Warm winters are associated to more intense West Nile virus circulation in southern Spain.

West Nile virus (WNV) is the most widely distributed mosquito-borne flavivirus in the world. This flavivirus can infect humans causing in some cases a fatal neurological disease and birds are the main reservoir hosts. WNV is endemic in Spain, and human cases have been reported since 2004. Although different studies analyse how climatic conditions can affect the dynamics of WNV infection, very few use long-term datasets. Between 2003 and 2020 a total of 2,724 serum samples from 1,707 common coots (Fulica atra) were analysed for the presence of WNV-specific antibodies. Mean (SD) annual seroprevalence was 24.67% (0.28) but showed high year-to-year variations ranging from 5.06% (0.17) to 68.89% (0.29). Significant positive correlations (p < 0.01) were observed between seroprevalence and maximum winter temperature and mean spring temperature. The unprecedented WNV outbreak in humans in the south of Spain in 2020 was preceded by a prolonged period of escalating WNV local circulation. Given current global and local climatic trends, WNV circulation is expected to increase in the next decades. This underscores the necessity of implementing One Health approaches to reduce the risk of future WNV outbreaks in humans. Our results suggest that higher winter and spring temperatures may be used as an early warning signal of more intense WNV circulation among wildlife in Spain, and consequently highlight the need of more intense vector control and surveillance in human inhabited areas.

Insulin-mediated endothelin signaling is antiviral during West Nile virus infection.

IMPORTANCE: Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identify potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

One antibody to treat them all.

Conserved flavivirus protein holds potential as target for versatile vaccines and therapies.

Seroepidemiological Survey of West Nile Virus Infections in Horses from Berlin/Brandenburg and North Rhine-Westphalia, Germany.

Following the introduction of the West Nile virus (WNV) into eastern Germany in 2018, increasing infections have been diagnosed in birds, equines, and humans over time, while the spread of WNV into western Germany remained unclear. We screened 437 equine sera from 2018 to 2020, excluding vaccinated horses, collected from convenience sampled patients in the eastern and western parts of Germany, for WNV-specific antibodies (ELISAs followed by virus/specific neutralization tests) and genomes (RT-qPCRs). Clinical presentations, final diagnoses, and demographic data were also recorded. In the eastern part, a total of eight horses were found WNV seropositive in 2019 (seroprevalence of 8.16%) and 27 in 2020 (13.77%). There were also two clinically unsuspected horses with WNV-specific antibodies in the western part from 2020 (2.63%), albeit travel history-related infections could not be excluded. None of the horse sera contained WNV-specific genomes. Eight horses in eastern Germany carried WNV-IgM antibodies, but only four of these showed typical clinical signs. These results underline the difficulty of detecting a WNV infection in a horse solely based on clinical signs. Thus, WNV circulation is established in the horse population in eastern Germany, but not yet in the western part.

Initial Seronegative West Nile Virus Encephalitis in an Immunocompromised Child.

We present a case of initial seronegative West Nile virus encephalitis in an immunocompromised child due to B-cell acute lymphoblastic leukemia. Although diagnostic guidelines for West Nile virus infection exist, we highlight that these may not be met in immunocompromised patients who may have a delayed immune response.

West Nile virus seroprevalence and associated risk factors among horses in Egypt.

Determination of the seroprevalence and risk factors that are associated with West Nile virus (WNV) in horses is essential for adoption of effective prevention strategies. Our objective in this study, therefore, was to determine the seroprevalence and to identify the risk factors associated with WNV infection in the most densely horse-populated governorates in Egypt. A cross-sectional study was conducted in 2018 on 930 horses, which were distributed over five governorates in the Nile delta of Egypt. The horses, which were randomly selected, were serologically tested through use of an ID screen West Nile competition enzyme-linked immunosorbent assay (ELISA) to detect anti-WNV immunoglobulin G (IgG) and plaque reduction neutralization tests (PRNT; gold standard) to confirm the seropositive status of animals and to avoid cross reaction with other flavi-viruses. Four variables (geographical location, breed, sex and age) were considered in the risk analysis. Univariable and stepwise forward multivariable logistic regression methods were used for risk-factor analysis. The odds ratio (OR) was used as an approximate measure of relative risk. A total of 156 (16.8%; 95% confidence interval (CI) 14.4-19.2; P < 0.001) serum samples were found to be serologically positive for WNV. The highest seroprevalence rate was detected in horses of age ≥ 15 years (68.1%; 95% CI 49.8-72.4), stallions (26.4%; 95% CI 22.7-30.4), and those of mixed breed (21.5%; 95% CI 17.7-27.5). Horses older than 15 years were found to be at increased risk of WNV infection with OR = 4.3 (95% CI 3.0-6.2, P < 0.001) compared with horses aged under 2.5 years. Also, when all the risk factors were considered, stallions were more likely than mares to be WNV seropositive (OR = 2.4, 95% CI 1.6-3.7, P < 0.001), and of the breeds, mixed-breed (OR = 1.9, 95% CI 1.2-2.8, P = 0.005) and Arabian horses (OR = 1.9, 95% CI 1.2-2.8, P = 0.005) were more likely to be seropositive. Geographical location seemed to have no impact on the seroprevalence of exposure to WNV among these horses. Due to these findings, we strongly recommend intensive surveillance and implementation of effective control and prevention strategies against WNV, especially in stallion, mixed-breed horses with ages ≥ 15 years.

Human Monoclonal Antibodies against NS1 Protein Protect against Lethal West Nile Virus Infection.

Envelope protein-targeted vaccines for flaviviruses are limited by concerns of antibody-dependent enhancement (ADE) of infections. Nonstructural protein 1 (NS1) provides an alternative vaccine target that avoids this risk since this protein is absent from the virion. Beyond its intracellular role in virus replication, extracellular forms of NS1 function in immune modulation and are recognized by host-derived antibodies. The rational design of NS1-based vaccines requires an extensive understanding of the antigenic sites on NS1, especially those targeted by protective antibodies. Here, we isolated human monoclonal antibodies (MAbs) from individuals previously naturally infected with WNV, mapped their epitopes using structure-guided mutagenesis, and evaluated their efficacy in vivo against lethal WNV challenge. The most protective epitopes clustered at three antigenic sites that are exposed on cell surface forms of NS1: (i) the wing flexible loop, (ii) the outer, electrostatic surface of the wing, and (iii) the spaghetti loop face of the ß-ladder. One additional MAb mapped to the distal tip of the ß-ladder and conferred a lower level of protection against WNV despite not binding to NS1 on the surface of infected cells. Our study defines the epitopes and modes of binding of protective anti-NS1 MAb antibodies following WNV infection, which may inform the development of NS1-based countermeasures against flaviviruses. IMPORTANCE Therapeutic antibodies against flaviviruses often promote neutralization by targeting the envelope protein of the virion. However, this approach is hindered by a possible concern for antibody-dependent enhancement of infection and paradoxical worsening of disease. As an alternative strategy, antibodies targeting flavivirus nonstructural protein 1 (NS1), which is absent from the virion, can protect against disease and do not cause enhanced infection. Here, we evaluate the structure-function relationships and protective activity of West Nile virus (WNV) NS1-specific monoclonal antibodies (MAbs) isolated from the memory B cells of a naturally infected human donor. We identify several anti-NS1 MAbs that protect mice against lethal WNV challenge and map their epitopes using charge reversal mutagenesis. Antibodies targeting specific regions in the NS1 structure could serve as the basis for countermeasures that control WNV infection in humans.

Infection-induced type I interferons critically modulate the homeostasis and function of CD8+ naïve T cells.

Naïve T (Tn) cells require two homeostatic signals for long-term survival: tonic T cell receptor:self-peptide-MHC contact and IL-7 stimulation. However, how microbial exposure impacts Tn homeostasis is still unclear. Here we show that infections can lead to the expansion of a subpopulation of long-lived, Ly6C+ CD8+ Tn cells with accelerated effector function. Mechanistically, mono-infection with West Nile virus transiently, and polymicrobial exposure persistently, enhances Ly6C expression selectively on CD5hiCD8+ cells, which in the case of polyinfection translates into a numerical CD8+ Tn cell increase in the lymph nodes. This conversion and expansion of Ly6C+ Tn cells depends on IFN-I, which upregulates MHC class I expression and enhances tonic TCR signaling in differentiating Tn cells. Moreover, for Ly6C+CD8+ Tn cells, IFN-I-mediated signals optimize their homing to secondary sites, extend their lifespan, and enhance their effector differentiation and antibacterial function, particularly for low-affinity clones. Our results thus uncover significant regulation of Tn homeostasis and function via infection-driven IFN-I, with potential implications for immunotherapy.

Nasal Immunization With Small Molecule Mast Cell Activators Enhance Immunity to Co-Administered Subunit Immunogens.

Mast cell activators are a novel class of mucosal vaccine adjuvants. The polymeric compound, Compound 48/80 (C48/80), and cationic peptide, Mastoparan 7 (M7) are mast cell activators that provide adjuvant activity when administered by the nasal route. However, small molecule mast cell activators may be a more cost-efficient adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. To identify novel mast cell activating compounds that could be evaluated for mucosal vaccine adjuvant activity, we employed high-throughput screening to assess over 55,000 small molecules for mast cell degranulation activity. Fifteen mast cell activating compounds were down-selected to five compounds based on in vitro immune activation activities including cytokine production and cellular cytotoxicity, synthesis feasibility, and selection for functional diversity. These small molecule mast cell activators were evaluated for in vivo adjuvant activity and induction of protective immunity against West Nile Virus infection in BALB/c mice when combined with West Nile Virus envelope domain III (EDIII) protein in a nasal vaccine. We found that three of the five mast cell activators, ST101036, ST048871, and R529877, evoked high levels of EDIII-specific antibody and conferred comparable levels of protection against WNV challenge. The level of protection provided by these small molecule mast cell activators was comparable to the protection evoked by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%). Thus, novel small molecule mast cell activators identified by high throughput screening are as efficacious as previously described mast cell activators when used as nasal vaccine adjuvants and represent next-generation mast cell activators for evaluation in mucosal vaccine studies.

Two Interferon-Stimulated Response Elements Cooperatively Regulate Interferon-Stimulated Gene Expression in West Nile Virus-Infected IFNAR-/- Mouse Embryo Fibroblasts.

We previously identified a subset of interferon-stimulated genes (ISGs) upregulated by West Nile virus (WNV) infection in wild-type mouse embryo fibroblasts (MEFs) after viral proteins had inhibited type I interferon (IFN)-mediated JAK-STAT signaling and also in WNV-infected RIG-I-/-, MDA5-/-, STAT1-/-, STAT2-/-, IFNAR-/-, IRF3-/-, IRF7-/-, and IRF3/7-/- MEFs. In this study, ISG upregulation by WNV infection in IFNAR-/- MEFs was confirmed by transcriptome sequencing (RNA-seq). ISG upregulation by WNV infection was inhibited in RIG-I/MDA5-/- MEFs. ISGs were upregulated in IRF1-/- and IRF5-/- MEFs but only minimally upregulated in IRF3/5/7-/- MEFs, suggesting redundant IRF involvement. We previously showed that a single proximal interferon-stimulated response element (ISRE) in the Oas1a and Oas1b promoters bound the ISGF3 complex after type I IFN treatment. In this study, we used wild-type and mutant promoter luciferase reporter constructs to identify critical regions in the Oas1b and Ifit1 promoters for gene activation in infected IFNAR-/- MEFs. Two ISREs were required in both promoters. Mutation of these ISREs in an Ifit1 promoter DNA probe reduced in vitro complex formation with infected nuclear extracts. An NF-κB inhibitor decreased Ifit1 promoter activity in cells and in vitro complex formation. IRF3 and p50 promoter binding was detected by chromatin immunoprecipitation (ChIP) for upregulated ISGs with two proximal ISREs. The data indicate that ISREs function cooperatively to upregulate the expression of some ISGs when type I IFN signaling is absent, with the binding complex consisting of IRF3, IRF5, and/or IRF7 and an NF-κB component(s) as well as other, as-yet-unknown factors. IMPORTANCE Type I IFN signaling in mammalian cells induces formation of the ISGF3 transcription factor complex, which binds to interferon stimulated response elements (ISREs) in the promoters of interferon-stimulated genes (ISGs) in the cell nucleus. Flavivirus proteins counteract type I IFN signaling by preventing either the formation or nuclear localization of ISGF3. A subset of ISRE-regulated ISGs was still induced in West Nile virus (WNV)-infected mouse embryo fibroblasts (MEFs), indicating that cells have an alternative mechanism for activating these ISGs. In this study, cellular components involved in this ISG upregulation mechanism were identified using gene knockout MEFs and ChIP, and critical promoter regions for gene activation were mapped using reporter assays. The data indicate a cooperative function between two ISREs and required binding of IRF3, IRF5, and/or IRF7 and an NF-κB component(s). Moreover, type I IFN signaling-independent ISG activation requires different additional promoter activation regions than type I IFN-dependent activation.

Obesity Enhances Disease Severity in Female Mice Following West Nile Virus Infection.

A rise in adiposity in the United States has resulted in more than 70% of adults being overweight or obese, and global obesity rates have tripled since 1975. Following the 2009 H1N1 pandemic, obesity was characterized as a risk factor that could predict severe infection outcomes to viral infection. Amidst the SARS-CoV-2 pandemic, obesity has remained a significant risk factor for severe viral disease as obese patients have a higher likelihood for developing severe symptoms and requiring hospitalization. However, the mechanism by which obesity enhances viral disease is unknown. In this study, we utilized a diet-induced obesity mouse model of West Nile virus (WNV) infection, a flavivirus that cycles between birds and mosquitoes and incidentally infects both humans and mice. Likelihood for severe WNV disease is associated with risk factors such as diabetes that are comorbidities also linked to obesity. Utilizing this model, we showed that obesity-associated chronic inflammation increased viral disease severity as obese female mice displayed higher mortality rates and elevated viral titers in the central nervous system. In addition, our studies highlighted that obesity also dysregulates host acute adaptive immune responses, as obese female mice displayed significant dysfunction in neutralizing antibody function. These studies highlight that obesity-induced immunological dysfunction begins at early time points post infection and is sustained through memory phase, thus illuminating a potential for obesity to alter the differentiation landscape of adaptive immune cells.

Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay.

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

West Nile Virus and Tick-Borne Encephalitis Virus Are Endemic in Equids in Eastern Austria.

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in addition to the autochthonous tick-borne encephalitis virus (TBEV) in Europe causes rising concern for public and animal health. The first equine case of West Nile neuroinvasive disease in Austria was diagnosed in 2016. As a consequence, a cross-sectional seroprevalence study was conducted in 2017, including 348 equids from eastern Austria. Serum samples reactive by ELISA for either flavivirus immunoglobulin G or M were further analyzed with the plaque reduction neutralization test (PRNT-80) to identify the specific etiologic agent. Neutralizing antibody prevalences excluding vaccinated equids were found to be 5.3% for WNV, 15.5% for TBEV, 0% for USUV, and 1.2% for WNV from autochthonous origin. Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect WNV nucleic acid in horse sera and was found to be negative in all cases. Risk factor analysis did not identify any factors significantly associated with seropositivity.

Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission.

The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention.

Levels of Circulating NS1 Impact West Nile Virus Spread to the Brain.

Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

Prevalence of arbovirus antibodies in young healthy adult population in Brazil.

BACKGROUND: The emergence and re-emergence of infectious diseases are a cause for worldwide concern. The introduction of Zika and Chikungunya diseases in the Americas has exposed unforeseen medical and logistical challenges for public health systems. Moreover, the lack of preventive measures and vaccination against known and emerging mosquito-transmitted pathogens, and the occurrence of unanticipated clinical complications, has had an enormous social and economic impact on the affected populations. In this study, we aimed to measure the seroprevalence of endemic and emerging viral pathogens in military personnel stationed in Manaus, Amazonas state. METHODS: We measured the seropositivity of antibodies against 19 endemic and emerging viruses in a healthy military personnel group using a hemagglutination inhibition assay (HIA). RESULTS: Overall, DENV positivity was 60.4%, and 30.9% of the individuals reacted against ZIKV. Also, 46.6%, 54.7%, 51.3% and 48.7% individuals reacted against West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilheus virus (ILHV) and Rocio virus (ROCV), respectively. Individuals with high DENV HIA titer reacted more frequently with ZIKV or WNV compared to those with low HIA titers. Observed cross-reactivity between Flaviviruses varied depending on the virus serogroup. Additionally, 0.6% and 0.3% individuals were seropositive for Oropouche virus (OROV) and Catu virus (CATUV) from the family Peribunyaviridae, respectively. All samples were negative for Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalomyelitis virus (WEEV), Mayaro virus (MAYV), Mucambo virus (MUCV) and CHIKV from the family Togaviridae. CONCLUSIONS: A high proportion of individuals in our high-risk population (~ 60%) lacked antibodies against major endemic and emerging viruses, which makes them susceptible for further infections. Military personnel serving in the Amazon region could serve as sentinels to strengthen global infectious disease surveillance, particularly in remote areas.

Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

Evaluating the Safety of West Nile Virus Immunity During Congenital Zika Virus Infection in Mice.

Antibody-dependent enhancement (ADE) is a phenomenon that occurs when cross-reactive antibodies generated from a previous flaviviral infection increase the pathogenesis of a related virus. Zika virus (ZIKV) is the most recent flavivirus introduced to the Western Hemisphere and has become a significant public health threat due to the unanticipated impact on the developing fetus. West Nile virus (WNV) is the primary flavivirus that circulates in North America, and we and others have shown that antibodies against WNV are cross-reactive to ZIKV. Thus, there is concern that WNV immunity could increase the risk of severe ZIKV infection, particularly during pregnancy. In this study, we examined the extent to which WNV antibodies could impact ZIKV pathogenesis in a murine pregnancy model. To test this, we passively transferred WNV antibodies into pregnant Stat2-/- mice on E6.5 prior to infection with ZIKV. Evaluation of pregnant dams showed weight loss following ZIKV infection; however, no differences in maternal weights or viral loads in the maternal brain, spleen, or spinal cord were observed in the presence of WNV antibodies. Resorption rates, and other fetal parameters, including fetal and placental size, were similarly unaffected. Further, the presence of WNV antibodies did not significantly alter the viral load or the inflammatory response in the placenta or the fetus in response to ZIKV. Our data suggest that pre-existing WNV immunity may not significantly impact the pathogenesis of ZIKV infection during pregnancy. Our findings are promising for the safety of implementing WNV vaccines in the continental US.

Seroprevalence of arboviruses in Ecuador: Implications for improved surveillance. / Seroprevalencia de arbovirus en Ecuador: implicaciones para mejorar su vigilancia

INTRODUCTION: Arthropod-borne viruses (arboviruses) cause morbidity and mortality in humans and domestic animals worldwide. The percentage of population immunity or susceptibility to these viruses in Ecuador is unknown. OBJECTIVES: To investigate the proportion of Ecuadorian populations with IgG antibodies (Abs) (past exposure/immunity) and IgM Abs (current exposure) against flaviviruses and alphaviruses and to study the activity of these viruses in Ecuador. MATERIALS AND METHODS: During 2009-2011, we conducted a serosurvey for selected arboviruses in humans (n=1,842), equines (n=149), and sentinel hamsters (n=84) at two coastal locations and one in the Amazon basin (Eastern Ecuador) using enzyme-linked immunosorbent assay and hemagglutination inhibition test. RESULTS: From 20.63% to 63.61% of humans showed IgG-antibodies for the flaviviruses: Dengue virus (DENV), yellow fever virus (YFV) Saint Louis encephalitis virus, and West Nile virus (WNV); from 4.67% to 8.63% showed IgG-Abs for the alphaviruses: Venezuelan equine encephalitis virus, eastern equine encephalitis virus, and western equine encephalitis virus. IgM-Abs were found for DENV and WNV. Equines and hamsters showed antibodies to alphaviruses in all locations; two hamsters seroconverted to YFV in the Amazonia. CONCLUSIONS: The results show a YFV vaccination history and suggest the activity of arboviruses not included in the current surveillance scheme. Enhanced arbovirus and mosquito surveillance, as well as continued YFV vaccination and evaluation of its coverage/effectiveness, are recommended.

Introducción. Los virus transmitidos por artrópodos (arbovirus) causan morbilidad y mortalidad en humanos y animales domésticos mundialmente. Se desconoce el porcentaje de inmunidad o vulnerabilidad de la población ecuatoriana ante estos virus. Objetivos. Investigar la proporción de poblaciones ecuatorianas con anticuerpos IgG (exposición o inmunidad pasada) y anticuerpos IgM (exposición reciente) contra flavivirus y alfavirus, e investigar su actividad en Ecuador. Materiales y métodos. Entre 2009 y 2011, se llevó a cabo una encuesta serológica para arbovirus en humanos (n=1.842), equinos (n=149) y hámsters centinela (n=84) en dos localidades costeras y en una en la Amazonía, utilizando la prueba ELISA (Enzyme-Linked ImmunoSorbent Assay) y la prueba de inhibición de la hemaglutinación. Resultados. Entre el 20,63 y el 63,61 % de los humanos registraron IgG contra el virus del dengue (DENV), el de la fiebre amarilla (YFV), el de la encefalitis de San Luis y el del Nilo Occidental (WNV); entre 4,67 y 8,63 % tenían IgG para los virus de la encefalitis equina venezolana, de la encefalitis equina del este y de la encefalitis equina del oeste. Se encontró IgM para DENV y WNV. En los equinos y en los hámsters se encontraron anticuerpos contra alfavirus en todas las localidades muestreadas; dos hámsters mostraron seroconversión a YFV en la Amazonía. Conclusiones. Los resultados del estudio evidenciaron los antecedentes de vacunación contra el YFV y sugieren la actividad de arbovirus no incluidos en el esquema de vigilancia actual. Se recomienda ampliar la vigilancia de arbovirus y mosquitos, continuar con la vacunación contra el YFV, y evaluar su cobertura y efectividad.

Identification of Anti-Premembrane Antibody as a Serocomplex-Specific Marker To Discriminate Zika, Dengue, and West Nile Virus Infections.

Although transmission of Zika virus (ZIKV) in the Americas has greatly declined since late 2017, recent reports of reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection underscore a critical need for serological tests that can discriminate past ZIKV, DENV, and/or other flavivirus infections and improve our understanding of the immune interactions between these viruses and vaccine strategy in endemic regions. As serological tests for ZIKV primarily focus on envelope (E) and nonstructural protein 1 (NS1), antibodies to other ZIKV proteins have not been explored. Here, we employed Western blot analysis using antigens of 6 flaviviruses from 3 serocomplexes to investigate antibody responses following reverse transcription-PCR (RT-PCR)-confirmed ZIKV infection. Panels of 20 primary ZIKV and 20 ZIKV with previous DENV infection recognized E proteins of all 6 flaviviruses and the NS1 protein of ZIKV with some cross-reactivity to DENV. While the primary ZIKV panel recognized only the premembrane (prM) protein of ZIKV, the ZIKV with previous DENV panel recognized both ZIKV and DENV prM proteins. Analysis of antibody responses following 42 DENV and 18 West Nile virus infections revealed similar patterns of recognition by anti-E and anti-NS1 antibodies, whereas both panels recognized the prM protein of the homologous serocomplex but not others. The specificity was further supported by analysis of sequential samples. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be used to delineate current and past flavivirus infections in endemic areas. IMPORTANCE Despite a decline in Zika virus (ZIKV) transmission since late 2017, questions regarding its surveillance, potential reemergence, and interactions with other flaviviruses in regions where it is endemic remain unanswered. Recent studies have reported reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection, highlighting a need for better serological tests to discriminate past ZIKV, DENV, and/or other flavivirus infections and improved understanding of the immune interactions and vaccine strategy for these viruses. As most serological tests for ZIKV focused on envelope and nonstructural protein 1, antibodies to other ZIKV proteins, including potentially specific antibodies, remain understudied. We employed Western blot analysis using antigens of 6 flaviviruses to study antibody responses following well-documented ZIKV, DENV, and West Nile virus infections and identified anti-premembrane antibody as a flavivirus serocomplex-specific marker to delineate current and past flavivirus infections in areas where flaviviruses are endemic.