Propagation and Harvest of Influenza Virus from a Fertilized Chicken Egg

Resumo

Source: Brauer, R. et al., Influenza Virus Propagation in Embryonated Chicken Eggs. J. Vis. Exp. (2015)

This video demonstrates the synthesis and harvest of the influenza virus in fertilized chicken eggs. The virus is inoculated into the allantoic cavity, where it replicates in epithelial cells and buds into the allantoic fluid, which is then collected and stored for downstream applications.

Protocolo

1. Influenza Virus Inoculation via the Allantoic Route
   1. On the day of virus inoculation (day 10 or 11), candled the eggs again and eliminated any dead embryos. Distinguish live embryos by looking for movement inside the egg.
   2. Remove three eggs from the incubator, and arrange the eggs in a separate holder with the air sac up.
   3. Mark the air space of the eggs with a permanent marker.
   4. Wash the eggshell above the air space with 70% ethanol.
   5. Put the eggs with the air space up in an egg carton and into a biosafety hood (biosafety level 2 or BSL-2).
   6. Use a sterile 18-G needle to punch a small hole in the shell over the air sac of each egg. Be careful not to insert the needle too deeply to avoid stabbing the embryo or yolk.
   7. Draw up the diluted influenza virus into a sterile 1 ml syringe and attach a 22G needle.
   8. Carefully advance the syringe with the needle at a 45° angle into the allantoic cavity.
   9. Inject 0.2 ml of the diluted influenza virus.
   10. Repeat the injection with the other two eggs, and then discard the syringe and needle.
   11. Seal the hole with a drop of glue from a glue gun (or melted paraffin wax). Be careful that the glue does not leak inside the egg.
   12. Place the eggs back into the egg incubator with the air space pointed upward.
   13. Repeat steps 1.2 – 1.12 until all the eggs have been inoculated.
2. Incubation Time
   1. Incubate the infected eggs without turning at 37 °C and ~60% humidity.
   2. Choose optimal incubation time depending on the influenza virus type. Incubate influenza A for 48 h; incubate influenza B and C for 72 hr.
3. Influenza Virus Harvest
   1. After the incubation period, chill the eggs at 4 °C for a minimum of 4 hr (or overnight) to kill the embryo and constrict the blood vessels to reduce the risk of contaminating the infected allantoic fluid with blood. Do this to prevent the red blood cells from absorbing the virus, which reduces the yield. Alternatively, chill eggs for 30 min at -20 °C; however, this increases the risk of blood contamination.
   2. After the eggs have been sufficiently chilled, transfer the eggs to a biosafety hood. Place the egg into a firm holder with the air sac facing up. Clean the egg surface with 70% ethanol.
   3. Use sterile scissors to open the eggshell above the air sac. Remove the shell around the air sac while being careful not to destroy the chorioallantoic membrane.
   4. Open the chorioallantoic membrane with sterile blunt forceps. Gently move aside the embryo and the yolk sac with a small spatula or spoon. Take care not to rupture the yolk.
   5. Using a 1 ml Eppendorf pipette, carefully collect the allantoic fluid. If needed, tilt the egg a bit to the side to collect as much fluid as possible.
   6. Combine the allantoic fluid from all the eggs into a 50 ml plastic conical tube. Keep tubes on ice at all times during virus harvest. One egg yields 5 – 10 ml of a slightly yellowish fluid.
   7. Spin the virus-containing allantoic fluid at 1,000 x g for 10 min at 4 °C to pellet debris. Transfer the clear fluid into a new 50 ml tube kept on ice. Dispose of eggs into a BSL-2 waste container.
4. Storage
   1. Immediately aliquot the clarified allantoic fluid (e.g., 50, 100 or 500 ul aliquots) and snap-freeze in liquid nitrogen. Transfer frozen virus stocks to -80 °C freezer for long-term storage.

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
Serum pathogen-free (SPF) fertilized chicken eggs	VALO BioMedia
Humidified egg incubator	FARM iNNOVATORS	Model 2100
Automatic egg turner	FARM iNNOVATORS	Model 3200
Egg candler	FARM iNNOVATORS	Model 3300
1 ml Syringe	BD Bioscience	309659
18G Needle	BD Bioscience	305196
Ethanol>99.5%	Sigma-Aldrich	459844	Diluted to 70% using water
Glue gun "Ad tech Hi Temp Project Pro"	Ad tech, Adhesive Technologies, Inc.	Model 1105
Glue "Ad tech MINI SIZE, Multi Temp"	Ad tech, Adhesive Technologies, Inc.	220-34ZIP30