Host genetic risk factors for West Nile virus infection and disease progression

West Nile virus (WNV), a category B pathogen endemic in parts of Africa, Asia and Europe, emerged in North America in 1999, and spread rapidly across the continental U.S. Outcomes of infection with WNV range from asymptomatic to severe neuroinvasive disease manifested as encephalitis, paralysis, and/or death. Neuroinvasive WNV disease occurs in less than one percent of cases, and although host genetic factors are thought to influence risk for symptomatic disease, the identity of these factors remains largely unknown. We tested 360 common haplotype tagging and/or functional SNPs in 86 genes that encode key regulators of immune function in 753 individuals infected with WNV including: 422 symptomatic WNV cases and 331 cases with asymptomatic infections. After applying a Bonferroni correction for multiple tests and controlling for population stratification, SNPs in IRF3 (OR 0.54, p?=?0.035) and MX1, (OR 0.19, p?=?0.014) were associated with symptomatic WNV infection and a single SNP in OAS1 (OR 9.79, p?=?0.003) was associated with increased risk for West Nile encephalitis and paralysis (WNE/P). Together, these results suggest that genetic variation in the interferon response pathway is associated with both risk for symptomatic WNV infection and WNV disease progression.     

West Nile Virus (WNV) protease and membrane interactions revealed by NMR spectroscopy

Although the importance of Wnt3a in melanocyte development has been well recognized, the effect of Wnt3a in normal HF melanocytes has not been clearly elucidated yet. Thus, we sought to examine the presence and location of Wnt3a in HF during hair cycle. By using melanocyte-targeted Dct-LacZ transgenic mice, we found that Wnt3a signaling is activated in mouse HF melanocytes during anagen of hair cycle. To further explore the potential functions of Wnt3a in mouse melanocytes, we infected melan-a cells with AdWnt3a to serve as the production source of Wnt3a protein. We demonstrated that Wnt3a promoted melanogenesis through upregulation of MITF and its downstream genes, tyrosinase and TRP1, in melanocytes. In vivo, AdWnt3a rescued the effects of AdsimMITF on HF melanocytes and promoted melanin synthesis. Our results suggest that Wnt3a plays an important role in mouse HF melanocytes homeostasis.     

Mosquito‐borne West Nile virus (WNV) surveillance in the Upper Rhine Valley,Germany

West Nile virus (WNV) could be introduced into Germany via migratory birds originating from Africa or southern Europe and subsequently transmitted to indigenous birds, humans, or horses by mosquitoes. Neither the virus itself nor antibodies against WNV have yet to be found in mosquitoes and horses, whereas antibodies have been detected in migrating birds and in humans that were in close contact with birds. At present, the West Nile virus itself has yet to be detected in Germany. This investigation was conducted primarily in major bird breeding, resting, and roosting habitats (hotspots) in the Upper Rhine Valley. Adult mosquitoes were trapped using CO₂‐baited Encephalitis Vector Surveillance (EVS)‐traps and were tested for WNV by the VecTest WNV Antigen Assay. In 2007 and 2008, a total of 11,073 host‐seeking adult female mosquitoes (13 species) were tested, and all tests were negative for WNV. Statistical calculations could be performed only where sufficient numbers of mosquitoes were trapped. For these sites, WNV infection among mosquitoes could be ruled out with 80% certainty. For the evaluation of the WNV situation in Germany, the results of this investigation are a further indication that the virus has not yet arrived.     

West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

Autophagy is a homeostatic process responsible for recycling cytosolic proteins and organelles. Moreover, this pathway contributes to the cell’s intrinsic innate defenses. While many viruses have evolved mechanisms to antagonize the antiviral effects of the autophagy pathway, others subvert autophagy to facilitate replication. Here, we have investigated the role of autophagy in West Nile virus (WNV) replication. Experiments in cell lines derived from a variety of sources, including the kidney, liver, skin, and brain, indicated that WNV replication does not upregulate the autophagy pathway. Furthermore, WNV infection did not inhibit rapamycin-induced autophagy, suggesting that WNV does not disrupt the authophagy signaling cascade. Perturbation of the autophagy pathway by depletion of the major autophagy factors Atg5 or Atg7 had no effect on WNV infectious particle production, indicating that WNV does not require a functional autophagy pathway for replication. Taken together, the results of our study provide evidence that WNV, unlike several other viruses of the family Flaviviridae, does not significantly interact with the conventional autophagy pathway in mammalian cells.     

Toll-Like Receptor-3 Is Dispensable for the Innate MicroRNA Response to West Nile Virus (WNV)

The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.     

Environmental risk factors associated with West Nile virus clinical disease in Florida horses

The objective of this study was to examine the extrinsic risk factors of West Nile virus (WNV) clinical disease in Florida horses as established from confirmed and negative horses tested within the state from 2001 to 2003. An Arboviral Case Information Form (ACF) was submitted by a referring veterinarian at the time of testing to the Florida Department of Agriculture and Consumer Services on every horse suspected of a viral encephalitis in Florida. A follow‐up survey that focused on arbovirus prevention and farm ecology was created and mailed to the owner of each tested horse. Data from the follow‐up survey indicated peak WNV prevalence in the late summer months in Florida. Quarter horses were the most commonly affected breed. The WNV vaccine was highly protective and natural water on the property also had a protective association. Factors that increased the risk of WNV to horses were the use of fans and a stable construction of solid wood or cement. Some risk indicators were dead birds on the property and other ill animals on the property. Data from this retrospective study have helped identify factors associated with WNV transmission in equines in Florida. Horses that have not been vaccinated and show clinical signs of arboviral infection from June to November should be tested for WNV. Horses that have been vaccinated and show clinical signs should be tested when the vaccination was administered within 1 month or greater than 6 months prior to the onset of clinical symptoms associated with WN infection.     

A genetic basis for human susceptibility to West Nile virus

West Nile virus (WNV) infects thousands of humans annually and causes a spectrum of disease ranging from an acute febrile illness to lethal encephalitis. A new study suggests a link between CCR5Delta32 (a common mutant allele of the chemokine and HIV receptor CCR5) and fatal WNV infection. The study highlights a possible risk in targeting this receptor for the prevention and/or treatment of infectious diseases.     

Inhibitors of Dengue virus and West Nile virus proteases based on the aminobenzamide scaffold

Dengue and West Nile viruses (WNV) are mosquito-borne members of flaviviruses that cause significant morbidity and mortality. There is no approved vaccine or antiviral drugs for human use to date. In this study, a series of functionalized meta and para aminobenzamide derivatives were synthesized and subsequently screened in vitro against Dengue virus and West Nile virus proteases. Four active compounds were identified which showed comparable activity toward the two proteases and shared in common a meta or para(phenoxy)phenyl group. The inhibition constants (K(i)) for the most potent compound 7n against Dengue and West Nile virus proteases were 8.77 and 5.55 μM, respectively. The kinetics data support a competitive mode of inhibition of both proteases by compound 7n. This conclusion is further supported by molecular modeling. This study reveals a new chemical scaffold which is amenable to further optimization to yield potent inhibitors of the viral proteases via the combined utilization of iterative medicinal chemistry/structure-activity relationship studies and in vitro screening.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.     

A continental risk assessment of West Nile virus under climate change

Since first introduced to North America in 1999, West Nile virus (WNV) has spread rapidly across the continent, threatening wildlife populations and posing serious health risks to humans. While WNV incidence has been linked to environmental factors, particularly temperature and rainfall, little is known about how future climate change may affect the spread of the disease. Using available data on WNV infections in vectors and hosts collected from 2003–2011 and using a suite of 10 species distribution models, weighted according to their predictive performance, we modeled the incidence of WNV under current climate conditions at a continental scale. Models were found to accurately predict spatial patterns of WNV that were then used to examine how future climate may affect the spread of the disease. Predictions were accurate for cases of human WNV infection in the following year (2012), with areas reporting infections having significantly higher probability of presence as predicted by our models. Projected geographic distributions of WNV in North America under future climate for 2050 and 2080 show an expansion of suitable climate for the disease, driven by warmer temperatures and lower annual precipitation that will result in the exposure of new and naïve host populations to the virus with potentially serious consequences. Our risk assessment identifies current and future hotspots of West Nile virus where mitigation efforts should be focused and presents an important new approach for monitoring vector‐borne disease under climate change.     

A dual-purpose synthetic colloidal platform for protease mapping: substrate profiling for Dengue and West Nile virus proteases

In a proof of concept study, we created a small focused fluorescent hexapeptide library onto 14 multiplexed barcoded sets of silica particles to probe the substrate recognition specificity of West Nile and Dengue virus proteases. A flow cytometric analysis demonstrated that the optical signature of each bead population remained distinguishable throughout the solid-phase peptide synthesis and proteolytic assay. As expected, both proteases displayed a narrow specificity for lysine and arginine residues in the P₁ and P₂ substrate positions. This open-ended platform enables the fast and simultaneous identification of peptide substrates and is applicable to other proteases.     

Mosquito (Diptera: Culicidae) fauna in an area endemic for West Nile virus

Mosquito collections with CDC light traps using dry ice and pigeon‐baited traps were carried out in south Moravia (Czech Republic) from April to October in 2007 and 2008 at two study sites. In 2007, 11 two‐day captures were carried out in two‐week intervals, and 1,490 female mosquitoes of nine species were caught. In 2008, 15 two‐day trappings of mosquitoes were carried out: 6,778 females of 22 species of mosquitoes were trapped. The results showed marked differences in abundance and species composition of mosquitoes between both study sites and between the trapping methods. In the floodplain forest ecosystem of the Soutok study area, Aedes vexans predominated. The species composition in the Nesyt study site was more varied and the most common species was Culex pipiens. At the latter study site, Anopheles hyrcanus (var. pseudopictus) and Uranotaenia unguiculata, mosquito species with largely southern Eurasian distribution, were repeatedly demonstrated. The largest capture of mosquitoes was in traps with CO₂ placed at a height 1 m above the ground. The capture of mosquitoes in the pigeon‐baited traps as well as in the traps with CO₂ placed in the canopy of trees was markedly lower in both study sites, with the predominant species being Culex pipiens.     

Ecological correlates of risk and incidence of West Nile virus in the United States

West Nile virus, which was recently introduced to North America, is a mosquito-borne pathogen that infects a wide range of vertebrate hosts, including humans. Several species of birds appear to be the primary reservoir hosts, whereas other bird species, as well as other vertebrate species, can be infected but are less competent reservoirs. One hypothesis regarding the transmission dynamics of West Nile virus suggests that high bird diversity reduces West Nile virus transmission because mosquito blood-meals are distributed across a wide range of bird species, many of which have low reservoir competence. One mechanism by which this hypothesis can operate is that high-diversity bird communities might have lower community-competence, defined as the sum of the product of each species’ abundance and its reservoir competence index value. Additional hypotheses posit that West Nile virus transmission will be reduced when either: (1) abundance of mosquito vectors is low; or (2) human population density is low. We assessed these hypotheses at two spatial scales: a regional scale near Saint Louis, MO, and a national scale (continental USA). We found that prevalence of West Nile virus infection in mosquito vectors and in humans increased with decreasing bird diversity and with increasing reservoir competence of the bird community. Our results suggest that conservation of avian diversity might help ameliorate the current West Nile virus epidemic in the USA     

Climatic,ecological, and socioeconomic factors associated with West Nile virus incidence in Atlanta,Georgia, U.S.A.

The integrated effects of the many risk factors associated with West Nile virus (WNV) incidence are complex and not well understood. We studied an array of risk factors in and around Atlanta, GA, that have been shown to be linked with WNV in other locations. This array was comprehensive and included climate and meteorological metrics, vegetation characteristics, land use / land cover analyses, and socioeconomic factors. Data on mosquito abundance and WNV mosquito infection rates were obtained for 58 sites and covered 2009–2011, a period following the combined storm water – sewer overflow remediation in that city. Risk factors were compared to mosquito abundance and the WNV vector index (VI) using regression analyses individually and in combination. Lagged climate variables, including soil moisture and temperature, were significantly correlated (positively) with vector index as were forest patch size and percent pine composition of patches (both negatively). Socioeconomic factors that were most highly correlated (positively) with the VI included the proportion of low income households and homes built before 1960 and housing density. The model selected through stepwise regression that related risk factors to the VI included (in the order of decreasing influence) proportion of houses built before 1960, percent of pine in patches, and proportion of low income households.     

A recombinant influenza A virus expressing domain III of West Nile virus induces protective immune responses against influenza and West Nile virus

West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 10(5) TCID(50) Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.     

The demographic and socioeconomic factors predictive for populations at high-risk for La Crosse virus infection in West Virginia

Although a large body of literature exists for the environmental risk factors for La Crosse virus (LACV) transmission, the demographic and socioeconomic risk factors for developing LACV infection have not been investigated. Therefore, this study investigated the demographic and socioeconomic risk factors for LACV infection in West Virginia from 2003 to 2007, using two forward stepwise discriminant analyses. The discriminant analyses were used to evaluate a number of demographic and socioeconomic factors for their ability to predict: 1) those census tracts with at least one reported case of LACV infection versus those census tracts with no reported cases of LACV infection and 2) to evaluate significantly high-risk clusters for LACV infection versus significantly low-risk clusters for LACV infection. In the first model, a high school education diploma or a general education diploma or less and a lower housing densitywere found to be predictive of those census tracts with at least one case of LACV infection. A high school or a general education diploma or less, lower housing density, and housing built in 1969 and earlier were all found to be predictive of those census tracts displaying high-risk clusters versus census tracts displaying low-risk clusters in the second model. The cluster discriminant analysis was found to be more predictive than the census tract discriminant analysis as indicated by the Eigenvalues, canonical correlation, and grouping accuracy. The results of this study indicate that socioeconomically disadvantaged populations are at the highest risk for LACV infection and should be a focus of LACV infection prevention efforts.     

Search for inhibitors of West Nile virus among antibiotics]

The activity of 24 antibiotics was studied in treatment of albino mice with experimental encephalitis caused by West Nile virus. The antiviral activity of gentamicin and kanamycin was stated. The survival rate of the animals 19. contaminated with 10-100 LD50 of the West Nile virus and treated parenterally with gentamicin in a dose of 80 to 400 micrograms/mouse was higher than that in the controls by 29.5 to 100 per cent and depended on the drug regimen. The efficacy of kanamycin was lower. The chemotherapeutic indices of gentamicin and kanamycin amounted to 100 and 10, respectively. Since there are no schemes for chemotherapy of the infection caused by the West Nile virus and the respective vaccines are not available the use of the antibiotics and gentamicin in particular appears to be promising in the disease prevention and treatment.