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West Nile virus (WNV) is a flavivirus of the family Flaviviridae endemic throughout Asia, Europe, Africa and Australia. A West Nile viral particle is spherical and measures approximately 50nm in diameter. Its genome consists of a single-stranded, positive-sense ribonucleic acid (RNA) that codes for 10 mature viral proteins: 3 structural and 7 non-structural proteins. The RNA is enclosed within an icosahedral capsid, which in turn, is surrounded by a host-derived lipid bilayer.
Most human hosts (80%) remain asymptomatic upon infection with WNV. The other 20% may experience self-limited febrile illness referred to as West Nile fever. West Nile fever manifests itself as a series of symptoms that mimic the flu, including body aches, chills and fatigue. Occasional gastrointestinal discomfort may also be present in some cases. Altogether, West Nile fever should resolve within 1-2 weeks. In rare cases (<1%), WNV can result in CNS disorders including meningitis, encephalitis and poliomyelitis; they are collectively known as West Nile neuroinvasive diseases. Radiographic images have revealed differences in the extent of WNV invasion within the CNS (1). Specifically, WNV appears to affect gray matter more than white matter, and inflammation in the brainstem is typically more severe than in the cortex or cerebellum.
WNV is presently the most widely disseminated arbovirus in the world, penetrating all continents except for Antarctica (2). The virus was first isolated in a febrile woman in Uganda back in the early 1930s. Since then, sporadic outbreaks were identified in distinct regions of the world and by the 1990s, epidemics began to surface more frequently. Between 1996 and 1999, three large epidemics took place in southeastern Romania, Russia and northeastern United States. Many WNV-positive individuals were diagnosed with severe neurological diseases and fatal infections. The introduction of WNV to New York in 1999 began the cascade of WNV outbreaks in the rest of North and South America, including Mexico and Canada.
In the temperate and subtropical regions, most WNV infections arise in the summer or early autumn. The tropics are usually burdened with WNV infections during the rainy seasons, when mosquitoes are highly abundant. While the incidence of WNV appears to have preference for neither age nor gender, the susceptibility to encephalitis and death due to WNV seems to increase with age, presumably as a consequence of declining immune function.
With the apparent persistence of WNV in many regions of the world, much effort is being put into trying to elucidate the molecular epidemiology of WNV to better understand its transmission dynamics, viral determinants of pathogenesis and propagation. There are presently two identified lineages of WNV: 1) Lineage 1 that is distributed across the world and 2) Lineage 2 that is fairly restricted to sub-Saharan Africa. The first strain of WNV isolated during the New York epidemic in 1999 resembled one that was previously identified in Israel the year before and strains collected in 2002 during a Texas outbreak was also akin (2). It was concluded that the WNV was genetically static. However, in 2003, a novel dominant strain of WNV was discovered that entirely replaced the original strain introduced to the United States in 1999. Its potency is hypothesized to be a result of a heightened transmission efficiency of the Culex spp. mosquitoes.
Mode of Transmission
The first West Nile outbreak in North America occurred in 1999, during which a similar epidemic was observed in Russia (1). Common to the two events was the employment of mosquitoes as vectors. Species of mosquitoes involved in the transmission cycle of WNV are specifically referred to as amplification vectors; they are ornitophilic, feeding primarily on avian blood (3). These amplification vectors are mainly of the Culex species and their efficiency in disease transmission largely depends on external factors such as rainfall and temperature. Other mosquitoes participate in the propagation of WNV to mammalian hosts; they are known as bridging vectors and are capable of transmitting the virus after feeding on virally-infected birds. To be deemed a significant contributor to the transmission cycle of WNV, a species of mosquito must: 1) be able to infect and transmit WNV after feeding with infectious blood meal under controlled conditions, 2) be found in abundance in the field, and 3) have WNV that can be isolated. Figure 1 shows a simplified overview of the transmission cycle of WNV. The primary enzootic cycle between birds and mosquitoes (Figure 1) maintains the WNV in nature and may be enhanced through bird-to-bird transmission.
Song birds are generally considered the principal reservoir host for WNV, with several other species identified, while pigeons and woodpeckers do not generate viraemias potent enough to infect mosquitoes. Certain mammals, such as eastern hamsters, chipmunks and fox squirrels, have been shown to produce viraemias of up to 107.8 pfu/mL, which is high enough to successfully infect birds (3). These experimental findings, however, have not yet been demonstrated in nature. Other studies have detected the presence of WNV antibodies in farmed crocodiles, lake frogs and other reptiles and amphibians, but their roles in the transmission cycle of the virus remain to be elucidated.
While the transmission of WNV to vertebrates primarily occurs through anthropod vectors, non-vector-borne modes of transmission have been observed especially between human hosts. This occurs through organ transplantation, breast feeding, intrauterine transmission, and blood transfusions. In alligators, additional modes of transmission include eating infected horse meat, or simply the sharing of tanks with infected mates. Taken altogether, it appears that the transmission of WNV need not depend solely on vectors (3).
Involvement of the Immune System
According to several mouse models, subcutaneous inoculation of WNV during mosquito feeding leads to the recruitment of Langerhans dendritic cells (DCs). The first round of viral replication is believed to occur within these DCs, before they migrate to the lymph nodes. The arrival of WNV in the lymph nodes then leads to an early immune response, which often involves the production of interferons (IFNs). Here, a second round of viral replication takes place in cells that are not yet definitively identified, but popular candidates include macrophages and follicular DCs (4). The infectious WNV then exits through the efferent lymphatic system and thoracic duct to enter the systemic circulation.
West Nile Virus & Innate Immunity
The innate immune system recognizes WNV using several detection mechanisms, including Toll-like receptors (TLRs) and cytoplasmic double-stranded RNA (dsRNA) sensors, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Binding of WNV to these pattern recognition molecules (PRMs) initiate immune cascades that involve the production of IFNs through the stimulation of IFN regulatory factors (IRFs). Type I IFNs triggers the activation of the Janus kinase (JAK)/STAT pathway and IFN-stimulated genes (ISGs), which results in an increase of IFN-Î² production, inhibition of viral translation and degradation of viral and cellular RNAs to prevent viral propagation (5).
The complement system also participates in WNV recognition and clearance. Its activation occurs via three different pathways: 1) classical, initiated by the binding of C1q, 2) lectin, initiated by mannose-binding lectins, and 3) alternative, initiated by the spontaneous hydrolysis of C3, the main complement component. Activation of the complement system contributes to survival by opsonisation, chemotaxis and modulation of the adaptive immune function. Curiously, the above three pathways control WNV infection through different mechanisms. For instance, alternative-pathway-deficient mice presented with normal humoral immune response but defected CD8+ T-cell responses, while classical- and lectin-pathway-deficient mice showed impairments in both cellular-mediated and humoral adaptive immune responses (5). Further analyses are required to explain the reasoning behind these observations.
The Î³Î´ T cells, a subset of T cells hypothesized to bridge the innate and adaptive immune responses, are thought to play a role in the clearance of WNV infection by way of their association with IFN-Î³. IFN-Î³ is produced by natural killer (NK) cells, Î³Î´ and CD8+ T cells to directly inhibit viral activity, enhance T helper cell responses and recruit phagocytes. Studies showed that mice deficient in Î³Î´ T cells had higher death rates and attenuated adaptive immune responses (5).
West Nile Virus & Adaptive Immunity
The importance of the adaptive immune system in controlling WNV infections was demonstrated using animal models. For instance, mice that lacked T-lymphocytes and those that suffer from Severe Combined Immunodeficiency Syndrome (SCID) succumbed to WNV infection (5). In accordance with these findings, mice under immunosuppressive drugs and younger rodents with immature adaptive immune systems failed to clear WNV infection. Recent studies have suggested that humoral immunity is largely responsible for eliminating WNV in the periphery, while T lymphocytes appear to be more critical in the clearing of WNV infection within the central nervous system (CNS).
T-cell mediated Immune Response
Although the mechanisms by which WNV successfully enters the brain is still unknown, studies suggest that WNV may cross the blood-brain barrier (BBB) through a hematogenous route (5), taking advantage of TNF-Î± or MMP-9-induced changes in permeability. In a pivotal paper, Wang et al. (6) showed that while immunosuppressed mice, specifically TLR-3-deficient, had impaired cytokine production and greater viral load during early infection, they had a higher survival rate than wild-type mice. This was attributed to the lack of viral penetration into the CNS in TLR-3 deficient mice. Thus, the findings of this particular study support the hypothesis presented above. Other proposed methods of CNS invasion include retrograde axonal spread through peripheral nerves, active replication in endothelial cells, or a "Trojan horse" phenomenon in which viral particles are transported into the brain within infected inflammatory cells (4).
Once in the CNS, WNV induces expression of CXCL10, a T cell chemoattractant, by infected neurons. Gene targeted deletion of CXCL10 resulted in a decreased recruitment of WNV-specific CD8+ T cells into the CNS, which led to increased mortality (5). As previously mentioned, CD8+ T cells produce IFN-Î³ that aids in the inhibition of viral activity and are important in the clearance of WNV in the CNS. CD8+ T cells, which are cytotoxic T lymphocytes, are able to directly kill virally-infected cells by releasing perforins and granzymes.
CD4+ T cells are essential in the maturation of IgG immune response (5) and are also required for fighting against WNV in the CNS. Mice deficient in these helper T cells, however, do not exhibit differences in the kinetics of WNV within the periphery (8). This suggests that peripheral immune responses are independent of direct intervention by helper T cells. Helper T cells may, on the other hand, enhance humoral immune responses and cytotoxic T cell activities in the periphery.
Humoral Immune Response
The WNV virions consist of 2 transmembrane proteins: a membrane (M) and an envelope (E). The E protein covers the surface of the viral particle and is composed of 3 domains: DI, DII, and DIII. The most potent WNV-neutralizing antibodies recognize epitopes in the DIII of the E protein. Upon activation of epidermal DCs by WNV, early induction of the adaptive immune system produces IgM to clear the virus from the bloodstream. The expression of IgG coincides with the clearance of WNV in infected cells.
Other anti-WNV neutralizing antibodies may also recognize the fusion loop of WNV, located at the tip of the DII region. In vitro and in vivo studies, however, showed decreased potency of these antibodies in neutralizing WNV virions (5). B-cell deficient mice were shown to have a lower LD50 of 100 PFU compared to 107 PFU for wild type mice (4). Natural antigen-antibody complexes are normally cleared through the spleen and lymph nodes, which may contribute to the prevention of viral propagation into the CNS.
Evasion of the Immune System
A study by Gale and Fredericksen (7) showed that WNV, specifically the strain that was isolated in the major New York epidemic, can evade immune system detection during early infection when it is most sensitive to host immune defences. It does so by inhibiting the activation of IRF-3 through its own replication, thereby allowing the accumulation of virions before the host immune system can initiate an antiviral response. The mechanism by which WNV avoids host immune detection in early stages of infection remains to be elucidated. Perhaps the amount of WNV virions required for detection and consequent IRF-3 activation is not high enough at early infection, thus allowing WNV to establish a productive infection before immune defences begin.
Two human genes have been identified as vulnerability loci of WNV: 1) the CCR5 chemokine receptor gene and 2) the OAS gene. In a study by Glass et al. (9) the role of CCR5 in human subjects was determined using the CCR5Î”32, a defective CCR5 allele found commonly in Caucasian population. The authors found that homozygotic subjects experienced fatal WNV infection outcome, leading them to conclude that the CCR5 gene is an important survival indicator of WNV infection in humans. Interestingly, CCR5 is also a coreceptor of HIV, which consequently has significant implications for the viability of CCR5 blockers being developed as therapeutic agents for HIV infections.
The OAS1 gene in humans codes for a component of the Type 1 IFN signalling cascade, and thus plays a critical role in immune defence. In a study by Lim et al. (10), individuals carrying two copies of the hypomorphic allele were shown to be more frequently infected by WNV, regardless of whether they are asymptomatic or symptomatic. Thus, the authors proposed that human OAS1 gene may confer protection against the WNV.
Currently, there is no single therapeutic method that has been proven effective in eliminating WNV. Instead, treatment is primarily supportive in nature, including pain management, antiemetic therapy, control of seizures in more severe cases, and prevention of secondary infections (11). A few antiviral agents have either been tested in vitro, administered to animals, or given empirically to humans with WNV encephalitis; some of these include: 1) purine and pyrimidine analogues, 2) IFNÎ± and 3) human antibodies.
Ribavirin is a guanosine analogue that competitively inhibits inosine monophosphate (IMP) dehydrogenase to decrease the intracellular levels of guanosine. As a result, RNA replication of WNV may be interfered, thereby controlling the spread of viral particles throughout the systemic circulation. In vitro cell culture studies using Ribavirin showed promising results at high doses of 60-100 µM (11), but administration in animals and humans led to high mortality rates. Similar findings were obtained with mycophenolic acid (MPA), another inhibitor of IMP dehydrogenase. MPA also acts as an immunosuppressive agent that has been extensively used to prevent rejection post-organ transplantation. Thus, it appears that although the above therapeutic agents assist in the reduction of viral replication, their negative effects on the immune system due to an inhibition of guanosine biosynthesis render them inappropriate treatments for WNV infections.
Type I IFNs (IFNÎ± and IFNÎ²) are essential components of the innate immune response. While IFNs appear to inhibit early WNV infection, this effect is attenuated after viral replication has begun. Viral replication allows non-structural proteins to antagonize type I IFNs by disrupting their downstream cascades, which involve JAK-STAT signalling. Nevertheless, type I IFNs are not without their merits. For instance, mice deficient in type I IFN receptors were shown to be more susceptible to death due to WNV. IFNÎ± has also been used to treat a few human cases of WNV encephalitis, though a large clinical trial involving the administration of IFNÎ± in children with WNV infection failed to produce favourable results (11).
The use of antibodies as therapeutic agents against WNV has not been extensively performed in human populations. In rodents, the transfer of immune serum protected all of wild type, B cell deficient and lymphocyte deficient mice from WNV infection (11). Administration of Î³-immunoglobulin into a small number of human hosts with neuroinvasive disease seems to improve their health status. However, the use of Î³-immunoglobulin as WNV treatment has its downsides: 1) it is purified from human plasma, which may carry other unknown infectious agents and 2) it must be administered at very large doses, which may adversely affect patients with cardiac or renal co-morbidities (11).
Although we have come a long way in our understanding of WNV, there are still mysteries left unsolved. For instance, the long-term effects of even mild forms of WNV infection, specifically the West Nile fever, are not yet known. More and more cases of neurological symptoms due to West Nile fever have been reported and nobody truly understands how and why this occurs. Additionally, scientists do not yet understand why certain species of birds, such as crows and blue jays, are more susceptible to WNV infection than others. The difficulty in determining avian migratory patterns further complicates matters.
The development of a vaccine for humans may also be an effective preventative method. A WNV vaccine could potentially be produced using attenuated or inactivated strains. Scientists have proposed using RNA interference technology but the enclosed compartment in which WNV replicates prevents its exposure to RNAi machinery (11). Research should focus on coming up with a delivery method that will allow siRNAs to penetrate intracellular membranes to come in contact with WNV.
Figure 1. The transmission cycle of West Nile Virus (Adapted from (1) and (3)).