Epidemiological Survey Of Swine Influenza In China From2011to2012and Establishment Of The Molecular Diagnostic Method

Swine influenza is a highly contagious respiratory disease caused by the swine influenza virus.Pigs have been proposed as the mixing vessel of new reassortment virus for both having thereceptors of avian and human. H1N1, H1N2and H3N2are the main subtypes of swine influenzaviruses. Therefore, it’s essential to carry out the epidemiological survey of swine influenza andestablishthe molecular diagnostic method.To investigate the prevalence of swine influenza virus (SIV) in China, serologicalsurveillance of SIV was conducted in different regions from2011to2012. A total of13,044pigserum samples were collected from slaughterhouse, large-scale pig farms and individual farm.These samples were tested by the HI test for antibody detection of the classic swine H1, Eurasianavian-like H1,2009A(H1N1) virus, H3subtype virus, avian H5and H9subtype virus. Serologicaltests showed that the positive rates and geometric mean antibody titer (GMT) of classic swine H1,the Eurasian avian-like H1and2009A(H1N1) were higher than other SIV subtypes. In addition,Eurasian avian-like H1positive were slightly increased from42.24%to45.89%during2011to2012. While, the GMT pH1N1/2009and classic swine H1were53.32and73.2which were highestin2011and2012, respectively. The results indicated that H1N1subtype SIV infection ispredominated subtype in China during2011to2012.In this study,3000samples were collected in pig farms and slaughterhouses from2011to2012.4strains of viruses were isolated by inoculation of10-day-old embryonated eggs viaallantoic cavities and identified as H1N1subtype by HI and RT-PCR. The eight gene fragments ofthese4isolates were sequenced and subjected to genetic analysis. The results showed that the4viruses belonged to2genetic lineages:2009A(H1N1) and Eurasian avian-like H1N1, respectively.SW/GX/133/11and SW/GX/547/11had close genetic relationship with the2009A(H1N1) virus,which were probably evolved from the pandemic H1N1in human in2009. The strains ofSW/HN/232/11and SW/HLJ/24/11which belonged to avian-like H1N1SIV. Above these resultsshowed that2009A (H1N1) and the avian-like H1N1were the main prevalent strains.To establish detect method for the2009A (H1N1) virus infection in swine, A TaqManReal-time PCR was developed based on primers and TaqMan probe derived from HA gene. A serial dilution of recombinant plasmids of pMD-H1-HA was prepared and used to generate standardcurves. The results showed that the sensitivity of the real-time RT-PCR was50TCID50, with acorrelation coefficient of0.999and no cross-reaction with classical swine H1, Eurasian avian-likeH1, human-like H1, or H1N2, H3N2, H5N1, H9N2influenza viruses. The results demonstrate thatthis assay is efficient for the diagnosis of2009A (H1N1) virus in both humans and animals.