Judith H. Edmonds

Characterization of Virulent West Nile Virus Kunjin Strain, Australia, 2011

An encephalitis outbreak among horses was caused by a pathogenic variant of Kunjin virus.

Effect of Wolbachia on replication of West Nile virus in a mosquito cell line and adult mosquitoes.

ABSTRACT Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.

A Novel Bacterium-Free Method for Generation of Flavivirus Infectious DNA by Circular Polymerase Extension Reaction Allows Accurate Recapitulation of Viral Heterogeneity

ABSTRACT A novel bacterium-free approach for rapid assembly of flavivirus infectious cDNAs using circular polymerase extension reaction was applied to generate infectious cDNA for the virulent New South Wales isolate of the Kunjin strain of West Nile virus (KUNV) that recently emerged in Australia. Recovered virus recapitulated the genetic heterogeneity present in the original isolate. The approach was utilized to generate viral mutants with designed phenotypic properties and to identify E protein glycosylation as one of the virulence determinants.

West Nile virus NS2A protein facilitates virus-induced apoptosis independently of interferon response

The flavivirus NS2A protein is a small, multifunctional protein, involved in replication, virion formation and regulation of the innate immune response. Using the Kunjin strain of West Nile virus (WNV(KUN)) we previously demonstrated that a single amino acid change from alanine to proline at position 30 of the NS2A protein (A30P) reduced viral cytopathicity in cells and virulence in mice. To further investigate functions of the NS2A protein we have substituted alanine at position 30 with different amino acids (A30 mutants) in a WNV(KUN) infectious clone. The virulence of mutant viruses in wild-type (WT) and IRF3/IRF7 double-knockout mice was influenced by the amino acid change and ranged from high to low in the order of WT>A30L>A30E>A30P/A30G. Moreover, infection of beta interferon (IFN-β)-deficient Vero cells with A30P virus showed less pronounced chromosomal DNA degradation and lower percentage of cells with positive TUNEL labelling than in WT virus infection, indicating a role for the WT NS2A protein in IFN-independent apoptotic cell death.

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

A variant Australian West Nile virus (WNV) strain, WNVNSW2011, 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 WNVNSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 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 WNVNSW2011, when substituted with that of WNVNY99, 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 WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNVNY99 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 WNVNSW2011 and WNVNY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNVNSW2011 strain to become significantly more virulent.

Comparison of attenuated and virulent West Nile virus strains in human monocyte-derived dendritic cells as a model of initial human infection

BackgroundThe human-pathogenic North American West Nile virus strain (WNVNY99), responsible for the outbreak in New York city in 1999, has caused 41000 infections and 1739 human deaths to date. A new strain of West Nile virus emerged in New South Wales, Australia in 2011 (WNVNSW2011), causing a major encephalitic outbreak in horses with close to 1000 cases and 10-15% mortality. Unexpectedly, no human cases have so far been documented.FindingsWe report here, using human monocyte-derived dendritic cells (MoDCs) as a model of initial WNV infection, that the pathogenic New York 99 WNV strain (WNVNY99) replicated better than WNVNSW2011, indicative of increased viral dissemination and pathogenesis in a natural infection. This was attributed to suppressed viral replication and type I interferon (IFN) response in the early phase of WNVNY99 infection, leading to enhanced viral replication at the later phase of infection. In addition, WNVNY99 induced significantly more pro-inflammatory cytokines in MoDCs compared to WNVNSW2011.ConclusionsOur results suggest that the observed differences in replication and induction of IFN response between WNVNY99 and WNVNSW2011 in MoDCs may be indicative of their difference in virulence for humans.

Virulence and evolution of West Nile Virus, Australia, 1960-2012

Despite the absence of disease in humans and animals, virulent virus strains have been circulating for >30 years.

Successful post-exposure prophylaxis of Ebola infected non-human primates using Ebola glycoprotein-specific equine IgG

Herein we describe production of purified equine IgG obtained from horses immunized with plasmid DNA followed by boosting with Kunjin replicon virus-like particles both encoding a modified Ebola glycoprotein. Administration of the equine IgG over 5 days to cynomolgus macaques infected 24 hours previously with a lethal dose of Ebola virus suppressed viral loads by more than 5 logs and protected animals from mortality. Animals generated their own Ebola glycoprotein-specific IgG responses 9–15 days after infection, with circulating virus undetectable by day 15–17. Such equine IgG may find utility as a post-exposure prophylactic for Ebola infection and provides a low cost, scalable alternative to monoclonal antibodies, with extensive human safety data and WHO-standardized international manufacturing capability available in both high and low income countries.

The in vitro and in vivo antiviral properties of combined monoterpene alcohols against West Nile virus infection

West Nile Virus (WNV) is a mosquito-borne flavivirus that can cause neuroinvasive disease in humans and animals for which no therapies are currently available. We studied an established combination of monoterpene alcohols (CMA) derived from Melaleuca alternifolia, against WNV infection. The in vitro results show that CMA exhibits virucidal activity, as well as reduces the viral titres and percentage of infected cells. The antiviral mechanism of action of CMA was studied. We found that CMA did not alter the intracellular pH, neither induced apoptosis, but did induce cell cycle arrest in the G0/G1-phase although that was not the antiviral mechanism. Furthermore, we tested CMA in vivo using IRF 3(-)(/)(-)/7(-/-)mice and it was found that CMA treatment significantly delayed morbidity due to WNV infection, reduced the loss of body weight and reduced the viral titres in brain. These findings suggest that CMA could be a therapeutic agent against WNV infection.