Treatment with Granulocyte-Macrophage Colony-Stimulating Factor Reduces Viral Titers in the Brains of West Nile Virus-Infected Mice and Improves Survival.

West Nile virus (WNV) is the leading cause of epidemic arboviral encephalitis in the United States. As there are currently no proven antiviral therapies or licensed human vaccines, understanding the neuropathogenesis of WNV is critical for rational therapeutic design. In WNV-infected mice, the depletion of microglia leads to enhanced viral replication, increased central nervous system (CNS) tissue injury, and increased mortality, suggesting that microglia play a critical role in protection against WNV neuroinvasive disease. To determine if augmenting microglial activation would provide a potential therapeutic strategy, we administered granulocyte-macrophage colony-stimulating factor (GM-CSF) to WNV-infected mice. Recombinant human GM-CSF (rHuGMCSF) (sargramostim [Leukine]) is an FDA-approved drug used to increase white blood cells following leukopenia-inducing chemotherapy or bone marrow transplantation. Daily treatment of both uninfected and WNV-infected mice with subcutaneous injections of GM-CSF resulted in microglial proliferation and activation as indicated by the enhanced expression of the microglia activation marker ionized calcium binding adaptor molecule 1 (Iba1) and several microglia-associated inflammatory cytokines, including CCL2 (C-C motif chemokine ligand 2), interleukin 6 (IL-6), and IL-10. In addition, more microglia adopted an activated morphology as demonstrated by increased sizes and more pronounced processes. GM-CSF-induced microglial activation in WNV-infected mice was associated with reduced viral titers and apoptotic activity (caspase 3) in the brains of WNV-infected mice and significantly increased survival. WNV-infected ex vivo brain slice cultures (BSCs) treated with GM-CSF also showed reduced viral titers and caspase 3 apoptotic cell death, indicating that GM-CSF specifically targets the CNS and that its actions are not dependent on peripheral immune activity. Our studies suggest that stimulation of microglial activation may be a viable therapeutic approach for the treatment of WNV neuroinvasive disease. IMPORTANCE Although rare, WNV encephalitis poses a devastating health concern, with few treatment options and frequent long-term neurological sequelae. Currently, there are no human vaccines or specific antivirals against WNV infections, so further research into potential new therapeutic agents is critical. This study presents a novel treatment option for WNV infections using GM-CSF and lays the foundation for further studies into the use of GM-CSF as a treatment for WNV encephalitis as well as a potential treatment for other viral infections.

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)

Surveillance for West Nile Virus Disease - United States, 2009-2018.

PROBLEM/CONDITION: West Nile virus (WNV) is an arthropodborne virus (arbovirus) in the family Flaviviridae and is the leading cause of domestically acquired arboviral disease in the contiguous United States. An estimated 70%-80% of WNV infections are asymptomatic. Symptomatic persons usually develop an acute systemic febrile illness. Less than 1% of infected persons develop neuroinvasive disease, which typically presents as encephalitis, meningitis, or acute flaccid paralysis. REPORTING PERIOD: 2009-2018. DESCRIPTION OF SYSTEM: WNV disease is a nationally notifiable condition with standard surveillance case definitions. State health departments report WNV cases to CDC through ArboNET, an electronic passive surveillance system. Variables collected include patient age, sex, race, ethnicity, county and state of residence, date of illness onset, clinical syndrome, hospitalization, and death. RESULTS: During 2009-2018, a total of 21,869 confirmed or probable cases of WNV disease, including 12,835 (59%) WNV neuroinvasive disease cases, were reported to CDC from all 50 states, the District of Columbia, and Puerto Rico. A total of 89% of all WNV patients had illness onset during July-September. Neuroinvasive disease incidence and case-fatalities increased with increasing age, with the highest incidence (1.22 cases per 100,000 population) occurring among persons aged ≥70 years. Among neuroinvasive cases, hospitalization rates were >85% in all age groups but were highest among patients aged ≥70 years (98%). The national incidence of WNV neuroinvasive disease peaked in 2012 (0.92 cases per 100,000 population). Although national incidence was relatively stable during 2013-2018 (average annual incidence: 0.44; range: 0.40-0.51), state level incidence varied from year to year. During 2009-2018, the highest average annual incidence of neuroinvasive disease occurred in North Dakota (3.16 cases per 100,000 population), South Dakota (3.06), Nebraska (1.95), and Mississippi (1.17), and the largest number of total cases occurred in California (2,819), Texas (2,043), Illinois (728), and Arizona (632). Six counties located within the four states with the highest case counts accounted for 23% of all neuroinvasive disease cases nationally. INTERPRETATION: Despite the recent stability in annual national incidence of neuroinvasive disease, peaks in activity were reported in different years for different regions of the country. Variations in vectors, avian amplifying hosts, human activity, and environmental factors make it difficult to predict future WNV disease incidence and outbreak locations. PUBLIC HEALTH ACTION: WNV disease surveillance is important for detecting and monitoring seasonal epidemics and for identifying persons at increased risk for severe disease. Surveillance data can be used to inform prevention and control activities. Health care providers should consider WNV infection in the differential diagnosis of aseptic meningitis and encephalitis, obtain appropriate specimens for testing, and promptly report cases to public health authorities. Public health education programs should focus prevention messaging on older persons, because they are at increased risk for severe neurologic disease and death. In the absence of a human vaccine, WNV disease prevention depends on community-level mosquito control and household and personal protective measures. Understanding the geographic distribution of cases, particularly at the county level, appears to provide the best opportunity for directing finite resources toward effective prevention and control activities. Additional work to further develop and improve predictive models that can foreshadow areas most likely to be impacted in a given year by WNV outbreaks could allow for proactive targeting of interventions and ultimately lowering of WNV disease morbidity and mortality.

A broadly protective antibody that targets the flavivirus NS1 protein.

There are no approved flaviviral therapies and the development of vaccines against flaviruses has the potential of being undermined by antibody-dependent enhancement (ADE). The flavivirus nonstructural protein 1 (NS1) is a promising vaccine antigen with low ADE risk but has yet to be explored as a broad-spectrum therapeutic antibody target. Here, we provide the structural basis of NS1 antibody cross-reactivity through cocrystallization of the antibody 1G5.3 with NS1 proteins from dengue and Zika viruses. The 1G5.3 antibody blocks multi-flavivirus NS1-mediated cell permeability in disease-relevant cell lines, and therapeutic application of 1G5.3 reduces viremia and improves survival in dengue, Zika, and West Nile virus murine models. Finally, we demonstrate that 1G5.3 protection is independent of effector function, identifying the 1G5.3 epitope as a key site for broad-spectrum antiviral development.

Structural basis for antibody inhibition of flavivirus NS1-triggered endothelial dysfunction.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the ß-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

Recruiting the innate immune system with GM-CSF to fight viral diseases, including West Nile Virus encephalitis and COVID-19.

As the coronavirus disease 2019 (COVID-19) pandemic grows throughout the world, it is imperative that all approaches to ameliorating its effects be investigated, including repurposing drugs that show promise in other diseases. We have been investigating an approach to multiple disorders that involves recruiting the innate immune system to aid the body's healing and regenerative mechanism(s). In the case of West Nile Virus encephalitis and potentially COVID-19, the proposed intervention to stimulate the innate immune system may give the adaptive immune response the necessary time to develop, finish clearing the virus, and provide future immunity. Furthermore, we have found that GM-CSF-induced recruitment of the innate immune system is also able to reverse brain pathology, neuroinflammation and cognitive deficits in mouse models of Alzheimer's disease and Down syndrome, as well as improving cognition in normal aging and in human patients with cognitive deficits due to chemotherapy, both of which exhibit neuroinflammation. Others have shown that GM-CSF is an effective treatment for both bacterial and viral pneumonias, and their associated inflammation, in animals and that it has successfully treated pneumonia-associated Acute Respiratory Distress Syndrome in humans. These and other data strongly suggest that GM-CSF may be an effective treatment for many viral infections, including COVID-19.

Delayed Diagnosis of West Nile Virus Infection in a Kidney Transplant Patient Due to Inaccuracies in Commonly Available Diagnostic Tests.

West Nile virus infection is more frequently associated with neuroinvasive disease and high morbidity and mortality in immunocompromised hosts. Here, we describe a 47-year-old Egyptian kidney transplant recipient who was admitted to our department in 2016 for persistent fever, altered mental status, and upper limb tremors. In addition, renal impairment, signs of acute thrombotic microangiopathy, pancreatitis, and slightly altered inflammatory indices were present. The patient was treated with antibacterial and antiviral therapy, and reduced immunosuppressive therapy was prescribed. After several biochemical and instrumental examinations, only slight blood positivity for West Nile virus immunoglobulin M in the absence of immunoglobulin G was found, whereas immunoglobulins M and G on cerebrospinal fluid and West Nile virus polymerase chain reaction were negative. Serology evaluated after 23 days of hospitalization confirmed immunoglobulin M positivity and detected weak immunoglobulin G positivity; however, according to the US Centers for Disease Control and Prevention diagnostic criteria, it was not sufficient to confirm diagnosis. During hospitalization, clinical recovery was observed, but severe renal insufficiency persisted. Renal biopsy performed after clinical recovery demonstrated chronic antibody-mediated rejection with advanced chronic lesions, without viral cytopathic signs. Four months later, we received confirmation of West Nile virus infection by plaque reduction neutralization test. The current case described severe West Nile virus infection with clinical neurologic involvement, thrombotic microangiopathy, and pancreatitis, resulting in irreversible loss of kidney function. Delayed diagnosis, based on US Centers for Disease Control and Prevention criteria, was due to absence of both characteristic radiologic features and sensitive and promptly available laboratory tests. This case stresses the need for accurate diagnostic tests and/or a partial revision of the diagnostic criteria. In fact, with an earlier diagnosis, we would have avoided diagnostic and therapeutic procedures that may have contributed to the loss of graft function in the described case.

West Nile encephalitis mimicking neuropsychiatric lupus in a patient with systemic lupus erythematosus.

A man in his 70s with known systemic lupus erythematosus (SLE) was admitted with confusion, worsening proteinuria and cutaneous vasculitis despite adherence to his home immunosuppressive regimen. Admission laboratories were consistent with active lupus. Despite treatment with pulse-dose glucocorticoids and intravenous immunoglobulin, he developed worsening mental status and meningeal signs. Investigations revealed cerebrospinal fluid (CSF) neutrophilic and plasmacytic pleocytosis and negative cultures. Empiric treatment for SLE flare with potential neuropsychiatric involvement was continued while workup for altered mental status was ongoing. Ultimately, West Nile encephalitis was diagnosed by CSF serologies, and steroids were tapered. Altered mental status in a patient with SLE has a broad differential, and primary neuropsychiatric SLE should be considered only after exclusion of secondary causes. Although evidence of end-organ SLE activity usually lends support to a neuropsychiatric SLE diagnosis, in this case, serological and clinical evidence of SLE activity may have been triggered by acute viral infection.

EEG in WNV Neuroinvasive Disease.

PURPOSE: Neuroinvasive West Nile virus (WNV) is rare, occurring in less than 1% of those infected, and may manifest as meningitis, encephalitis, and/or acute flaccid paralysis. Patients may present initially with nonspecific symptoms including fevers. Although rare, neuroinvasive WNV is associated with significant morbidity and mortality. The mainstay of treatment is supportive care. Electroencephalography (EEG) allows for identification of nonconvulsive status epilepticus and other epileptiform and nonepileptiform patterns suggestive of underlying cognitive dysfunction. Our aim was to describe specific EEG patterns observed in WNV neuroinvasive disease. METHODS: A retrospective chart review was conducted. West Nile virus was confirmed with serum and/or cerebrospinal fluid markers. Patients with a history of abnormal EEG were excluded. Electroencephalography reports were classified into categories based on the presence of epileptiform activity, focal slowing, generalized periodic discharges with triphasic morphology, and frontally predominant generalized rhythmic delta activity. RESULTS: In our cohort of 34 patients, 60% of focal EEG abnormalities were anterior-predominant, seen as epileptiform discharges, focal slowing, or frontally predominant generalized rhythmic delta activity. Nonepileptiform EEG patterns included nonspecific slowing and generalized periodic discharges with triphasic morphology. Two patients had electrographic seizures, one arising from the frontocentral head region. CONCLUSIONS: EEGs are important in the evaluation of WNV infection to rule out seizures or alternative causes of encephalopathy, and because of the risk of nonconvulsive seizures or status epilepticus in encephalitis. Although an anterior predominance of EEG abnormalities was seen in our cohort, this most likely is more correlative to encephalopathy than WNV itself. Although a specific correlative EEG pattern may not accompany all cases of WNV neuroinvasive disease, WNV should be considered as a possible etiology in patients presenting with an encephalitic or meningitic syndrome in the presence of abnormal EEG findings including encephalopathic patterns, particularly those with anterior predominant EEG changes.

Pediatric West Nile Virus-Associated Neuroinvasive Disease: A Review of the Literature.

Over the past two decades, West Nile virus has become the most common arbovirus in North America, leading to several outbreaks and infecting thousands of people. Mosquitos help transmit the virus in the majority of cases, but transmission occurs via blood transfusions, organ transplantation, and possibly pregnancy and breastfeeding. While most infected patients experience mild to no symptoms, thousands of West Nile virus-associated neuroinvasive cases have been reported in the United States, with over 700 cases occurring in children from 2003 to 2016. Neuroinvasive disease presents as meningitis, encephalitis, or acute flaccid paralysis, and carries a high likelihood of poor outcome, including severe neurological disability or death. To date, no pharmacologic treatment has proven effective. Therapeutic clinical trials have not been successfully completed due to the sporadic nature of viral outbreaks and resultant poor study enrollment. Although older age and chronic disease are risk factors for neuroinvasive West Nile virus disease in adults, the specific factors that influence the risk in pediatric populations have not been fully elucidated. This review summarizes the most recent literature regarding West Nile virus-associated neuroinvasive disease, especially as it pertains to the pediatric population. Moreover, the review describes the epidemiology, clinical, laboratory, and radiographic findings, and outlines the various therapies that have been trialed and potential future research directions.

A protective human monoclonal antibody targeting the West Nile virus E protein preferentially recognizes mature virions.

West Nile virus (WNV), a member of the Flavivirus genus, is a leading cause of viral encephalitis in the United States1. The development of neutralizing antibodies against the flavivirus envelope (E) protein is critical for immunity and vaccine protection2. Previously identified candidate therapeutic mouse and human neutralizing monoclonal antibodies (mAbs) target epitopes within the E domain III lateral ridge and the domain I-II hinge region, respectively3. To explore the neutralizing antibody repertoire elicited by WNV infection for potential therapeutic application, we isolated ten mAbs from WNV-infected individuals. mAb WNV-86 neutralized WNV with a 50% inhibitory concentration of 2 ng ml-1, one of the most potently neutralizing flavivirus-specific antibodies ever isolated. WNV-86 targets an epitope in E domain II, and preferentially recognizes mature virions lacking an uncleaved form of the chaperone protein prM, unlike most flavivirus-specific antibodies4. In vitro selection experiments revealed a neutralization escape mechanism involving a glycan addition to E domain II. Finally, a single dose of WNV-86 administered two days post-infection protected mice from lethal WNV challenge. This study identifies a highly potent human neutralizing mAb with therapeutic potential that targets an epitope preferentially displayed on mature virions.

WEST NILE VIRUS INFECTION WITH NEUROLOGICAL DISORDERS: A CASE REPORT AND A BRIEF REVIEW OF THE SITUATION IN BULGARIA.

A case of a 66-year-old man with West Nile neuroinvassive disease manifested with fever, weakness, fatigue, consciousness disorders and underlying diabetes mellitus type 2 and cardiovascular diseases is presented. Laboratory data showed elevated erythrocyte sedimentation rate and fibrinogen. Serological tests revealed West Nile virus specific antibodies of class IgM and IgG in serum. West Nile virus RNA was detected in urine sample. Supportive therapy was applied.

West Nile virus induces a post-infectious pro-inflammatory state that explains transformation of stable ocular myasthenia gravis to myasthenic crises.

West Nile virus (WNV) infection has been reported to promote myasthenia gravis (MG) and various other diseases that have a presumed autoimmune pathogenesis. Molecular mimicry between WNV proteins and host proteins has been postulated as the major mechanism for WNV-triggered breaking of immunological self-tolerance. We present a patient with stable ocular MG and positive anti-acetylcholine receptor antibodies who progressed to myasthenic crisis after WNV neuroinvasive disease. In this case of stable autoimmune disease with proven auto-antibodies, transformation to generalized disease cannot be attributed to molecular mimicry, which requires that an immune response first be generated against an infectious agent. Rather, the evidence supports the concept of a post-infectious pro-inflammatory state that may contribute to the amplification and promotion of autoimmune disease in some WNV survivors.

Outcomes in Patients With Severe West Nile Neuroinvasive Disease.

OBJECTIVE: To assess the long-term outcomes of patients hospitalized with severe West Nile neuroinvasive disease. DESIGN: Retrospective cohort. SETTING: Patients admitted to a referral center (Saint Mary's Hospital, Mayo Clinic). PARTICIPANTS: Twenty-six patients with West Nile neuroinvasive disease were identified by retrospective search of electronic database of Saint Mary's Hospital from January 1999 to November 2016. INTERVENTIONS: Retrospective electronic medical records review and prospective telephone follow-up. MEASUREMENTS AND MAIN RESULTS: Functional disability and cognitive outcomes were evaluated with the modified Rankin Scale and the Telephone Interview for Cognitive Status scores. Data on the time that the patient returned home after the hospitalization for West Nile neuroinvasive disease and the time of return to work were also collected. We identified 26 patients (81% males), 59 ± 17 years old. After a median hospital stay of 14.5 days (3-126), four patients died and 90% of survivors had a modified Rankin Scale of 3-5. Two additional patients died, and 80% of survivors had a modified Rankin Scale of 0-2 after a median follow-up of 73 months (1-144). Seven patients had cognitive impairment, which was severe in two of them. The combination of encephalitis and acute flaccid paralysis at presentation was associated with lower likelihood of returning home within 1 month after discharge (p < 0.01). Patients who required mechanical ventilation were more likely to have a modified Rankin Scale of 3-5 at last follow-up (p = 0.03), less likely to return home within 1 month of discharge (p < 0.01), less likely to return to their jobs (p < 0.01), and showed a trend toward having cognitive impairment (p = 0.05). CONCLUSIONS: Despite having poor outcomes at discharge, most West Nile neuroinvasive disease survivors with severe early disability can recover functional independence in the long term, justifying aggressive support during the acute phase and extensive rehabilitation efforts.

Human monoclonal antibodies against West Nile virus from Japanese encephalitis-vaccinated volunteers.

West Nile virus (WNV) is a positive-sense single-stranded RNA flavivirus belonging to the Japanese encephalitis virus (JEV) serocomplex of the Flaviviridae family and causes mosquito-borne infections. Although most human infection cases are asymptomatic, approximately one in 150 infected individuals develops meningoencephalitis, with a mortality rate of 4-14%. While the development of human neutralizing antibody therapeutics against WNV is strongly anticipated, WNV is difficult to study in conventional laboratories due to its high safety level requirement. In this study, we established fully human WNV-neutralizing monoclonal antibodies from the peripheral blood mononuclear cells of inactivated-JEV-vaccinated individuals, and these antibodies exhibited WNV neutralization both in vitro and in vivo. Our results demonstrate a new antibody cross-reactivity strategy to develop immunological therapeutic reagents for WNV and other JEV serotype viruses.

Critical West Nile Neuroinvasive Disease.

BACKGROUND: Data to guide neurointensivists seeing patients with West Nile Neuroinvasive disease (WNND) are lacking. We present a comparatively large series of patients with WNND admitted to the intensive care unit (ICU) and provide data on their early diagnosis, triage to the ICU and predictors of short-term outcomes. METHODS: We retrospectively identified patients aged ≥ 18 years old with WNND from January 1999 to November 2016. Demographic and clinical data, the modified Rankin Scale at discharge and disposition were collected. Univariate analysis was performed to find predictors of ICU admission and to assess the impact of ICU admission on the short-term outcomes. P values < 0.05 were considered significant. RESULTS: Among 26 patients, 16 were admitted to the ICU. Age < 60 years and the presentation with encephalitis and acute flaccid paralysis predicted ICU admission (P = 0.044 and 0.0007). Among patients requiring ICU admission, four died and no one was discharged home. ICU admission predicted longer hospital stay (P = 0.021), inhospital death (P = 0.034), survival with inability to walk independently (P = 0.0094), and discharge disposition other than home (P = 0.007). In the ICU group, older age was associated with longer hospital stay (P = 0.0001) and inhospital death (P = 0.035). CONCLUSION: WNND requiring ICU care has a high morbidity and mortality, especially among older patients. Survivors are highly disabled at discharge, but many improve over time. Therefore, more data on the long-term prognosis of survivors are needed to guide the goals of care in the acute setting.

Latitudinal Diversity of Culex pipiens Biotypes and Hybrids in Farm, Peri-Urban, and Wetland Habitats in Europe.

Despite the presence of Culex (Cx.) pipiens mosquitoes and circulation of West Nile virus (WNV), WNV outbreaks have so far not occurred in northern Europe. The species Cx. pipiens consists of two morphologically identical biotypes, pipiens and molestus, which can form hybrids. Until now, population dynamic studies of Cx. pipiens have not differentiated between biotypes and hybrids at the European scale, nor have they used comparative surveillance approaches. We therefore aimed to elucidate the relative abundance of Cx. pipiens biotypes and hybrids in three habitat types at different latitudes across Europe, using two different surveillance traps. BG-Sentinel and Mosquito-Magnet Liberty Plus traps were placed in three habitat types (farms, peri-urban, wetlands), in three European countries (Sweden, The Netherlands, Italy). Collected Cx. pipiens mosquitoes were identified to biotype with real-time PCR. Both trap types collected equal ratios of the biotypes and their hybrids. From northern to southern latitudes there was a significant decrease of pipiens and an increase of molestus. Habitat types influenced the relative ratios of biotypes and hybrids, but results were not consistent across latitudes. Our results emphasize the need to differentiate Cx. pipiens to the biotype level, especially for proper future WNV risk assessments for Europe.

You're the Flight Surgeon.

Martin BR. You're the flight surgeon: West Nile virus neuroinvasive disease. Aerosp Med Hum Perform. 2016; 87(11):976-979.