| Swine Influenza Virus(SIV) belongs to the family Orthomyxoviridae, influenza A virus, negative, single-strand RNA vuirus whose genome consists of eight segments, and it causes swine respiratory disease.The H1N1, H1N2 and H3N2 are main subtypes in worldwild swine population. The European avian-like H1N1 and classic H1N1(cH1N1) subtype are mainly epidemic strains.The researches showed the cH1N1 SIVs emerged in humans as a reassortant virus and caused the H1N1 influenza virus pandemic in 2009, and the European avian-like H1N1 SIVs has caused several human infections in European countries and Asian countries. So it is very important to prevent SIV from spreading among the swine population, and contributing to guide and control human influenza as well as avian influenza.The European Avian-like swine H1N1 subtype influenza virus A/swine/Shanghai/1/2014(H1N1)(abbreviated as SH1) was selected and the HA and NA segments were respectively amplified by RT-PCR and inserted into the PBD vector, the HA and NA recombinant plasmids were constructed successfully. The six plasmids from PR8 virus A/Puerto Rico/8/34(H1N1)(abbreviated as PR8) which could replicate efficiently and HA, NA recombinant plasmids from SH1 virus were transfected into 293 T cells, and the reassortant European Avian-like H1N1 subtype influenza virus vaccine strain(abbreviated as SH1/PR8) was rescued successfully. After infecting novel isolated SH1 virus and recombinant SH1/PR8 viruses into MDCK cells, its growth curve showed the recombinant virus SH1/PR8 could reach highest viral titers at MOI of 0.001 at 48 h after infection, with 1×108TCID50/ml and HA titers of 29 /50μl, Compared with original virus SH1 whose titers were 1×106TCID50/ml and the HA titers of 27 /50μl had higher viral titers. Furthermore, plaque assays showed that the recombinant virus SH1/PR8 has the higher replication ability in MDCK cell. The recombinant virus G11/PR8 inoculated into MDCK cell had been constructed successfully in our laboratory as well as original virus G11, and the growth curve showed the recombinant virus G11/PR8 has higher replication ability with the titers of 2×108TCID50/ml as well as the HA titer of 210 /50μl than the original virus G11.The reassortant virus SH/PR8 was served as the European avian-like inactivated univalent vaccine with Oil emulsion adjuvant and its efficacy was evaluated in mice and pigs. It indicated the HI antibodies, IgG antibodies and neutralization antibodies titers were dramatically increased after inoculation and reached highest titer level at fourth week in mice. The results in challenging experiment in mice showed the vaccinated group mice did not lose weight obviously and survival proportion was 100%, however, the group mice without vaccination losed weight drastically and all died at 6d after inoculation. The lungs were used for virus titers detection and histopathological observation after challenging. The result showed immunization of inactived vaccine could limit the viral replication effectively and attenuated histopathological damages. Meanwhile Inoculation the inactived vaccine can induce high antibodies titer in pigs. The detection of viral titers in nasal swabs, BALF and turbinate at fifth day after challenge. It was showed the virus from vaccined pigs samples was not detected, and the vaccine can protect animals from virus damage from macroscopic lung lesions and microscopic lung lesions.The reassortant virus SH1/PR8 and G11/PR8 was served as the inactivated bivalent vaccine with Oil emulsion adjuvant and its efficacy was evaluated in mice. It was demonstrated inoculating inactivated bivalent vaccine can induce high antibodies by challenging SH1 and G11-MA virus and provide well protective effection. In comparison inoculating unit price vaccine induce low antibodies titer and provide weak protective efficacy by challenging heterologous virus, with mice losing their weight continuously and survival rate was 66.7%. In conclusion, the mice inoculated inactived bivalent vaccine can be protected well from cH1N1 SIV and European avian-like H1N1 SIV. But inoculating inactivated unit price vaccine can only provide partly protective efficacy and has weak cross-protection. |
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