Molecular epidemiology and biology of Kunjin and West Nile viruses

Abstract

Until recently, West Nile (WN) virus and Kunjin (KUN) virus were classified as distinct virus types within the Flavivirus genus. However genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To properly define the relationship between KUN and WN viruses, sequence analyses were performed on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN virus isolate from the recent outbreak in New York. Two independent 500 - 550 nucleotide regions of the genome were sequenced: (i) a region coding for a portion of the envelope protein surrounding the potential glycosylation site at amino acid 154; and (ii) a region encompassing the 3' end of the non-structural (NS) 5 gene and 5' end of the 3' untranslated region (UTR). Sequence comparisons from both regions revealed that the KUN virus isolates from Australia were tightly grouped whereas the WN virus isolates exhibited significant divergence and could be differentiated into three distinct groups. Some WN virus isolates shared greater sequence identity with Australian KUN virus isolates than with other WN virus isolates, while a KUN virus isolate from Sarawak, Malaysia, showed greatest sequence identity with WN virus strains. Virus isolates were also tested for antigenic variation using a panel of seven monoclonal antibodies (Mab) produced to either KUN or WN viruses. The binding patterns of these antibodies in ELISA demonstrated that KUN virus isolates from Australia were antigenically homologous and distinct from the WN virus isolates and the Malaysian KUN virus isolate. Similarly, all WN virus isolates except one displayed a distinct Mab binding profile. The remaining WN virus isolate (Sarafend), the Malaysian KUN virus isolate and Koutango virus also displayed distinct Mab binding patterns. The results in this thesis suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into a number of subgroups on the basis of genetic and antigenic analyses.During the course of this study, viral cultures were identified that contained both a KUN-like virus and a WN-like virus. The observation that the KUN virus population grew more efficiently in a mosquito cell line (C6/36) while the WN virus population replicated more effectively in mammalian cells (Vero) allowed enrichment for either virus by culturing the mixture in the appropriate cell line. A novel strategy was then designed to separate the two virus populations. Limit dilution of the enriched virus preparations was then performed in the appropriate cell line by infecting microtitre cultures with serial ten fold dilutions. Culture wells that contained a pure population of virus were then identified by removing and retaining the culture fluid from each well and immunostaining fixed cell monolayers with virus-specific Mabs. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero cells and analysis of the infected cultures by specific Mab staining, PCR and nucleotide sequencing confirmed the identity of the virus and that in each case an homogenous 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.