Virus isolation in suckling mice • Many viruses will grow satisfactorily in chick embryos or newborn mice but neither is now commonly used because cell culture is generally the simpler option. • Suckling mice less than 24 hours old are injected intracerebrally and/or intraperitoneally, then observed for up to 2 weeks for the development of pathognomonic signs before euthanizing them for examination by various means such as histopathology, immunofluorescence, immunohistochemistry, or serology.
Virus isolation in embryonated eggs • Embryonated hens' eggs are used for the isolation of some avian viruses. • According to the virus of interest, the diagnostic specimen is inoculated in the amniotic cavity, the allantoic cavity, the yolk sac, or on the chorioallantoic membrane. Evidences of viral growth may be seen on the chorioallantoic membrane (e.g., characteristic pocks caused by poxviruses) but otherwise other means are used to detect virus growth (e.g., HA, immunofluorescence).
Egg candling • For propagation of the virus, specific pathogen-free (SPF) eggs are used 11-12 days after fertilisation. The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac. This is marked with a pencil. After all the eggs have been ‘candled’ in this way, a small nick is made in the shell.
Virus inoculation • After all the eggs have been nicked, they are inoculated with virus using a tuberculin syringe – a 1 ml syringe fitted with a 1/2 inch, 27 gauge needle. • The needle passes through the hole in the shell, through the chorioallantoic membrane. The hole in the shell is sealed with melted paraffin, and the eggs are placed at 37 °C for 48 hours
Virus harvesting • 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. • The shell membrane and chorioallantoic membrane are pierced with a pipette which is then used to remove the allantoic fluid – about 10 ml per egg.