Setoh, Y.X.Prow, N.A.Rawle, D.J.Tan, C.S.E.Edmonds, J.H.Hall, R.A.Khromykh, A.A.2025-12-182025-12-182015Journal of General Virology, 2015; 96(6):1297-13080022-13171465-2099https://hdl.handle.net/11541.2/137964Data source: supplementary tables, https://doi.org/10.1099/vir.0.000069A variant Australian West Nile virus (WNV) strain, WNV<inf>NSW2011</inf>, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However, no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNV<inf>NSW2011</inf> was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNV<inf>NY99</inf>). To identify viral genes and mutations responsible for the difference in virulence between WNV<inf>NSW2011</inf> and WNV<inf>NY99</inf> strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes and untranslated regions, as well as seven individual non-structural gene chimeras, using a modified circular polymerase extension cloning method. Our results showed that the complete non-structural region of WNV<inf>NSW2011</inf>, when substituted with that of WNV<inf>NY99</inf>, significantly enhanced viral replication and the ability to suppress type I IFN response in cells, resulting in higher virulence in mice. Analysis of the individual non-structural gene chimeras showed a predominant contribution of WNV<inf>NY99</inf> NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNV<inf>NY99</inf> non-structural proteins (NS2A, NS4B and NS5) were shown to contribute to the modulation of IFN response. Thus a combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNV<inf>NSW2011</inf> and WNV<inf>NY99</inf> strains, and accumulative mutations within these proteins would likely be required for the Australian WNV<inf>NSW2011</inf> strain to become significantly more virulent.enCopyright 2015 The Authors. This article is open access and allows authors and their funding bodies to make published articles freely available online from the time of publication, upon payment of an Article Processing Charge (APC) (https://jgv.microbiologyresearch.org/about/open-access-policy)Australian West Nile virusWest Nile virusJournal article10.1099/vir.0.0000692-s2.0-84929386184