Isolation of homologous arbovirus cultures from heterologous mixtures using limit dilution and virus-specific enzyme immunoassays.

Viral cultures were identified recently that contained both Kunjin virus and the closely related flavivirus West Nile. The observation that the KUN virus population grew more efficiently in a mosquito cell line (C6/36) while the WN population replicated more effectively in mammalian cells (Vero) allowed enrichment for either virus by culturing the mixture in the appropriate cell line. Limit dilution of the enriched virus preparations was then performed by infecting microtitre cultures with serial ten fold dilutions. Culture wells that contained a pure population of virus were then identified by immunostaining fixed cell monolayers with virus-specific monoclonal antibodies. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero cells and analysis of the infected cultures by specific monoclonal antibody staining, PCR and nucleotide sequencing confirmed the identity of the virus and that in each case an homogeneous virus population had been obtained. This procedure is particularly useful for isolating virus populations from heterogeneous mixtures that fail to develop discrete plaques in infected cell monolayers.