Nucleic acid-based detection of influenza A virus subtypes H7 and N9 with a special emphasis on the avian H7N9 virus

D Kalthoff,J Bogs,B Hoffmann,A Pohlmann,M Beer,C Grund,T Harder

doi:10.2807/1560-7917.es2014.19.10.20731

Abstract

In 2013, a novel influenza A virus of subtype H7N9 was transmitted from avian sources to humans in China, causing severe illness and substantial mortality. Rapid and sensitive diagnostic approaches are the basis of epidemiological studies and of utmost importance for the detection of infected humans and animals. We developed various quantitative reverse transcriptase PCR (RT-qPCR) assays for (i) the generic detection of the haemagglutinin (HA) gene of H7 viruses or the neuraminidase (NA) gene of N9 viruses, and (ii) the specific detection of HA and NA of the novel avian H7N9/2013 virus. The sensitivity of the newly developed assays was compared with previously published PCRs, and the specificity of all RT-qPCRs was examined using a panel of 42 different H7 and 16 different N9 isolates. Furthermore, we analysed the performance of the RT-qPCR assays with dilution series and diagnostic samples obtained from animal experiments. Our study provides a comprehensive set of RT-qPCR assays for the reliable detection of the novel avian H7N9 virus, with high sensitivity and improved and tailored specificity values compared with published assays. Finally, we also present data about the robustness of a duplex assay for the simultaneous detection of HA and NA of the avian influenza H7N9/2013 virus.

Highlights

Multiple reassortment events, trans-species transmissions, and viral adaptation of influenza A viruses (IAV) in non-human host species shaped the latest human pandemic influenza virus that emerged in 2009 [1]
We developed a set of real-time quantitative reverse transcriptase polymerase chain reactions (RT-qPCR), which target different fragments of the haemagglutinin (HA) and the neuraminidase (NA) genes of influenza A viruses, with special emphasis on the novel avian H7N9/2013 virus and with explicit advantages
In order to develop primer and probes for the generic detection of the HA of H7 viruses or the NA of N9 viruses, 910 nearly complete H7 sequences as well as 181 nearly complete N9 sequences were analysed in silico

Summary

Introduction

Trans-species transmissions, and viral adaptation of influenza A viruses (IAV) in non-human host species shaped the latest human pandemic influenza virus that emerged in 2009 [1]. Most recently, another animal influenza virus, this time of purely avian origin, was introduced into the human population in the east of China: influenza A subtype H7N9 [2], hereafter referred to as avian H7N9 virus. At least 354 people were infected, most probably after contact with infected poultry, other avian species, or contaminated environment [3,4]. The avian reservoir of this virus has remained obscure.

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