Lessons Learned from West Nile Virus Infection:Vaccinations in Equines and Their Implications for One Health Approaches.

Humans and equines are two dead-end hosts of the mosquito-borne West Nile virus (WNV) with similar susceptibility and pathogenesis. Since the introduction of WNV vaccines into equine populations of the United States of America (USA) in late 2002, there have been only sporadic cases of WNV infection in equines. These cases are generally attributed to unvaccinated and under-vaccinated equines. In contrast, due to the lack of a human WNV vaccine, WNV cases in humans have remained steadily high. An average of 115 deaths have been reported per year in the USA since the first reported case in 1999. Therefore, the characterization of protective immune responses to WNV and the identification of immune correlates of protection in vaccinated equines will provide new fundamental information about the successful development and evaluation of WNV vaccines in humans. This review discusses the comparative epidemiology, transmission, susceptibility to infection and disease, clinical manifestation and pathogenesis, and immune responses of WNV in humans and equines. Furthermore, prophylactic and therapeutic strategies that are currently available and under development are described. In addition, the successful vaccination of equines against WNV and the potential lessons for human vaccine development are discussed.

Immunoinformatics assisted profiling of West Nile virus proteome to determine immunodominant epitopes for the development of next-generation multi-peptide vaccine.

Emerging infectious diseases represent a significant threat to global health, with West Nile virus (WNV) being a prominent example due to its potential to cause severe neurological disorders alongside mild feverish conditions. Particularly prevalent in the continental United States, WNV has emerged as a global concern, with outbreaks indicating the urgent need for effective prophylactic measures. The current problem is that the absence of a commercial vaccine against WNV highlights a critical gap in preventive strategies against WNV. This study aims to address this gap by proposing a novel, multivalent vaccine designed using immunoinformatics approaches to elicit comprehensive humoral and cellular immune responses against WNV. The objective of the study is to provide a theoretical framework for experimental scientists to formulate of vaccine against WNV and tackle the current problem by generating an immune response inside the host. The research employs reverse vaccinology and subtractive proteomics methodologies to identify NP_041724.2 polyprotein and YP_009164950.1 truncated flavivirus polyprotein NS1 as the prime antigens. The selection process for epitopes focused on B and T-cell reactivity, antigenicity, water solubility, and non-allergenic properties, prioritizing candidates with the potential for broad immunogenicity and safety. The designed vaccine construct integrates these epitopes, connected via GPGPG linkers, and supplemented with an adjuvant with the help of another linker EAAAK, to enhance immunogenicity. Preliminary computational analyses suggest that the proposed vaccine could achieve near-universal coverage, effectively targeting approximately 99.74% of the global population, with perfect coverage in specific regions such as Sweden and Finland. Molecular docking and immune simulation studies further validate the potential efficacy of the vaccine, indicating strong binding affinity with toll-like receptor 3 (TLR-3) and promising immune response profiles, including significant antibody-mediated and cellular responses. These findings present the vaccine construct as a viable candidate for further development and testing. While the theoretical and computational results are promising, advancing from in-silico predictions to a tangible vaccine requires comprehensive laboratory validation. This next step is essential to confirm the vaccine's efficacy and safety in eliciting an immune response against WNV. Through this study, we propose a novel approach to vaccine development against WNV and contribute to the broader field of immunoinformatics, showcasing the potential to accelerate the design of effective vaccines against emerging viral threats. The journey from hypothesis to practical solution embodies the interdisciplinary collaboration essential for modern infectious disease management and prevention strategies.

Accelerating targeted mosquito control efforts through mobile West Nile virus detection.

BACKGROUND: Different mosquito control strategies have been implemented to mitigate or prevent mosquito-related public health situations. Modern mosquito control largely relies on multiple approaches, including targeted, specific treatments. Given this, it is becoming increasingly important to supplement these activities with rapid and mobile diagnostic capacities for mosquito-borne diseases. We aimed to create and test the applicability of a rapid diagnostic system for West Nile virus that can be used under field conditions. METHODS: In this pilot study, various types of adult mosquito traps were applied within the regular mosquito monitoring activity framework for mosquito control. Then, the captured specimens were used for the detection of West Nile virus RNA under field conditions with a portable qRT-PCR approach within 3-4 h. Then, positive samples were subjected to confirmatory RT-PCR or NGS sequencing in the laboratory to obtain genome information of the virus. We implemented phylogenetic analysis to characterize circulating strains. RESULTS: A total of 356 mosquito individuals representing 7 species were processed in 54 pools, each containing up to 20 individuals. These pools were tested for the presence of West Nile virus, and two pools tested positive, containing specimens from the Culex pipiens and Anopheles atroparvus mosquito species. As a result of subsequent sequencing, we present the complete genome of West Nile virus and Bagaza virus. CONCLUSIONS: The rapid identification of infected mosquitoes is the most important component of quick response adulticide or larvicide treatments to prevent human cases. The conceptual framework of real-time surveillance can be optimized for other pathogens and situations not only in relation to West Nile virus. We present an early warning system for mosquito-borne diseases and demonstrate its application to aid rapid-response mosquito control actions.

Do we protect ourselves against West Nile Virus? A systematic review on knowledge, attitudes, and practices and their determinants.

BACKGROUND: West Nile virus (WNV) is a mosquito-borne flavivirus. In humans, 80% of infections are asymptomatic, while approximately 20% experience influenza-like symptoms. Fewer than 1% develop the neuroinvasive form which can lead to encephalitis, meningitis, acute flaccid paralysis, and even death. The global spread of the virus to areas where it was not previously present has become a growing concern. Since the 2000 s, there have been numerous outbreaks affecting local and travelling populations worldwide. Given the lack of a vaccine, preventative measures are primarily focused on surveillance, vector control, and the use of personal protective behaviours (PPBs). The importance of PPBs is central to public health recommendations. However, translating these messages into coherent action by the public can prove challenging, as the uptake of such measures is inevitably influenced by socio-economic factors, awareness, knowledge, and risk perception. METHODS: A PRISMA-based systematic research was conducted on EMBASE, PubMed/MEDLINE, and Web of Science databases. PROSPERO registration number CRD42023459714. Quality of studies included in the final stage was evaluated using the Critical Appraisal Checklist for Cross-Sectional Study (CEBMa). RESULTS: 2963 articles were screened, and 17 studies were included in the final round. Out of these, six were deemed of high quality, ten were of medium quality, and one was of low quality. In almost all studies considered, both awareness and knowledge of WNV transmission were above 90%, while concern about WNV ranged from 50% to 80%. Concern about the safety of repellents, either with or without DEET, ranged from 27% to 70%. The percentage of people actually using repellents ranged from 30% to 75%, with the lowest usage reported among individuals over 60 years old (29%) and pregnant women (33%), and the highest among students aged 9-11 (75%). Concern for West Nile Virus (WNV) was consistently linked to an increase in taking preventative measures, including the use of repellents, by two to four times across studies. The school-based intervention was effective in increasing the practice of removing standing water (AOR=4.6; 2.7-8.0) and wearing long clothing (AOR=2.4; 95%CI: 1.3-4.3), but did not have a significant impact on the use of repellents. CONCLUSIONS: The present systematic review provides an overview of the knowledge, attitudes, and practices (KAP) of WNV and their determinants. While concern about West Nile Virus (WNV) and its effects can be a significant motivator, it is important to promote evidence-based personal protective behaviours (PPBs) to counter unwarranted fears. For example, the use of repellents among the most vulnerable age groups. Given the geographical expansion of WNV, it is necessary to target the entire population preventively, including those who are difficult to reach and areas not yet endemic. The findings of this investigation could have significant implications for public health and support well-informed and effective communication strategies and interventions.

From mosquitoes to menace: Unveiling the dangers of West Nile virus in Pakistan.

Pakistan, a subtropical nation, has seen a rapid rise in Arboviral transmission in the past decade. Environmental hazards such as intense monsoon rains and yearly floods in addition to unsanitary living conditions pose an increased risk of arboviral infections. In recent years, the emergence and spread of West Nile virus (WNV), which can lead to a life-threatening meningoencephalitis, in Pakistan has alarmed the health care authorities of an impending outbreak. Health professionals and policymakers should give paramount importance to prevent its transmission across Pakistan as another arboviral outbreak would wreak havoc on the already fragile health infrastructure of the country. Proactive surveillance and prompt reporting are crucial for mitigating this threat as there are no vaccines available to prevent WNV infection.

A Chimeric Classical Insect-Specific Flavivirus Provides Complete Protection Against West Nile Virus Lethal Challenge in Mice.

West Nile virus (WNV), an arthropod-borne flavivirus, can cause severe symptoms, including encephalitis, and death, posing a threat to public health and the economy. However, there is still no approved treatment or vaccine available for humans. Here, we developed a novel vaccine platform based on a classical insect-specific flavivirus (cISF) YN15-283-02, which was derived from Culicoides. The cISF-WNV chimera was constructed by replacing prME structural genes of the infectious YN15-283-02 cDNA clone with those of WNV and successfully rescued in Aedes albopictus cells. cISF-WNV was nonreplicable in vertebrate cells and nonpathogenic in type I interferon receptor (IFNAR)-deficient mice. A single-dose immunization of cISF-WNV elicited considerable Th1-biased antibody responses in C57BL/6 mice, which was sufficient to offer complete protection against lethal WNV challenge with no symptoms. Our studies demonstrated the potential of the insect-specific cISF-WNV as a prophylactic vaccine candidate to prevent infection with WNV.

Predicted reduction in transmission from deployment of ivermectin-treated birdfeeders for local control of West Nile virus.

Ivermectin (IVM)-treated birds provide the potential for targeted control of Culex mosquitoes to reduce West Nile virus (WNV) transmission. Ingestion of IVM increases mosquito mortality, which could reduce WNV transmission from birds to humans and in enzootic maintenance cycles affecting predominantly bird-feeding mosquitoes and from birds to humans. This strategy might also provide an alternative method for WNV control that is less hampered by insecticide resistance and the logistics of large-scale pesticide applications. Through a combination of field studies and modeling, we assessed the feasibility and impact of deploying IVM-treated birdfeed in residential neighborhoods to reduce WNV transmission. We first tracked 105 birds using radio telemetry and radio frequency identification to monitor their feeder usage and locations of nocturnal roosts in relation to five feeder sites in a neighborhood in Fort Collins, Colorado. Using these results, we then modified a compartmental model of WNV transmission to account for the impact of IVM on mosquito mortality and spatial movement of birds and mosquitoes on the neighborhood level. We found that, while the number of treated lots in a neighborhood strongly influenced the total transmission potential, the arrangement of treated lots in a neighborhood had little effect. Increasing the proportion of treated birds, regardless of the WNV competency status, resulted in a larger reduction in infection dynamics than only treating competent birds. Taken together, model results indicate that deployment of IVM-treated feeders could reduce local transmission throughout the WNV season, including reducing the enzootic transmission prior to the onset of human infections, with high spatial coverage and rates of IVM-induced mortality in mosquitoes. To improve predictions, more work is needed to refine estimates of daily mosquito movement in urban areas and rates of IVM-induced mortality. Our results can guide future field trials of this control strategy.

Assessing the risk of West Nile Virus seasonal outbreaks and its vector control in an urbanizing bird community: An integrative R0-modelling study in the city of Merida, Mexico.

Urbanization is a global trend associated with key socio-economic issues, one of them being to control the transmission of infectious diseases to a urban fraction of the world's population that shall reach 68% in 2050. While urban growth has been shown to favor mosquito species responsible for the transmission of the West Nile Virus (WNV), a major human arbovirosis, the effects of concomitant changes in the host bird communities remain hard to anticipate albeit essential to quantify disease risk and to plan control initiatives. We developed a R0 modelling of WNV transmission in a urban bird community to assess the risk of outbreak in Merida, one of the cities with the highest growth rate in Mexico. The model was parameterized using ecological and epidemiological data collected over the past 15-years on the local vector, Culex quinquefasciatus, and avian community. We identified a 3-weeks summer period during which the vector population strongly amplifies the WNV enzootic transmission and lead to a significant risk of outbreaks in humans. Extensive sensitivity analyses showed that urbanization induced changes in the bird community could lead to an up-to 6-fold increase in the duration of the risk period, while the daily risk could rise by 40%. Interestingly, the increase in Quiscalus mexicanus abundance had 4-5 times larger impact than any other change in the bird community. In such a context, annihilating the current and future risk of WNV outbreaks in Merida requires reducing the mosquito population by 13% and up to 56%, respectively. This study provides an integrative assessment of the current and future risks of WNV outbreak in the fast urbanizing city of Merida, and points toward the implementation of epidemiological monitoring combined with preemptive measures targeting both C. quinquefasciatus and Q. mexicanus populations, as they are expected to have synergistic effects.

Immunization with Usutu virus and with a chimeric West Nile virus (WNV) harboring Usutu-E protein protects immunocompetent adult mice against lethal challenges with different WNV lineage 1 and 2 strains.

West Nile virus (WNV) and Usutu virus (USUV), two antigenically related flaviviruses co-circulating in Europe, can cause severe neurological disease in animals and humans. The immune response against USUV and WNV and their immunopathogenesis are still poorly investigated. Here we present results upon sequential infections of adult immunocompetent CD-1 and BALB/c mice primed with two different doses (high dose, HD or low dose, LD) of an USUV isolate and challenged with HD or LD of three different WNV isolates. CD-1 and BALB/c LD USUV-primed mice, regardless of the dose, are largely protected from lethal WNV challenges despite showing no detectable neutralizing antibodies. Furthermore, mice immunized with a chimeric virus harboring the E protein of USUV within the WNV backbone (WNVE-USUV) are protected against a lethal challenge with WNV. We believe these findings could contribute to understanding the dynamics of the interaction during sequential infection of these two flaviviruses.

Safety Procedures to Work with West Nile Virus in Biosafety Level 3 Facilities.

West Nile virus (WNV) can cause severe and sometimes fatal disease, but we do not have treatments or therapeutics to manage these outcomes. Since its introduction to the USA in 1999, WNV has been handled in a biosafety level 3 laboratory to decrease risk to researchers, requiring strict safety protocols and important considerations with planning experiments. Recent changes in US guidelines suggest that WNV can be handled at a lower biosafety level due to its endemicity in the USA and generally minor symptoms, but some research still requires the use of the agent at biosafety level 3. This chapter will briefly discuss the considerations of biosafety when working with WNV.

Development of Antibody-Based Therapeutics Against West Nile Virus in Plants.

Since its discovery in 1937 in the West Nile district of Uganda, West Nile virus (WNV) has been one of the leading causes of mosquito-transmitted infectious diseases (Smithburn, Burke, Am J Trop Med 20:22, 1940). Subsequently, it spread to Europe, Asia, Australia, and finally North America in 1999 (Sejvar, Ochsner 5(3):6-10, 2003). Worldwide outbreaks have continued to increase since the 1990s (Chancey et al, Biomed Res Int 2015:376230, 2015). According to the Center for Disease Control and Prevention, more than 51,000 cases of WNV infection and nearly 2400 cases of WNV-related death were reported in the USA from 1999 to 2019. The estimated economic impact of WNV infections is close to 800 million dollars in the USA from 1999 to 2012 (Barrett, Am J Trop Med Hyg 90:389, 2014).

Assessment of Truck-Mounted Area-Wide S-methoprene Applications to Manage West Nile Virus Vector Species in the Suburbs of Chicago, IL, USA.

West Nile virus remains the leading cause of arboviral neuroinvasive disease in the United States, despite extensive efforts to control the mosquito vectors involved in transmission. In this study, we evaluated the effectiveness of Altosid SR-20 (active ingredient, S-methoprene 20%) larvicide applications using truck-mounted ultra-low volume (ULV) dispersal equipment to target Culex pipiens Linnaeus (Diptera: Culicidae) and Cx. restuans (Theobald)larvae. A combination of emergence bioassays, open-field measurements of deposited S-methoprene and spray distribution using gas chromatography-mass spectrometry, and assessments of adult Culex spp. populations in response to applications were conducted over the summer of 2020 within the North Shore Mosquito Abatement District (IL, USA). Open-field applications revealed that dispersed Altosid SR-20 using ULV equipment was effective (75% emergence inhibition in susceptible lab strain Cx. pipiens larvae) up to 53 m. In suburban neighborhood applications, we found that S-methoprene deposition and larval emergence inhibition (EI) in front yards did not differ significantly from backyards. An overall EI of 46% and 28% were observed for laboratory strain Cx. pipiens and wild Cx. restuans larvae respectively, and both had an EI significantly higher than the untreated control group. The EI of exposed wild Cx. pipiens larvae did not differ from the untreated controls, suggesting an increased tolerance to S-methoprene. No difference in abundance of gravid or host-seeking adult Culex spp. post-application was detected between treated and untreated sites. These results document the ability of area-wide application to distribute S-methoprene, but this strategy will need further modifications and evaluation for Culex spp. management.

Febre do Nilo Ocidental / West Nile fever

A Febre do Nilo Ocidental (FNO) é uma infecção viral transmitida por meio da picada de mosquitos, principalmente do gênero Culex (pernilongo) infectados pelo agente etiológico, cujos hospedeiros naturais são algumas espécies de aves silvestres, que atuam como amplificadoras do vírus e como fonte de infecção para os vetores. Tal doença pode também infectar humanos, equinos, primatas e outros mamíferos sendo que, homem e equídeos são considerados hospedeiros acidentais e terminais, uma vez que a contaminação pelo vírus se dá por um curto período de tempo e em níveis insuficientes para infectar mosquitos, encerrando o ciclo de transmissão (WHO, 2017; ECDC , 2022a; CDC, 2017; BRASIL, 2021)

West Nile Fever (WNF) is a viral infection transmitted through the bite of mosquitoes, mainly of the Culex genus (legged mosquito) infected by the etiological agent, whose natural hosts are some species of wild birds, which act as amplifiers of the virus and as source of infection for the vectors. Such a disease can also infect humans, horses, primates and other mammals, and humans and horses are considered accidental and terminal hosts, since contamination by the virus occurs for a short period of time and at levels insufficient to infect mosquitoes, ending the transmission cycle (WHO, 2017; ECDC, 2022a; CDC, 2017). ; BRAZIL, 2021)

Evaluation of the effectiveness of the California mosquito-borne virus surveillance & response plan, 2009-2018.

Local vector control and public health agencies in California use the California Mosquito-Borne Virus Surveillance and Response Plan to monitor and evaluate West Nile virus (WNV) activity and guide responses to reduce the burden of WNV disease. All available data from environmental surveillance, such as the abundance and WNV infection rates in Culex tarsalis and the Culex pipiens complex mosquitoes, the numbers of dead birds, seroconversions in sentinel chickens, and ambient air temperatures, are fed into a formula to estimate the risk level and associated risk of human infections. In many other areas of the US, the vector index, based only on vector mosquito abundance and infection rates, is used by vector control programs to estimate the risk of human WNV transmission. We built models to determine the association between risk level and the number of reported symptomatic human disease cases with onset in the following three weeks to identify the essential components of the risk level and to compare California's risk estimates to vector index. Risk level calculations based on Cx. tarsalis and Cx. pipiens complex levels were significantly associated with increased human risk, particularly when accounting for vector control area and population, and were better predictors than using vector index. Including all potential environmental components created an effective tool to estimate the risk of WNV transmission to humans in California.

Correlation Between Serum Amyloid A and Antibody Response to West Nile Virus Vaccine Antigen in Healthy Horses.

The purpose of this study was to establish if peak serum amyloid A (SAA) concentrations can be used to determine an appropriate immune response to a vaccine containing West Nile Virus (WNV) antigen. A pilot study with 20 clinically healthy horses was performed to identify peak SAA concentration postvaccination with a commercial multivalent WNV vaccine. Blood was collected for SAA at 0, 24, 48, 72, 96, 168 hours postvaccination. Serum for WNV serum neutralization antibody testing was obtained immediately prior to and 30 days postvaccination. An additional 40 horses underwent the study protocol, but with SAA measurements acquired at 0-hours and 72-hours postvaccination. Ninety percent of the population had an increase in SAA in response to WNV vaccination, though no significant correlation was identified between SAA peak and antibody titer fold changes. WNV antibody titer fold changes between pre- and post-vaccination revealed 57% of horses had increased fold changes, 30% had no fold changes and 13% had negative fold changes. There was a negative correlation between age and SAA response (P = .0008). The main conclusions were SAA response postvaccination against WNV does not appear to mirror antibody response. Age appears to significantly affect SAA response. Further, vaccination with WNV antigen may not consistently induce a positive increase in WNV antibodies.

Human Monoclonal Antibodies against NS1 Protein Protect against Lethal West Nile Virus Infection.

Envelope protein-targeted vaccines for flaviviruses are limited by concerns of antibody-dependent enhancement (ADE) of infections. Nonstructural protein 1 (NS1) provides an alternative vaccine target that avoids this risk since this protein is absent from the virion. Beyond its intracellular role in virus replication, extracellular forms of NS1 function in immune modulation and are recognized by host-derived antibodies. The rational design of NS1-based vaccines requires an extensive understanding of the antigenic sites on NS1, especially those targeted by protective antibodies. Here, we isolated human monoclonal antibodies (MAbs) from individuals previously naturally infected with WNV, mapped their epitopes using structure-guided mutagenesis, and evaluated their efficacy in vivo against lethal WNV challenge. The most protective epitopes clustered at three antigenic sites that are exposed on cell surface forms of NS1: (i) the wing flexible loop, (ii) the outer, electrostatic surface of the wing, and (iii) the spaghetti loop face of the ß-ladder. One additional MAb mapped to the distal tip of the ß-ladder and conferred a lower level of protection against WNV despite not binding to NS1 on the surface of infected cells. Our study defines the epitopes and modes of binding of protective anti-NS1 MAb antibodies following WNV infection, which may inform the development of NS1-based countermeasures against flaviviruses. IMPORTANCE Therapeutic antibodies against flaviviruses often promote neutralization by targeting the envelope protein of the virion. However, this approach is hindered by a possible concern for antibody-dependent enhancement of infection and paradoxical worsening of disease. As an alternative strategy, antibodies targeting flavivirus nonstructural protein 1 (NS1), which is absent from the virion, can protect against disease and do not cause enhanced infection. Here, we evaluate the structure-function relationships and protective activity of West Nile virus (WNV) NS1-specific monoclonal antibodies (MAbs) isolated from the memory B cells of a naturally infected human donor. We identify several anti-NS1 MAbs that protect mice against lethal WNV challenge and map their epitopes using charge reversal mutagenesis. Antibodies targeting specific regions in the NS1 structure could serve as the basis for countermeasures that control WNV infection in humans.

Development of peptides targeting receptor binding site of the envelope glycoprotein to contain the West Nile virus infection.

West Nile virus (WNV), re-emerging neurotropic flavivirus, can cross the blood-brain barrier (BBB) and cause fatal encephalitis and meningitis. Infection of the human brain microvascular endothelial cells (hBMECs), building blocks of the BBB, represents the pivotal step in neuroinvasion. Domain III (DIII) of the envelope (E) glycoprotein is a key receptor-binding domain, thus, it is an attractive target for anti-flavivirus strategies. Here, two combinatorial phage display peptide libraries, Ph.D.-C7C and Ph.D.-12, were panned against receptor-binding site (RBS) on DIII to isolate peptides that could block DIII. From series of pannings, nine peptides (seven 7-mer cyclic and two 12-mer linear) were selected and overexpressed in E. coli SHuffle T5. Presence of disulfide bond in 7-mer peptides was confirmed with thiol-reactive maleimide labeling. Except for linear peptide 19 (HYSWSWIAYSPG), all peptides proved to be DIII binders. Among all peptides, 4 cyclic peptides (CTKTDVHFC, CIHSSTRAC, CTYENHRTC, and CLAQSHPLC) showed significant blocking of the interaction between DIII and hBMECs, and ability to neutralize infection in cultured cells. None of these peptides showed toxic or hemolytic activity. Peptides identified in this study may serve as potential candidates for the development of novel antiviral therapeutics against WNV.