Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains

Abstract

A variant Australian West Nile virus (WNV) strain, WNV NSW2011 , 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 NSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNV NY99 ). To identify viral genes and mutations responsible for the difference in virulence between WNV NSW2011 and WNV NY99 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 NSW2011 , when substituted with that of WNV NY99 , 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 NY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNV NY99 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 NSW2011 and WNV NY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNV NSW2011 strain to become significantly more virulent.

Development of a human live attenuated West Nile infectious DNA vaccine

“…The identified unique amino acid differences, which likely contribute to the highly attenuated profile of W956, are distinct from previously described mutations in flavivirus nonstructural proteins that affect the virus viability or virus replication properties in general. While conclusions drawn on the basis of comparative sequence analysis await experimental confirmation, for example by examining biological properties of chimeric WN viruses Setoh et al, 2015), the provided evidence strengthens the support for using attenuated W956 as a vector in the design of a live chimeric WN vaccine.…”

“…Yet with the exception of changes in NS1 and NS2A, the unique to W956 mutations in NS4A, NS4B and NS5 result in replacement of smaller amino acids with bulkier residues, implying that increased steric hindrance in protein-protein interaction may contribute to the attenuated profile of W956. Reported evidence indicates that several nonstructural proteins of NY99 are involved in inhibition of IFN signaling pathways (Guo et al, 2005;Keller et al, 2006;Liu et al, 2005;Munoz-Jordan et al, 2005;Samuel and Diamond, 2005;Scholle and Mason, 2005;Setoh et al, 2015). These aspects of virus-host interaction may be affected by the above mutations yielding much less pathogenic W956.…”

“…The role of this change in attenuation is uncertain. The NY99 virulence and cytopathicity are associated with its nonstructural protein region of its genome Setoh et al, 2015). W956 also has no unique amino acid changes in NS2B and NS3, which could affect function of the viral protease.…”

Chimeric viruses between Rocio and West Nile: the role for Rocio prM-E proteins in virulence and inhibition of interferon-α/β signaling

“…1a). Equimolar amounts of the 7 RT-PCR fragments and a flavi-UTR-linker fragment containing CMV promoter, conserved first and last 22 nucleotides of viral sequence and hepatitis delta virus ribozyme26, were used in CPEC reaction (Fig. 1a, Supplementary Table S1).…”

“…Recently, we have described a simple and efficient method for generation of WNV infectious cDNAs, termed circular polymerase extension cloning (CPEC), and used it to produce chimeric viruses between attenuated Australian and virulent American WNV strains in order to identify viral genetic determinants of virulence262728. In the current study, we employed the CPEC method to generate infectious cDNA of ROCV and reciprocal prM-E chimeric viruses between ROCV and Australian strain of WNV.…”