A Duplex Real-time RT-PCR For H1 And H3 Subtype Swine Influenza Viruses And The Study On Its Genetic Evolution And Biological Characteristics

Pig is considered to be an important place for gene reassortment between human and avian influenza viruses,as the respiratory epithelium simultaneously expressing both avian(?-2,3)and human(?-2.6)type receptors for influenza viruses.It has been reported that the mainly prevalent swine influenza virus(SIV)in the past decade in China include Eurasian avian-like H1N1(EA H1N1),pandemic 2009 H1N1(Pdm09 H1N1)and human-like H3N2(HL H3N2).Moreover,these three distinct SIV lineages constantly reassort with each other in swine herds,generating numerous genotypes.Notably,the genotype 4(G4)EA H1N1 reported by Jinhua Liu et al has not merely acquired the dominate prevalence in pigs but also possessed high potential to cross host species,posing threats to both pig industry and public health.In this study,based on the current situation of dominance of H1 and H3 subtype SIV in Chinese pig herds,we established an one-step real-time quantative reverse transcription PCR(RT-qPCR)assay for simultaneously detection and differentiation of the two SIV subtypes,and the method was applied to the SIV epidemiological survey and virus isolation and identification,and the genetic evolution and biological characteristics of SIV clinical isolates were further analyzed.The results of this study will provide technical support for SIV rapid detection,and offer theoretical reference for deep understanding of SIV epidemic dynamics and pathogenic feature.1.Establishment and application of an one-step duplex RT-qPCR for H1 and H3 subtype swine influenza virusBy aligning the HA gene sequences of SIV circulating in China in recent 10 years.a H1 primer-probe set targeting both EA H1N1 and Pdm09 H1N1 lineages plus a H3 prime-probe set targeting the prevalent HL H3N2 subtype were designed,respectively,Further,based on the TaqMan-MGB probe,an one-step duplex RT-qPCR assay was established and evaluated.The duplex RT-qPCR possessed the detection limit of 5 copies/?L HA gene plasmid and 2.1 EIDso/mL of viral RNA.and matched an overall detection accuracy of 97.9%(47/48)with traditional virus isolation through chicken embryo inoculation using experimentally infected mice lung samples.Besides,the method showed that the coefficient of variation(CV)was all less than 3%both within-run and between-runs,suggesting high repeatability of RT-qPCR.Furthermore,the duplex RT-qPCR method revealed a relatively higher prevalent rate of H1 than H3 subtype SIV in the clinical application of nasal swabs collected in a slaughterhouse in autumn and winter from 2019 to 2020,which was consistent with previously reported studies.It was indicated that the qRT-PCR had a good application prospect in rapid detection of H1 and H3 subtype SIV.2.Isolation and identification of swine influenza virus and the phylogenetic analysisCombining with the RT-qPCR detection method established in Chapter 1,9 strains of EA H1N1 and 4 strains of HL H3N2 viruses with hemagglutination activity were successfully isolated from the 598 nasal swabs collected from a slaughterhouse.Additionally,1 strain of EA H1N1 virus was isolated from 170 test samples that were sent by pig farms.Next,based on the result of HA gene sequencing of the 14 strains,3 strains of EA H1N1 and 2 strains of HL H3N2 subtype SIV were selected to perform whole-genome sequencing and genetic evolution analysis.The results showed that the 5 SIV strains all possessed Pdm09 HIN1-like PB2,PB1,PA,NP,M genes and triple reassortant(TR)SIV-like NS gene.In other words,they had the same internal gene constellation of G4 EAH1N1 which was dominantly prevailing in current Chinese pig herds.Furthermore,it was worth noting that the EA H1N1 isolate A/swine/Huadong/11/2020(H1N1)[HD11]was highly homogeneous with a human influenza strain A/Shandong/00204/2021(H1N1).The nucleotide identity between the two viruses was as high as 99.35%?99.8%,suggesting that the two H1N1 strains may have a common ancestor or may exist interspecies transmission.3.Biological characteristics of 5 swine influenza virusesFurther analysis of antigenic difference and determination of receptor binding preference,replication capability in vitro and pathogenicity to mice of the 5 SIV strains sequenced in Chapter 2 were conducted.The results showed that there were no obvious antigenic changes among the 3 isolates of G4 EA H1N1,and between G4 and genotype 1(G1)or genotype 5(G5),as well as between the 2 isolates of HL H3N2 or between HL H3N2 and human H3N2 viruses.But certain antigenic variation was observed between some EA HlN1 strains and Pdm09 H1N1.The 5 SIV isolates all possessed double receptor binding property of ?-2,3 and ?-2,6,in which the strain HD11(H1N1)evidently preferred ?-2,6 receptors.In addition,5 SIV isolates all replicated well in MDCK cells,in which HD11(H1N1)possessed the relatively highest viral titers at each sampling time points.At the challenge dosage of 106.0EID50,HD11(H1N1)and A/swine/shandong/SG03(H1N1)showed apparent pathogenicity to mice with 100%and 80%mortality,respectively,and possessed the relatively highest viral load in tissues and expression level of cell inflammatory factors in the 5 tested isolates.It was further suggested that the threat to public health caused by the interspecies transmission potential of G4 EA H1N1 should not be ignored and require enhanced surveillance.