BACKGROUND: Multiplex PCR assay is a sensitive tool for the detection of various respiratory viruses. Seeplex RV12 ACE Detection (Seegene, Korea) assay is a multiplex RT-PCR assay for detection of 12 respiratory viruses. We had observed several cases with faint bands in the test. Those results were investigated in this study. METHODS: A total of 163 specimens were tested with Seeplex RV12 ACE Detection assay. The amplicons showing faint band in electrophoresis were reamplified and sequenced. RESULTS: A total of 99 viruses were detected in 80 specimens (49.1%). Twenty-four amplicons showed faint band in eletrophoresis. All of influenza virus A, parainfluenza viruses (PIV), coronavirus OC43, human metapneumovirus (HMPV) and adenovirus amplicons were reamplified, but 4 of 12 human rhinovirus amplicons were not reamplified. Sequences of reamplified PCR products showed homology of 95-99% to those of corresponding viruses in the National Center for Biotechnology Information database. CONCLUSIONS: Faint bands of influenza virus A, PIV-1, PIV-3, coronavirus OC43, HMPV and adenovirus in Seeplex RV12 ACE Detection assay are specific bands and seems to be weak positive results.
Adenoviridae ; Biotechnology ; Coronavirus ; Coronavirus OC43, Human ; Electrophoresis ; Humans ; Metapneumovirus ; Multiplex Polymerase Chain Reaction ; Orthomyxoviridae ; Paramyxoviridae Infections ; Polymerase Chain Reaction ; Rhinovirus

BACKGROUND: Direct antigen test (DAT) and culture are primary tests to diagnose infections by respiratory viruses, but are mainly available for the traditional viral pathogens such as respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), and adenovirus in clinical laboratories. The objective of this study was to evaluate a multiplex reverse transcriptase-PCR method using Seeplex(TM) RV Detection kit (Seegene, Korea) for the detection of rhinovirus, coronavirus, and human metapneumovirus (hMPV). METHODS: From January to May 2007, nasopharyngeal aspirates (NPAs) from pediatric patients negative for culture and DAT of traditional viral pathogens were tested with Seeplex(TM). All the amplicons were directly sequenced and homology of the sequences was searched in the National Center for Biotechnology Information (NCBI) database. Patients' medical records were reviewed for clinical and demographic features. RESULTS: Forty-seven (26.4%) of 178 NPAs were positive: 18 rhinovirus, 15 hMPV, 4 RSV A, 3 coronavirus OC43, 3 influenza virus A, 2 adenovirus, 1 coronavirus NL63, and 1 RSV B. Based on maximum identity, each of the sequences indicating rhinovirus, hMPV, and coronavirus OC43 matched to the corresponding viruses with homology of 94-98%, 96-99%, and 98-100%, respectively. Seeplex(TM) positive patients were 0-11 yr old with a male:female ratio of 1.5:1. Clinical diagnoses included 9 pneumonia, 6 bronchiolitis, 2 cold, 1 asthma exacerbation for rhinovirus; 10 pneumonia, 4 bronchiolitis, and 1 clinical sepsis for hPMV; and 1 pneumonia, 2 croup, and 1 cold for coronavirus. CONCLUSIONS: Multiplex reverse transcriptase-PCR method using Seeplex(TM) RV Detection kit is a reliable test to detect rhinovirus, hMPV, and coronavirus. It may improve the diagnostic sensitivity for RSV, influenza virus, PIV, and adenovirus.
Adolescent ; Child ; Child, Preschool ; Coronavirus/classification/*isolation & purification ; Coronavirus 229E, Human/classification/genetics/isolation & purification ; Coronavirus OC43, Human/classification/genetics/isolation & purification ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Metapneumovirus/classification/genetics/*isolation & purification ; Phylogeny ; Reagent Kits, Diagnostic ; Respiratory Tract Infections/*diagnosis/virology ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Rhinovirus/classification/genetics/*isolation & purification ; Sequence Analysis, DNA

Canine coronavirus is a single-stranded RNA virus that causes enteritis in dogs of any age. Coronaviral enteritis is seldom definitively diagnosed, since it is usually much less severe than many other types of enteritis and is self-limiting. Conventional diagnostics for the canine coronaviral enteritis such as polymerase chain reaction (PCR), virus isolation, and electron microscopic examination are inappropriate for small animal clinics due to the complicated experimental processes involved. Therefore, a commercially available lateral flow test kit based on chromatographic immunoassay techniques was tested to evaluate its performance as a first-line diagnostic test kit that could be used in clinics. The coronavirus antigen test kit detected canine coronavirus-infected dogs with 93.1% sensitivity and 97.5% specificity. The detection limit of the test kit was between 1.97 × 10⁴/mL and 9.85 × 10³/mL for samples with a 2-fold serial dilution from 1.25 × 10⁶ TCID₅₀ (TCID₅₀, 50% tissue culture infectious dose). Additionally, the test kit had no cross-reactivity with canine parvovirus, distemper virus, or Escherichia coli. Overall, the commercially available test kit showed good diagnostic performance in a clinical setting, with results similar to those from PCR, confirming their potential for convenient and accurate use in small animal clinics.
Animals ; Coronavirus ; Coronavirus, Canine ; Diagnostic Tests, Routine ; Distemper ; Dogs ; Enteritis ; Escherichia coli ; Immunoassay ; Limit of Detection ; Parvovirus, Canine ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; RNA Viruses ; Sensitivity and Specificity

Objective: To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. Methods: A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID Results: The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.
Capsid Proteins/genetics* ; Enterovirus A, Human/genetics* ; Enterovirus B, Human/genetics* ; Enterovirus C, Human/genetics* ; Enterovirus D, Human/genetics* ; Humans ; Molecular Epidemiology/methods* ; Molecular Typing/methods* ; Reverse Transcriptase Polymerase Chain Reaction/methods*

Canine respiratory coronavirus (CRCoV) is commonly associated with canine kennel cough worldwide. Clinically infected dogs present coughing, sneezing, and nasal discharge. Severe infections may progress to pneumonia. Through serological surveys, CRCoV has been identified as a worldwide pathogen found in the respiratory tracts of dogs suffering from mild or severe respiratory disease. In this study, three dogs were obtained from a dog kennel. Over the previous 5 days, the dogs showed coughing, sneezing, and nasal discharge. To detect the etiologic pathogen, we performed multiplex RT-PCR (mRT-PCR) to amplify the genes encoding canine influenza virus matrix protein, canine distemper virus nucleocapsid protein, and CRCoV spike protein. Dot blotting was achieved with a CRCoV-specific probe. Nasal-secreting CRCoV was detected by the 442 bp CRCoV-positive PCR reaction in the nasal swabbing samples from dogs. Further, CRCoV-positive reactions by dot blot hybridization were detected in the nasal swabbing samples from dogs. In conclusion, we detected CRCoV in kenneled dogs with respiratory disease in Korea. Multiplex RT-PCR was able to detect successfully CRCoV infection in dogs. We suggest that mRT-PCR would be useful and effective for monitoring CRCoV infection in various kinds of dogs.
Animals ; Coronavirus* ; Cough ; Distemper Virus, Canine ; Dogs* ; Korea ; Nucleocapsid Proteins ; Orthomyxoviridae ; Pneumonia ; Polymerase Chain Reaction ; Respiratory System ; Sneezing

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.
Antibodies, Viral/*immunology ; Blotting, Western ; Coronavirus 229E, Human/*immunology ; Coronavirus OC43, Human/*immunology ; Cross Reactions ; Humans ; Nucleocapsid Proteins/genetics/*immunology ; Recombinant Proteins/immunology ; SARS Virus/genetics/*immunology ; Severe Acute Respiratory Syndrome/diagnosis/*immunology

<b>OBJECTIVEb>To prepare and characterize monoclonal antibodies (mAbs) against the recombinant nucleocapsid (N) protein of 3 human coronaviruses SARS-CoV, 229E and OC43 and study the antigenic relationship between the 3 N proteins.

<b>METHODSb>BALB/c mice were immunized with the recombinant N proteins of SARS-CoV, 229E and OC43 to obtain the mAbs by means of hybridoma. Screening and identification of the mAbs were performed using indirect enzyme-linked immunosorbent assay (ELISA), Western blotting and indirect immunofluorescence assay. Cross-reactivity between the N proteins of the 3 coronaviruses was analyzed with the prepared mAbs.

<b>RESULTSb>The mAbs against the recombinant N proteins of SARS-CoV, 229E and OC43 were obtained, which reacted specifically with the corresponding viral N protein as shown by indirect ELISA, Western blotting and indirect immunofluorescence assay. No cross-reactivity was found between the 3 N proteins.

<b>CONCLUSIONb>The prepared mAbs against the recombinant N proteins may provide valuable assistance in studying antigenic relationships of N proteins between the 3 human coronaviruses.

Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Coronavirus 229E, Human ; genetics ; immunology ; Coronavirus OC43, Human ; genetics ; immunology ; Cross Reactions ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; genetics ; immunology ; Recombinant Proteins ; immunology ; SARS Virus ; genetics ; immunology

OBJECTIVE: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. METHODS: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. RESULTS: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. CONCLUSION A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.
Encephalitis Viruses, Tick-Borne ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; RNA, Viral ; analysis

Viruses are the most common cause of lower respiratory tract infections (LRTIs) in infants and young children and are a major public health problem in this age group. Viruses were identified in 54.9-70.4% of hospitalized infants and children with LRTIs in Korea. The viral pathogens identified included respiratory syncytial virus (RSV) A and RSV B, influenza (Inf) A, Inf B, parainfluenza (PIV)1, PIV2, human bocavirus (hBoV), human rhinovirus (hRV), adenovirus (ADV), human metapneumovirus (hMPV), human coronavirus (hCoV)-OC 43, hCoV-229E, hCoV-NL63, hCoV-HKU1, and human enterovirus (hEV). Coinfections with > or =2 viruses were observed in 11.5-22.8% of children. The occurrence of LRTIs was the highest in the first year of life. The specific viruses are frequently associated with specific clinical syndromes of LRTIs. LRTIs caused by RSV were predominant among younger infants. hRV accounted for a larger proportion of LRTIs in young infants than ADV and hBoV. hMPV was frequently detected in children >24 months old. The number of hMPV infections peaked between February and May, whereas hRV was detected throughout the year. Thus far, hCoV is a less common respiratory pathogen in cases of ALRI and URI in Korean children.
Adenoviridae ; Child ; Coinfection ; Coronavirus ; Coronavirus NL63, Human ; Enterovirus ; Human bocavirus ; Humans ; Infant ; Influenza, Human ; Korea ; Metapneumovirus ; Paramyxoviridae Infections ; Public Health ; Respiratory Syncytial Viruses ; Respiratory System ; Respiratory Tract Infections ; Rhinovirus

PURPOSE: During the late autumn to winter season (October to December) in the Republic of Korea, respiratory syncytial virus (RSV) is the most common pathogen causing lower respiratory tract infections (LRTIs). Interestingly, in 2014, human coronavirus (HCoV) caused not only upper respiratory infections but also LRTIs more commonly than in other years. Therefore, we sought to determine the epidemiology, clinical characteristics, outcomes, and severity of illnesses associated with HCoV infections at a single center in Korea. MATERIALS AND METHODS: We retrospectively identified patients with positive HCoV respiratory specimens between October 2014 and December 2014 who were admitted to Severance Children’s Hospital at Yonsei University Medical Center for LRTI. Charts of the patients with HCoV infection were reviewed and compared with RSV infection. RESULTS: During the study period, HCoV was the third most common respiratory virus and accounted for 13.7% of infections. Coinfection was detected in 43.8% of children with HCoV. Interestingly, one patient had both HCoV-OC43 and HCoV-NL63. Mild pneumonia was most common (60.4%) with HCoV, and when combined with RSV, resulted in bronchiolitis. Two patients required care in the intensive care unit. However, compared with that of RSV infection, the disease course HCoV was short. CONCLUSION: Infections caused by HCoVs are common, and can cause LRTIs. During an epidemic season, clinicians should be given special consideration thereto. When combined with other medical conditions, such as neurologic or cardiologic diseases, intensive care unit (ICU) care may be necessary.
Child ; Child, Preschool ; Coronavirus/*isolation & purification ; Coronavirus Infections/epidemiology/*virology ; Coronavirus OC43, Human/isolation & purification ; Female ; Hospitalization ; Humans ; Infant ; Male ; Republic of Korea/epidemiology ; Respiratory Tract Infections/epidemiology/*virology ; Retrospective Studies ; Seasons