Molecular Mechanism Of Roles Of NDV Infection On Immune Function Of Goose

The waterfowl breeding in China ranked the first in the world. A large number of birds, including goose, died of infectious diseases, such as Newcastle disease(ND) every year. ND is a highly contagious and pathogenic disease caused by Newcastle disease virus(NDV). Vaccination frequently does not provide a sufficiently protection to prevent infection of geese. As a consequence, alternatives to suppress infection with the virus are needed. In recent years, defensins have been recognized as key mediators of the innate immune response in many vertebrate species, and they provide the first line of defense against potential pathogens. Therefore, improvement poultry’s innate immune response and development of new green, efficient and antimicrobial agents has become a research focus.Four novel goose β-defensins(Av BDs, 4, 7, 12, and 16), inducible nitric oxide synthase(i NOS), 7 Toll-like receptors(TLRs, 1-5, 7, and 15), and 8 cytokines, including: interleukin(IL)-1β, IL-2, IL-6, IL-8, IL-18, interferon-γ(IFN-γ), major histocompatibility complex(MHC class I), and FAS ligand(FASLG) were isolated from goose by RT-PCR. Sequence analysis showed the gene fragment of goose Av BD4 consisted of 171 bp encoding 56 amino acids, the full length c DNA of goose Av BD7, Av BD12, Av BD16 consisted of 201 bp, 198 bp, 180 bp encoding 66, 65, 59 amino acids, respectively. In addition, phylogenetic relationships between the four goose Av BDs genes, Av BDs from other avian species, and some mammalian beta-defensin were analyzed. It was demonstrated that goose Av BD4 shared high amino acid homology 80.9% with chicken Av BD4. The goose Av BD7 shared 84.4% amino acid homology with chicken or duck Av BD7. goose Av BD12 shared 87.4 % amino acid homology with its orthologue from chicken. Howhere goose Av BD16 shared 66.3 % amino acid homology with its orthologue from duck.The c DNA of goose Av BD4 were cloned into Eco R I and Sal I sites of p Pro EX HTa vector. The c DNA of goose Av BD7, Av BD12, Av BD16 were cloned into Eco R I and Xhol I sites of p Pro EX HTa vector, repectively. The four recombinant expression plasmids were translated into E. coli Rosseta and the bacteria were expression induced by IPTG. It was demonstrated by Tricine-SDS-PAGE that the recombinant proteins were produced as insoluble bodies in the cells. Additionally, these four recombinant proteins were purified. Micrococcus tetragenus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella pullorum were used to research the antibacterial activities of Av BDs by colony counting method. The results indicated that the recombinant proteins have different degrees of antibacterial activity. Furthermore, the effect of salt concentration on the antibacterial activity and hemolytic activity of the four recombinant proteins were investigated. The results indicated that in high salt ions conditions, antibacterial activity of recombinant proteins was significantly decreased. None of the goose Av BDs found significant hemolytic activity.In order to assess host responses of goose against NDV infection, twenty 40-day-old NDV-maternal antibodies free geese were randomly divided into two groups of 10 per group. Geese in one of the groups were inoculated intranasally with a NDV strain(106 EID50 of go/CH/LHLJ/01/06). Geese in the control group were mock inoculated with phosphate-buffered saline(PBS). Ten tissues, namely trachea, glandular stomach, liver, lung, kidney, brain, bursa of Fabricius, Harderian glands, cecal tonsil, and spleen, were collected from geese on 36 and 72 hours post infection(hpi) to measure the mRNA levels of Av BDs, TLRs, i NOS, and cytokines by the Real-time RT-PCR. The results showed that viral loads were detected in seven tissues, including glandular stomach, liver, lung, kidney, bursa of Fabricius, cecal tonsil, and spleen of geese post infection. Viral RNA in spleen and liver of infected geese was significantly greater than that of control.The result of Correlation between relative gene expressions of goose Av BDs and viral RNA copies in spleen showed that a significantly positive correlation between Av BD12 expression and viral RNA copies in the spleen of geese on 36 hpi, and between Av BD4, Av BD5, Av BD6, Av BD9, Av BD10, Av BD12 and viral RNA copies in the spleen of geese on 72 hpi. The results of gene expression showed that the expression of Av BD4 was significantly down-regulation in brain, trachea and spleen of infected geese(P<0.05). The m RNA of Av BD16 was significantly up-regulated in cecal tonsil, harders glands and significantly down-regulated in brain(P<0.05). The expression of TLR1, TLR2, TLR7 were significantly changed some tissues. Little significant difference was found in the m RNA of TLR3, TLR4, TLR5 and TLR15 from geese(P>0.05). In addition, the m RNAs of i NOS and cytokines were found to be distributed widely in tissues of geese. The expression of i NOS, IFN-γ, IL-8 and MHC class I were significantly up-regulated in trachea, bursa of fabricius and cecal tonsil of infected geese(P<0.05). The present results suggested that Av BD4, Av BD16, TLR1, TLR2, TLR7, i NOS, IFN-γ, IL-8, MHC class I may play a pivotal role in the initiation of the immune response to protect these tissues from NDV infection. Based on the above results, both Av BD4 and 16 were chosn for further anti-NDV study. The results suggested that both recombinant Av BD4 and 16 have little obvious antiviral activity against NDV in vitro.In conclusion, four novel goose Av BDs were isolated from goose by RT-PCR in the study. These four novel oose Av BDs possess potent antibacterial activity. In addition, none of them showed significant hemolytic activity. Other than the four Av BDs, Goose i NOS, TLRs(TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR15)and cytokines(IL-1β, IL-2, IL-6, IL-8, IL-18, IFN-γ, MHC class I, FASLG) were isolated from goose by RT-PCR. The results showed that the expression of Av BD4, Av BD16, TLR1, TLR2, TLR7, IL-2, IL-8, IFN-γ in tissues of geese was significantly regulated in response to NDV infection. Recombinant proteins have little obvious antiviral activity against NDV in vitro.