1.Introduction

Swine influenza, normally caused by influenza A virus, is a highly contagious respiratory disease of swine.1 Pigs independently infected with swine influenza virus (SIV) often exhibit typical respiratory symptoms, such as fever, coughing and difficulty breathing.2However, more severe clinical symptoms could be observed when other pathogens co-infected or secondarily-infected with SIV.3,4 In addition, the fact that both avian and human influenza virus receptors are expressed in pig respiratory epithelial cells paves the way for influenza reassortants to spread across host barriers. Currently, SIV of H1N1, H1N2 and H3N2 subtypes are dominantly circulating in swine worldwide.5 Especially in China, Eurasian avian-like H1N1 (EA H1N1), Pandemic 2009 H1N1 (Pdm09 H1N1) and human-like H3N2 (HL H3N2) are the three main prevalent lineages of H1 and H3 subtype SIV in the past 10 years.6,7
Recently, EA H1N1 has been the overwhelmingly prevailing SIV in China and classified into six genotypes (G1-G6) according to its gene constellation.8 Of extraordinary importance, G4 has been assessed to possess the highest possibility to transmit from swine to human and cause an influenza pandemic.6,8 Actually, several human cases have been confirmed as directly infected with EA H1N1, such as that the 9-year-old boy suffering from fever and headache was tested SIV-RNA positive by the Tianjin Centers for Disease Control and Prevention in 2019.9 In addition, HL H3N2 SIV could also pose a threat to human health as the pathogen was once isolated from a 10-year-old girl in Guangdong province in 2018.10 Therefore, it is urgent to carry out systematical surveillance of SIV since swine could yet serve as ‘mixing vessel’ for producing novel influenza reassortants from mammalian and avian viruses. And, to develop a sort of rapid, sensitive and specific method for SIV detection is of great significance not only for the prevention and control in swine, but also for the early warning of cross-host transmission in human.
The classical laboratory diagnostic methods of SIV infection include virus isolation through inoculation into specific-pathogen-free (SPF) embryonated chicken eggs or Madin-Darby canine kidney (MDCK) cells, and then followed by haemagglutinin (HA) and neuraminidase (NA) inhibition assays via defined antisera to subtype HA and NA, respectively.11,12 However, this method is time-consuming and labor-intensive to conduct a large-scale survey of SIV detection.13,14 Although some immunological assays like immunofluorescent staining and enzyme-linked immunosorbent assay that have been used to detect the viral nucleoprotein antigen provide results more quickly than viral isolation, the sensitivity is not stable while mainly depends on the affinity and compatibility of the antibodies used.14 By contrast, the real-time RT-PCR (RRT-PCR) method for detection of PCR products in real time through fluorescent dyes or fluorescent labeled specific probes is endowed all the combined qualities of high sensitivity, specificity and speediness. Especially, the probe with its 3’ termini tagged of minor groove binder (MGB) instead of the traditional Tamra as fluorescence quencher has been widely applied in the detection of various pathogens involving equine herpes virus 5, infectious bursal disease virus, bovine viral diarrhea virus and so on.15-17
In this study, based on the Taqman-MGB probes targeting conserved HA gene regions, an one-step duplex RRT-PCR method for rapid detection and differentiation of H1 and H3 subtype SIV was successfully established. The sensitivity, specificity, repeatability and the primary clinical applications of the developed assay was also determined.