Antiviral Cytokine Response in Neuroinvasive and Non-Neuroinvasive West Nile Virus Infection

Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification of 13 human cytokines. The panel included cytokines associated with innate and early pro-inflammatory immune responses (TNF-α/IL-6), Th1 (IL-2/IFN-γ), Th2 (IL-4/IL-5/IL-9/IL-13), Th17 immune response (IL-17A/IL-17F/IL-21/IL-22) and the key anti-inflammatory cytokine IL-10. Elevated levels of IFN-γ were detected in 71.7% of CSF and 22.7% of serum samples (p = 0.003). Expression of IL-2/IL-4/TNF-α and Th1 17 cytokines (IL-17A/IL-17F/IL-21) was detected in the serum but not in the CSF (except one positive CSF sample for IL-17F/IL-4). While IL-6 levels were markedly higher in the CSF compared to serum (CSF median 2036.71, IQR 213.82–6190.50; serum median 24.48, IQR 11.93–49.81; p < 0.001), no difference in the IL-13/IL-9/IL-10/IFN-γ/IL-22 levels in serum/CSF was found. In conclusion, increased concentrations of the key cytokines associated with innate and early acute phase responses (IL-6) and Th1 type immune responses (IFN-γ) were found in the CNS of patients with WNV infection. In contrast, expression of the key T-cell growth factor IL-2, Th17 cytokines, a Th2 cytokine IL-4 and the proinflammatory cytokine TNF-α appear to be concentrated mainly in the periphery.     

Characteristics of Chimeric West Nile Virus Based on the Japanese Encephalitis Virus SA14-14-2 Backbone

West Nile virus disease (WND) is an arthropod-borne zoonosis responsible for nonspecific fever or severe encephalitis. The pathogen is West Nile virus belonging to the genus Flavivirus, family Flaviviridae. Every year, thousands of cases were reported, which poses significant public health risk. Here, we constructed a West Nile virus chimera, ChiVax-WN01, by replacing the prMΔE gene of JEV SA14-14-2 with that of the West Nile virus NY99. The ChiVax-WN01 chimera showed clear, different characters compared with that of JEV SA14-14-2 and WNV NY99 strain. An animal study indicated that the ChiVax-WN01 chimera presented moderate safety and immunogenicity for 4-week female BALB/c mice.     

A Potential Role for Substance P in West Nile Virus Neuropathogenesis

Of individuals who develop West Nile neuroinvasive disease (WNND), ~10% will die and >40% will develop long-term complications. Current treatment recommendations solely focus on supportive care; therefore, we urgently need to identify novel and effective therapeutic options. We observed a correlation between substance P (SP), a key player in neuroinflammation, and its receptor Neurokinin-1 (NK1R). Our study in a wild-type BL6 mouse model found that SP is upregulated in the brain during infection, which correlated with neuroinvasion and damage to the blood–brain barrier. Blocking the SP/NK1R interaction beginning at disease onset modestly improved survival and prolonged time to death in a small pilot study. Although SP is significantly increased in the brain of untreated WNND mice when compared to mock-infected animals, levels of WNV are unchanged, indicating that SP likely does not play a role in viral replication but may mediate the immune response to infection. Additional studies are necessary to define if SP plays a mechanistic role or if it represents other mechanistic pathways.     

Large Human Outbreak of West Nile Virus Infection in North-Eastern Italy in 2012

Human cases of West Nile virus (WNV) disease have been reported in Italy since 2008. So far, most cases have been identified in north-eastern Italy, where, in 2012, the largest outbreak of WNV infection ever recorded in Italy occurred. Most cases of the 2012 outbreak were identified in the Veneto region, where a special surveillance plan for West Nile fever was in place. In this outbreak, 25 cases of West Nile neuroinvasive disease and 17 cases of fever were confirmed. In addition, 14 WNV RNA-positive blood donors were identified by screening of blood and organ donations and two cases of asymptomatic infection were diagnosed by active surveillance of subjects at risk of WNV exposure. Two cases of death due to WNND were reported. Molecular testing demonstrated the presence of WNV lineage 1 in all WNV RNA-positive patients and, in 15 cases, infection by the novel Livenza strain was ascertained. Surveillance in other Italian regions notified one case of neuroinvasive disease in the south of Italy and two cases in Sardinia. Integrated surveillance for WNV infection remains a public health priority in Italy and vector control activities have been strengthened in areas of WNV circulation.     

Infection with Non-Lethal West Nile Virus Eg101 Strain Induces Immunity that Protects Mice against the Lethal West Nile Virus NY99 Strain

Herein we demonstrate that infection of mice with West Nile virus (WNV) Eg101 provides protective immunity against lethal challenge with WNV NY99. Our data demonstrated that WNV Eg101 is largely non-virulent in adult mice when compared to WNV NY99. By day 6 after infection, WNV-specific IgM and IgG antibodies, and neutralizing antibodies were detected in the serum of all WNV Eg101 infected mice. Plaque reduction neutralization test data demonstrated that serum from WNV Eg101 infected mice neutralized WNV Eg101 and WNV NY99 strains with similar efficiency. Three weeks after infection, WNV Eg101 immunized mice were challenged subcutaneously or intracranially with lethal dose of WNV NY99 and observed for additional three weeks. All the challenged mice were protected against disease and no morbidity and mortality was observed in any mice. In conclusion, our data for the first time demonstrate that infection of mice with WNV Eg101 induced high titers of WNV specific IgM and IgG antibodies, and cross-reactive neutralizing antibodies, and the resulting immunity protected all immunized animals from both subcutaneous and intracranial challenge with WNV NY99. These observations suggest that WNV Eg101 may be a suitable strain for the development of a vaccine in humans against virulent strains of WNV.     

West Nile Virus Drug Discovery

The outbreak of West Nile virus (WNV) in 1999 in the USA, and its continued spread throughout the Americas, parts of Europe, the Middle East and Africa, underscored the need for WNV antiviral development. Here, we review the current status of WNV drug discovery. A number of approaches have been used to search for inhibitors of WNV, including viral infection-based screening, enzyme-based screening, structure-based virtual screening, structure-based rationale design, and antibody-based therapy. These efforts have yielded inhibitors of viral or cellular factors that are critical for viral replication. For small molecule inhibitors, no promising preclinical candidate has been developed; most of the inhibitors could not even be advanced to the stage of hit-to-lead optimization due to their poor drug-like properties. However, several inhibitors developed for related members of the family Flaviviridae, such as dengue virus and hepatitis C virus, exhibited cross-inhibition of WNV, suggesting the possibility to re-purpose these antivirals for WNV treatment. Most promisingly, therapeutic antibodies have shown excellent efficacy in mouse model; one of such antibodies has been advanced into clinical trial. The knowledge accumulated during the past fifteen years has provided better rationale for the ongoing WNV and other flavivirus antiviral development.     

The Innate Immune Playbook for Restricting West Nile Virus Infection

West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes annual epidemics of encephalitic disease throughout the world. Despite the ongoing risk to public health, no approved vaccines or therapies exist for use in humans to prevent or combat WNV infection. The innate immune response is critical for controlling WNV replication, limiting virus-induced pathology, and programming protective humoral and cell-mediated immunity to WNV infection. The RIG-I like receptors, Toll-like receptors, and Nod-like receptors detect and respond to WNV by inducing a potent antiviral defense program, characterized by production of type I IFN, IL-1β and expression of antiviral effector genes. Recent research efforts have focused on uncovering the mechanisms of innate immune sensing, antiviral effector genes that inhibit WNV, and countermeasures employed by WNV to antagonize innate immune cellular defenses. In this review, we highlight the major research findings pertaining to innate immune regulation of WNV infection.     

Role of Natural Killer and Gamma-Delta T cells in West Nile Virus Infection

Natural Killer (NK) cells and Gamma-delta T cells are both innate lymphocytes that respond rapidly and non-specifically to viral infection and other pathogens. They are also known to form a unique link between innate and adaptive immunity. Although they have similar immune features and effector functions, accumulating evidence in mice and humans suggest these two cell types have distinct roles in the control of infection by West Nile virus (WNV), a re-emerging pathogen that has caused fatal encephalitis in North America over the past decade. This review will discuss recent studies on these two cell types in protective immunity and viral pathogenesis during WNV infection.     

Vaccines in Development against West Nile Virus

West Nile encephalitis emerged in 1999 in the United States, then rapidly spread through the North American continent causing severe disease in human and horses. Since then, outbreaks appeared in Europe, and in 2012, the United States experienced a new severe outbreak reporting a total of 5,387 cases of West Nile virus (WNV) disease in humans, including 243 deaths. So far, no human vaccine is available to control new WNV outbreaks and to avoid worldwide spreading. In this review, we discuss the state-of-the-art of West Nile vaccine development and the potential of a novel safe and effective approach based on recombinant live attenuated measles virus (MV) vaccine. MV vaccine is a live attenuated negative-stranded RNA virus proven as one of the safest, most stable and effective human vaccines. We previously described a vector derived from the Schwarz MV vaccine strain that stably expresses antigens from emerging arboviruses, such as dengue, West Nile or chikungunya viruses, and is strongly immunogenic in animal models, even in the presence of MV pre-existing immunity. A single administration of a recombinant MV vaccine expressing the secreted form of WNV envelope glycoprotein elicited protective immunity in mice and non-human primates as early as two weeks after immunization, indicating its potential as a human vaccine.     

A brief report of West Nile Virus neuroinvasive disease in the summer of 2012 in Hamilton,Ontario

West Nile neuroinvasive disease is a severe infectious disease that is associated with a high mortality rate, especially in immunocompromised hosts. Physicians who are aware of its clinical presentations may be able to order diagnostic tests more appropriately and avoid inappropriate treatment. In the present series, the cases of seven patients admitted to Hamilton Health Sciences (Hamilton, Ontario) in the summer of 2012 with a diagnosis of West Nile neuroinvasive disease were retrospectively reviewed based on available medical records. According to the clinical and laboratory criteria published by the Centers for Disease Control and Prevention, five cases were diagnosed as encephalitis, one case as meningitis and one case as meningomyelitis. Patients were managed supportively. Forty-three percent (three of seven) presented with rash, 71% (five of seven) did not report headache despite exhibiting neurological symptoms, 43% (three of seven) did not have fever on presentation and 37.5% of cerebrospinal fluid samples exhibited a neutrophil predominance. The mortality rate in the present series was 14.3% (one of seven), and 57.1% (four of seven) of the patients had residual symptoms on discharge and at follow-up.     

West Nile Virus Vaccination Protects against Usutu Virus Disease in Mice

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV-specific antibodies and USUV-specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV infection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD-1 and Ifnar1^−/− mice cross-neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD-1 mice, there was no difference in USUV titers between vaccinated and mock-vaccinated mice. However, in the Ifnar1^−/− model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock-vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development.     

Epidemiology and Clinical Manifestation of West Nile Virus Infections of Equines in Hungary, 2007–2020

West Nile virus (WNV) is an emerging pathogen in Hungary, causing severe outbreaks in equines and humans since 2007. The aim of our study was to provide a comprehensive report on the clinical signs of West Nile neuroinvasive disease (WNND) in horses in Hungary. Clinical details of 124 confirmed equine WNND cases were collected between 2007 and 2019. Data about the seasonal and geographical presentation, demographic data, clinical signs, treatment protocols, and disease progression were evaluated. Starting from an initial case originating from the area of possible virus introduction by migratory birds, the whole country became endemic with WNV over the subsequent 12 years. The transmission season did not expand significantly during the data collection period, but vaccination protocols should be always reviewed according to the recent observations. There was not any considerable relationship between the occurrence of WNND and age, breed, or gender. Ataxia was by far the most common neurologic sign related to the disease, but weakness, behavioral changes, and muscle fasciculation appeared frequently. Apart from recumbency combined with inappetence, no other clinical sign or treatment regime correlated with survival. The survival rate showed a moderate increase throughout the years, possibly due to the increased awareness of practitioners.     

Pathogenesis of West Nile Virus Lineage 2 in Domestic Geese after Experimental Infection

West Nile virus (WNV) is an emerging infectious pathogen circulating between mosquitoes and birds but also infecting mammals. WNV has become autochthonous in Germany, causing striking mortality rates in avifauna and occasional diseases in humans and horses. We therefore wanted to assess the possible role of free-ranging poultry in the WNV transmission cycle and infected 15 goslings with WNV lineage 2 (German isolate). The geese were monitored daily and sampled regularly to determine viremia, viral shedding, and antibody development by molecular and serological methods. Geese were euthanized at various time points post-infection (pi). All infected geese developed variable degrees of viremia from day 1 to day 10 (maximum) and actively shed virus from days 2 to 7 post-infection. Depending on the time of death, the WN viral genome was detected in all examined tissue samples in at least one individual by RT-qPCR and viable virus was even re-isolated, except for in the liver. Pathomorphological lesions as well as immunohistochemically detectable viral antigens were found mainly in the brain. Furthermore, all of the geese seroconverted 6 days pi at the latest. In conclusion, geese are presumably not functioning as important amplifying hosts but are suitable sentinel animals for WNV surveillance.     

Seroprevalence and Risk Factors for Equine West Nile Virus Infections in Eastern Germany, 2020

West Nile virus (WNV) infections were first detected in Germany in 2018, but information about WNV seroprevalence in horses is limited. The study’s overall goal was to gather information that would help veterinarians, horse owners, and veterinary-, and public health- authorities understand the spread of WNV in Germany and direct protective measures. For this purpose, WNV seroprevalence was determined in counties with and without previously registered WNV infections in horses, and risk factors for seropositivity were estimated. The cohort consisted of privately owned horses from nine counties in Eastern Germany. A total of 940 serum samples was tested by competitive panflavivirus ELISA (cELISA), and reactive samples were further tested by WNV IgM capture ELISA and confirmed by virus neutralization test (VNT). Information about potential risk factors was recorded by questionnaire and analyzed by logistic regression. A total of 106 serum samples showed antibodies against flaviviruses by cELISA, of which six tested positive for WNV IgM. The VNT verified a WNV infection for 54 samples (50.9%), while 35 sera neutralized tick-borne encephalitis virus (33.0%), and eight sera neutralized Usutu virus (7.5%). Hence, seroprevalence for WNV infection was 5.8% on average and was significantly higher in counties with previously registered infections (p = 0.005). The risk factor analysis showed breed type (pony), housing in counties with previously registered infections, housing type (24 h turn-out), and presence of outdoor shelter as the main significant risk factors for seropositivity. In conclusion, we estimated the extent of WNV infection in the resident horse population in Eastern Germany and showed that seroprevalence was higher in counties with previously registered equine WNV infections.     

Evidence of West Nile Virus Circulation in Lebanon

West Nile virus (WNV) has never been reported from Lebanon. Yet, this country is located on the flyway of migratory birds in the Middle East region. Serological screening was conducted to assess the potential circulation of this virus. Human, horse, and chicken sera were collected from the Bekaa and North districts. Specific IgG and IgY were first screened by ELISA. Then, positive samples were confirmed by plaque reduction neutralization test (PRNT). Besides this, adult mosquitoes were collected and tested for the presence of WNV RNA using conventional RT-PCR. Sera screening revealed a seroprevalence rate reaching 1.86% among humans and 2.47% among horses. Cross-reactions revealed by ELISA suggested the circulation of flaviviruses other than WNV. None of the tested mosquitoes was positive for WNV. The observed results constitute strong evidence of local exposure of the Lebanese population to this virus and the first report of equine WNV in Lebanon.     

The Effect of Fluctuating Incubation Temperatures on West Nile Virus Infection in Culex Mosquitoes

Temperature plays a significant role in the vector competence, extrinsic incubation period, and intensity of infection of arboviruses within mosquito vectors. Most laboratory infection studies use static incubation temperatures that may not accurately reflect daily temperature ranges (DTR) to which mosquitoes are exposed. This could potentially compromise the application of results to real world scenarios. We evaluated the effect of fluctuating DTR versus static temperature treatments on the infection, dissemination, and transmission rates and viral titers of Culex tarsalis and Culex quinquefasciatus mosquitoes for West Nile virus. Two DTR regimens were tested including an 11 and 15 °C range, both fluctuating around an average temperature of 28 °C. Overall, no significant differences were found between DTR and static treatments for infection, dissemination, or transmission rates for either species. However, significant treatment differences were identified for both Cx. tarsalis and Cx. quinquefasciatus viral titers. These effects were species-specific and most prominent later in the infection. These results indicate that future studies on WNV infections in Culex mosquitoes should consider employing realistic DTRs to reflect interactions most accurately between the virus, vector, and environment.     

西尼罗病毒感染免疫应答反应研究进展

西尼罗病毒病是由西尼罗病毒引起的一种人兽共患传染病,给人类和动物健康带来重大危害。虽现已有疫苗处在研究阶段,但仍没有人用疫苗获批上市。通过感染动物模型,有关西尼罗病毒免疫反应的研究已经开展。本文对固有免疫和获得性免疫在抵抗西尼罗病毒感染中的作用进行综述,为进一步研究西尼罗病毒激发免疫应答反应的机制和新型疫苗研制提供依据。