Purification of infectious bronchitis virus propagated in embryonated chicken eggs and its confirmation by RT-PCR
Sylvester S. Arthur, Kataria J.M.*, Dhama K., Rahul S., Bhardwaj Nitin, Tomar S.
Division of Avian Diseases, Indian Veterinary Research Institute, Izatnagar - 243 122 (U.P.)
The Massachusetts strain (M41) of avian infectious bronchitis virus (IBV), was obtained in freeze dried ampoule and reconstituted in 1 ml phosphate buffered saline (PBS) (pH 7.4), of which 0.2 ml was inoculated in ten 9 day old specific pathogen free (SPF) embryonated chicken eggs (ECE). At 48 hours of incubation, the embryos were chilled and the allantoic fluid harvested with titre of the virus determined to be 103.108 EID50/0.1 ml, 103.508 EID50/0.1 ml, and between 104.508-5.508 EID50/0.1 ml of allantoic fluid after 1st, 2nd and 3rd passages, respectively. Characteristic signs of IBV like curling and dwarfing of the embryos were obtained at 7 days post infection. The virus growth was also confirmed by in vitro specific amplification of RNA from the infected allantoic fluid using reverse transcription - polymerase chain reaction (RT-PCR). The high titred virus at the third passage level was used as a seed inoculum for bulk virus propagation in fifty 9 day old ECE, following the protocol described earlier. Along with the allantoic fluid, the chorioallantoic membrane (CAM) was also carefully collected separately from each inoculated embryo and washed once with PBS, and centrifuged to remove RBCs. The pooled CAMs were homogenized thoroughly and the homogenate clarified by centrifugation at 1500 rpm for 10 minutes. For purification of the virus, 20 ml of the allantoic fluid and CAM suspension were layered separately over 5 ml of discontinuous 30% and 55% sucrose in GNTE buffer (0.025M Tris HCl, 0.2M Glycine, 0.002M EDTA; pH 7.0) and centrifuged at 90,000 g for 4 hrs at 4°C. The virus specific band was collected from the interface of sucrose gradient and diluted 1:2 in GNTE buffer. Further, the above suspension was layered over 15 ml of continuous gradient of 30% and 50% sucrose and centrifuged at 90,000 g for 6 hrs. The virus band was collected, diluted as described before and pelleted at 90,000 g for 3 hrs through a 5 ml of 30% sucrose cushion. The virus pellet was resuspended in GNTE buffer and the protein concentration estimated at 260/280 nm optical density (OD) ratio and the virus preparation having an OD of 1.2 was considered as pure in nature. IBV purified by density gradient ultra-centrifugation from allantoic fluid had a protein concentration of 2.56 mg/ml and the CAM virus had 4.36 mg/ml. Higher yield of the virus was obtained from the CAM membranes than from the allantoic fluid. The presence of the virus was confirmed using RT-PCR testing of the purified virus suspension. Further work on the use of the purified IB virus as an antigen in ELISA is underway.