Isolation Of Avian Influenza Virus And Study On Construction And Immunogenicity Of Multi-epitopes Recombinants

Avian influenza (AI) has caused enormous economic loss and grand challenges forthe public health. Further more, there is new situation that many subtypes avianinfluenza viruses (AIV) are coexistent. Vaccines are still the main means to prevent AI.But traditional vaccines can't play a efficient role to prevent the AI for the characteristicof AIV for the variation and more subtypes. So it's an important question how to designand study new vaccines, especially polyvalent vaccines. Based on genetic analysis of anavian influenza virus, a multi-epitopes gene cassette was designed and synthesized. ThenDNA and fowl pox virus recombinant were constructed for the prevention of H5, H7, H9,H3 subtype influenza virus. After the mouse and chicken were inoculated with theserecombinants, the indexes of humoral and cell immunity were detected. This studyestablished the base for prevention of multitude influenza viruses. The more informationas follows:The H5N1 subtype avian influenza virus (AIV) A/Chicken/Jilin/9/2004 (H5N1)(JL9/04) was isolated from Jilin Province. Then its was sequenced and analyzed. Theresults showed that the sequences of JL9/04 include eight full-length genes fragmentswhich contains the whole open reading frame of each gene. NCBI BLAST analysisshowed that HA, PA, PB1 and NS gene of JL9/04 shared the highest homology withH5N1 subtypes from swine. PB2 gene shared the highest homology with H5N1 subtypesfrom human. But NP and M gene shared the highest homology with H9N2 subtypesfrom fowl. JL9/04 has the characteristic of High Pathogenic Avian Influenza Viruses.The sequencing results of the HA gene cDNA of JL9/04 revealed that there was aninsertion of 6 basic amino acids in the cleavage site between the HA1 and HA2, whichwas the characterization of the H5 subtype HPAIV. Further more, no difference ofnucleotide related to all the 6 potential glycosylation sites, the 2 receptor-binding sitesand the basic amino acid insert within the HA existed among JL9/04 and other referenceviruses. The homology of HA gene between JL9/04 and A/Turkey/Ireland/1378/83(Ire/83) was 86.9% (<89%). The results suggest that JL9/04 might be a new genotype ofAIV, and both the H5N1 subtype AIVs from swine and the H9N2 subtype AIVs fromfowl may play a important role in the recent H5N1 outbreaks.According to the independence of epitopes of influenza virus and the regulation ofKozak, a gene cassette (Epi), which included epitopes of H3 and H9 influenza virus HAgene and epitopes of other influenza virus antigen (NP, NA, M) and some enzyme cutsites, was designed and synthesized. Length of Epi is 765 bp. Based on the HA genesequence of JL9/04, HA gene of H5 AIV was cloned. According to the HA gene ofA/African Starling/983/79 (H7N1) , the HA gene of H7 AIV was synthesized. Based onthe mRNA of AA flesh, the IL-18 gene of fowl was cloned. After recombinating theDNA fragments Epi with plasmid pRIES1neo, the recombinant plasmid pRI-Epi wasconstructed. After inserting Epi into the co-expressed gene of H5-H7 HA, therecombinant plasmid pRI-H57-Epi was constructed. Then its antigenicity was detectedby IFA in Hela cells. The Western-blot, IFA results showed that these recombinantplasmids could expressed the object proteins. These results identified that it's goodantigenicity.H5HA-H7HA-Epi cDNA and chicken Interleukin-18 cDNA were inserted intotransfer vector plasmid pUTA-16-LacZ, and respectively regulated by ATI-P7.5×20promoter and P7.5 promoter and the transfer vector plasmid pUTA-L-H57-Epi wasconstructed. Then its was cotransfected with FPV in CEF under the selection pressure ofBrdU. After identified with restricted enzyme cutting, PCR, IFA and Western-blot, arecombinant FPV virus (vFA7935L18) was gained. The results of Western-blot and IFAshowed that the vFA7935L18 could express the object proteins. Its suggest that therecombinant FPV was successfully designed and constrcted.SPF chicken, which was used as experimental animal model, were innoculated byDNA recombinant and fowlpox viral recombinant strain being constructed above, thenthe humoral and cell immunity index were detected. HI antibody of H3, H5, H7 and H9subtype influenza viruses were detected after recombinant fowlpox strain vFA7935L18and recombinant pIRC-H57-Epi groups were inocculated a week later, it still increasedafter 21 days .DNA recombinant pIRE-Epi can induce organism create HI antibodyaiming at H3 and H5 subtype.SPF chicken produced higher HI antibody level aiming atH5 subtype AIV after 14 day innoculated by inactivated vaccination, but HI antibodyaiming at H3, H5 and H7 subtype was a bit lower. The proliferation ability of spleniclymphocyte stimulated by Con-A was detected and the result showed lymphocyte Con-Astimulating index in group vFA7935L18, rFPV-H5HA-H7HA-IL18, pIRE-H57-Epi andpIRE-Epi is higher than that in group inactivated vaccination and wt-FPV. It indicatedthat IL-18 expressed by vFA7935L18 and rFPV-H5HA-H7HA-IL18 maybe enhance thecell immunity level of chicken, or it maybe related to the NP antigenic epitopes ofcombination multi-epitopes box gene. The detection of inhibition in bodyweight showedthat IL-18 expressed by vFA7935L18 and rFPV-H5HA-H7HA-IL18 can lessen thefowlpox viral vector inhibit chicken weight increase.BALB/c mice, which was used as mammal experimental model, were innoculatedby DNA recombinants and fowlpox viral recombinant strain as before. The humoral andcell immunity level were detected. The results showed that special antibody aiming atH3 subtype could be generated in group of pIRE-H57-Epi, pIRE-Epi and vFA7935L18,and antibody titer (1:1600～1:6400) were lower than that of inactivated vaccinationgroup (1:12800) , but is all higher than that of control group (p<0.01) . The antibody titeragainst H5HA, H7HA in group pIRE-H57-Epi is 1:12800 and 1:6400, which was higherthan other groups (p<0.05). The antibody titers against H5HA and H7HA was just sameas contrast between groups vFA7935L18 and rFPV-H5HA-H7HA-IL18. The antibodytiters against H5HA and H7HA of group pIRE-Epi is lower (1:200,1:100) than othergroups except blank group. The antibody titer against AIV H9 HA of groupspIRE-H57-Epi, vFA7935L18 and pIRE-Epi (1:6400,1:6400,1:3200) was higher thanother groups (p<0.05). The subtype numbers of T lymphocyte of groups pIRE-H57-Epi,pIRE-Epi, vFA7935L18 and rFPV-H5HA-H7HA-IL18 were higher (p<0.05) than groupsof PBS, pIRE1neo and In addition, ratios of all groups were stabile between 1.5-20,which indicate there is no abnormalities during the immune response. The results ofELISPOT detection for number of IFN-γ blot showed the number (>70) of groupspIRE-H57-Epi, vFA7935L18 and rFPV-H5HA-H7HA-IL18 were higher (p<0.05) thanother2 groups, which include group of split vaccine (<40).The results above indicated that the recombinant DNA and fowl pox virus whichwere constructed above have good immunogenicity. The study had established thefoundation for the multivalent vaccine of influenza viruses.