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Virus Res. A Novel Pyrosequencing Assay for the Detection of Neuraminidase Inhibitor Resistance-Conferring Mutations among Clinical Isolates of Avian H7N9 Influenza Virus.
[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]
Virus Res. 2013 Nov 5. pii: S0168-1702(13)00373-0. doi: 10.1016/j.virusres.2013.10.026. [Epub ahead of print]
A Novel Pyrosequencing Assay for the Detection of Neuraminidase Inhibitor Resistance-Conferring Mutations among Clinical Isolates of Avian H7N9 Influenza Virus.
Qi Y, Fan H, Qi X, Zhu Z, Guo X, Chen Y, Ge Y, Zhao K, Wu T, Li Y, Shan Y, Zhou M, Shi Z, Wang H, Cui L.
Source: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Rd, Nanjing 210009, P. R. China.
Abstract
A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February, 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.
Copyright ? 2013 The Authors. Published by Elsevier B.V. All rights reserved.
KEYWORDS: Avian influenza A (H7N9) virus, E120V mutation, NAIs resistance, Pyrosequencing
PMID: 24211668 [PubMed - as supplied by publisher]
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Virus Res. 2013 Nov 5. pii: S0168-1702(13)00373-0. doi: 10.1016/j.virusres.2013.10.026. [Epub ahead of print]
A Novel Pyrosequencing Assay for the Detection of Neuraminidase Inhibitor Resistance-Conferring Mutations among Clinical Isolates of Avian H7N9 Influenza Virus.
Qi Y, Fan H, Qi X, Zhu Z, Guo X, Chen Y, Ge Y, Zhao K, Wu T, Li Y, Shan Y, Zhou M, Shi Z, Wang H, Cui L.
Source: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Rd, Nanjing 210009, P. R. China.
Abstract
A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February, 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.
Copyright ? 2013 The Authors. Published by Elsevier B.V. All rights reserved.
KEYWORDS: Avian influenza A (H7N9) virus, E120V mutation, NAIs resistance, Pyrosequencing
PMID: 24211668 [PubMed - as supplied by publisher]
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