Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

Yan, Fang; Zhao, Yujun; Hu, Yongting; Qiu, Jianyang; Lei, Wenxin; Ji, Wenhui; Li, Xuying; Wu, Qian; Shi, Xiumin; Li, Zhong

Plasmids pVAX1-16S1, pVAX1-16N, and pVAX1-16M encoding the S1, N, and M proteins of the virulent IBVSX16 strain, respectively, were described in our previous publication [22] . The virulent IBVSX16 strains used to challenge immunized chickens in this study were isolated from the kidneys of IB-infected chickens from the Shanxi province by Department of infectious Disease and immunology, College of Animal Science and Technology, Shanxi Agricultural University [21] . Virus stocks containing 1 × 10 3 egg infective dosage (EID 50 ) of IBVSX16 with 100 μL were used to inoculate the allantoic cavities of 10-day-old specific pathogen free (SPF) embryonated chicken eggs (Shandong Specific-Pathogen-Free Chicken Research Center, China) that were then kept at 37 o C for 48 h. Allantoic fluid containing the virus was harvested after 48 h post-inoculation, stored at −80 o C until use. Chill eggs at 4 o C for at least 2 h to kill the embryo and to reduce the contamination of the allantoic fluid with blood during harvesting. Remove sticky tape and swab each egg with cotton wool soaked with 70% alcohol to disinfect and remove condensation from the shells. Dip the forceps or scissors in absolute alcohol and flame to sterilize. Remove the eggshell above the air space. Discard embryos that are visibly contaminated. Remove a sample of allantoic fluid from each egg. The 50% EID 50 /mL of the viral stocks were calculated as previously described by Reed and Muench [25] . Titer of the IBVSX16 strains was 10 9 EID 50 /mL. A total of 140 seven-day-old SPF chickens (Shandong Specific-Pathogen-Free Chicken Research Center, China) were housed in SPF environment at the Laboratory Animal and Resources Facility, Shanxi Agricultural University.