Kunjin (KUN) is a flavivirus in the Japanese encephalitis antigenic complex that was first isolated from Culex annulirostris mosquitoes captured in northern Australia in 1960. It is the etiological agent of a human disease characterized by febrile illness with rash or mild encephalitis and, occasionally, of a neurological disease in horses. KUN virus shares a similar epidemiology and ecology with the closely related Murray Valley encephalitis (MVE) virus, the major causative agent of arboviral encephalitis in Australia. Based on traditional antigenic methods, KUN was initially found to be similar to, but distinct from, reference strains of West Nile (WN) virus and designated as a new species. However, more recent phylogenic analyses have revealed that some strains of WN virus, including the isolates from New York, are more similar to KUN virus and form a separate lineage to other WN viruses. An unusual KUN isolate from Malaysia and the African virus Koutango appear to form additional lineages within the WN group of viruses. While these findings are in agreement with the Seventh Report of the International Committee for the Taxonomy of Viruses that designates KUN as a subtype of West Nile, they also suggest that the species should be further subdivided into additional subtypes.
Japanese encephalitis virus (JEV) and West Nile virus (WNV) provide some of the most important examples of emerging zoonotic viral encephalitides. For these flaviviruses, only a small proportion of those infected develop clinical features, and these may range from a non-specific flu-like illness to a severe fatal meningoencephalitis, often with Parkinsonian features, or a poliomyelitis-like flaccid paralysis. The factors governing the clinical presentations, and outcome of flavivirus infections are poorly understood, but studies have looked at viral virulence determinants and the host immune response. Previous studies on JEV have suggested that the distribution of the four genotypes across Asia may relate to the differing clinical epidemiology (epidemic disease in the north, endemic disease in the south). However, new data based on the complete nucleotide sequence of a virus representing one of the oldest lineages, and phylogenetic analyses of all JEV strains for which genetic data are available, suggest that the distribution is best explained in terms of the virus' origin in the Indonesia-Malaysia region (where all genotypes have been found), and the spread of the more recent genotypes to new geographical areas. Clinical studies have shown that innate immunity, as manifested by interferon alpha levels, is important in JEV and other flaviviruses, but treatment with interferon alpha did not improve the outcome. A failure of the humoral immune response, is associated with death from encephalitis caused by JEV and WNV. Cellular immunity has been less well characterized, but CD8+ and CD4+ T cells are thought to be important.
Rodent models have been used extensively to study West Nile virus (WNV) infection because they develop severe neurological symptoms similar to those observed in human WNV neuroinvasive disease. Most of this research has focused on old lineage (L) 1 strains, while information about pathogenicity is lacking for the most recent L1 and L2 strains, as well as for newly defined lineages. In this study, 4-week-old Swiss mice were inoculated with a collection of 12 WNV isolates, comprising 10 old and recent L1 and L2 strains, the putative L6 strain from Malaysia and the proposed L7 strain Koutango (KOU). The intraperitoneal inoculation of 10-fold dilutions of each strain allowed the characterization of the isolates in terms of LD50, median survival times, ID50, replication in neural and extraneural tissues and antibody production. Based on these results, we classified the isolates in three groups: high virulence (all L1a strains, recent L2 strains and KOU), moderate virulence (B956 strain) and low virulence (Kunjin and Malaysian isolates). We determined that the inoculation of a single dose of 1000 p.f.u. would be sufficient to classify WNV strains by pathotype. We confirmed the enhanced virulence of the KOU strain with a high capacity to cause rapid systemic infection. We also corroborated that differences in pathogenicity among strains do not correlate with phylogenetic lineage or geographic origin, and confirmed that recent European and African WNV strains belonging to L1 and L2 are highly virulent and do not differ in their pathotype profile compared to the prototype NY99 strain.
Defective interfering (DI) particles of the flavivirus West Nile (WN) were generated after as few as two high multiplicity serial passages in Vero and LLC-MK2 cells. Six cell lines (Vero, LLC-MK2, L929, HeLa, BHK-21 and SW13) were used to assay interference by DI particles in a yield reduction assay. Interference was found to vary depending on the cell type used. The highest levels of interference were obtained in LLC-MK2 cells, whereas no detectable effect was observed in BHK-21 and SW13 cells. The ability of DI virus to be propagated varied depending on the cell line used; no detectable propagation of DI virus was observed in SW13 cells. Optimum interference was obtained following co-infection of cells with DI virus and standard virus at a multiplicity of 5. Interference between DI and standard viruses occurred only when they were co-infected or when cells were infected with DI virus 1 h before standard virus. Investigation of heterotypic interference by DI particles of WN virus strains from Sarawak, India and Egypt revealed that interference was dependent on the strain of WN virus or flavivirus used as standard virus. A measure of the similarity between five strains of WN virus and other flaviviruses was made on the basis of interference by DI viruses, and was found to be similar to that based on haemagglutination inhibition tests using a panel of monoclonal antibodies.
Until recently, West Nile (WN) and Kunjin (KUN) viruses were classified as distinct types in the Flavivirus genus. However, genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To better define this relationship, we performed sequence analyses on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN isolate from the recent outbreak in New York. Sequence comparisons showed that the KUN virus isolates from Australia were tightly grouped but that the WN virus isolates exhibited substantial divergence and could be differentiated into four distinct groups. KUN virus isolates from Australia were antigenically homologous and distinct from the WN isolates and a Malaysian KUN virus. Our results suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into subgroups on the basis of genetic and antigenic analyses.