Posted 30 August 2012 - 07:49 AM
I am a masters student working on the Sendai virus pathogenesis. I was wondering if anyone had any experience propagating virus in chicken eggs. As this was my first time inoculating and collecting allantoic fluid; Not surprisingly, I ran into a problem.
While collecting the virus from the allantoic fluid the yolk sac ruptured and some of my collections have yolk in suspension (I collected roughly 15-20mls of allantoic fluid) making the collected allantoic fluid yellowish in color.
I centrifuged the 50ml tubes at 4000 rpm for 5min 2X. I only got a small pellet (consisting of blood cells and some other stuff), the yolk was still thoroughly mixed in the allantoic fluid.
I am planning to filter the allantoic fluid next. But I am wondering if anyone has a better idea on how to remove the yolk from the allantoic fluid. Or let me know if the yolk will have any detrimental effect on the virus (I read that chick antibody production starts in the yolk).
Also I have read a few protocol with inconsistencies concerning whether the inoculated eggs should be rotated or not before harvesting virus. So if anyone has experience propagating virus (it would be super helpful if the virus was Sendai) in chicken eggs, I would like to know if inoculated eggs should be rotated or not before virus harvest.
Any help would be amazing, Thanks!
Posted 13 September 2012 - 11:07 PM
Posted 17 September 2012 - 09:31 AM
If you have a digital protocol of your egg infection/virus harvest, I could I bother you to email it to me. I’d like to review what your lab does differently than ours.
Posted 19 September 2012 - 01:31 AM
Briefly, virus titer was measured by haemagglutination assays (HA) and around 20 HA of it was diluted in 0.1 mL ice-cold phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2PO4, 1.47 mM KH2PO4, pH 7.4) (Dulbecco & Vogt, 1954) and injected into the allantoic fluid of 9-day-old specific pathogen-free (SPF) embryonated chicken eggs. The eggs were sealed and further incubated for two more days at 37°C in a humidified incubator. Later, the upper part of the egg shell was aseptically opened and clear allantoic fluid was collected and clarified by spinning at 6,000 xg for 20 min at 4°C. The fluid was then further centrifuged at 40,000 xg for 2 h at 4°C and the pellet was resuspended in 1 mL ice-cold PBS. At the same time, a gradient of different sucrose concentrations (20%, 30%, 40%, 50% and 60%) was made in an ultra-centrifuge tube and incubated for at least 1 h at 4°C to stabilize.Later, about 0.6-0.8 mL of the resuspended virus was added on top of the gradient and ultra-centrifuged at 161,000 xg for 4 h at 4°C. The virus band was clearly apparent at the lower middle of the tube. Furthermore, the virus band was pipetted into a fresh tube and topped up with ice-cold PBS, to further pellet by spinning at 161,000 xg for 4 h at 4C. The pellet was resuspended in 0.1-1 mL of ice-cold PBS, aliquoted and kept in -80°C