| Infectious bronchitis (IB) is a highly contagious viral disease of the chicken caused by infectious bronchitis virus (IBV), which results in significant mortality of young chickens, and a decline in egg production and quality in layers and poor weight gain and reduction of the feedstuff repay in broilers. In addition, the ability of IBV to genetically mutate and recombine results in antigenic shift and drift. This means that vaccine program is constantly challenged. In present study, we have attempted to find a novel control strategy to combat infections from innate immunity.Avian defensins (AvBDs) are cationic cystine-rich antimicrobial peptides that play a pivotal role in the innate immune system of avians. In order to understand wether the mRNA expression of AvBDs in tissues of chicken was constitutive or inducible in response to velogenic and lentogenic IBV infection, the levels of the mRNA which include18sRNA, AvBD1-13,10toll-like receptors (TLRs) and inducible nitric oxide synthase (iNOS) in tissues from chicken with the real-time RT-PCR method.The recombinant plasmids, which encoded the chicken AvBD2,6,12were amplified by PCR, inserted into the pProEX HTa, translated into E. coli Rosetta. The bacteria were induced with IPTG. Colony counting assays were performed to investigate the antimicrobial activities of non-recombinant protein (served as control) and recombinant AvBDs (rAvBD) from chicken against bacteria. The antiviral activity of chicken AvBD2,6, and12against IBV was determined according to methods described for neutralization assays.The results showed as follows:(1) In order to investigate whether the expression of AvBDs mRNA in tissues from chicken was constitutive or inducible in response to velogenic and lentogenic IBV infection, chicken was infected with IBV. The results showed that highly significant downregulation of the level of expression AvBD1,2,4,5,6, and9in most of tissues of chicken infected with lentogenic IBV. In contrast, expression of these genes was increased in respect to velogenic IBV infection (P<0.05). The expression of AvBD2,11,12, and13was increased significantly (P<0.05). The results showed that expression of TLRs and iNOS mRNA were induced or up-regulated in most of tissues in response to velogenic IBV infection (P<0.05), and down-regulated by lentogenic IBV infection.(2) All of the rAvBDs (molecular weight,10-15kD) were produced as inclusion bodies after induction with IPTG.. The results of the neutralization assays show that these3rAvBDs could inhibit the replication of IBV.(3) Five pathogenic bacterial strains were used to investigate the antibacterial activities of these3rAvBDs. It was showed that non-recombinant proteins has no antibacterial activities against all of bacteria investigated, three rAvBDs have high antibacterial activities including Gram-positive and Gram-negative strains investigated except for S.anreus. AvBD12has high antibacterial activities against S.anreus, Lactobacillus, S.choleraesuis, and B.Bronchiseptica (P<0.05). In addition, it is found that rAvBDs were sensitive to salt concentration. The antibacterial activities of chicken rAvBD against E. Coli and Mtetragenus were decreased with the salt concentration elevation (0～150mM)(P<0.05).(4) Little hemolysis of chicken erythrocytes was observed at any concentration of any of rAvBDs, when compared with the negative control (P>0.05).In conclusion, the data from the present study revealed that chicken AvBDs possess potent antiviral activity against IBV. An AvBD-mediated immune system exists in various tissues of chicken and IBV stimulate and modulates the expression of AvBDs mRNA. These data suggest that the antiviral activity is at least partially mediated by a direct effect on the viron. These peptides probably play a role in the recognition of pathogens such as IBV, and in the initiation of the immune response to protect these tissues from microbial pathogens. As only mRNA of AvBDs was detected in the current study, these production will be evaluated by ELISA in the futher study. |
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