Neuroinvasive Disease Research Articles

West Nile Virus: An Update Focusing on Southern Europe

West Nile Virus (WNV) is a zoonotic, vector-borne pathogen affecting humans and animals, particularly in Europe. The virus is primarily transmitted through mosquitoes that infect birds, which serve as the main reservoirs. Humans and horses are incidental hosts. This review focuses on the epidemiology of WNV in southern Europe, particularly its increasing prevalence. Methods included an extensive literature review and analysis of recent outbreaks. WNV is largely asymptomatic in humans, but a small percentage can develop West Nile neuroinvasive disease (WNND), leading to severe neurological symptoms and fatalities. Horses can also suffer from neurological complications, with high mortality rates. Climate change, migratory birds, and mosquito population dynamics contribute to the virus spread across Europe. Control efforts focus on vector management, and while vaccines are available for horses, none has been approved for humans. Surveillance, particularly of bird and mosquito populations, and further research into the virus molecular structure are crucial for understanding and mitigating future outbreaks.

Short-term effect of temperature and precipitation on the incidence of West Nile Neuroinvasive Disease in Europe: a multi-country case-crossover analysis

Short-term effect of temperature and precipitation on the incidence of West Nile Neuroinvasive Disease in Europe: a multi-country case-crossover analysis

The TLR7/8 agonist INI-4001 enhances the immunogenicity of a Powassan virus-like-particle vaccine.

Powassan virus (POWV) is a pathogenic tick-borne flavivirus that causes fatal neuroinvasive disease in humans. There are currently no approved therapies or vaccines for POWV infection. Here, we develop a POW virus-like-particle (POW-VLP) based vaccine adjuvanted with the novel synthetic Toll-like receptor 7/8 agonist INI-4001. We demonstrate that INI-4001 outperforms both alum and the Toll-like receptor 4 agonist INI-2002 in enhancing the immunogenicity of a dose-sparing POW-VLP vaccine in mice. INI-4001 increases the magnitude and breadth of the antibody response as measured by whole-virus ELISA, induces neutralizing antibodies measured by FRNT, reduces viral burden in the brain of infected mice measured by RT qPCR, and confers 100% protection from lethal challenge with both lineages of POWV. We show that the antibody response induced by INI-4001 is more durable than standard alum, and 80% of mice remain protected from lethal challenge 9-months post-vaccination. Lastly, we show that the protection elicited by INI-4001 adjuvanted POW-VLP vaccine is unaffected by either CD4+ or CD8+ T cell depletion and can be passively transferred to unvaccinated mice indicating that protection is mediated through humoral immunity. This study highlights the utility of novel synthetic adjuvants in VLP-based vaccines.

Cache Valley virus isolation from a horse in Veracruz State, Mexico

Cache Valley virus (CVV) was isolated from a clinically presenting horse in the Mexican Gulf region. CVV is linked to neuroinvasive disease in humans and fetal demise and defects in sheep. This is the first association between CVV and disease in horses. Implications for public and veterinary health are discussed.

In vitro and in vivo characterization of a novel West Nile virus lineage 2 strain

Over recent decades, West Nile virus (WNV) has continued to expand its geographical range, emerging in previously non-endemic areas, including northern Europe. In Europe, WNV lineage 2 strains are most prevalent and cause sporadic outbreaks of WNV disease in humans each transmission season. Here, we assessed the virulence of a newly emerged WNV lineage 2 strain that was isolated in the Netherlands in 2020 (WNV-NL20) and caused several cases of West Nile disease in humans and used a WNV lineage 2 strain related to major outbreaks of neuroinvasive disease in humans in central and south-eastern Europe in 2010 (WNV-578/10) as a reference. Infection of primary human cells of the blood-brain barrier in vitro did not show major differences in replication kinetics between WNV-578/10 and WNV-NL20. Experimental infection of mice showed that both WNV strains induced significant weight loss, neurological signs, and lethal disease. Neurological involvement was confirmed for both WNV strains by the presence of infectious virus and viral antigen in the brain. In conclusion, we show that the recent WNV-NL20 strain that emerged in the Netherlands is neurovirulent in mice. The use of in vitro and in vivo models to characterize the pathogenesis of emerging WNV strains may aid in predicting the neurovirulence of WNV infections in humans during potential future outbreaks.

Oropouche Virus: An Emerging Neuroinvasive Arbovirus.

Oropouche virus (OROV) is an arthropod-borne virus (arbovirus) in the Orthobunyavirus genus and Peribunyaviridae viral family that is endemic to parts of South America, Central America, and the Caribbean. It has recently emerged in Cuba, and travel-imported cases are recently being reported in the United States and Europe. Typically maintained in a sylvatic cycle between certain forest sloths, non-human primates, birds, and mosquitoes, OROV disease outbreaks can occur in an urban cycle between certain biting midges and/or mosquitoes and humans. Clinically, approximately 60% of infections are symptomatic with an abrupt fever and non-specific influenza-like illness within 3 to 10 days. Many initial OROV infections can present similarly to chikungunya, dengue, and Zika virus infections. Interestingly, OROV infections can follow a biphasic course with recurrence of symptoms approximately 1 week after initial symptom onset. Concerningly, similar to Zika virus, it appears that vertical transmission of OROV may occur with potentially adverse effects on fetal development including miscarriages. Neuroinvasion of OROV occurs in animal models, and human cases of meningitis, encephalitis, and peri-infectious Guillain-Barré syndrome have all been reported. Diagnosis is either through detection of OROV nucleic acid, OROV immunoglobulin M, or OROV neutralizing antibodies in the serum and/or cerebrospinal fluid. No antiviral treatments are available, and there are no current vaccines. Preventing mosquito and biting midge bites is key. Neurologists should be aware of and report any potential neuroinvasive OROV disease cases to local/state/territorial health departments. ANN NEUROL 2024.

Lrp1 facilitates infection of neurons by Jamestown Canyon virus.

Jamestown Canyon virus (JCV) is a bunyavirus and arbovirus responsible for neuroinvasive disease in the United States. Little is known about JCV pathogenesis, and no host factors required for cellular infection have been identified. Recently, we identified low-density lipoprotein receptor related protein 1 (Lrp1) as a host entry factor for two other bunyaviruses Rift Valley fever virus (RVFV) and Oropouche virus (OROV). Here, we assessed the role of Lrp1 in mediating JCV cellular infection of neurons. Both neuronal and non-neuronal immortalized cell lines deficient for Lrp1 displayed reduction in infection with JCV, and early stages of infection such as binding and internalization were impacted by lack of Lrp1. In primary rat neurons, Lrp1 was highly expressed, and the neurons were highly permissive for JCV infection. Treatment of primary neurons with recombinant receptor-associated protein (RAP), a high affinity ligand for Lrp1, resulted in reduced infectivity with JCV. In addition, pretreatment of cells with RVFV Gn inhibited JCV infection, suggesting that the two viruses may share overlapping binding sites. These results provide compelling evidence that Lrp1 is an important cellular factor for efficient infection by JCV, and thus multiple bunyaviruses with varying clinical manifestations and tissue tropism are facilitated by the host cell Lrp1. Reliance of multiple bunyaviruses on Lrp1 makes it a promising target for pan-bunyaviral antivirals and therapeutics.

The Re-Emergence of Neuroinvasive Flaviviruses in Croatia During the 2022 Transmission Season.

(Re-)emerging arboviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Usutu virus (USUV), are continuously increasing in incidence. We analyzed the epidemiological characteristics of flavivirus infections in humans, sentinel animals, and mosquitoes detected in the 2022 transmission season in Croatia. From April to November 2022, 110 hospitalized patients with neuroinvasive diseases (NID) were tested for the presence of arboviruses. RT-qPCR was used to detect TBEV, WNV, and USUV RNA. An ELISA and virus neutralization tests were used for the detection of flavivirus antibodies. TBEV infection was confirmed in 22 patients with NID. WNV NID was detected in six patients. TBE showed male predominance (81.8%; male-to-female ratio of 4.5:1). All but one WNV patients were males. TBE occurred from April to August, with the majority of patients (83.3%) being detected during the May-June-July period. WNV infections were recorded in August and September. In addition to human cases, asymptomatic WNV infections (IgM positive) were reported in 10 horses. For the first time in Croatia, WNV NID was observed in one horse that presented with neurological symptoms. Furthermore, USUV was confirmed in one dead blackbird that presented with neurological symptoms. A total of 1984 mosquitoes were collected in the City of Zagreb. Two Ae. albopictus pools tested positive for flavivirus RNA: one collected in July (USUV) and the other collected in August (WNV). A phylogenetic analysis of detected human and avian strains confirmed WNV lineage 2 and the USUV Europe 2 lineage. The presented results confirm the endemic presence of neuroinvasive flaviviruses in continental Croatia. The continuous monitoring of virus circulation in humans, sentinel animals, and mosquitoes is needed to reduce the disease burden.

Evidence of Lineage 1 and 3 West Nile Virus in Person with Neuroinvasive Disease, Nebraska, USA, 2023.

West Nile virus (WNV) is the most common cause of human arboviral disease in the contiguous United States, where only lineage 1 (L1) WNV had been found. In 2023, an immunocompetent patient was hospitalized in Nebraska with West Nile neuroinvasive disease and multisystem organ failure. Testing at the Centers for Disease Control and Prevention indicated an unusually high viral load and acute antibody response. Upon sequencing of serum and cerebrospinal fluid, we detected lineage 3 (L3) and L1 WNV genomes. L3 WNV had previously only been found in Central Europe in mosquitoes. The identification of L3 WNV in the United States and the observed clinical and laboratory features raise questions about the potential effect of L3 WNV on the transmission dynamics and pathogenicity of WNV infections. Determining the distribution and prevalence of L3 WNV in the United States and any public health and clinical implications is critical.

Differential susceptibility of human motor neurons to infection with Usutu and West Nile virus

West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion.

Laboratory Testing for Powassan Virus: Past, Present, and Future.

Powassan virus is a tick-borne flavivirus that can cause severe neuroinvasive disease, with areas of endemicity in the Northeast and Midwest United States, Canada, and Russia. Diagnosis is challenging and relies on a high index of suspicion and choosing the right test based on duration of infection and the patient's immune status. This review covers laboratory testing for Powassan virus, including historical considerations, modern options, and methods being developed in the research space.

CD11b maintains West Nile virus replication through modulation of immune response in human neuroblastoma cells

BackgroundWest Nile virus (WNV) is a rapidly spreading mosquito-borne virus accounted for neuroinvasive diseases. An insight into WNV-host factors interaction is necessary for development of therapeutic approaches against WNV infection. CD11b has key biological functions and been identified as a therapeutic target for several human diseases. The purpose of this study was to determine whether CD11b was implicated in WNV infection.MethodsSH-SY5Y cells with and without MEK1/2 inhibitor U0126 or AKT inhibitor MK-2206 treatment were infected with WNV. CD11b mRNA levels were assessed by real-time PCR. WNV replication and expression of stress (ATF6 and CHOP), pro-inflammatory (TNF-α), and antiviral (IFN-α, IFN-β, and IFN-γ) factors were evaluated in WNV-infected SH-SY5Y cells with CD11b siRNA transfection. Cell viability was determined by MTS assay.ResultsCD11b mRNA expression was remarkably up-regulated by WNV in a time-dependent manner. U0126 but not MK-2206 treatment reduced the CD11b induction by WNV. CD11b knockdown significantly decreased WNV replication and protected the infected cells. CD11b knockdown markedly increased TNF-α, IFN-α, IFN-β, and IFN-γ mRNA expression induced by WNV. ATF6 mRNA expression was reduced upon CD11b knockdown following WNV infection.ConclusionThese results demonstrate that CD11b is involved in maintaining WNV replication and modulating inflammatory as well as antiviral immune response, highlighting the potential of CD11b as a target for therapeutics for WNV infection.

Usutu virus NS4A suppresses the host interferon response by disrupting MAVS signaling

Usutu virus (USUV) is an emerging flavivirus that can infect birds and mammals. In humans, in severe cases, it may cause neuroinvasive disease. The innate immune system, and in particular the interferon response, functions as the important first line of defense against invading pathogens such as USUV. Many, if not all, viruses have developed mechanisms to suppress and/or evade the interferon response in order to facilitate their replication. The ability of USUV to antagonize the interferon response has so far remained largely unexplored. Using dual-luciferase reporter assays we observed that multiple of the USUV nonstructural (NS) proteins were involved in suppressing IFN-β production and signaling. In particular NS4A was very effective at suppressing IFN-β production. We found that NS4A interacted with the mitochondrial antiviral signaling protein (MAVS) and thereby blocked its interaction with melanoma differentiation-associated protein 5 (MDA5), resulting in reduced IFN-β production. The TM1 domain of NS4A was found to be essential for binding to MAVS. By screening a panel of flavivirus NS4A proteins we found that the interaction of NS4A with MAVS is conserved among flaviviruses. The increased understanding of the role of NS4A in flavivirus immune evasion could aid the development of vaccines and therapeutic strategies.

Early rise of West Nile fever in Israel, June 2024.

This report describes an unusual surge of West Nile fever in Israel in June 2024, during which 125 cases were diagnosed, compared with 4 cases on average during June in previous years (2014-23). Of the cases, 64 (62.1%) had neuroinvasive disease and 12 (9.6%) died; the 2024 case fatality rate was not significantly elevated vs the average rate in 2014-23. The early rise could be related to a temperature increase in spring and early summer of 2024.

Interspecies co-feeding transmission of Powassan virus between a native tick, Ixodes scapularis, and the invasive East Asian tick, Haemaphysalis longicornis

BackgroundPowassan virus, a North American tick-borne flavivirus, can cause severe neuroinvasive disease in humans. While Ixodes scapularis are the primary vectors of Powassan virus lineage II (POWV II), also known as deer tick virus, recent laboratory vector competence studies showed that other genera of ticks can horizontally and vertically transmit POWV II. One such tick is the Haemaphysalis longicornis, an invasive species from East Asia that recently established populations in the eastern USA and already shares overlapping geographic range with native vector species such as I. scapularis. Reports of invasive H. longicornis feeding concurrently with native I. scapularis on multiple sampled hosts highlight the potential for interspecies co-feeding transmission of POWV II. Given the absence of a clearly defined vertebrate reservoir host for POWV II, it is possible that this virus is sustained in transmission foci via nonviremic transmission between ticks co-feeding on the same vertebrate host. The objective of this study was to evaluate whether uninfected H. longicornis co-feeding in close proximity to POWV II-infected I. scapularis can acquire POWV independent of host viremia.MethodsUsing an in vivo tick transmission model, I. scapularis females infected with POWV II (“donors”) were co-fed on mice with uninfected H. longicornis larvae and nymphs (“recipients”). The donor and recipient ticks were infested on mice in various sequences, and mouse infection status was monitored by temporal screening of blood for POWV II RNA via quantitative reverse transcription polymerase chain reaction (q-RT-PCR).ResultsThe prevalence of POWV II RNA was highest in recipient H. longicornis that fed on viremic mice. However, nonviremic mice were also able to support co-feeding transmission of POWV, as demonstrated by the detection of viral RNA in multiple H. longicornis dispersed across different mice. Detection of viral RNA at the skin site of tick feeding but not at distal skin sites indicates that a localized skin infection facilitates transmission of POWV between donor and recipient ticks co-feeding in close proximity.ConclusionsThis is the first report examining transmission of POWV between co-feeding ticks. Against the backdrop of multiple unknowns related to POWV ecology, findings from this study provide insight on possible mechanisms by which POWV could be maintained in nature.Graphical

West Nile Virus Seroprevalence in Wild Birds and Equines in Madrid Province, Spain.

West Nile virus (WNV) is a re-emerging flavivirus, primarily circulating among avian hosts and mosquito vectors, causing periodic outbreaks in humans and horses, often leading to neuroinvasive disease and mortality. Spain has reported several outbreaks, most notably in 2020 with seventy-seven human cases and eight fatalities. WNV has been serologically detected in horses in the Community of Madrid, but to our knowledge, it has never been reported from wild birds in this region. To estimate the seroprevalence of WNV in wild birds and horses in the Community of Madrid, 159 wild birds at a wildlife rescue center and 25 privately owned equines were sampled. Serum from thirteen birds (8.2%) and one equine (4.0%) tested positive with a WNV competitive enzyme-linked immunosorbent assay (cELISA) designed for WNV antibody detection but sensitive to cross-reacting antibodies to other flaviviruses. Virus-neutralization test (VNT) confirmed WNV antibodies in four bird samples (2.5%), and antibodies to undetermined flavivirus in four additional samples. One equine sample (4.0%) tested positive for WNV by VNT, although this horse previously resided in a WN-endemic area. ELISA-positive birds included both migratory and resident species, juveniles and adults. Two seropositive juvenile birds suggest local flavivirus transmission within the Community of Madrid, while WNV seropositive adult birds may have been infected outside Madrid. The potential circulation of flaviviruses, including WNV, in birds in the Madrid Community raises concerns, although further surveillance of mosquitoes, wild birds, and horses in Madrid is necessary to establish the extent of transmission and the principal species involved.

Persistent spatial clustering and predictors of pediatric La Crosse virus neuroinvasive disease risk in eastern Tennessee and western North Carolina, 2003–2020

The combined region of eastern Tennessee and western North Carolina has a persistently high risk of pediatric La Crosse virus neuroinvasive disease (LACV-ND). To guide public health intervention in this region, the objectives of this retrospective ecological study were to investigate the geographic clustering and predictors of pediatric LACV-ND risk at the ZIP code tabulation area (ZCTA) level. Data on pediatric cases of LACV-ND reported between 2003 and 2020 were obtained from Tennessee Department of Health and North Carolina Department of Health and Human Services. Purely spatial and space-time scan statistics were used to identify ZCTA-level clusters of confirmed and probable pediatric LACV-ND cases from 2003–2020, and a combination of global and local (i.e., geographically weighted) negative binomial regression models were used to investigate potential predictors of disease risk from 2015–2020. The cluster investigation revealed spatially persistent high-risk and low-risk clusters of LACV-ND, with most cases consistently reported from a few high-risk clusters throughout the entire study period. Temperature and precipitation had positive but antagonistic associations with disease risk from 2015–2020, but the strength of those relationships varied substantially across the study area. Because LACV-ND risk clustering in this region is focally persistent, retroactive case surveillance can be used to guide the implementation of targeted public health intervention to reduce the disease burden in high-risk areas. Additional research on the role of climate in LACV transmission is warranted to support the development of predictive transmission models to guide proactive public health interventions.

Endemic La Crosse Virus Neuroinvasive Disease in North Carolina Residents: 2000-2020.

La Crosse virus neuroinvasive disease (LACVND) is the most common cause of arboviral encephalitis in children within the United States; in North Carolina, it is the most prevalent endemic mosquito-borne disease in humans. Here we report a surveillance summary of confirmed and probable LACVND during 2000-2020 using North Carolina Electronic Disease Surveillance System data, and we describe associated demographic characteristics, spatiotemporal distribution, clinical features, and mortality rates. A total of 355 cases (74.9% confirmed) were reported from 41 North Carolina counties; most cases (92%) occurred in 19 Western North Carolina counties. An average of 17 cases were reported annually with the majority (94%) of cases occurring between mid-June and early October (epiweeks 25-41). The median case age was nine years (range: 1-95 years), 79% were aged ≤ 18 years, and 56.6% were male. Cases commonly presented with headache (95.4%), fever (95.1%), and altered mental status (80.8%); encephalitis (82.0%) and meningitis (45.9%) were frequently diagnosed. Encephalitis was more common in children (87%) than adults (62%) (P < .001). Similarly, seizures were more common in children (54%) than adults (27%) (P < .01). The case fatality rate was 1.4%; however, differences in age-specific rates were observed. Changes in case definitions and reporting requirements, missing data, different reporting sources, and the retrospective nature of this study are all important limitations of our study. Given the persistent endemicity of La Crosse virus in Western North Carolina, clinicians and public health providers should consider La Crosse virus disease in all individuals, especially children, with compatible symptoms and a travel history to endemic counties. Prevention measures and educational outreach/public health messaging should focus on caregivers and children in Western North Carolina counties and during times when transmission risk is higher (i.e., summer and early fall).

West Nile virus meningoencephalitis and cytokines analysis: review and case report

West Nile virus (WNV) is transmitted via mosquito bites and causes ubiquitous zoonosis. Most infections in humans are asymptomatic, approximately 20% present as fever, and less than 1% as neuroinvasive disease. Central nervous system involvement is presented as meningitis, encephalitis, acute flaccid paralysis, or a combination of them. West Nile neuroinvasive disease (WNND) has a severe clinical course, potentially fatal outcome and frequent neurological sequelae in survivors. Risk factors for neurological impairment are advanced age and immunosuppression. Here we present a clinical case of meningoencephalitis caused by WNV and a brief literature review. Clinical and epidemiological data, laboratory, microbiological, molecular methods and imaging techniques were used. The cytokine levels of IL-6, IL-8, IL-10, IL-12(p40) and TNF-α in cerebrospinal fluid (CSF) and serum were also measured. We present a 55-year-old man with a sudden onset of headache, vomiting and fever. The symptoms appeared after a recent trip to Türkiye and involved multiple mosquito bites. Neck stiffness, disturbances in consciousness, signs of cerebral edema and subsequently focal neurological deficits were observed. The etiological diagnosis was verified by positive polymerase chain reaction for WNV and the presence of specific IgM antibodies in the CSF. After a 28-day hospital stay, the patient was discharged and referred to a Physiotherapy Unit due to residual motor deficits. WNV etiology should be suspected in patients with clinical and laboratory signs of viral neuroinfection, mosquito bites, and/or travel to endemic regions.

Exploring Climate-Disease Connections in Geopolitical Versus Ecological Regions: The Case of West Nile Virus in the United States.

Many infectious disease forecasting models in the United States (US) are built with data partitioned into geopolitical regions centered on human activity as opposed to regions defined by natural ecosystems; although useful for data collection and intervention, this has the potential to mask biological relationships between the environment and disease. We explored this concept by analyzing the correlations between climate and West Nile virus (WNV) case data aggregated to geopolitical and ecological regions. We compared correlations between minimum, maximum, and mean annual temperature; precipitation; and annual WNV neuroinvasive disease (WNND) case data from 2005 to 2019 when partitioned into (a) climate regions defined by the National Oceanic and Atmospheric Administration (NOAA) and (b) Level I ecoregions defined by the Environmental Protection Agency (EPA). We found that correlations between climate and WNND in NOAA climate regions and EPA ecoregions were often contradictory in both direction and magnitude, with EPA ecoregions more often supporting previously established biological hypotheses and environmental dynamics underlying vector-borne disease transmission. Using ecological regions to examine the relationships between climate and disease cases can enhance the predictive power of forecasts at various scales, motivating a conceptual shift in large-scale analyses from geopolitical frameworks to more ecologically meaningful regions.