A multiplex real-time RT-PCR method for detecting H5, H7 and H9 subtype avian influenza viruses in field and clinical samples
Introduction
Avian influenza viruses (AIVs), composed of eight single-stranded RNA fragments, belong to the Orthomyxoviridae family (Webster et al., 1992). Two envelope proteins, hemagglutinin (HA) and neuraminidase (NA), are present on the virus surface and form the basis of AIV classification. Since most AIV subtypes have been isolated from waterfowl, birds are considered natural reservoirs of AIVs (Kawaoka et al., 1988).
Highly pathogenic (HP-) H5N1 AIVs have been separated into 10 clades (clades 0−9) and various subclades (Uyeki, 2008). The currently circulating strains, H5N1 AIVs from clade 2.3.4.4, are continuously reassorted with other NA genes, producing novel H5N1, H5N2, H5N3, H5N5, H5N6 and H5N8 subtype AIVs (Antigua et al., 2019; Bi et al., 2015; Hassan et al., 2020). At least one outbreak has been caused by H5 AIVs occurring in domestic and/or wild birds in more than 61 countries or regions. As of April 2021, there were 862 human infection cases with H5N1 AIV reported from 17 countries, 31 laboratory-confirmed human infection cases with H5N6 AIV, and 7 human infection cases with H5N8 AIV (Liang et al., 2021). Regarding H7 subtype HPAIVs, sporadic human infection cases with H7N2, H7N3 and H7N7 AIVs emerged in the USA, Canada, Italy and England before 2013 (Belser et al., 2009; Philippon et al., 2020). A novel reassortant H7N9 influenza virus was identified in Eastern China in 2013 with a mortality rate of ∼40%, causing 609 human deaths (Chen et al., 2013; Lam et al., 2013). To date, five H7N9 epidemic waves have emerged in China. The occurrence of HP-H7N9 viruses in poultry and humans in the fifth wave has attracted wide attention (Liu et al., 2021). In 2018, the first isolation of H7N4 virus from a human being in China was reported (Tong et al., 2018).
H9N2 low pathogenic AIVs (LPAIVs) typically cause mild flu-like symptoms and are easily overlooked, which provides ideal conditions for adaptive mutations (Song and Qin, 2020). Previous reports showed that H9N2 AIV can donate its internal genes for reassortant with other subtype AIVs (such as H5, H6, H7 and H10), leading to a potential influenza pandemic (Chen et al., 2014; Guan et al., 1999; Pu et al., 2015). Long-term monitoring of AIVs has become an important part of the prevention and control of influenza viruses.
A variety of diagnostic methods have been developed for detecting AIVs, of which virus isolation is the most traditional (Yang et al., 2018a). Other rapid and convenient commercial influenza virus detection kits include enzyme-linked immunoassays assay (ELISA) for detecting viral nucleoprotein antigen, and the real-time reverse transcriptase PCR (RRT-PCR) for detecting matrix genes of influenza A viruses. However, virus isolation is time-consuming, the specificity and sensitivity of ELISA are influenced by different monoclonal antibodies in different kits, and kits are expensive (Vemula et al., 2016). RT-PCR and multiplex RRT-PCR assays are now mature enough to be widely applied for rapid diagnosis of influenza viruses (Kim et al., 2013; Lee et al., 2008).
Live poultry markets (LPMs) provide an opportunity for the reassortment and transmission of AIVs, and human exposure to poultry. In previous reports, several RRT-PCR assays were developed to detect H6, H10 and H5N8 AIVs with very high specificity and sensitivity (Yang et al., 2018a; Yang et al., 2018b; Yang et al., 2020b). Due to the widespread distribution of H5, H7 and H9 AIVs in LPMs and the potential threat to human health (Hu et al., 2020; Qiu et al., 2019; Wu et al., 2015a), we developed a TaqMan minor-groove binder RRT-PCR method to rapidly screen and differentiate influenza A virus and H5, H7 and H9 subtype AIVs in a single reaction.
Section snippets
Ethics and biosafety statement
All research protocols were approved by the First Affiliated Hospital, School of Medicine, Zhejiang University (No. 2019–39). The researches followed the requirements of the biosafety management manual strictly when conducting experiments involving highly pathogenic influenza virus in biosafety level 3 laboratories.
Viruses and viral rna
A variety of AIVs were analysed in this study, including representative H5, H7 and H9 subtype AIVs isolated in eastern China in the past 10 years (Table 1). All AIVs were amplified
Design of primer and probe sequences
Primers and probes were carefully designed based on the conserved regions of influenza A virus M-, H5-, H7- and H9-HA genes. To ensure that the detected signal could be distinguished in a single reaction, probes targeting influenza A M-, H5-, H7- and H9-HA genes were labelled with FAM, Vic, Cy5 and Texas Red reporters, respectively (Table 1). A total of 2000 M genes from Asian countries were retrieved from the GenBank database, 1200 H5-HA, 1200 H7-HA and 1200 H9-HA gene sequences from Eurasian
Discussion
During the continuous surveillance of AIVs in LPMs, several subtypes, including H5N1, H5N2, H5N6, H5N8, H7N3, H7N6, H7N7, H7N9 and H9N2, are frequently identified from LPMs in Eastern China (Bo et al., 2021; Wu et al., 2017b; Wu et al., 2015b). According to previous research, only H5 and H7 subtype HPAIVs have caused poultry outbreaks (Alexander, 2000), the most notable of which are H5N1 and H7N9 AIVs, resulting in huge economic losses and even human deaths (Gao et al., 2013; Yu et al., 2014).
Funding
This study was funded by Grants from the National Science and Technology Major Project for the Control and Prevention of Major Infectious Diseases in China (2018ZX10711001, 2018ZX10102001 and 2020ZX10001016–004–002), Zhejiang Provincial Natural Science Foundation of China (LY19H260006), and the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (2021ZZ24).
CRediT authorship contribution statement
Fan Yang: Conceptualization, Methodology, Writing – original draft. Dalu Dong: Methodology. Danna Wu: Data curation. Linwei Zhu: Formal analysis. Fumin Liu: Software. Hangping Yao: Resources. Nanping Wu: Visualization. Chunsheng Ye: Investigation, Project administration. Haibo Wu: Funding acquisition, Project administration, Writing – review & editing.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
Not applicable.
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