- Short report
- Open Access
Comparison of attenuated and virulent West Nile virus strains in human monocyte-derived dendritic cells as a model of initial human infection
© Rawle et al.; licensee BioMed Central. 2015
- Received: 22 January 2015
- Accepted: 12 March 2015
- Published: 22 March 2015
The 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.
We 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.
Our 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.
- West Nile virus
- Dendritic cells
West Nile virus (WNV) is the leading cause of arboviral encephalitis in the Americas with over 41000 infections and 1739 human deaths (http://www.cdc.gov/westnile/index.html, accessed 25/11/2014) since the outbreak of a human virulent strain in New York in 1999 (WNVNY99 strain) [1,2]. There is currently no effective treatment or approved WNV vaccine for use in humans. A strain of WNV called Kunjin (WNVKUN) has been circulating in Australia since it was discovered in 1960, and have caused very few symptomatic infections in humans or horses . This was until a new strain of WNV emerged in New South Wales in 2011 (WNVNSW2011) causing a major encephalitic outbreak in over 1000 horses [4,5]. WNVNSW2011 has likely emerged through mutations of previously circulating WNVKUN, and gained at least two known virulence determinants found in the human-pathogenic WNVNY99 strain ; i) the N-linked glycosylation on the E protein associated with increased virulence in mice , and ii) the phenylalanine residue at position 653 of NS5 which is a potent inhibitor of STAT1 phosphorylation .
The horses affected by the WNVNSW2011 outbreak presented with similar clinical symptoms to horses infected with WNVNY99, therefore it was rather unexpected that the WNVNSW2011 outbreak had no reported human cases. This prompted us to investigate the viral growth kinetics and immune induction profiles of WNVNSW2011 compared to WNVNY99 using cultures of primary human dendritic cells (DCs) as a model of initial infection in humans [8-11]. Human MoDCs were used as an ex vivo model of initial WNV infection in this study, because it has been shown that large numbers of bone marrow monocytes differentiate into DCs soon after WNV infection in the dermis . Human MoDCs have been previously used to show that WNV replication was required for type I IFN induction, and this was a result of IRF3 translocation to the nucleus after dsRNA stimulation of RIG-1, MDA5 or TLR3 . While comparison of WNV strains has been previously performed in DCs to show that WNV strains with glycosylated envelope (E) protein have increased infection and replication rates , WNVNY99 and WNVNSW2011 both have glycosylated E , and are therefore expected to be equally efficient in viral entry into MoDCs through attachment to DC-SIGN or DC-SIGNR receptors . Therefore, additional differences must exist that result in either productive infection of DCs or effective viral clearance by the innate immune response.
To investigate this, we used MoDCs by isolating peripheral blood mononuclear cells (PBMCs) from human buffy coat from three donors, which were tested to be negative for arboviral infections, then performed CD14+ magnetic selection to isolate monocytes. The isolated monocytes were cultured with GM-CSF and IL-4 for 6 days to differentiate into immature MoDCs, which were then matured upon WNV infection represented by increased CD80 and CD86 expression. Briefly, at 48 hours post infection (hpi), 78.7% of WNVNY99-infected MoDCs and 84.5% of WNVNSW2011-infected MoDCs showed CD80 upregulation, and 93.9% of WNVNY99-infected MoDCs, 95.8% of WNVNSW2011-infected MoDCs showed CD86 upregulation.
The upregulation of TNFα and IL-6 is likely to occur via viral dsRNA activation of TLR3 [9,17,18]. Studies in vivo have shown that TLR3 knockout mice were more resistant against WNV encephalitis, and this was linked to decreased TNFα and IL-6 expression . The proposed mechanism of this is that TNFα and IL-6 secreted into the bloodstream from infected leukocytes may induce endocytosis and degradation of tight junction proteins (Claudin-1, JAM-1 and occludin) of the blood brain barrier (BBB), causing breakdown of the BBB and allowing WNV entry into the brain [18-22].
Our finding that WNVNY99, but not WNVNSW2011, suppresses early viral RNA replication in MoDCs is supported by previous reports. Scherbik et al.  showed that the pathogenic lineage 1 strain of WNV (Eg101), but not the non-pathogenic lineage 2 strain (W956IC), suppressed early viral RNA replication (but increased viral protein expression), resulting in reduced host innate immune responses at the early phase of infection. This effect of early IFN suppression was also shown for pathogenic vs. non-pathogenic Tick Borne Encephalitis virus (TBEV) strains . These two studies suggest that pathogenic viral strains more effectively suppress early viral RNA replication but increase translation, which has three outcomes that favour virulence; i) decreasing the amount of viral dsRNA to decrease IFN induction, ii) increased viral non-structural protein production to block the JAK/STAT signalling cascade, and iii) more effective viral protein-dependent remodelling of cellular membranes which house replication complexes [23,25].
Taken together, our results have identified that a possible factor leading to higher human virulence of WNVNY99 is likely to be decreased viral RNA replication and lower induction of IFNβ, OAS1 and MxA early in MoDCs infection. This likely facilitates the enhanced replication of WNVNY99 vRNA and dissemination of particles later in infection.
We acknowledge the help of the Australian Red Cross Blood Service for providing buffy coat, and Jesse Fyrk (Australian Red Cross Blood Service Research Assistant) for organising and distributing the buffy coat on request. This work was supported by the National Health and Medical Research Council of Australia (APP1045188).
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