A multiplex real-time RT-PCR method for detecting H5, H7 and H9 subtype avian influenza viruses in field and clinical samples

Highlights

• A quadruplex real-time RT-PCR method for detecting H5, H7 and H9 subtype AIVs was developed with high sensitivity and specificity.

• The clinical performance of the method was evaluated by various specimens from poultries and humans.

• The multiplex RRT-PCR method is a fast, convenient and practical method for AIV clinical detection and epidemiological analysis.

Summary

In recent years, H5 and H7 subtypes of highly pathogenic avian influenza viruses (HPAIVs) have been identified in poultry worldwide, resulting in large economic losses to poultry production. Furthermore, H9N2 low pathogenic AIVs are reported to provide internal genes for generating novel reassortant AIVs, leading to potential pandemic risks. To establish an accurate, sensitive and convenient diagnostic method for H5, H7 and H9 subtype AIVs in Eurasian lineage, four groups of specific primers and probes were designed based on the conserved fragments of M, H5, H7 and H9 genes, and a multiplex real-time RT-PCR (RRT-PCR) method was established. High sensitivity was achieved for the multiplex RRT-PCR approach, with a detection limit of 1−10 copies (plasmid DNA) per reaction. The specificity of the method was evaluated using diverse subtypes of AIVs and other avian respiratory viruses isolated in eastern China over the last 9 years. Compared with virus isolation, a higher consistency was achieved when assessing 135 field samples and 126 clinical samples. The results showed that the multiplex RRT-PCR method is a fast, convenient and practical method for AIV clinical detection and epidemiological analysis.

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.