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)

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.

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 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.

Not the Usual Viral Suspects: Parvovirus B19, West Nile Virus, and Human T-Cell Lymphotrophic Virus Infections After Kidney Transplantation.

Kidney transplant recipients are at increased risk of developing clinical disease due to uncommon opportunistic viral pathogens. Refractory anemia is classically associated with parvovirus B19 infection. West Nile virus has the propensity to cause fever and neurologic symptoms, while spastic paresis and lymphoma can be triggered by human T cell lymphotrophic virus. In this review article, the epidemiology, clinical manifestations, diagnosis and treatment of less common viruses are discussed in the setting of kidney transplantation.

West nile virus infection and myasthenia gravis.

INTRODUCTION: Viruses are commonly cited as triggers for autoimmune disease. It is unclear if West Nile virus (WNV) initiates autoimmunity. METHODS: We describe 6 cases of myasthenia gravis (MG) that developed several months after WNV infection. All patients had serologically confirmed WNV neuroinvasive disease. None had evidence of MG before WNV. RESULTS: All patients had stable neurological deficits when they developed new symptoms of MG 3 to 7 months after WNV infection. However, residual deficits from WNV confounded or delayed MG diagnosis. All patients had elevated acetylcholine receptor (AChR) antibodies, and 1 had thymoma. Treatment varied, but 4 patients required acetylcholinesterase inhibitors, multiple immunosuppressive drugs, and intravenous immune globulin or plasmapheresis for recurrent MG crises. CONCLUSIONS: The pathogenic mechanism of MG following WNV remains uncertain. We hypothesize that WNV-triggered autoimmunity breaks immunological self-tolerance to initiate MG, possibly through molecular mimicry between virus antigens and AChR subunits or other autoimmune mechanisms.

Neglected infections of poverty in Texas and the rest of the United States: management and treatment options.

In the poorest regions of the United States, especially along the Gulf Coast and in South Texas, are a group of endemic parasitic and related infections known as the neglected infections of poverty. Such infections are characterized by their chronicity, disabling features, and disproportionate impact on the estimated 46 million people who live below the U.S. poverty line. Today more Americans live in poverty than ever before in the half-century that the Census Bureau has been recording poverty rates. In association with that poverty, a group of major neglected infections of poverty have emerged in the United States. Here we describe the major neglected infections of poverty in the United States, with a brief overview of their significant epidemiological features, their links with poverty, and our approaches to their diagnosis, management, and treatment.

Monoclonal antibody produced in plants efficiently treats West Nile virus infection in mice.

Over the past decade, West Nile virus (WNV) has spread to all 48 of the lower United States as well as to parts of Canada, Mexico, the Caribbean, and South America, with outbreaks of neuroinvasive disease occurring annually. At present, no therapeutic or vaccine is available for human use. Epidemics of WNV and other emerging infectious disease threats demand cost-efficient and scalable production technologies that can rapidly transfer effective therapeutics into the clinical setting. We have previously reported that Hu-E16, a humanized anti-WNV mAb, binds to a highly conserved epitope on the envelope protein, blocks viral fusion, and shows promising postexposure therapeutic activity. Herein, we generated a plant-derived Hu-E16 mAb that can be rapidly scaled up for commercial production. Plant Hu-E16 was expressed at high levels within 8 days of infiltration in Nicotiana benthamiana plants and retained high-affinity binding and potent neutralizing activity in vitro against WNV. A single dose of plant Hu-E16 protected mice against WNV-induced mortality even 4 days after infection at rates that were indistinguishable from mammalian-cell-produced Hu-E16. This study demonstrates the efficacy of a plant-produced mAb against a potentially lethal infection several days after exposure in an animal challenge model and provides a proof of principle for the development of plant-derived mAbs as therapy against emerging infectious diseases.

Fusion loop peptide of the West Nile virus envelope protein is essential for pathogenesis and is recognized by a therapeutic cross-reactive human monoclonal antibody.

West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human mAb, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Ab mAb11 cross-reacts with all four dengue viruses and provides protection against dengue (serotypes 2 and 4) viruses. In contrast to the parental West Nile virus, a neutralization escape variant failed to cause lethal encephalitis (at higher infectious doses) or induce the inflammatory responses associated with blood-brain barrier permeability in mice, suggesting an important role for the fusion loop in viral pathogenesis. Our data demonstrate that an intact West Nile virus fusion loop is critical for virulence, and that human mAb11 targeting this region is efficacious against West Nile virus infection. These experiments define the molecular determinant on the envelope protein recognized by mAb11 and demonstrate the importance of this region in causing West Nile encephalitis.

Development of resistance to passive therapy with a potently neutralizing humanized monoclonal antibody against West Nile virus.

Previous studies have established the therapeutic efficacy of humanized E16 (hE16) monoclonal antibody against West Nile virus in animals. Here, we assess the potential for West Nile virus strains encoding mutations in the hE16 epitope to resist passive immunotherapy and for the selection of neutralization escape variants during hE16 treatment. Resistance to hE16 in vivo was less common than expected, because several mutations that affected neutralization in vitro did not significantly affect protection in mice. Moreover, the emergence of resistant variants after infection with fully sensitive virus occurred but was relatively rare, even in highly immunocompromised B and T cell-deficient RAG mice.

Identification of novel small-molecule inhibitors of West Nile virus infection.

West Nile virus (WNV) has spread throughout the United States and Canada and now annually causes a clinical spectrum of human disease ranging from a self-limiting acute febrile illness to acute flaccid paralysis and lethal encephalitis. No therapy or vaccine is currently approved for use in humans. Using high-throughput screening assays that included a luciferase expressing WNV subgenomic replicon and an NS1 capture enzyme-linked immunosorbent assay, we evaluated a chemical library of over 80,000 compounds for their capacity to inhibit WNV replication. We identified 10 compounds with strong inhibitory activity against genetically diverse WNV and Kunjin virus isolates. Many of the inhibitory compounds belonged to a chemical family of secondary sulfonamides and have not been described previously to inhibit WNV or other related or unrelated viruses. Several of these compounds inhibited WNV infection in the submicromolar range, had selectivity indices of greater than 10, and inhibited replication of other flaviviruses, including dengue and yellow fever viruses. One of the most promising compounds, AP30451, specifically blocked translation of a yellow fever virus replicon but not a Sindbis virus replicon or an internal ribosome entry site containing mRNA. Overall, these compounds comprise a novel class of promising inhibitors for therapy against WNV and other flavivirus infections in humans.

Mapping and analysis of West Nile virus-specific monoclonal antibodies: prospects for vaccine development.

Seasonal epidemics of West Nile virus (WNV) infection now occur throughout North America, causing clinical symptoms ranging from fever to encephalitis. There are no specific treatment options or licensed vaccines. Several classically developed vaccine candidates are being evaluated in clinical trials. However, questions of safety and/or immunogenicity may limit their usefulness. Mapping of human and murine antibody repertoires against the WNV envelope protein after WNV infection have revealed important insights into the protective immune response against the virus. This review will give an overview of vaccines under development and summarize current data on E-protein antigenicity that could aid in the design of next generation WNV vaccines.

Antiviral peptides targeting the west nile virus envelope protein.

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 muM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 muM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.

El virus del Oeste del Nilo: una realidad en México / West Nile virus: a reality in Mexico

El virus del Oeste del Nilo (VON) es un virus ARN perteneciente a la familia Flaviviridae del género Flavivirus que causa infección en aves, equinos y humanos. La infección por VON es transmitida por el mosquito Culex sp. El ciclo de vida del virus incluye a los mosquitos como vectores y a las aves como huéspedes naturales. El virus mantiene un ciclo de transmisión mosquito–ave-mosquito. Los seres humanos son huéspedes accidentales. Se han reportado epidemias en Rumania, Nueva York e Israel. Mediante el programa de vigilancia epidemiológica en nuestro país, se han reportado 90 muestras positivas en 1,223 casos estudiados en aves hasta el 15 de Septiembre del 2005. La enfermedad por el VON se presenta con fiebre, malestar general, anorexia, nausea, vómito, cefalea, mialgia, erupción cutánea y linfadenopatía. La principal entidad clínica descrita es la encefalitis y la parálisis flácida. A mayor edad, es mayor el riesgo de enfermedad neurológica y muerte. Los métodos diagnósticos incluyen determinación de anticuerpos IgM e IgG en suero y/o liquido cerebroespinal. No existe tratamiento antiviral para la infección por VON. Algunas terapias que se han utilizado incluyen interferón α2b e inmunoglobulina específica contra VON. La prevención juega un papel crucial.

West Nile virus (WNV) is a RNA virus of the Flaviridae, genus flavivirus family. It is a neuropathogenic virus causing disease in birds, horses and humans. WNVis transmitted by the vector mosquito Culex sp. The virus life 's cycle includes mosquitoes as vectors and birds as natural hosts. Humans are accidental hosts. Since the introduction of the Epidemiological Surveillance Program at the Ministry ofHealth. we have documented 90 positive test results among birds out of 1,223 cases studied in Mexico as of September IS. 2005. The incubation period in humans after a mosquito bite ranges from 3 to 14 days. Disease is characterized by early onset fever, general malaise, decreased appetite, nausea, vomiting, headaches, myalgias, enlarged lymph nodes andrash. Neurological manifestations include encephalitis andflaccid paralysis, which are present in less than 1% of subjects infected with WNV. Older patients display more adverse outcomes including death. The diagnosis is made by the determination of specific IgM and JgG antibodies in serum and/or cerebrospinal fluid. There is no antiviral treatment to date against WNV but interferon ?2b, and WNVspec4ic-immunoglobulin have been used Prevention is therefore the key to control the infection.

Stroke associated with central nervous system vasculitis after West Nile virus infection.

We report the association of West Nile virus infection, isolated vasculitis, and stroke in a 9-year-old girl. West Nile virus is of growing epidemiologic importance and should be considered in the differential diagnosis of stroke etiologies, especially during late summer and in patients with a history of exposure in areas where West Nile virus transmission is present.

Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus.

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.