Characterization of virulent West Nile virus Kunjin strain, Australia, 2011

Abstract

To determine the cause of an unprecedented outbreak of encephalitis among horses in New South Wales, Australia, in 2011, we performed genomic sequencing of viruses isolated from affected horses and mosquitoes. Results showed that most of the cases were caused by a variant West Nile virus (WNV) strain, WNV(NSW2011), that is most closely related to WNV Kunjin (WNV(KUN)), the indigenous WNV strain in Australia. Studies in mouse models for WNV pathogenesis showed that WNV(NSW2011) is substantially more neuroinvasive than the prototype WNV(KUN) strain. In WNV(NSW2011), this apparent increase in virulence over that of the prototype strain correlated with at least 2 known markers of WNV virulence that are not found in WNV(KUN). Additional studies are needed to determine the relationship of the WNV(NSW2011) strain to currently and previously circulating WNV(KUN) strains and to confirm the cause of the increased virulence of this emerging WNV strain.

Figure 1

Known distribution of West Nile virus infection and disease caused by Kunjin strain (A) and distribution of encephalitis cases among equids (B), New South Wales, Australia, 2011. Dashed line indicates the Great Dividing Range.

Figure 2

Maximum-likelihood tree based on nucleotide sequences of the complete open reading frame of genomes of West Nile virus (WNV) NSW2011 (boldface) and representative strains of WNV from the different lineages and clades. All published complete Kunjin (KUN) virus sequences are included. Bootstrap values are shown on the nodes and are expressed as a percentage of 1,000 replicates. Sequences downloaded from GenBank were WNV_{Russia88–90}, AY277251; WNV_Rabensburg, AY765264; WNV_Sarafend, AY688948; WNV_Uganda, AY532665; WNV_India, DQ256376; WNV_NY99, AF196835; WNV₂₀₀₂, GU827998; WNV_KUNV-MRM16, GQ851602; WNV_KUNV-MRM61C, AY274504; and WNV_KUNV-K6453, GQ851603. NY, New York; NSW, New South Wales. Horizontal branch lengths indicate genetic distance proportional to the scale bar.

Figure 3

Studies of West Nile virus (WNV) properties in cell cultures and mice. A) Plaque morphology of WNV_NY99, prototype WNV_KUN, and WNV_NSW2011 in Vero cells. Cells in 6-well plates were infected with specified virus and overlaid with 0.75% low melting point agarose in Dulbecco modified minimum essential medium (Life Technologies, Carlsbad, CA, USA) containing 2% fetal bovine serum. Four days after infection, the cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet. B) Assessment of envelope (E) protein glycosylation of WNV_NSW2011, WNV_KUN and WNV_NY99 by endoglycosidase digestion (PNGase F; Roche Diagnostics, Basel, Switzerland). Viral proteins in culture supernatant were digested by PNGase F (+) or undigested (−) and then resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The migration rate of the E protein in each sample was determined by Western blot with E glycoprotein–specific monoclonal antibodies. C) Young adult (4 weeks old) or D) weanling (18–19 days old) Swiss outbred mice survival after intraperitoneal injection with 1,000 PFU (adult) or 10 PFU (weanling) of WNV_NY99, WNV_KUN, or WNV_NSW2011. The mice were monitored for 21 days after injection for signs of encephalitis and then euthanized. The differences in virulence in weanling and adult mice between different pairs of viruses were all highly significant, as calculated by log rank Mantel-Cox algorithm with exact p values: for adult mice, WNV_NY99 vs. WNV_KUN p<0.0001, WNV_NY99 vs. WNV_NSW2011 p = 0.0001, and WNV_KUN vs. WNV_NSW2011 p = 0.0012; and for weanling mice, WNV_NY99 vs. WNV_KUN p<0.0001, WNV_NY99 vs. WNV_NSW2011 p =0.0004, and WNV_KUN vs. WNV_NSW2011 p = 0.0006. NY, New York; KUN, Kunjin; NSW, New South Wales.