| Swine influenza is an acute respiratory disease caused by swine influenza virus(SIV),which leads to a rapid onset of high fever, dullness, loss of appetite, labored abdominal breathing and coughing and thus harms the swine industry all over the world. Pig's epithelial cells are susceptible to both human and avian influenza viruses and have been proposed as the intermediate hosts of influenza transmission from avian to human, which may act as"mixing vessels"for the generation of novel pandemic influenza viruses through reassortment or adaption, and the swine-origin 2009 A (H1N1) pandemic proved so. Vaccination is the primary strategy for the prevention and control of influenza. Current influenza vaccines have poor heterosubtypic protective immunity. In addition, frequent mutation of the viral surface proteins HA and NA largely restricts the efficiency of the present vaccines. We investigated the immunity and protection induced by inactivated reassortant H3N2 swine influenza and combined vaccination with M2e based synthetic peptide vaccine and the research was conducted as follows:The real-time TaqMan fluorescence quantitative reverse transcription polymerase chain reaction (rRT-PCR) assay based on conserved region of matrix gene was developed for rapid detection of type A influenza virus. The target sequence of M gene was cloned into the pMD-18T vector and a series of diluted recombinant plasmids were used to generate standard curve. The rRT-PCR assay has a detection limit of 10 copies of target RNA per reaction and can detect 1 50% egg infected dose of virus, with a correlation coefficient of 0.998. The assay showed good specificity and did not cross react with other viruses including PRRSV, CSFV, PCV2 and TTV. The coefficients of variation were both below 3% intra-assay or inter-assay respectively, which indicated good reproducibility. 40 clinical infected samples were validated by the assay and compared with the regular RT-PCR test, the rRT-PCR assay showed better sensitivity and specificity.The extracellular-domain of influenza Matrix 2 protein (M2e) is considered as a putative target for designing universal influenza vaccines. In this study, we designed a tetra-brabched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusion of four copies of M2e to one copy of foreign T helper (Th) cell epitope and we found it highly immunogenic. The M2e-MAP induced strong M2e-specific IgG antibody responses following 2 doses immunization in the presence of Freund and limited viral replication and attenuated histopathological damage in the challenged mice lungs and was able to counteract weight loss and protected mice from lethal challenge with PR8 virus. These results provide useful information for the development of M2e-based influenza vaccine.Infuenza A viruses of subtypes H3N2 have been reported widely in pigs. In the present study, a high growth reassortant H3N2 (rgH3N2) influenza virus was generated by reverse genetics, which contained the HA and NA genes from the H3N2 virus and the other 6 internal genes from PR8 virus. Inactivated rgH3N2 vaccine candidate could provoke complete protection of H3N2 virus challenge in mice. However, it provided poor cross protectivity against heterosutypic viruses. The combined vaccination with M2e-MAP was able to induce similar strain-specific hemagglutinin inhibition (HI) antibodies and neutralization antibodies as the inactivated rgH3N2 vaccinated alone. And the combinated vaccination also provoked high titer of M2e-specific and rgH3N2-specific IgG antibodies. Furthermore, we found that the addition of M2e peptide greatly enhanced the cross-protective efficacy of rgH3N2 in Freund and conferred efficient cross-protection against heterosubtypic influenza virus. According to the results, we suggest that the M2e-MAP should be added into inactivated vaccine in the strategy of future vaccine research and development.
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