Reassortant Of The Swine Influenza Virus And Preparation Of HA Protein Special Serum

Swine influenza(SI)is caused by swine influenza virus(SIV)with acute and chronic respiratory diseases in pigs.The symptom of this disease is outbreak rapidly,high fever and difficulty in breathing,etc.Swine influenza virus(SIV)is an orthomyxovirus of the influenza A group and this virus have the hemagglutinating antigen H and neuraminidase antigen N(such as H1N1,et al).At the same time,pig is a gene recombination "mixer" of influenza virus and maybe act as a "incubation" for new pandemic strain recombined with avain influenza virus and human influenza viruses.So pig who serve as intermediate hosts plays an important role for cross-species transmission of the influenza virus in "birds and mammals".These characteristics of SIVs and pigs determine the irreplaceable position in the pandemic process of the influenza virus.So to study the SIVs have great significance to understand the epidemiological characteristics,evolution and spread across species.In this study,the influenza virus 8 plasmid system were used successfully and it has been verified it can save as a reassortant strain for H1N1 SIV.Through comparing with parent strain,the reassortant strains have specificity of directional selection.This study provide a basis for assess the risks of SIVs,spread across species and produce of reverse genetic vaccine and optimize H1 ?H3 subtype HA special serum for SIVs epidemiological investigation and viruses subtype identification.Test 1: H9N2 avian influenza virus A/chicken/Gansu/SS01/2014(H9N2)(GS/SS01/14)have analysised by its biological characteristics,the whole genome sequencing and evolutionary,and the SPF chicken embryos and BALB/c mice infected with the virus titration.Sequence analysis showed that the isolates of HA of GS/SS01/14 cleavage site is333 PARSSR/GL340,and there is no continuous basic amino acid belong pathogenic avian influenza virus(LPAIV)features.The different genes of virus come from the same subtype,and has high homology with A(H9N2)/chicken/Jiangsu / 245/2004.HA test and EID50 test show that the virus has high titer of hemagglutinin and high chicken embryo growth ability.Virus infection test showed that GS/SS01/14 can't effectively replication in mice.Above studies have suggested that the GS/SS01/14 can reconstruct the swine influenza virus as a candidate skeleton strain,for subsequent research in reverse genetics technology as the template.Test 2: In order to understand the influenza virus propagation regular,pathogenic mechanism and reason of the cross-species transmission,this experiment has established the reassortant SIV using reverse genetic system.8 genes of H9N2 AIV GS/SS01/14 were obtained with RT-PCR method and the HA,NA genes of popular H1N1,H3N2 subtypes SIVs were synthesised,then connected each gene using a homologous recombination technology to vector p BD respectively and H9N2 AIV GS/SS01/14 and " 6 + 2 " reassortant H1N1 and H3N2 influenza virus 8 plasmid system were established.Then,three groups of 8plasmid system transferred to 293 T cells,after 2 days incubation,the supernatant fluid were inoculated in to the 10 days chicken embryos,and the virus from chicken embryo allantoic fluid identified.Results showed that H9N2 AIV GS/SS01/14 and reassortant H1N1 SIV have been rescueed successful after 8 plasmid transfection system of 293 T cells.And the hemagglutination titer of the ressaorant viruses reached 10log2-8log2 respectively,and growth ability is similar as wild type virus.Therefore,the success of the test set up H9N2 AIV GS/SS01/14 and reassortant H1N1 SIV reverse genetic system and reassortant H1N1 SIV can be used as a vaccine candidate strain.Test 3: In order to detect H1 and H3 subtypes swine influenza viruses by serological methods quickly and exactly,SIV hemagglutinin subtype-specific serums against European avian-like H1N1 and human-like H3N2 were prepared in this study.The selected HA genes were first synthesized and cloned into eukaryotic expression p CAGGS vector.Then the two recombinant plasmids were confirmed by enzyme digestion identification and indirect immunofluorescent assay(IFA).SPF chickens were injected by the recombinant plasmids in a volume of 200 ?g and the serum of the chickens were collected after the third immunization.Hemagglutinin inhibition test(HI)indicated that the antibody levels reached at 4log2 to 6log2 with no cross-reaction.The subtype-specific serums prepared in this study could be used in identify for H1 and H3 subtypes SIV and provide the reference for swine influenza vaccine application in pig herds in China.