A simplified new method for the identification of arboviruses: virus detection using enzyme immunoassay on cultured cells

A simple new method is reported for the identification of arbovirus isolates and the titration of arboviruses. Horseradish peroxidase conjugated Staphylococcus aureus protein A was used for the indirect staining of virus antigens grown in an Aedes albopictus cell line, C6/36 cells. Thirty-five isolates from Culex tritaeniorhynchus mosquitoes were examined by this method and 20 of these were identified as Japanese encephalitis virus (JEV). These results were confirmed by immunofluorescent assay and plaque neutralization test. Comparative titration of JEV, Murray Valley encephalitis (MVE) and Kunjin viruses showed that this method was as sensitive as the chick embryo plaque assay. Specific enzymatic reactions on these virus-infected cells began to appear before day 3 and reached the end-point on day 5 post-infection of C6/36 cells, whereas the cytopathic effect (CPE) appeared about 2 days later than the positive enzymatic reactions. Fixation with 10% formalin for 0.5-8 h did not damage the positive reaction in infected cells and did not increase the background colour of uninfected cells.