Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction

Vabret, Astrid; Mouthon, Franck; Mourez, Thomas; Gouarin, S.; Petitjean, J.; Freymuth, F.

doi:10.1016/s0166-0934(01)00343-3

Cited by 83 publications

(104 citation statements)

References 17 publications

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“…For the multiplex PCR and hemi-nested PCR assays, Taq DNA polymerase (Promega, San Luis Obispo, CA) was used. Twenty-eight primers used were established in this study and 11 were from the published studies (3)(4)(5).…”

Section: Methodsmentioning

confidence: 99%

Viral Pathogens Associated With Acute Respiratory Infections in Central Vietnamese Children

Yoshida

Suzuki

Yamamoto

et al. 2010

Pediatric Infectious Disease Journal

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Hospitalized Vietnamese children with acute respiratory infection (ARI) were investigated for 13 viral pathogens using multiplex-polymerase chain reaction. We enrolled 958 children of whom 659(69%) had documented viral infection: rhinovirus (28%), respiratory syncytial virus (23%), influenza virus (15%), adenovirus (5%), human metapneumo virus (4.5%), parainfluenza virus (5%) and bocavirus (2%). These Vietnamese children had a range of respiratory viruses which underscores the need for enhanced ARI surveillance in tropical developing countries. Positive templates were used in each assay for quality control. RESULTSDuring the 14-months study period, a total of 1,014 pediatric patients from the catchment area were admitted to KHGH, of which 958 (95%) were enrolled in the study.Males comprised 58% of patients and 94% of the patients were less than 5 years old (median age: 1.4 years). The results showed that one or more respiratory viruses were found in 69% of patients: 11% had dual and 1.4% had triple infection. Eighty six percent of the viral ARI patients were less than 3 years old (detail information of age breakdown is shown in supplementary table 2, online only).Major viruses detected were rhinovirus (28%), RSV (23%) and influenza A (15%). This was followed by adenovirus (5%), hMPV (5%), PIV3 (4%) and bocavirus (2%). Other viruses (PIV1, PIV2 and influenza B) were detected in a small proportion (1.5%) of ARI patients. Across age, sex, and case categories, there were no significant differences between proportion of virus positive and negative patients.The pattern of virus detection did not differ between URTI and LRTI patients. A total of 268 radiologically-confirmed pneumonia (RCP) patients and 195 bronchiolitis 7 patients were identified. PIV3 detection was significantly associated with hospitalized LRTI (p=0.016) and bronchiolitis (p=<0.001). Similar to previous reports, we found that RSV infection was significantly associated with bronchiolitis (p=0.002) (6). We also found that a significantly higher proportion of patients (n=119)

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Section: Methodsmentioning

confidence: 99%

Viral Pathogens Associated With Acute Respiratory Infections in Central Vietnamese Children

Yoshida

Suzuki

Yamamoto

et al. 2010

Pediatric Infectious Disease Journal

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“…Ad, hRSV and some hRV strains are likely to replicate in this cellular type. When samples were negative in immunofluorescence assay, we also attempted to isolate them using HuH7 cells (Nakabayashi et al, 1977) that had been grown in 48-well tissue culture plates, as described previously (Vabret et al, 2001). After 4 days of incubation, cultures were examined for cytopathogenic effects and the cells were scraped and tested by immunofluorescence assay.…”

Section: Isolation and Identification Of Viruses By Immunofluorescencmentioning

confidence: 99%

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

Bellau-Pujol

Vabret

Legrand

et al. 2005

Journal of Virological Methods

282

261

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Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.

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“…Briefl y, RNA was extracted from 50 l of NPs applying the guanidinium-isothiocyanate method as described elsewhere [15] and subsequently reverse transcribed into cDNA using primers MF3 for hCoV-OC43 and Pcon4.2 for hCoV-229E. The resulting cDNA was amplifi ed by PCR using primer MF1 as a sense primer, and again MF3 as an antisense primer for hCoV-OC43 [16] . For a replicase gene, of hCoV-229E, Pcon3 was used as an outer sense primer, Pcon4.2 as an outer antisense primer, Pcon1 as an inner sense primer, and Pcon2.2 as an inner antisense primer base on the sequence accession No.…”

Section: Samplesmentioning

confidence: 99%

Human Coronavirus Infection among Children with Acute Lower Respiratory Tract Infection in Thailand

Theamboonlers¹,

Samransamruajkit²,

Thongme³

et al. 2006

Intervirology

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Objective: This study was performed to further identify the previously uncharacterized human coronavirus 229E (hCoV-229E) and human coronavirus OC43 (hCoV-OC43) in Thailand by using the RT-PCR technique. In addition, we performed this study in order to delineate the prevalence, the potential clinical impacts and evaluation of the genetic characterization of this pathogen in young children who presented with acute lower respiratory tract infections (ALRI). Methods: We obtained nasopharyngeal secretions (NPs) from 226 children <5 years of age who were either attending the outpatient department or hospitalized with ALRI from March 2002 to July 2003. All clinical, laboratory, RT-PCR, direct sequencing and phylogenetic analysis data were collected and analyzed. Results: Of the 226 NPs samples from infants and young children presented with ALRI, 8 (3.54%) were positive for hCoV-229E, 2 (0.88%) were positive for hCoV-OC43, and 1 (0.44%) had co-infection. The following clinical presentations were noted: fever (100%), rhinitis (44%), acute bronchiolitis (44%), viral pneumonia (33%), viral pneumonia triggering asthma exacerbation (11%) as well as viral pneumonia causing BPD exacerbation (11%). All positive samples were subjected to direct sequencing. The amino acid sequences had 82–99% similarity to previous sequences stored in the GenBank database. Conclusion: The molecular technique we applied to detect human coronavirus appears justified as a valuable diagnostic approach to elucidate the prevalence, cause and clinical implications of ALRI among infants and young children.

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Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction

Cited by 83 publications

References 17 publications

Viral Pathogens Associated With Acute Respiratory Infections in Central Vietnamese Children

Viral Pathogens Associated With Acute Respiratory Infections in Central Vietnamese Children

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

Human Coronavirus Infection among Children with Acute Lower Respiratory Tract Infection in Thailand

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