For example, in Australia and Papua New Guinea, the antigenically closely related virus of Murray Valley encephalitis occurs. Mix one volume of each diluted serum with an equal volume of diluted virus. Include virus control with culture medium, negative serum control and positive serum control in each plate. Wear rubber gloves when handling the fixing solution. Discard the stain and rinse the cells with tap water.
Air dry the cells and count the plaques.
The HI test is widely used for the diagnosis of Japanese encephalitis, but has cross-reactivity with other Flaviviruses. For this test, the sera must first be treated with acetone or kaolin, and then absorbed with homotypic RBCs to remove any nonspecific haemagglutinins. Extract and weigh the brains of the inoculated dead mice. Add to the pellet the same volume of cold acetone as used in step ii above, and mix well. Repeat steps iv and v twice using cold ether.