| Swine influenza (SI) is an acute respiratory diseases caused by swine influenza virus (SIV), which belong to Orthomyxoviridae, type A influenza virus group. This disease is manifested with high morbidity and low mortality. The severity of this disease depends on secondary infection with other bacteria and viruses. Moreover, pigs can be infected not only by human influenza virus but also by avian influenza virus, suggesting the pigs as an intermediate host of interspecies transmission or as mixing vessels, play significant role in the public health implications. By far, there are 11 subtypes of SIV, including H1N1, H1N2, H1N7, H2N3, H3N1, H3N2, H3N6, H4N6, H5N1, H5N2 and H9N2, had been isolated from pigs. The results of epidemiological study of SI showed that H1N1, H1N2 and H3N2 SIVs were widely spread in China.To consrtuct recombinant adenovirus shuttle plasimd pDC315-H3HA-EGFP and pDC315-NP-EGFP, HA and NP gene of A/Swine/Guangdong/9/2005(H3N2) amplified by PCR from the recombinant plasmid pMD18-H3HA and pMD18-NP, was subcloned into pIRES2-EGFP. The gene fragment containing interest genes and EGFP was then inserted into adenovirus shuttle plasmid pDC315. The replication-defective recombinant adenovirus expressing HA and NP gene were generated by co-transfecting the recombinant shuttle plasmid and the backbone plasmid pBHGlox△E1/E3Cre in HEK293 cells. The recombinant adenovirus was confirmed by the typical cytopathic effect and expression of EGFP gene in HEK293 cells. The results of identification by PCR and Western blot indicated that the recombinant adenovirus could efficiently express HA and NP protien of SIV in vitro. The TCID50 of the rAd-H3HA-EGFP and rAd-NP-EGFP were evaluated as 1.58×1010/mL and 1.26×1010/mL, respectively, after propagation and purification.6-week-old female Balb/c mice were randomly divided into 6 groups(20 per group), three of which were vaccinated intramuscularly with the recombinants rAd-H3HA-EGFP,rAd-NP-EGFP and a mix of rAd-H3HA-EGFP and rAd-NP-EGFP. Three weeks after the primary vaccination, mice were vaccinated intramuscularly again with the same dose as the first vaccination, and simultaneously two groups vaccinated with adenovirus vector rAd-EGFP, PBS and one group having not been vaccinated and challenged were included in the animal trial as control groups. 3 weeks after the second vaccination, the first five groups were divided equally into two groups, and were challenged with SIV of different subtype which were A/Swine/Guangdong/9/2005(H3N2) and A/Swine/Henan/11/2005(H1N1). The immune efficacy of the recombinant adenoviruses in mice was evaluated by detecting the antibody levels after immunization, weight changes and virus secretion of organ after challenge. High level HI antibody titers were detected in the mice vaccinated with rAd-H3HA-EGFP or the mix of rAd-H3HA-EGFP and rAd-NP-EGFP. After challenge with the same subtype SIV, mice of the above two groups gained weight, and no virus was detected. The results showed that recombinant adenovirus rAd-H3HA-EGFP could provide complete protection against the same subtype of SIV. ELISA antibodies were detected in sera of mice vaccinated with rAd-NP-EGFP alone or combined with rAd-H3HA-EGFP. When challenged with the different subtype SIV, the results showed that rate of weight loss and viral titers were lower than other groups. These results showed that rAd-NP-EGFP could induce effective immunity against different subtype SIV.Two replication-defective recombinant adenovirus expressing HA and NP, respectively, were obtained in this study. The results of further animal experiments demonstrated that these two viruses could effectively induce immune efficacy in mice, which laid the foundation for developing genetical engineered SI vaccine.
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