Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance

Yang Yang,Qi Zhao,George F Gao,Sufang Ma,Yuhai Bi,Haixia Zheng,Wenjun Liu,Jingyan Lin,Hong Li,Shanqin Li,Yingxia Liu,Gary Wong,Zhixiang Xu,Xiaonan Zhao,Wen Xu

doi:10.1186/s12879-018-3302-7

Abstract

BackgroundDuring the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. Moreover, a neuraminidase inhibitor (NAI) resistance (R292K in NA) mutation was found in H7N9 isolates from humans, poultry and the environment. In this study, we set out to develop and validate a multiplex quantitative reverse transcript polymerase chain reaction (qRT-PCR) to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations.MethodsA quadruple qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations was designed based on the analyses of the HA and NA genes of H7N9. This assay was further tested for specificity and sensitivity, and validated using clinical samples.ResultsThe assay was highly specific and able to detect low pathogenic (LP)- or HP-H7N9 with/without the NAI-resistance mutation. The detection limit of the assay was determined to be 50 genome-equivalent copies and 2.8 × 10− 3 50% tissue culture infectious doses (TCID50) of live H7N9 per reaction. Clinical validation was confirmed by commercial kits and Sanger sequencing with ten clinical samples.ConclusionsWe developed and validated a rapid, single-reaction, one-step, quadruple real-time qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP- and NAI-resistance strains with excellent performance in specificity and sensitivity. This assay could be used to monitor the evolution of H7N9 viruses in the laboratory, field and the clinic for early-warning and the prevention of H7N9 infections.

Highlights

During the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site
During the first four waves, the H7N9 viruses circulating in China were classified as low pathogenic (LP) avian influenza virus (AIV) carrying amino acid residues PKGR/G at the HA cleavage site, causing asymptomatic infection in poultry [4, 6]
A universal probe (H7-P-W) targeting both the HP- and LP-H7N9 within the same region was designed to identify the presence of H7

Summary

Introduction

During the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. During the first four waves, the H7N9 viruses circulating in China were classified as low pathogenic (LP) avian influenza virus (AIV) carrying amino acid residues PKGR/G at the HA cleavage site, causing asymptomatic infection in poultry [4, 6]. A highly pathogenic (HP) H7N9 AIV variant emerged during the fifth wave in 2017, and has since been identified in 28 human infections [7,8,9,10] These HP-H7N9 isolates contained polybasic amino acids with a PKGKRTAR/G, PKGKRIAR/G, PKRKRAAR/G or PKRKRTAR/G in the cleavage site of HA proteins [2], which are associated with enhanced virulence in chickens [2, 11]

Methods

Results

Discussion

Conclusion