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 an d the closely related flavivirus West Nile. The observation that the KUN vi rus population grew more efficiently in a mosquito cell line (C6/36) while the WN population replicated more effectively in mammalian cells (Vero) all owed enrichment for either virus by culturing the mixture in the appropriat e cell line. Limit dilution of the enriched virus preparations was then per formed by infecting microtitre cultures with serial ten fold dilutions. Cul ture wells that contained a pure population of virus were then identified b y immunostaining fixed cell monolayers with virus-specific monoclonal antib odies. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero c ells 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. T his procedure is particularly useful for isolating virus populations from h eterogeneous mixtures that fail to develop discrete plaques in infected cel l monolayers. (C) 1999 Elsevier Science B.V. All rights reserved.