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A novel pyrosequencing assay for the detection of neuraminidase inhibitor resistance-conferring mutations among clinical isolates of avian H7N9 influenza virus

Authors :
Lunbiao Cui
Yan Li
Yin Chen
Zheng Zhu
Zhiyang Shi
Minghao Zhou
Huan Fan
Xiling Guo
Hua Wang
Yunfeng Shan
Tao Wu
Xian Qi
Yiyue Ge
Kangchen Zhao
Yuhua Qi
Source :
Virus Research
Publication Year :
2013
Publisher :
Elsevier B.V., 2013.

Abstract

Highlights • Drug-resistant influenza viruses present a major public health problem. • We present an assay for the identification of resistance-conferring mutations. • We tested 26 clinical samples of an infectious H7N9 influenza virus for mutations. • The assay successfully identified a mixed population of E120/E120V associated with drug-resistant mutant. • Parallel testing of clinical specimens and cultured samples may avoid misinterpreting culture-induced mutations.<br />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.

Details

Language :
English
ISSN :
18727492 and 01681702
Volume :
179
Database :
OpenAIRE
Journal :
Virus Research
Accession number :
edsair.doi.dedup.....18f3c5d497e0960676bee7927b99eb56