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.