Cultivation of virus

Influenza virus in eggs

Before the development of cell culture, many viruses were propagated in embryonated chicken eggs. Today this method is most commonly used for growth of influenza virus. The excellent yield of virus from chicken eggs has led to their widespread use in research laboratories and for vaccine production. In fact the vast majority of influenza vaccines – both inactivated and infectious – are produced in chicken eggs. How is influenza virus propagated in eggs?

The illustration below shows a cutaway view of an embryonated chicken egg. The different routes of inoculation into the egg are shown, as well as the different compartments in which viruses replicate.

For propagation of influenza virus, pathogen-free eggs are used 11-12 days after fertilization. The egg is placed in front of a light source to locate a non-veined area of the allantoic hollow just below the air sac. This is marked with a pencil. Next, a hole is drilled at the top of the egg with a Dremel motorized tool.

After all the eggs have been nicked and drilled, they are inoculated with virus using a tuberculin syringe. The needle passes through the hole in the shell, through the chorioallantoic membrane, and the virus is placed in the allantoic cavity, which is filled with allantoic fluid. The two holes in the shell are sealed with melted paraffin, and the eggs are placed at 37 degrees C for 48 hours.
During the incubation period, the virus replicates in the cells that make up the chorioallantoic membrane. As new virus particles are produced by budding, they are released into the allantoic fluid.

To harvest the virus, the top of the egg shell – the part covering the air sac – is removed. We used to have a special tool to do this, which was placed over the egg. When the handle of this tool is squeezed, it makes a neat crack around the top of the egg. It was then easy to remove the flap of shell with tweezers. The shell membrane and chorioallantoic membrane are pierced with a pipette which is then used to remove the allantoic fluid.

Cultivation of plant virus

The leaves of the plant are mechanically inoculated by rubbing with a mixture of viruses and an abrasive. The virus would directly intact the plasma membrane and infects the exposed host cells when the cell walls are broken by the abrasive. The role of the abrasive is often done by insects that suck of crush plant leaves and thus transmit viruses. Due to rapid death of cells in the infected area, a localized necrotic lesion would develop and even if lesions does not occurs, the infected plant may show symptoms like change in pigmentation, etc. A picture of a plant infected with tobacco mosaic virus is shown