1 Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, St Lucia, Queensland 4072, Australia
2 Arbovirus Diseases Branch, Division of Vector-borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80522, USA
3 Tropical Public Health Unit, Cairns, Queensland 4870, Australia
4 School of Public Health and Tropical Medicine, James Cook University, PO Box 6811, Cairns, Queensland 4870, Australia
Correspondence
Roy A. Hall
roy.hall{at}uq.edu.au
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are available as supplementary material in JGV Online.
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MAIN TEXT |
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KOKV and the closely related Stratford virus (STRV) were originally classified as separate species in the Japanese encephalitis virus (JEV) complex within the genus Flavivirus. Important members of this complex include JEV, Murray Valley encephalitis virus (MVEV), West Nile virus and Saint Louis encephalitis virus (Mackenzie et al., 2002). However, recent studies have indicated that although KOKV and STRV are related closely to each other, they are antigenically and genetically distinct from the other viruses in the JEV group (Hall et al., 1991
; Poidinger et al., 1996
; Kuno et al., 1998
). This has led to the reclassification of KOKV into a separate virus complex within the genus, with STRV listed as a subtype of KOKV (Heinz et al., 2000
). This paper describes the characterization of two novel flaviviruses in the Kokobera virus complex and examines their phylogenetic relationship with other viruses within the genus Flavivirus.
Virus isolates were obtained from mosquitoes collected in 1998 on Cape York Peninsula and in 2000 on Saibai Island, as described previously (van den Hurk et al., 2001; Johansen et al., 2003
, 2004
). Virus stocks were prepared by inoculating confluent monolayers of PS-EK cells (Gorman et al., 1975
) and harvesting culture supernatants when at least 70 % of the cells exhibited cytopathic effect.
Six isolates were initially identified as KOKV based on their reaction with a panel of monoclonal antibodies (mAbs) in an ELISA (Broom et al., 1998). Further investigations with a panel of non-neutralizing mAbs prepared to the E and NS1 proteins of KOKV isolate MRM32 (Hall et al., 1991
) revealed that four isolates showed an identical mAb-binding profile to that of KOKV MRM32 (Table 1
). However, one isolate (TS5273) was recognized by only five of the seven mAbs, showing a similar binding pattern to that reported previously for a genetically divergent KOKV isolate from PNG (MK7979) (Poidinger et al., 2000
). A second isolate (CY1014) and the prototype STRV strain (C338) also showed distinct binding patterns, the former being recognized by only three of the seven mAbs and the latter by only two (Table 1
).
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To determine their relationship to other flaviviruses, CY1014 and TS5273 were also sequenced by using the primer pair cFD3 and FU1 (Kuno et al., 1998) or a degenerate version of these primers (cFD3PM and FU1PM). These primers amplify approximately 1 kb of the NS5 gene, using the same conditions as described above. Multiple sequence alignments of a 677 nt sequence (corresponding to nt 93129988 of the KUNV genome; Coia et al., 1988
) of CY1014, TS5273 and previously published nucleotide sequences of this region from a range of flaviviruses (Kuno et al., 1998
) were performed by using CLUSTAL W. This analysis confirmed that CY1014 was related most closely to KOKV, but shared only 69 % similarity with this virus and 67 % with STRV. In contrast to the sequence alignments obtained by using the EMF/VD8 primers, nucleotide sequence from the cFD3 and FU1 region of NS5 revealed that TS5273 had a slightly closer relationship to KOKV (76 % nucleotide similarity) than to STRV (74 % nucleotide similarity). However, TS5273 showed a closer relationship to STRV in the deduced amino acid sequence of this region (94 %) than to KOKV (91 %).
Phylogenetic analysis was then performed on multiple sequence alignments of nucleotide sequence obtained from the cFD3 and FU1 region by using the PHYLIP package (Felsenstein, 1989) and a phylogram was created by using the neighbour-joining method (Fig. 1
). Confidence values were placed on branches by using bootstrap analysis of 100 replicates. The tree reveals that CY1014 branches with KOKV, STRV and TS5273, but is the most evolutionary distant member of the group. TS5273, on the other hand, clusters with STRV, but the bootstrap value is very low. This reflects the equivocal data that were obtained from both antigenic studies and nucleotide sequencing, some of which suggest a closer relationship between TS5273 and STRV (cross-neutralization, EMF/VD8 nucleotide sequence and cFD3 and FU1 amino acid sequence) and some indicate higher similarity of TS5273 to KOKV (mAb binding and cFD3 and FU1 nucleotide sequence). In summary, it can be concluded that TS5273 is closely related to, but distinct from, both KOKV and its subtype STRV.
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The isolation of two novel viruses from northern Australia was unexpected, given the considerable effort expended on surveillance for mosquito-borne viruses throughout Australia over many years. Whether they were always present, perhaps in cryptic foci or in areas or niches that were not investigated previously, or whether they were introduced, is unknown.
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ACKNOWLEDGEMENTS |
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Received 19 June 2004;
accepted 9 October 2004.
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