Identification of Salivary Gland Escape Barriers to Western Equine Encephalitis Virus in the Natural Vector, Culex tarsalis

Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.

Cross-Strain Neutralizing and Protective Monoclonal Antibodies against EEEV or WEEV

The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.

Seroprevalence of arboviruses in Ecuador: Implications for improved surveillance

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.

Stabilization of a Broadly Neutralizing Anti-Chikungunya Virus Single Domain Antibody

A single domain antibody (clone CC3) previously found to neutralize a vaccine strain of the chikungunya virus (PRNT50 = 2. 5 ng/mL) was found to be broadly neutralizing. Clone CC3 is not only able to neutralize a wild-type (WT) strain of chikungunya virus (CHIKV), but also neutralizes WT strains of Mayaro virus (MAYV) and Ross River virus (RRV); both arthralgic, Old World alphaviruses. Interestingly, CC3 also demonstrated a degree of neutralizing activity against the New World alphavirus, Venezuelan equine encephalitis virus (VEEV); albeit both the vaccine strain, TC-83, and the parental, WT Trinidad donkey strain had PRNT50 values ~1,000-fold higher than that of CHIKV. However, no neutralization activity was observed with Western equine encephalitis virus (WEEV). Ten CC3 variants designed to possess a range of isoelectric points, both higher and lower, were constructed. This approach successfully identified several lower pI mutants which possessed improved thermal stabilities by as much as 10°C over the original CC3 (Tm = 62°C), and excellent refolding abilities while maintaining their capacity to bind and neutralize CHIKV.

Immune Responses in Mice Vaccinated with Virus-Like Particles of Western Equine Encephalitis Virus from an Insect Cell-Based Expression System

Western equine encephalitis virus (WEEV) can cause lethal encephalitis in humans and equines and represents a serious public health threat in many countries. Therefore, development of efficient vaccines against WEEV remains an important challenge in the field of disease control. This study described for the first time successful production of WEEV virus-like particles (VLPs) in insect cells using recombinant baculoviruses. This well-established expression system is very suitable for production of WEEV VLPs. The immune experiment herein in mice showed that the VLPs formulated with 206-adjuvant were responsible for the stronger-VLP-specific cellular immune response, and were able to induce the secretion of IL-2, IL-4, IFN-γ and production of high titer antibodies that can effectively neutralize the WEEV pseudoviruses. The WEEV VLPs from insect cells could provide a new, safe, non-replicating and effective vaccine candidate against WEEV infections.

First Record of Mosquito-Borne Kyzylagach Virus in Central Europe

RNA of Kyzylagach virus (KYZV), a Sindbis-like mosquito-borne alphavirus from Western equine encephalitis virus complex, was detected in four pools (out of 221 pools examined), encompassing 10,784 female Culex modestus mosquitoes collected at a fishpond in south Moravia, Czech Republic, with a minimum infection rate of 0.04%. This alphavirus was never detected in Central Europe before.

Transmission of West Nile and five other temperate mosquito-borne viruses peaks at temperatures between 23°C and 26°C

The temperature-dependence of many important mosquito-borne diseases has never been quantified. These relationships are critical for understanding current distributions and predicting future shifts from climate change. We used trait-based models to characterize temperature-dependent transmission of 10 vector–pathogen pairs of mosquitoes (Culex pipiens, Cx. quinquefascsiatus, Cx. tarsalis, and others) and viruses (West Nile, Eastern and Western Equine Encephalitis, St. Louis Encephalitis, Sindbis, and Rift Valley Fever viruses), most with substantial transmission in temperate regions. Transmission is optimized at intermediate temperatures (23–26°C) and often has wider thermal breadths (due to cooler lower thermal limits) compared to pathogens with predominately tropical distributions (in previous studies). The incidence of human West Nile virus cases across US counties responded unimodally to average summer temperature and peaked at 24°C, matching model-predicted optima (24–25°C). Climate warming will likely shift transmission of these diseases, increasing it in cooler locations while decreasing it in warmer locations.