Evaluation Of Immune Efficacy Of A Reassortant Swine H1N1Vaccine Constructed By Reverse Genetic Manipulation

Swine influenza (SI) is an acute and highly contagious respiratory disease caused by influenza Avirus. The Classcical H1N1subtype swine influenza viruses are still circulating in the domestic swinepopulation. Among all subtypes of influenza viruses, H1N1remains as one of the dominate subtypes indomestic swine population. The HA, NP and NS gene segments of the2009pandemic H1N1influenzavirus were proved to be originated from the classical swine lineage, the PB2, PA and PB1wereoriginated from the tripple reassortant swine lineage, the NA and M segment were originated from theEuropean or Eurasian avian-like swine lineage. The emergence of2009novel pandemic influenzaA/H1N1(pH1N1) raised widely public concern on SI, and pigs were seemed as the“mixing vessel” forgenerating new subtypes of influenza virus with pandemic potential. Therefor, developing H1N1vaccines for pigs is necessary for both pig production industry and public health.In2011，an influenza virus strain was isolated from tissue samples collected in pig with respiratorytract problems in a pig farm of south China．It was propagated in SPF embryonated chicken s eggs, thevirus was determined to be a H1N1subtype SIV by HI and NI after purification. We designated it asA/swine/Guangdong/1/11(H1N1)(abbreviated as GD/11). All the genes of GD/11were respectivelycloned into pMD18-T vector and sequenced. It was found that GD/11was classified to be the classicalSIV. Several H1N1subtype SIV strains that isolated from pigs recently were then chosen as referencestrains to perform genomic sequence alignment with GD/11. Results demonstrated that HA segment ofGD/11have high homology with those reference strains，which hints that GD/11may could serve as theseed virus for H1N1subtype influenza vaccine production. However, the results of hemagglutinin assayand EID50detection of GD/11was26,and105EID50/ml respectively, which denotes GD/11could notreplicate efficiently in embryonated chicken s eggs. In order to get a seed virus for vaccine production,we appplied reverse genetics to generate the reassortant influenza virus. The HA and NA segments ofGD/11were respectively cloned into the bidirection transcriptional vector PBD. In this study, we selectintern segments(PB2, PB1, PA, NP, M, NS) of A/Puerto Rico/8/34(H1N1)(abbreviated as PR8), whichcould replicate efficiently but could not infect humans, as the backbone of the influenza reverse geneticsystem. After the plasmids were co-transfected into293T cells, the cell suspension was collected andinjected into SPF embryonated eggs, the reassortant virus (GD/PR8) that could agglutinate erythrocyteswere rescued, and all the HA, NA genes were proved to be derived from the GD/11and the other sixgenes were derived form PR8by necleotide sequencing after three generations in eggs. The EID50ofGD/PR8was determined to be107EID50/ml which was102higher than GD/11. Then we inoculated100EID50of GD/PR8and GD/11respectively to SPF embryonated chicken s eggs and carried outhemagglutinin assay at different time points. The results showed that the HA titers of GD/PR8got210at48h which was much higher than that of GD/11. No eggs was found dead by72h after the inoculation.These results demonstrated that GD/PR8was stable and could replicate to high titers in embryonatedeggs. Considering the superiority of the reassortant virus, GD/PR8was served as the seed virus for thepreparation of inactivated SI vaccine and its efficacy was evaluated in mice and pigs. High titers ofspecific IgG and HI antibodies were detected in both mice and pigs after prime innoculation. The titerswere dramatically increased after the booster immunilization. No virus was detected by real-time PCRand EID50in the lungs of mice and pigs in the vaccinated group, which showed that the GD/PR8vaccinecould inhibit replication of virus in the lungs of animals. Nasal swabs were collected for five days afterthe challege. No virus were dected in the nasal swabs of pigs that received two dose of vaccine, whichdemonstrated that the GD/PR8vaccine could limit the shedding of virus in pigs. Then we performedMacroscopic and Microscopic observation of lungs to determin the lesions of lungs that caused byGD/11. The results showed that the GD/PR8vaccine could obviously limit the lesions to the lungs. Thepresent study indicated that GD/PR8vaccine could induce high level of HI and IgG antibodies in miceand pigs. Protection against viral replication, pathological change was evident in animal. In conclusion,the reassortant GD/PR8virus could serve as an ideal seed virus for preparation of an inactivated vaccineto provide best protection against classical H1N1subtype influenza virus for the swine population.