Diagnosis of West Nile virus infections:Evaluation of different laboratory methods

BACKGROUND The diagnosis of West Nile virus(WNV)is challenging due to short-term and low-level viremia,flavivirus cross-reactivity,and long immunoglobulin M(IgM)persistence.AIM To evaluate different methods for WNV detection[reverse transcription-polymerase chain reaction(RT-PCR),IgM/IgG antibodies,IgG avidity]in serum,cerebrospinal fluid(CSF),and urine samples of patients with confirmed WNV infection.METHODS The study included patients with confirmed WNV neuroinvasive infection(n=62),asymptomatic WNV seropositive individuals(n=22),and individuals with false-positive WNV IgM antibodies(n=30).WNV RNA was detected using RT-PCR.A commercial ELISA was used to detect WNV IgM/IgG antibodies with confirmation of cross-reactive samples using a virus neutralization test(VNT).IgG-positive samples were tested for IgG avidity.RESULTS The WNV-RNA detection rates were significantly higher in the urine(54.5%)/serum(46.4%)than in CSF(32.2%).According to the sampling time,the WNV-RNA detection rates in urine collected within 7 days/8-14/≥15 days were 29.4/66.6/62.5%(P=0.042).However,these differences were not observed in the CSF.The median RT-PCR cycle threshold values were significantly lower in urine(32.5,IQR=28-34)than in CSF(34.5,IQR=33-36).The frequency of positive WNV IgM and IgG significantly differed according to the sampling time in serum but not in CSF.Positive IgM/IgG antibodies were detected in 84.3/9.3%of serum samples collected within 7 days,100/71.1%of samples collected 8-14,and 100%samples collected after≥15 days.Recent WNV infection was confirmed by low/borderline avidity index(AI)in 13.6%of asymptomatic individuals.A correlation between ELISA and AI was strong negative for IgM and strong positive for IgG.No significant correlation between ELISA IgG and VNT was found.CONCLUSION The frequency of WNV RNA and antibody detection depends on the sampling time and type of clinical samples.IgG avidity could differentiate recent WNV infections from long-persisting IgM antibodies.

Mutagenesis of D80-82 and G83 Residues in West Nile Virus NS2B: Effects on NS2B-NS3 Activity and Viral Replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D80DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D80DDG in NS2B on protease activity and viral replication, the negatively charged region D80DD and the conserved residue G83 of NS2B were mutated (D80DD/E80EE, D80DD/K80KK, D80DD/A80AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D80DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D80DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.

Development and Characterization of West Nile Virus Replicon Expressing Secreted Gaussia Luciferase

We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.

MATHEMATICAL ANALYSIS OF WEST NILE VIRUS MODEL WITH DISCRETE DELAYS

The paper presents the basic model for the transmission dynamics of West Nile virus （WNV）. The model, which consists of seven mutually-exclusive compartments representing the birds and vector dynamics, has a locally-asymptotically stable disease- free equilibrium whenever the associated reproduction number （R0） is less than unity. As reveal in [3, 20], the analyses of the model show the existence of the phenomenon of backward bifurcation （where the stable disease-free equilibrium of the model co-exists with a stable endemic equilibrium when the reproduction number of the disease is less than unity）. It is shown, that the backward bifurcation phenomenon can be removed by substituting the associated standard incidence function with a mass action incidence. Analysis of the reproduction number of the model shows that, the disease will persist, whenever R0 〉 1, and increase in the length of incubation period can help reduce WNV burden in the community if a certain threshold quantities, denoted by △b and △v are negative. On the other hand, increasing the length of the incubation period increases disease burden if △b 〉 0 and △v 〉 0. Furthermore, it is shown that adding time delay to the corresponding autonomous model with standard incidence （considered in [2]） does not alter the qualitative dynamics of the autonomous ：system （with respect to the elimination or persistence of the disease）.

Cauda equina arachnoiditis–a rare manifestation of West Nile virus neuroinvasive disease:A case report

BACKGROUND Data regarding the neuroradiology features of the West Nile virus neuroinvasive disease(WNV NID)is rather scarce.To contribute to the knowledge of the WNV NID,we present a patient with a combination of encephalitis and acute flaccid paresis,with cauda equina arachnoiditis as the main magnetic resonance(MR)finding.CASE SUMMARY A 72-year-old female patient was admitted due to fever,headache and gait instability.During the first several days she developed somnolence,aphasia,urinary incontinence,constipation,and asymmetric lower extremities weakness.Cerebrospinal fluid analysis indicated encephalitis.Native brain computed tomography and MR were unremarkable,while spinal MR demonstrated cauda equina enhancement without cord lesions.Virology testing revealed WNV IgM and IgG antibodies in serum and cerebrospinal fluid,which confirmed acute WNV NID.The treatment was supportive.After two months only a slight improvement was noticed but cognitive impairment,loss of sphincter control and asymmetric inferior extremities weakness remained.The patient died after a month on chronic rehabilitation.CONCLUSION Cauda equina arachnoiditis is a rare,but possible neuroradiological feature in acute flaccid paresis form of WNV NID.

Using a Collateral Damage Model to Explain Survival Data in West Nile Virus Infections

Simulation code for a model of the adaptive immune response seen in flavivirus infections is used to explain the immunopathological consequences seen in West Nile Virus virus (WNV) infections. We use a model that specifically handles the differences in how the virus infects resting cells, the G0 state, versus dividing cells, the G1 state, which includes vastly increased MHC-I upregulation for resting cells over dividing cells. The simulation suggests how the infection progresses in a one host model and the results shed insight into the unusual survival curve data obtained for this infection: there is an increase in health even though viral load has increased.

Impact of climate change on the global circulation of West Nile virus and adaptation responses:a scoping review

Background West Nile virus(WNV),the most widely distributed flavivirus causing encephalitis globally,is a vector-borne pathogen of global importance.The changing climate is poised to reshape the landscape of various infectious diseases,particularly vector-borne ones like WNV.Understanding the anticipated geographical and range shifts in disease transmission due to climate change,alongside effective adaptation strategies,is critical for mitigating future public health impacts.This scoping review aims to consolidate evidence on the impact of climate change on WNV and to identify a spectrum of applicable adaptation strategies.Main body We systematically analyzed research articles from PubMed,Web of Science,Scopus,and EBSCOhost.Our criteria included English-language research articles published between 2007 and 2023,focusing on the impacts of climate change on WNV and related adaptation strategies.We extracted data concerning study objectives,populations,geographical focus,and specific findings.Literature was categorized into two primary themes:1)climate-WNV associations,and 2)climate change impacts on WNV transmission,providing a clear understanding.Out of 2168 articles reviewed,120 met our criteria.Most evidence originated from North America(59.2%)and Europe(28.3%),with a primary focus on human cases(31.7%).Studies on climate-WNV correlations(n=83)highlighted temperature(67.5%)as a pivotal climate factor.In the analysis of climate change impacts on WNV(n=37),most evidence suggested that climate change may affect the transmission and distribution of WNV,with the extent of the impact depending on local and regional conditions.Although few studies directly addressed the implementation of adaptation strategies for climate-induced disease transmission,the proposed strategies(n=49)fell into six categories:1)surveillance and monitoring(38.8%),2)predictive modeling(18.4%),3)cross-disciplinary collaboration(16.3%),4)environmental management(12.2%),5)public education(8.2%),and 6)health system readiness(6.1%).Additionally,we developed an accessible online platform to summarize the evidence on climate change impacts on WNV transmission(https://2xzl2o-neaop.shinyapps.io/WNVScopingReview/).Conclusions This review reveals that climate change may affect the transmission and distribution of WNV,but the literature reflects only a small share of the global WNV dynamics.There is an urgent need for adaptive responses to anticipate and respond to the climate-driven spread of WNV.Nevertheless,studies focusing on these adaptation responses are sparse compared to those examining the impacts of climate change.Further research on the impacts of climate change and adaptation strategies for vector-borne diseases,along with more comprehensive evidence synthesis,is needed to inform effective policy responses tailored to local contexts.

Modeling West Nile Virus transmission in birds and humans:Advantages of using a cellular automata approach

In Canada,the periodic circulation of West Nile Virus(WNV)is difficult to predict and,beyond climatic factors,appears to be related to the migratory movements of infected birds from the southern United States.This hypothesis has not yet been explored in a spatially distributed model.The main objective of this work was to develop a spatially explicit dynamic model for the transmission of WNV in Canada,that allows us to explore non-climate related hypotheses associated with WNV transmission.A Cellular Automata(CA)approach for multiple hosts(birds and humans)is used for a test region in eastern Ontario,Canada.The tool is designed to explore the role of host and vector spatial heterogeneity,host migration,and vector feeding preferences.We developed a spatialized compartmental SEIRDS-SEI model for WNV transmission with a study region divided into 4 rectangular cells.We used 2010–2021 bird data from the eBird project and 2010–2019 mosquito data collected by Ontario Public Health to mimic bird and mosquito seasonal variation.We considered heterogeneous bird densities(high and low suitability areas)and homogeneous mosquito and human densities.In high suitability areas for birds,we identified 5 entry points for WNV-infected birds.We compared our simulations with pools of WNV-infected field collected mosquitoes.Simulations and sensitivity analyses were performed using MATLAB software.The results showed good correspondence between simulated and observed epidemics,supporting the validity of our model assumptions and calibration.Sensitivity analysis showed that a 5%increase or decrease in each parameter of our model except for the biting rate of bird by mosquito(c^(B,M)and mosquito natural mortality rate(d^(M)),had a very limited effect on the total number of cases(newly infected birds and humans),prevalence peak,or date of occurrence.We demonstrate the utility of the CA approach for studying WNV transmission in a heterogeneous landscape with multiple hosts.

Isolated Hyperacute T-Waves in West Nile Encephalitis Indicating Atypical Variant of Stress-Induced Cardiomyopathy

Several cardiac outcomes have been reported with West Nile-encephalitis;however, the underlying pathophysiology remains complex. We present a 42-year-old female, with multiple sclerosis, whose neurological symptoms and respiratory decline were finally explained by the diagnosis of West Nile-encephalitis. During her admission, the isolated peaked T-waves indicated the underlying stress-induced cardiomyopathy. The absence of all other causes of hyperacute T-waves, their subsequent resolution with the resolution of infection and improvement in wall motion abnormalities, further supported the association. This case highlights the importance of considering hyperacute T-waves in an approach towards the diagnosis of WNV-encephalitis related atypical variant of stress-induced cardiomyopathy.

The recombinant truncated envelope protein of West Nile virus adjuvanted with Alum/CpG induces potent humoral and T cell immunity in mice

West Nile virus(WNV)is a mosquito-transmitted flavivirus distributed globally for decades and can cause disease in humans and animals.So far,no WNV vaccine has been licensed for human use.Therefore,the development of novel candidate vaccines and the improvement of vaccination strategies is imperative.As the WNV envelope(E)glycoprotein plays an important role in mediating viral binding to cellular receptors and virus-cell membrane fusion,it is a critical target for the host humoral response.Here,we prepared a recombinant truncated envelope protein of WNV(rWNV-80E)and developed a WNV subunit vaccine formulation with a combination of aluminum hydroxide(alum)and a synthetic oligonucleotide CpG as adjuvants.C57BL/6 mice were immunized twice intramuscularly at 28-day intervals with 5μg purified rWNV-80E adjuvanted with Alum/CpG.WNV E-specific IgG was detected by enzyme-linked immunosorbent assay and neutralizing antibodies(nAbs)was detected using single-round infectious particles of WNV.Furthermore,T cell immunity was detected by enzyme-linked immunospot assay and intracellular cytokine staining assay.Notably,rWNV-80E was highly immunogenic and elicited potent humoral and cell immunity,as evidenced by significant levels of IFN-γ and TNF-αsecretion in the T cells of mice.In summary,the Alum/CpG-adjuvanted rWNV-80E subunit vaccine elicited potent and balanced B-and T-cell immunity in mice,and therefore it is a promising candidate vaccine that warrants further investigation for use in human or veterinary applications.

Development and evaluation of neutralizing antibodies for cross-protection against West Nile virus and Japanese encephalitis virus

Background:West Nile virus is a severe zoonotic pathogen that can cause severe central nervous system symptoms in humans and horses,and is fatal for birds,chickens and other poultry.With no specific drugs or vaccines available,antibody-based therapy is a promising treatment.This study aims to develop neutralizing antibodies against West Nile virus and assess their cross-protective potential against Japanese encephalitis virus.Methods:Monoclonal antibodies against WNV and JEV were isolated by hybridoma technology.The therapeutic efficacy of these antibodies was evaluated using a mouse model,and a humanized version of the monoclonal antibody was generated for potential human application.Results:In this study,we generated eight monoclonal antibodies that exhibit neutralizing activity against WNV.Their therapeutic effects against WNV were validated both in vivo and in vitro.Among these antibodies,C9-G11-F3 also exhibited cross-protective activity against JEV.We also humanized the antibody to ensure that it could be used for WNV infection treatment in humans.Conclusion:This study highlights the importance of neutralizing antibodies as a promising approach for pro-tection against West Nile virus infection and suggests their potential utility in the development of therapeutic interventions.

Spreading and vanishing in a West Nile virus model with expanding fronts

We study a simplified version of a West Nile virus （WNv） model discussed by Lewis et al. （2006）, which was considered as a first approximation for the spatial spread of WNv. The basic reproduction number R0 for the non-spatial epidemic model is defined and a threshold parameter RD for the corresponding problem with null Dirichlet boundary condition is introduced. We consider a free boundary problem with a coupled system, which describes the diffusion of birds by a PDE and the movement of mosquitoes by an ODE. The risk index R0^F（t） associated with the disease in spatial setting is represented. Sufficient conditions for the WNv to eradicate or to spread are given. The asymptotic behavior of the solution to the system when the spreading occurs is considered. It is shown that the initial number of infected populations, the diffusion rate of birds and the length of initial habitat exhibit important impacts on the vanishing or spreading of the virus. Numerical simulations are presented to illustrate the analytical results.

West Nile virus:North American experience

West Nile virus,a mosquito-vectored flavivirus of the Japanese encephalitis serogroup,was first detected in North America following an epizootic in the New York City area in 1999.In the intervening 11 years since the arrival of the virus in North America,it has crossed the contiguous USA,entered the Canadian provinces bordering the USA,and has been reported in the Caribbean islands,Mexico,Central America and,more recently,South America.West Nile virus has been reported in over 300 species of birds in the USA and has caused the deaths of thousands of birds,local population declines of some avian species,the clinical illness and deaths of thousands of domestic horses,and the clinical disease in over 30000 Americans and the deaths of over 1000.Prior to the emergence of West Nile virus in North America,St.Louis encephalitis virus and Dengue virus were the only other known mosquito-transmitted flaviviruses in North America capable of causing human disease.This review will discuss the North American experience with mosquito-borne flavivirus prior to the arrival of West Nile virus,the entry and spread of West Nile virus in North America,effects on wild bird populations,genetic changes in the virus,and the current state of West Nile virus transmission.

Asymptotic periodicity in a diffusive West Nile virus model in a heterogeneous environment

This paper is concerned with a diffusive West Nile virus model （WNv） in a heterogeneous environment. The basic reproduction number R0 for spatially homogeneous model is first introduced. We then define a threshold parameter R0N for the corresponding diffusive WNv model in a heterogeneous environment. It is shown that if R0^N 〉 1, the model admits at least one nontrivial T-periodic solution, whereas if RN 〈 1, the model has no nontrivial T-periodic solution. By means of monotone iterative schemes, the true solution can be obtained and the asymptotic behavior of periodic solutions is presented. The paper is closed with some numerical simulations to illustrate our theoretical results.

The diffusive model for West Nile virus with advection and expanding fronts in a heterogeneous environment

In this paper,we investigate a reaction-diffusion-advection model with expanding fronts,which models the spatial transmission of West Nile virus(WNv)in a heterogeneous environment.A free boundary problem is formulated and the global existence and uniqueness of the solution is presented.In addition to a classical basic reproduction number,the spatial-temporal basic reproduction number for the model with null Dirichlet boundary condition is introduced and the risk index associated with the virus in spatial setting is defined,and their properties are discussed.Sufficient conditions for the WNv to vanish or spread are given,and the asymptotic behavior of the solution to the free boundary problem when the spreading occurs is established.Our results show that the initial number of infected populations and the expanding capability of the expanding fronts exhibit important impacts on the extinction or persistence of the virus.

Comparative studies for the fractional optimal control in transmission dynamics of West Nile virus

In this paper, optimal control for a novel West Nile virus （WNV） model of fractional order derivative is presented. The proposed model is governed by a system of fractional differential equations （FDEs）, where the fractional derivative is defined in the Caputo sense. An optimal control problem is formulated and studied theoretically using the Pon- tryagin maximum principle. Two numerical methods are used to study the fractional- order optimal control problem. The methods are, the iterative optimal control method （OCM） and the generalized Euler method （GEM）. Positivity, boundedness and conver- gence of the IOCM are studied. Comparative studies between the proposed methods are implemented, it is found that the IOCM is better than the GEM.

A parsimonious Bayesian predictive model for forecasting new reported cases of West Nile disease

Upon researching predictive models related toWest Nile virus disease,it is discovered that there are numerous parameters and extensive information in most models,thus contributing to unnecessary complexity.Another challenge frequently encountered is the lead time,which refers to the period for which predictions are made and often is too short.This paper addresses these issues by introducing a parsimonious method based on ICC curves,offering a logistic distribution model derived from the vector-borne SEIR model.Unlike existing models relying on diverse environmental data,our approach exclusively utilizes historical and present infected human cases(number of new cases).With a yearlong lead time,the predictions extend throughout the 12 months,gaining precision as new data emerge.Theoretical conditions are derived to minimize Bayesian loss,enhancing predictive precision.We construct a Bayesian forecasting probability density function using carefully selected prior distributions.Applying these functions,we predict monthspecific infections nationwide,rigorously evaluating accuracy with probabilistic metrics.Additionally,HPD credible intervals at 90%,95%,and 99%levels is performed.Precision assessment is conducted for HPD intervals,measuring the proportion of intervals that does not include actual reported cases for 2020e2022.

A Virus-type Specific Serological Diagnosis of Flavivirus Infection Using Virus-like Particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus （WNV）, dengue virus （DENV）, yellow fever virus （YFV）, and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test （PRNT） is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety levet-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles （VLPs） that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps： （i） VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; （ii）the neutralized VLPs are used to infect Vero cells; and （iii） the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen （as quantified by the luciferase activities） is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the ＂gold standard＂ of the classic PRNT; importantly, it shortens the assay time from 〉10 days to 〈1 day, and can be performed in biosafety level-2 facility.

Adjusting Second Moment Bias in Eigenspace Using Bayesian Empirical Estimators, Dirichlet Tessellations and Worldview I Data for Predicting Culex Quinquefasciatus Habitats in Trinidad

Temporally weighted regression models with a spatial autoregressive component may estimate nonlinearities in spatiotemporal-sampled data of Culex quinquefasciatus, a major vector of West Nile Virus (WNV) which can help implement control strategies by determining optimal predictors associated to prolific habitats. The design of this kind of mixed model can specifically incorporate spatial autocorrelation whilst including the influence of other aspatial predictor variables. Currently, the lack of an estimation theory that allows for het- eroscedasticity and corresponding joint hypothesis testing in the presence of spatial dependence in georefer- enced Cx. quinquefasciatus habitat data is a serious shortcoming in WNV research. In this paper we used spatially lagged and simultaneous autoregressive models based on multiple predictor variables of immature Cx. quinquefasciatus and Worldview 1 (WV-1) data to help implant a remote habitat-based surveillance sys- tem in Trinidad. Initially, we used Geomatica Ortho Engine? v. 10.2 for extracting a Digital Elevation Model (DEM) from the WV-1 raw imagery. Results of the DEM analyses indicated a statistically significant inverse linear relationship between total sampled Cx. quinquefasciatus data and elevation (m) (R2 = -0.439;p < 0.0001), with a standard deviation of 10.41. Additional field-sampled information was derived using data from an or-thogonal grid-matrix constructed in an ArcInfo 9.3? and overlaid onto the WV-1 data. A unique identifier was placed in the centroid of each grid cell. Univariate statistics and Poisson regression models were then generated using the georeferenced covariates in SAS/GIS?. Coefficient estimates were also used to define expectations for prior distributions in a Bayesian estimation matrix using Markov Chain Monte Carlo (MCMC) specifications. A spatial residual trend analyses was then performed using autocorrelation indices which linked tabular data in SAS PROCLMIXED? with the egg-raft count data in ArcInfo?. The estimation matrix identified prolific habitats based on the covariate distance to the nearest house. An Ordinary kriged-based interpolator was then constructed in Geostatistical Analyst Extension of ArcGIS 9.3? based on the adjusted Bayesian estimates. For total Cx. quinquefasciatus egg-raft count, first order trend was fitted to the semivariogram at a partial sill of 5.931 km, nugget of 6.374 km, lag size of 7.184 km, and a range of 31.02 km using 12 lags. We assessed the performance accuracy of the interpolation procedures based on the magnitude and distribution of errors between observed and model-predicted values using Voroni tessella- tions. These residuals divided the space between the individual georeferenced Cx. quinquefasciatus habitats by XY coordinates in 2-dimenisional space which revealed that the geophysical parameter error residuals in the interpolation model were within normal statistical limitations. Newer GIS software and WV-1 data can generate highly accurate predictive Cx. quinquefasciatus habitat distribution models which can target prolific habitats of based on field-sampled count data. Our results suggest it may be unnecessary to manage all Cx. quinquefasciatus habitats to obtain significant reductions in incidence and prevalence of WNV in Trinidad.

Arbovirus and seizures

The high prevalence and spread of arthropod-borne viruses(arboviruses)make them an important cause of viral encephalitis in humans.Most epidemic viral encephalitides have an etiology associated with arboviruses.Among various arboviruses,the Japanese encephalitis virus,West Nile virus,Zika virus,Dengue virus and Chikungunya virus can induce seizures.Arboviruses of the genus Flavivirus are usually transmitted by mosquitoes and other host animals.These vector-borne pathogens can cause epidemic viral encephalitis.Seizures may not be the major manifestation in these viral encephalitides,but may predict a poor prognosis.In this article,we discuss the relationships between these viruses and seizures from perspectives of clinical characteristics,pathogenesis,prognosis and treatments of each.