Development of a multiplex PCR and recombinant vaccine against infectious bronchitis virus infection.

摘要

Infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease in chickens, which results in significant economic losses in commercial flocks. One of the major hurdles associated with the control of the disease is the continued emergence of new serotypes/variants due to mutation or recombination. This also makes the diagnosis of disease using conventional serological methods difficult. Therefore, there is a need for alternative vaccines and serotyping methods.; A multiplex PCR for Massachusetts and Arkansas serotypes of IBV was developed. Two serotype specific PCR products, 1026 bp for Massachusetts and 896 bp for Arkansas were amplified; the detection limits for both were 5 pg of viral RNA. By testing different isolates/serotypes of IBV and other avian bacterial and viral pathogens, the high specificity of the multiplex PCR was demonstrated. In addition, its ability to detect Massachusetts and Arkansas as co-infections was revealed.; To explore the potential use of recombinant vaccines in the control of IBV infections, recombinant fowlpox virus containing the S1 gene (rFPV-S1), and DNA vaccines containing the S1 (pBHCX402-S1) and S gene (pCMV-S) of IBV Massachusetts serotype were constructed. Expressions of the inserted genes were examined by SDS-PAGE and immunoblotting techniques and their protective immunity was evaluated in chickens. Results indicated that both S1 and S gene were expressed and IBV specific IgG and virus neutralization antibodies were detected in all groups vaccinated with (rFPV-S1), pBHCX402-S1 and pCMV-S. Although earlier immune response was detected in pCMV-S gene gun delivered group, no significant difference in the magnitude of immune response was found after three immunizations or 5 days post IBV challenge among the groups. Certain levels of protection manifested as preventing severe disease was achieved in all vaccinated groups, but not control groups. Furthermore, pCMV-S delivered by gene gun was much more efficacious, suggesting the DNA vaccination using S gene instead of S1 might provide better protection. The consecutive immunization of chickens with DNA vaccines and recombinant fowlpox virus of IBV did not have any enhanced effect on protective immunity. Overall, the potential use of the developed recombinant FPV and DNA vaccines against IBV infections was demonstrated.