory tract infections in chickens and turkeys. Although the swollen head syndrome (SHS) associated with aMPV in chickens has been reported in Korea since 1992, this is the study isolating aMPV from chickens in this country. We examined 780 oropharyngeal swab or nasal turbinate samples collected from 130 chicken flocks to investigate the prevalence of aMPV and to isolate aMPV from chickens from 2004-2008. Twelve aMPV subtype A and 13 subtype B strains were detected from clinical samples by the aMPV subtype A and B multiplex real-time reverse transcription polymerase chain reaction (RRT-PCR). Partial sequence analysis of the G glycoprotein gene confirmed that the detected aMPVs belonged to subtypes A and B. Two aMPVs subtype A out of the 25 detected aMPVs were isolated by Vero cell passage. In animal experiments with an aMPV isolate, viral RNA was detected in nasal discharge, although no clinical signs of SHS were observed in chickens. In contrast to chickens, turkeys showed severe nasal discharge and a relatively higher titer of viral excretion than chickens. Here, we reveal the co-circulation of aMPV subtypes A and B, and isolate aMPVs from chicken flocks in Korea.
Animals ; Antibodies, Viral/blood ; Base Sequence ; *Chickens ; Glycoproteins/chemistry/genetics ; Metapneumovirus/immunology/*isolation & purification ; Molecular Sequence Data ; Paramyxoviridae Infections/immunology/*veterinary/virology ; *Phylogeny ; Poultry Diseases/immunology/*virology ; RNA, Viral/chemistry/genetics ; Respiratory Tract Infections/immunology/*veterinary/virology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sequence Alignment ; Sequence Analysis, DNA ; Serotyping ; Specific Pathogen-Free Organisms ; Turkeys