Eastern Equine Encephalitis Research Articles

West Nile Virus and Other Domestic Nationally Notifiable Arboviral Diseases - United States, 2018.

Arthropodborne viruses (arboviruses) are transmitted to humans primarily through the bites of infected mosquitoes and ticks. West Nile virus (WNV) is the leading cause of domestically acquired arboviral disease in the continental United States (1). Other arboviruses, including eastern equine encephalitis, Jamestown Canyon, La Crosse, Powassan, and St. Louis encephalitis viruses, cause sporadic cases of disease and occasional outbreaks. This report summarizes surveillance data reported to CDC for 2018 on nationally notifiable arboviruses. It excludes dengue, chikungunya, and Zika viruses because they are primarily nondomestic viruses typically acquired through travel. In 2018, 48 states and the District of Columbia (DC) reported 2,813 cases of domestic arboviral disease, including 2,647 (94%) WNV disease cases. Of the WNV disease cases, 1,658 (63%) were classified as neuroinvasive disease (e.g., meningitis, encephalitis, and acute flaccid paralysis), for a national incidence of 0.51 cases of WNV neuroinvasive disease per 100,000 population. Because arboviral diseases continue to cause serious illness and have no definitive treatment, maintaining surveillance is important to direct and promote prevention activities. Health care providers should consider arboviral infections in patients with aseptic meningitis or encephalitis, perform appropriate diagnostic testing, and report cases to public health authorities.

Five Emerging Neuroinvasive Arboviral Diseases: Cache Valley, Eastern Equine Encephalitis, Jamestown Canyon, Powassan, and Usutu

There are many arthropod-borne viruses (arboviruses) capable of neuroinvasion, with West Nile virus being one of the most well known. In this review, we highlight five rarer emerging or reemerging arboviruses capable of neuroinvasion: Cache Valley, eastern equine encephalitis, Jamestown Canyon, Powassan, and Usutu viruses. Cache Valley and Jamestown Canyon viruses likely circulate throughout most of North America, while eastern equine encephalitis and Powassan viruses typically circulate in the eastern half. Usutu virus is not currently circulating in North America, but has the potential to be introduced in the future given similar climate, vectors, and host species to Europe (where it has been circulating). Health care providers should contact their state or local health departments with any questions regarding arboviral disease surveillance, diagnosis, treatment, or prevention. To prevent neuroinvasive arboviral diseases, use of insect repellent and other mosquito and tick bite prevention strategies are key.

Potential Viroporin Candidates From Pathogenic Viruses Using Bacteria-Based Bioassays

Viroporins are a family of small hydrophobic proteins found in many enveloped viruses that are capable of ion transport. Building upon the ability to inhibit influenza by blocking its archetypical M2 H+ channel, as a family, viroporins may represent a viable target to curb viral infectivity. To this end, using three bacterial assays we analyzed six small hydrophobic proteins from biomedically important viruses as potential viroporin candidates. Our results indicate that Eastern equine encephalitis virus 6k, West Nile virus MgM, Dengue virus 2k, Dengue virus P1, Variola virus gp170, and Variola virus gp151 proteins all exhibit channel activity in the bacterial assays, and as such may be considered viroporin candidates. It is clear that more studies, such as patch clamping, will be needed to characterize the ionic conductivities of these proteins. However, our approach presents a rapid procedure to analyze open reading frames in other viruses, yielding new viroporin candidates for future detailed investigation. Finally, if conductivity is proven vital to their cognate viruses, the bio-assays presented herein afford a simple approach to screen for new channel blockers.

SEROLOGICAL SURVEY FOR SELECT INFECTIOUS AGENTS IN WILD MAGELLANIC PENGUINS (SPHENISCUS MAGELLANICUS) IN ARGENTINA, 1994–2008

Despite being the most numerous penguin species in South America, exposure of the Magellanic Penguin (Spheniscus magellanicus) to pathogens has not yet been thoroughly assessed. We collected serum from 1,058 Magellanic Penguins at 10 breeding colonies along the entire latitudinal range of this species in Argentina. The work spanned 10 breeding seasons over 15 yr (1994-2008). Sera were tested for antibodies to select infectious agents. Antibodies reacting against 16 pathogens were detected (seroprevalence): Aspergillus sp. (15.1%), Chlamydia psittaci (6.5%), Salmonella Pullorum (3.1%), Salmonella Typhimurium (81.3%), Aviadenovirus sp. (18.1%), Duck atadenovirus A (23.6%), Anatid herpesvirus 1 (0.7%), Avian orthoreovirus (3.3%), Avian coronavirus M41 (43.5%), Avian coronavirus C46 (59.8%), Avian coronavirus A99 (37.4%), Avian coronavirus JMK (40.2%), Tremovirus A (0.3%), Avian avulavirus 1 (44.0%), Avian avulavirus 2 (43.8%), and Avian avulavirus 3 (46.6%). No antibodies were detected against nine infectious agents: Gallid alphaherpesvirus 1, Gallid alphaherpesvirus 2, Infectious bursal disease virus, Avastrovirus 2, West Nile virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Influenza A virus. While restricted by limitations inherent to serological methods, our results provide baseline knowledge for a key species in the South Atlantic Ocean. This information is valuable for adaptive conservation management in a time of increasing environmental stressors affecting the Patagonian Sea, one of the world's richest pelagic seabird communities.

Weather-based forecasting of mosquito-borne disease outbreaks in Canada.

Early warning systems to predict infectious disease outbreaks have been identified as a key adaptive response to climate change. Warming, climate variability and extreme weather events associated with climate change are expected to drive an increase in frequency and intensity of mosquito-borne disease (MBD) outbreaks globally. In Canada, this will mean an increased risk of endemic and emerging MBD outbreaks such as West Nile virus and other MBDs. The availability of timely information on the risk of impending MBD outbreaks has important public health implications, by allowing implementation of mosquito control measures and targeted communications regarding the need for increased personal protective measures-before an outbreak occurs. In Canada, both mechanistic and statistical weather-based models have been developed to predict West Nile virus outbreaks. These include models for different species of mosquitoes that transmit West Nile virus in different geographical areas of Canada. Although initial results have been promising, further validation and assessment of forecasting skill are needed before wide scale implementation. Weather-based forecasting for other emerging MBDs in Canada, such as Eastern equine encephalitis, may also be feasible.

Complex Epidemiological Dynamics of Eastern Equine Encephalitis Virus in Florida.

Eastern equine encephalitis virus (EEEV) infection results in high mortality in infected horses and humans. Florida has been identified as an important source of EEEV epidemics to other states in the United States. In this study, we further characterized the epidemiological and evolutionary dynamics of EEEV in Florida. Epidemiological analysis of sentinel chicken seroconversion rates to EEEV infections during 2005-2016 suggested significant seasonality of EEEV activity in Florida. We observed significant annual activity of EEEV in the North and North Central regions, with little significant seasonality in the Panhandle region. Phylogenetic analysis of complete EEEV genome sequences from different host sources and regions in Florida during 1986-2014 revealed extensive genetic diversity and spatial dispersal of the virus within Florida and relatively more clustering of the viruses in the Panhandle region. We found no significant association between EEEV genetic variation and host source. Overall, our study revealed a complex epidemiological dynamic of EEEV within Florida, implicating the Panhandle region as a possible source of the virus with sustained year-round transmission. These findings will help in implementing targeted control measures that can have the most impact in reducing or eliminating EEEV and other mosquito-borne viral infections within Florida and in the rest of the United States.

The transmission dynamic of Madariaga Virus by bayesian phylogenetic analysis: Molecular surveillance of an emergent pathogen

Madariaga Virus (MADV) is an emergent Alphavirus of the eastern equine encephalitis virus (EEEV) strain complex causing epizootic epidemics. In this study the genetic diversity and the transmission dynamics of Madariaga virus has been investigated by Bayesian phylogenetics and phylodynamic analysis. A database of 32 sequences of MADV group structural polyprotein were downloaded from GenBank, aligned manually edited by Bioedit Software. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Neighbor-joining tree was generated using MEGA7. The phylogenetic signal of the dataset was tested by the likelihood mapping analysis. The Bayesian phylogenetic tree was built using BEAST. Selective pressure analysis revealed one positive selection site. The phylogenetic trees showed two main clusters. In particular, Lineage II showed an epizootic infection in monkeys and Lineage III, including 2 main clusters (IIIa and IIIB), revealing an epizootic infection in humans in Haiti and an epizootic infection in humans in Venezuela during the 2016, respectively. The Bayesian maximum clade credibility tree and the time of the most common recent ancestor estimates, showed that the root of the tree dated back to the year 346 with the probable origin in Brazil. Gene flow analysis revealed viral exchanges between different neighbor countries of South America. In conclusion, Bayesian phylogenetic and phylodynamic represent useful tools to follow the transmission dynamic of emergent pathogens to prevent new epidemics spreading worldwide.

Efficacy of a ML336 derivative against Venezuelan and eastern equine encephalitis viruses

Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5 mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48 h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25 mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase.

Increased risk of endemic mosquito-borne diseases in Canada due to climate change.

There are currently over 80 species of mosquito endemic in Canada-although only a few of these carry pathogens that can cause disease in humans. West Nile virus, Eastern equine encephalitis virus and the California serogroup viruses (including the Jamestown Canyon and snowshoe hare viruses) are mosquito-borne viruses that have been found to cause human infections in North America, including in Canada. Over the last 20 years, the incidence of most of these endemic mosquito-borne diseases (MBD) has increased approximately 10% in Canada, due in large part to climate change. It is anticipated that both the mosquito lifecycle and virus transmission patterns will be affected by climate change, resulting in an increase in both the range and local abundance of several important mosquito species. Laboratory studies and mathematical modelling suggest that increased ambient temperatures, changes in precipitation and extreme weather events associated with climate change will likely continue to drive mosquito vector and MBD range expansion, increasing the duration of transmission seasons and leading to MBD-related epidemics. Furthermore, Canada's endemic MBDs have complex transmission cycles, involving multiple reservoir hosts (birds and mammals), multiple pathogens and multiple mosquito species-all of which may be sensitive to climate and other environmental changes, and making forecasting of potential emerging trends difficult. These expected climate-induced changes in mosquitoes and MBDs underline the need for continued (and expanded) surveillance and research to ensure timely and accurate evaluation of the risks to the public health of Canadians.

Facts About Wildlife Diseases: Eastern Equine Encephalitis

The Eastern equine encephalitis virus (EEEV) causes Eastern equine encephalitis (EEE) disease in birds, humans, horses, and other animals in the eastern United States, including Florida, which is especially receptive to the virus because of its freshwater hardwood swamps and wetlands, which provide good niches for the mosquito vectors of the disease. This 5-page fact sheet written by Samantha Wisely and Karen Hood and published by the UF/IFAS Department of Wildlife Ecology and Conservation describes the disease and includes strategies for limiting its spread. http://edis.ifas.ufl.edu/uw453

Rational design of a live-attenuated eastern equine encephalitis virus vaccine through informed mutation of virulence determinants.

Live attenuated vaccines (LAVs), if sufficiently safe, provide the most potent and durable anti-pathogen responses in vaccinees with single immunizations commonly yielding lifelong immunity. Historically, viral LAVs were derived by blind passage of virulent strains in cultured cells resulting in adaptation to culture and a loss of fitness and disease-causing potential in vivo. Mutations associated with these phenomena have been identified but rarely have specific attenuation mechanisms been ascribed, thereby limiting understanding of the attenuating characteristics of the LAV strain and applicability of the attenuation mechanism to other vaccines. Furthermore, the attenuated phenotype is often associated with single nucleotide changes in the viral genome, which can easily revert to the virulent sequence during replication in animals. Here, we have used a rational approach to attenuation of eastern equine encephalitis virus (EEEV), a mosquito-transmitted alphavirus that is among the most acutely human-virulent viruses endemic to North America and has potential for use as an aerosolized bioweapon. Currently, there is no licensed antiviral therapy or vaccine for this virus. Four virulence loci in the EEEV genome were identified and were mutated individually and in combination to abrogate virulence and to resist reversion. The resultant viruses were tested for virulence in mice to examine the degree of attenuation and efficacy was tested by subcutaneous or aerosol challenge with wild type EEEV. Importantly, all viruses containing three or more mutations were avirulent after intracerebral infection of mice, indicating a very high degree of attenuation. All vaccines protected from subcutaneous EEEV challenge while a single vaccine with three mutations provided reproducible, near-complete protection against aerosol challenge. These results suggest that informed mutation of virulence determinants is a productive strategy for production of LAVs even with highly virulent viruses such as EEEV. Furthermore, these results can be directly applied to mutation of analogous virulence loci to create LAVs from other viruses.

Structural Insights into Entry and Antibody Neutralization of Eastern Equine Encephalitis Virus

Alphaviruses are enveloped pathogens that cause arthritis and encephalitis. Host entry of alphaviruses involves binding to plasma and endosomal membranes followed by low pH-triggered membrane fusion. Here we present a 3.5-6.5Å resolution (average 4.4Å) cryo-electron microscopy (cryoEM) structure of Eastern Equine Encephalitis virus (EEEV), an alphavirus that often causes fatal encephalitis in humans. Motifs for potential binding of host membrane receptors were identified. The capsid protein structure showed an RNA genome-binding segment and an inherently unstable nucleocapsid core primed for disassembly. CryoEM structures of five different Fab-EEEV complexes derived from neutralizing antibodies showed how the icosahedral structure of alphaviruses poses steric restrictions on interactions with antibodies. These results also identified molecular mechanisms of host cell entry that likely are conserved among enveloped icosahedral viruses.

Investigation of serological cross-reactivity within the alphavirus genus using IFA biochip mosaics

Purpose: The alphavirus genus comprises several human pathogens, e.g. chikungunya virus (CHIKV), Ross River virus (RRV) and Eastern equine encephalitis virus (EEEV). Depending on the antigenic relatedness cross-reactivity in serological assays is commonly observed. Due to overlapping geographical distribution and similar clinical pictures a serological differentiation is desirable. Methods & Materials: Human serum or plasma samples from different geographical areas and precharacterised for IgG and IgM antibodies against various alphaviruses were investigated with multiplex indirect immunofluorescence assays. The method is based on a mosaic of up to nine biochips which carry infected or transfected cell lines each expressing antigens of another alphavirus. The samples were tested for the presence of specific IgG and IgM antibodies and endpoint titers were determined for every virus in parallel. Results: Investigation of samples from patients with different alphavirus infections led to a significant amount of cross-reactivities on IFA biochip mosaics, especially regarding IgG antibodies. Using parallel endpoint titration it was possible to determine the causative alphavirus in the vast majority of samples. For example, investigating samples from RRV infected patients, 93% and 11% reacted positive on CHIKV infected cells regarding IgG and IgM antibodies, respectively. Regarding samples from Barmah-Forest virus (BFV) infected patients, 28% and 3% were positive on CHIKV for IgG and IgM, respectively. By comparison of the endpoint titers for the respective viruses resulting from the same incubation on a biochip mosaic, 88% of RRV IgG samples and all IgM samples showed an at least ten times higher titer on the RRV biochip than on the CHIKV biochip. Regarding BFV patients, all samples showed at least ten times higher titers for BFV than for CHIKV in both IgG and IgM investigation. Conclusion: Cross-reactivity of IgG and IgM antibodies plays an important role in serological diagnostics of alphavirus infections. Parallel investigation of a sample using different alphavirus antigens in a biochip mosaic can identify the causative virus in most of the cases, depending on the antigenic relatedness of the viruses.

The role of Arbovirus genome untranslated regions on neurotropism

Purpose: Arboviruses are a group of viruses transmitted by arthropods, mostly ticks, and mosquitoes. Among the members of this group are the families Togaviridae and Flaviviridae, (+)ssRNA viruses, such as Chikungunya, Dengue, and Zika. Multiple of these viruses have demonstrated their ability to cause neuropathology in humans. In Zika, a protein binding motif known as Musashi binding element (MBE) has been attributed to promoting replication, neurotropism, and pathology. Musashi-1 (MSI1) is an RNA-binding protein involved in the maintenance and self-renewal of stem cells and a translational regulator in many biological systems. MSI1 predominantly binds single-stranded UAG motifs in the 3’ untranslated region (UTR) of RNA. We have recently analyzed Musashi binding elements (MBEs) in the 3’UTR of flaviviruses (FV) in silico. In this study, we could show that MBEs in the 3’UTR of neurotropic viruses such as Zika, West Nile, and Powassan virus are highly accessible, and mostly occur in an unpaired structural context, which renders them optimal Musashi binding targets and corroborates previous experimental studies by a theoretical model. Methods & Materials: In order to expand to other related viruses, we systematically analyzed the properties of Musashi binding elements (MBEs) in the 3’UTR of Alphaviruses (AV) based on a thermodynamic model for RNA folding and correlated to the currently described pathogenicity in literature. Results: Our preliminary results indicate that MBEs in the 3’UTR of Chikungunya, Venezuelan Equine Encephalitis, and Eastern Equine Encephalitis viruses occur predominantly in paired, double-stranded structural context, suggesting that the mechanism to cause neurotropism in these AV must be a different one. Analyses in other AV are currently being performed to investigate the role of MBEs in a more broad dataset. Our initial analysis suggests that the MBE role in neuropathology may be an exclusive feature of FV. Conclusion: Our study addresses the broader question whether other emerging arboviruses can cause similar neurotropic effects. We expanded our study from a previous Flavivirus dataset to all (+)ssRNA arboviruses to evaluate the link between MBE and currently described neuropathology. Moreover, our thermodynamic model can be the initial indicative for in vivo studies to evaluate the potential neurotropic effect of MBEs on different viral families.

Emergence of Madariaga virus as a cause of acute febrile illness in children, Haiti, 2015-2016.

Madariaga virus (MADV), also known as South American eastern equine encephalitis virus, has been identified in animals and humans in South and Central America, but not previously in Hispaniola or the northern Caribbean. MADV was isolated from virus cultures of plasma from an 8-year-old child in a school cohort in the Gressier/Leogane region of Haiti, who was seen in April, 2015, with acute febrile illness (AFI). The virus was subsequently cultured from an additional seven AFI case patients from this same cohort in February, April, and May 2016. Symptoms most closely resembled those seen with confirmed dengue virus infection. Sequence data were available for four isolates: all were within the same clade, with phylogenetic and molecular clock data suggesting recent introduction of the virus into Haiti from Panama sometime in the period from October 2012-January 2015. Our data document the movement of MADV into Haiti, and raise questions about the potential for further spread in the Caribbean or North America.

Self-Amplifying RNA Vaccines for Venezuelan Equine Encephalitis Virus Induce Robust Protective Immunogenicity in Mice

Venezuelan equine encephalitis virus (VEEV) is a known biological defense threat. A live-attenuated investigational vaccine, TC-83, is available, but it has a high non-response rate and can also cause severe reactogenicity. We generated two novel VEE vaccine candidates using self-amplifying mRNA (SAM). LAV-CNE is a live-attenuated VEE SAM vaccine formulated with synthetic cationic nanoemulsion (CNE) and carrying the RNA genome of TC-83. IAV-CNE is an irreversibly-attenuated VEE SAM vaccine formulated with CNE, delivering a TC-83 genome lacking the capsid gene. LAV-CNE launches a TC-83 infection cycle in vaccinated subjects but eliminates the need for live-attenuated vaccine production and potentially reduces manufacturing time and complexity. IAV-CNE produces a single cycle of RNA amplification and antigen expression without generating infectious viruses in subjects, thereby creating a potentially safer alternative to live-attenuated vaccine. Here, we demonstrated that mice vaccinated with LAV-CNE elicited immune responses similar to those of TC-83, providing 100% protection against aerosol VEEV challenge. IAV-CNE was also immunogenic, resulting in significant protection against VEEV challenge. These studies demonstrate the proof of concept for using the SAM platform to streamline the development of effective attenuated vaccines against VEEV and closely related alphavirus pathogens such as western and eastern equine encephalitis and Chikungunya viruses.

Aerosol infection of Balb/c mice with eastern equine encephalitis virus; susceptibility and lethality

BackgroundEastern equine encephalitis virus is an alphavirus that naturally cycles between mosquitoes and birds or rodents in Eastern States of the US. Equine infection occurs by being bitten by cross-feeding mosquitoes, with a case fatality rate of up to 75% in humans during epizootic outbreaks. There are no licensed medical countermeasures, and with an anticipated increase in mortality when exposed by the aerosol route based on anecdotal human data and experimental animal data, it is important to understand the pathogenesis of this disease in pursuit of treatment options. This report details the clinical and pathological findings of mice infected with EEEV by the aerosol route, and use as a model for EEEV infection in humans.MethodsMice were exposed by the aerosol route to a dose range of EEEV to establish the median lethal dose. A pathogenesis study followed whereby mice were exposed to a defined dose of virus and sacrificed at time-points thereafter for histopathological analysis and virology.ResultsClinical signs of disease appeared within 2 days post challenge, culminating in severe clinical signs within 24 h, neuro-invasion and dose dependent lethality. EEEV was first detected in the lung 1 day post challenge, and by day 3 peak viral titres were observed in the brain, spleen and blood, corresponding with severe meningoencephalitis, indicative of encephalitic disease. Lethality follows severe neurological signs, and may be linked to a threshold level of virus replication in the brain. Effective medical countermeasures for EEEV may necessitate early inoculation to inhibit infection of the brain in zoonotic incidents, and be able to traverse the blood-brain barrier to sufficiently interrupt replication in the brain in cases of aerosol infection.ConclusionsThere is little human data on the hazard posed by aerosol infection with encephalitic alphaviruses, and use of EEEV as a bioweapon may be by the aerosol route. A well characterized model of aerosol exposure that recapitulates some of the most severe human clinical features is necessary to evaluate the efficacy of putative medical countermeasures, and to increase our understanding about how this route of infection induces such rapid neuro-invasion and resulting disease.

Broad-spectrum monoclonal antibodies against chikungunya virus structural proteins: Promising candidates for antibody-based rapid diagnostic test development.

In response to the aggressive global spread of the mosquito-borne chikungunya virus (CHIKV), an accurate and accessible diagnostic tool is of high importance. CHIKV, an arthritogenic alphavirus, comprises three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. A previous rapid immunochromatographic (IC) test detecting CHIKV E1 protein showed promising performance for detection of the ECSA genotype. Unfortunately, this kit exhibited lower capacity for detection of the Asian genotype, currently in circulation in the Americas, reflecting the low avidity of one of the monoclonal antibodies (mAbs) in this IC kit for the E1 protein of the Asian-genotype because of a variant amino acid sequence. To address this shortcoming, we set out to generate a new panel of broad-spectrum mouse anti-CHIKV mAbs using hybridoma technology. We report here the successful generation of mouse anti-CHIKV mAbs targeting CHIKV E1 and capsid proteins. These mAbs possessed broad reactivity to all three CHIKV genotypes, while most of the mAbs lacked cross-reactivity towards Sindbis, dengue, and Zika viruses. Two of the mAbs also lacked cross-reactivity towards other alphaviruses, including O'nyong-nyong, Ross River, Mayaro, Western Equine Encephalitis, Eastern Equine Encephalitis, and Venezuelan Equine Encephalitis viruses. In addition, another two mAbs cross-reacted weakly only with most closely related O'nyong-nyong virus. Effective diagnosis is one of the keys to disease control but to date, no antibody-based rapid IC platform for CHIKV is commercially available. Thus, the application of the mAbs characterized here in the rapid diagnostic IC kit for CHIKV detection is expected to be of great value for clinical diagnosis and surveillance purposes.

THE ARTHROPOD-BORNE ENCEPHALITIS VIRUSES, THE NEGLECTED SERIOUS DISEASES

Arthropod-borne encephalitis viruses are highly adapted to a particular reservoir host. Viruses spread from animal to animal by an infected specific mosquito or tick species. Mosquito or tick becomes infected when feeding on the viremic animal blood. Virus then replicates in the vector, ultimately infecting the salivary glands, which transmits the virus to a new host with infective saliva while taking a blood meal. Infected patients may become ill; they usually do not develop sufficient viremia to infect feeding vectors, and thus do not contribute to the transmission cycle. Among mosquito-borne encephalitis, the greatest public health threat in North America and is posed by the West Nile, St. Louis encephalitis, and La Crosse encephalitis viruses. Venezuelan equine encephalitis virus is of concern in Central and South America, while Japanese encephalitis virus affects residents or visitors to parts of Asia. Among the tick-borne encephalitis, tick-borne Encephalitis virus causes the greatest public health threat among residents or visitors to Eastern Europe and Asia. Eastern equine encephalitis (EEE) virus is widely distributed throughout North, Central, and South America and the Caribbean. EEE virus is the most severe one, with mortality up to 30% .Western equine encephalitis (WEE) virus is a found in North and South America and is a potential agent of bioterrorism through the aerosol route. Case fatality rate is 3 to 7%, .La Crosse virus (LAC) is the most pathogenic member of California encephalitis serogroup in central and eastern United States, mostly in school-aged children. Most infections are asymptomatic with low mortality rates. Treatment is supportive, with emphasis on control of cerebral edema and seizures. Murray Valley encephalitis (MVE) virus occurs in Australia, New Guinea, and probably islands in the eastern part of the Indonesian archipelago. MVE virus is maintained in a natural cycle involving water birds and Culex annulirostris. Only 1 in 1000 to 2000 infections had clinical illness; but, about one-third of patients die and about half the survivors suffered from neurologic deficits. Venezuelan equine encephalitis (VEE) widelyspread from Florida to South America, occur periodically, but occasionally, large epidemics occur among equine and humans. VEE is infectious via aerosols, making it an occupational risk to certain laboratory workers and a potential agent of bioterrorism. Effective prevention by immunizing equines, which serve as the primary amplification hosts for the epizootic VEE viruses. Tick-borne encephalitis (TBE) exists over a wide geographical area, including Russia and Europe. Human exposure occurs through work or recreational activities when the ticks are most active. TBE virus is transmitted from the saliva of an infected tick while taking blood meal. Case fatality rates range from 2 to 8%, Treatment is mainly supportive. In Middle Eastern Countries, no proper reported statistics, although the climatic environment favors the spreading of both the vectors and the viruses. So, it is advisable to make diagnostic techniques readily available and more public health awareness of those risky, potentially fatal infectious diseases. Already Dengue viral hemorrhagic fever is mostly responsible for major worldwide fatalities as well as West Nile Fever already present there from before, without specific treatment or vaccine available.

Cryo-EM Structures of Eastern Equine Encephalitis Virus Reveal Mechanisms of Virus Disassembly and Antibody Neutralization

SummaryAlphaviruses are enveloped pathogens that cause arthritis and encephalitis. Here, we report a 4.4-Å cryoelectron microscopy (cryo-EM) structure of eastern equine encephalitis virus (EEEV), an alphavirus that causes fatal encephalitis in humans. Our analysis provides insights into viral entry into host cells. The envelope protein E2 showed a binding site for the cellular attachment factor heparan sulfate. The presence of a cryptic E2 glycan suggests how EEEV escapes surveillance by lectin-expressing myeloid lineage cells, which are sentinels of the immune system. A mechanism for nucleocapsid core release and disassembly upon viral entry was inferred based on pH changes and capsid dissociation from envelope proteins. The EEEV capsid structure showed a viral RNA genome binding site adjacent to a ribosome binding site for viral genome translation following genome release. Using five Fab-EEEV complexes derived from neutralizing antibodies, our investigation provides insights into EEEV host cell interactions and protective epitopes relevant to vaccine design.