Genome Sequencing of West Nile Virus from Human Cases in Greece, 2012

A West Nile Virus (WNV) lineage 2 strain, named Nea Santa-Greece-2010, has been demonstrated to be responsible for the large outbreaks of neuroinvasive disease (WNND) that have been occurring in Greece since 2010, based on sequence similarities of viral isolates identified between 2010–2012. However, knowledge on the evolution of this strain is scarce because only partial WNV genome sequences are available from Greece. The aim of this study was to get the complete genome sequence of WNV from patients with infection. To this aim, plasma and urine samples collected during the 2012 Greek outbreak were retrospectively investigated. Full WNV genome sequence was obtained from a patient with WNND. The genome had 99.7% sequence identity to Nea Santa, higher than to other related WNV lineage 2 strains, and five amino acid changes apparently not relevant for viral pathogenicity or fitness. In addition, infection by WNV lineage 2 was confirmed in additional nine patients with WNND; in three of them the infection with WNV Nea Santa was demonstrated by sequencing. In conclusion, this study characterized for the first time a WNV full genome from a patient with WNND from Greece, demonstrated the persistence of the Nea Santa strain, and suggested that the virus might have locally evolved.     

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.     

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.     

West Nile Virus Neuroinfection in Humans: Peripheral Biomarkers of Neuroinflammation and Neuronal Damage

Among emerging arthropod-borne viruses (arbovirus), West Nile virus (WNV) is a flavivirus that can be associated with severe neuroinvasive infections in humans. In 2018, the European WNV epidemic resulted in over 2000 cases, representing the most important arboviral epidemic in the European continent. Characterization of inflammation and neuronal biomarkers released during WNV infection, especially in the context of neuronal impairments, could provide insight into the development of predictive tools that could be beneficial for patient outcomes. We first analyzed the inflammatory signature in the serum of WNV-infected mice and found increased concentrations of several inflammatory cytokines. We next analyzed serum and cerebrospinal-fluid (CSF) samples from a cohort of patients infected by WNV between 2018 and 2019 in Hungary to quantify a large panel of inflammatory cytokines and neurological factors. We found higher levels of inflammatory cytokines (e.g., IL4, IL6, and IL10) and neuronal factors (e.g., BDNF, GFAP, MIF, TDP-43) in the sera of WNV-infected patients with neuroinvasive disease. Furthermore, the serum inflammatory profile of these patients persisted for several weeks after initial infection, potentially leading to long-term sequelae and having a deleterious effect on brain neurovasculature. This work suggests that early signs of increased serum concentrations of inflammatory cytokines and neuronal factors could be a signature underlying the development of severe neurological impairments. Biomarkers could play an important role in patient monitoring to improve care and prevent undesirable outcomes.     

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.     

Clinical Manifestations and Outcomes of West Nile Virus Infection

Since the emergence of West Nile virus (WNV) in North America in 1999, understanding of the clinical features, spectrum of illness and eventual functional outcomes of human illness has increased tremendously. Most human infections with WNV remain clinically silent. Among those persons developing symptomatic illness, most develop a self-limited febrile illness. More severe illness with WNV (West Nile neuroinvasive disease, WNND) is manifested as meningitis, encephalitis or an acute anterior (polio) myelitis. These manifestations are generally more prevalent in older persons or those with immunosuppression. In the future, a more thorough understanding of the long-term physical, cognitive and functional outcomes of persons recovering from WNV illness will be important in understanding the overall illness burden.     

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。     

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.     

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.     

Epidemiology of human West Nile virus infections in the European Union and European Union enlargement countries, 2010 to 2018

BackgroundWest Nile virus (WNV) circulates in an enzootic cycle involving mosquitoes and birds; humans are accidental hosts.AimWe analysed human WNV infections reported between 2010 and 2018 to the European Centre for Disease Prevention and Control to better understand WNV epidemiology.MethodsWe describe probable and confirmed autochthonous human cases of WNV infection reported by European Union (EU) and EU enlargement countries. Cases with unknown clinical manifestation or with unknown place of infection at NUTS 3 or GAUL 1 level were excluded from analysis.ResultsFrom southern, eastern and western Europe, 3,849 WNV human infections and 379 deaths were reported. Most cases occurred between June and October. Two large outbreaks occurred, in 2010 (n = 391) and in 2018 (n = 1,993). The outbreak in 2018 was larger than in all previous years and the first cases were reported unusually early. The number of newly affected areas (n = 45) was higher in 2018 than in previous years suggesting wider spread of WNV.ConclusionReal-time surveillance of WNV infections is key to ensuring that clinicians and public health authorities receive early warning about the occurrence of cases and potential unusual seasonal patterns. Human cases may appear shortly after first detection of animal cases. Therefore, public health authorities should develop preparedness plans before the occurrence of human or animal WNV infections.     

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.     

Seroprevalence of West Nile virus infection in solid organ transplant recipients

A.G. Freifeld, J. Meza, B. Schweitzer, L. Shafer, A.C. Kalil. A.R. Sambol. Seroprevalence of West Nile virus infection in solid organ transplant recipients.
Transpl Infect Dis 2010: 12: 120–126. All rights reserved Background. Of people infected with mosquito‐borne West Nile virus (WNV), <1% develop neuroinvasive disease (NID). Population studies suggest that people older than 65 years may be at higher risk for neurologic symptoms. It has been suggested that solid organ transplant (SOT) recipients are also at higher risk for WNV NID, but definitive serologic and epidemiologic data are lacking. Methods. A serologic screening survey, using a US Food & Drug Administration‐approved enzyme‐linked immunosorbant assay to detect WNV immunoglobulin‐G (IgG) antibody responses in cohorts of SOT recipients and non‐immunocompromised controls, was undertaken at a large Midwestern university organ transplant center in the aftermath of the summer 2003 WNV regional outbreak. Hemagglutination‐inhibition testing was used to confirm WNV IgG‐positive results and differentiate them from positive results caused by Saint Louis encephalitis virus, another flavivirus that is endemic in the Midwestern US. Findings. The rate of WNV IgG‐seropositive responses did not differ between SOT recipients and non‐immunocompromised controls, and were 12% and 10%, respectively. Retrospective chart review showed no documented WNV NID in the seropositive SOT recipients, suggesting an incidence of WNV NID may be as low as 0.7% in this population. Interpretation. Asymptomatic WNV infection is common among immunocompromised SOT patients, occurring as often as it does in non‐immunocompromised controls. Our data indicated that severe WNV NID is less frequent in SOT patients, contrary to what has been suggested in other studies.     

Unprecedented increase of West Nile virus neuroinvasive disease,Spain, summer 2020

Cases of West Nile neuroinvasive disease (WNND) in Spain increased in summer 2020. Here we report on this increase and the local, regional and national public health measures taken in response. We analysed data from regional surveillance networks and the National Epidemiological Surveillance Network, both for human and animal West Nile virus (WNV) infection. During the 2020 season, a total of 77 human cases of WNV infection (median age 65 years; 60% males) were detected in the south-west of Spain; 72 (94%) of these cases developed WNND, presenting as meningoencephalitis, seven of which were fatal. In the previous two decades, only six human cases of WNND were detected in Spain. Reduced activities for vector control this season, together with other factors, might have contributed to the massive increase. Public health measures including vector control, campaigns to raise awareness among physicians and the general population, and interventions to ensure the safety of donations of blood products, organs, cells and tissues were effective to reduce transmission. Going forward, maintenance of vector control activities and an update of the vector-borne diseases response plan in Spain is needed.     

West Nile and Usutu Virus Introduction via Migratory Birds: A Retrospective Analysis in Italy

The actual contribution of migratory birds in spreading West Nile (WNV) and Usutu virus (USUV) across Europe and from Africa to old countries is still controversial. In this study, we reported the results of molecular and serological surveys on migrating birds sampled during peaks of spring and autumn migration at 11 Italian sites located along important flyways, from 2012 to 2014. A total of 1335 specimens made of individual or pooled sera, and organs from 275 dead birds were tested for WNV and USUV RNA by real time PCR (RT-PCR). Furthermore, sera were tested by serum neutralization assay for detecting WNV and USUV neutralizing antibodies. Molecular tests detected WNV lineage 2 RNA in a pool made of three Song Thrush (Turdus philomelos) sera sampled in autumn, and lineage 1 in kidneys of six trans-Saharan birds sampled in spring. Neutralizing antibodies against WNV and USUV were found in 5.80% (n = 72; 17 bird species) and 0.32% (n = 4; 4 bird species) of the tested sera, respectively. Our results do not exclude the role of migratory birds as potential spreaders of WNV and USUV from Africa and Central Europe to Mediterranean areas and highlight the importance of a more extensive active surveillance of zoonotic viruses.     

West Nile and Usutu Viruses’ Surveillance in Birds of the Province of Ferrara,Italy, from 2015 to 2019

West Nile (WNV) and Usutu (USUV) viruses are mosquito-borne flaviviruses. Thanks to their importance as zoonotic diseases, a regional plan for surveillance of Arboviruses was implemented in Emilia-Romagna in 2009. The province of Ferrara belongs to the Emilia-Romagna region, and it is an endemic territory for these viruses, with favorable ecological conditions for abundance of mosquitoes and wild birds. From 2015 to 2019, we collected 1842 dead-found birds at a wildlife rehabilitation center, which were analysed by three different PCRs for the detection of WNV and USUV genomes. August was characterized by the highest infection rate for both viruses. Columbiformes scored the highest USUV prevalence (8%), while Galliformes and Strigiformes reported the highest prevalence for WNV (13%). Among Passeriformes (the most populated Order), Turdus merula was the most abundant species and scored the highest prevalence for both viruses. To optimize passive surveillance plans, monitoring should be focused on the summer and towards the avian species more prone to infection by both viruses.     

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.     

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.