scholarly journals Hypervariable Domain of nsP3 of Eastern Equine Encephalitis Virus Is a Critical Determinant of Viral Virulence

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Chetan D. Meshram ◽  
Nikita Shiliaev ◽  
Elena I. Frolova ◽  
Ilya Frolov
Keyword(s):  
Viral Replication ◽  
Capsid Protein ◽  
Host Factors ◽  
Eastern Equine Encephalitis Virus ◽  
Wild Type ◽  
Protein Families ◽  
Host Proteins ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus

ABSTRACT Eastern equine encephalitis virus (EEEV) is the most pathogenic member of the Alphavirus genus in the Togaviridae family. This virus continues to circulate in the New World and has a potential for deliberate use as a bioweapon. Despite the public health threat, to date no attenuated EEEV variants have been applied as live EEEV vaccines. Our previous studies demonstrated the critical function of the hypervariable domain (HVD) in EEEV nsP3 for the assembly of viral replication complexes (vRCs). EEEV HVD contains short linear motifs that recruit host proteins required for vRC formation and function. In this study, we developed a set of EEEV mutants that contained combinations of deletions in nsP3 HVD and clustered mutations in capsid protein, and tested the effects of these modifications on EEEV infection in vivo. These mutations had cumulative negative effects on viral ability to induce meningoencephalitis. The deletions of two critical motifs, which interact with the members of cellular FXR and G3BP protein families, made EEEV cease to be neurovirulent. The additional clustered mutations in capsid protein, which affect its ability to induce transcriptional shutoff, diminished EEEV’s ability to develop viremia. Most notably, despite the inability to induce detectable disease, the designed EEEV mutants remained highly immunogenic and, after a single dose, protected mice against subsequent infection with wild-type (wt) EEEV. Thus, alterations of interactions of EEEV HVD and likely HVDs of other alphaviruses with host factors represent an important direction for development of highly attenuated viruses that can be applied as live vaccines. IMPORTANCE Hypervariable domains (HVDs) of alphavirus nsP3 proteins recruit host proteins into viral replication complexes. The sets of HVD-binding host factors are specific for each alphavirus, and we have previously identified those specific for EEEV. The results of this study demonstrate that the deletions of the binding sites of the G3BP and FXR protein families in the nsP3 HVD of EEEV make the virus avirulent for mice. Mutations in the nuclear localization signal in EEEV capsid protein have an additional negative effect on viral replication in vivo. Despite the inability to cause a detectable disease, the double HVD and triple HVD/capsid mutants induce high levels of neutralizing antibodies. Single immunization protects mice against infection with the highly pathogenic North American strain of EEEV. High safety, the inability to revert to wild-type phenotype, and high immunogenicity make the designed mutants attractive vaccine candidates for EEEV infection.

scholarly journals Hypervariable Domain of Eastern Equine Encephalitis Virus nsP3 Redundantly Utilizes Multiple Cellular Proteins for Replication Complex Assembly

2017 ◽  
Vol 91 (14) ◽  
Author(s):  
Ilya Frolov ◽  
Dal Young Kim ◽  
Maryna Akhrymuk ◽  
James A. Mobley ◽  
Elena I. Frolova
Keyword(s):  
New World ◽  
Encephalitis Virus ◽  
Host Factors ◽  
Eastern Equine Encephalitis Virus ◽  
Protein Families ◽  
Eastern Equine Encephalitis ◽  
Replication Complex ◽  
Equine Encephalitis Virus ◽  
Public Health Threat ◽  
Efficient Replication

ABSTRACT Eastern equine encephalitis virus (EEEV) is a representative member of the New World alphaviruses. It is pathogenic for a variety of vertebrate hosts, in which EEEV induces a highly debilitating disease, and the outcomes are frequently lethal. Despite a significant public health threat, the molecular mechanism of EEEV replication and interaction with hosts is poorly understood. Our previously published data and those of other teams have demonstrated that hypervariable domains (HVDs) of the alphavirus nsP3 protein interact with virus-specific host factors and play critical roles in assembly of viral replication complexes (vRCs). The most abundantly represented HVD-binding proteins are the FXR and G3BP family members. FXR proteins drive the assembly of vRCs of Venezuelan equine encephalitis virus (VEEV), and G3BPs were shown to function in vRC assembly in the replication of chikungunya and Sindbis viruses. Our new study demonstrates that EEEV exhibits a unique level of redundancy in the use of host factors in RNA replication. EEEV efficiently utilizes both the VEEV-specific FXR protein family and the Old World alphavirus-specific G3BP protein family. A lack of interaction with either FXRs or G3BPs does not affect vRC formation; however, removal of EEEV's ability to interact with both protein families has a deleterious effect on virus growth. Other identified EEEV nsP3 HVD-interacting host proteins are also capable of supporting EEEV replication, albeit with a dramatically lower efficiency. The ability to use a wide range of host factors with redundant functions in vRC assembly and function provides a plausible explanation for the efficient replication of EEEV and may contribute to its highly pathogenic phenotype. IMPORTANCE Eastern equine encephalitis virus (EEEV) is one of the most pathogenic New World alphaviruses. Despite the continuous public health threat, to date, the molecular mechanisms of its very efficient replication and high virulence are not sufficiently understood. The results of this new study demonstrate that North American EEEV exhibits a high level of redundancy in using host factors in replication complex assembly and virus replication. The hypervariable domain of the EEEV nsP3 protein interacts with all of the members of the FXR and G3BP protein families, and only a lack of interaction with both protein families strongly affects virus replication rates. Other identified HVD-binding factors are also involved in EEEV replication, but their roles are not as critical as those of FXRs and G3BPs. The new data present a plausible explanation for the exceptionally high replication rates of EEEV and suggest a new means of its attenuation and new targets for screening of antiviral drugs.

scholarly journals The Structural Biology of Eastern Equine Encephalitis Virus, an Emerging Viral Threat

2021 ◽  
Author(s):  
S. Saif Hasan ◽  
Debajit Dey ◽  
Suruchi Singh ◽  
Matthew Martin
Keyword(s):  
Structural Biology ◽  
The United States ◽  
Encephalitis Virus ◽  
Eastern Equine Encephalitis Virus ◽  
Host Proteins ◽  
Structural Investigations ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Fundamental Biology ◽  
Functional Analyses

Alphaviruses are arboviruses that cause arthritis and encephalitis in humans. Eastern Equine Encephalitis Virus (EEEV) is a mosquito transmitted alphavirus that is implicated in severe encephalitis in humans with high mortality. However, limited insights are available into its fundamental biology of EEEV and residue-level details of its interactions with host proteins. In recent years, outbreaks of EEEV have been reported mainly in the United States, raising concerns about public safety. This review article summarizes recent advances in the structural biology of EEEV based mainly on recent single particle cryogenic electron microscopy (cryoEM) structures. Together with functional analyses of EEEV and related alphaviruses, these structural investigations provide clues to how EEEV interacts with host proteins, which may open avenues for the development of therapeutics.

scholarly journals The Structural Biology of Eastern Equine Encephalitis Virus, an Emerging Viral Threat

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 973
Author(s):  
S. Saif Hasan ◽  
Debajit Dey ◽  
Suruchi Singh ◽  
Matthew Martin
Keyword(s):  
Structural Biology ◽  
The United States ◽  
Encephalitis Virus ◽  
Eastern Equine Encephalitis Virus ◽  
Host Proteins ◽  
Structural Investigations ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Fundamental Biology ◽  
Functional Analyses

Alphaviruses are arboviruses that cause arthritis and encephalitis in humans. Eastern Equine Encephalitis Virus (EEEV) is a mosquito-transmitted alphavirus that is implicated in severe encephalitis in humans with high mortality. However, limited insights are available into the fundamental biology of EEEV and residue-level details of its interactions with host proteins. In recent years, outbreaks of EEEV have been reported mainly in the United States, raising concerns about public safety. This review article summarizes recent advances in the structural biology of EEEV based mainly on single-particle cryogenic electron microscopy (cryoEM) structures. Together with functional analyses of EEEV and related alphaviruses, these structural investigations provide clues to how EEEV interacts with host proteins, which may open avenues for the development of therapeutics.

scholarly journals Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus

2019 ◽  
Author(s):  
Derek W. Trobaugh ◽  
Chengqun Sun ◽  
Nishank Bhalla ◽  
Christina L. Gardner ◽  
Matthew Dunn ◽  
...  
Keyword(s):  
Virus Replication ◽  
Binding Sites ◽  
Untranslated Region ◽  
Myeloid Cells ◽  
Encephalitis Virus ◽  
Eastern Equine Encephalitis Virus ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Western Equine Encephalitis

AbstractEastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3’ untranslated region (3’ UTR) of the virus. Three of the sites are “canonical” with all 8 seed sequence residues complimentary to miR-142-3p while one is “non-canonical” and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites.Author SummaryEastern equine encephalitis virus (EEEV) is one of the most acutely virulent mosquito-borne viruses in the Americas. A major determinant of EEEV virulence is a mammalian microRNA (miRNA) that is primarily expressed in myeloid cells, miR-142-3p. Like miRNA suppression of host mRNA, miR-142-3p binds to the 3’ untranslated region (UTR) of the EEEV genome only in myeloid cells suppressing virus replication and the induction of the innate immune response. In this study, we used point mutations in all four miR-142-3p binding sites in the EEEV 3’ UTR to understand the mechanism behind this miRNA suppression. We observed that decreasing the number of miR-142-3p binding sites leads to virus escape and ultimately attenuation in vivo. Furthermore, another virus, western equine encephalitis virus, also encodes miR-142-3p binding sites that contribute to virulence in vivo. These results provide insight into the mechanism of how cell-specific miRNAs can mediate suppression of virus replication.

scholarly journals Variation in Interferon Sensitivity and Induction among Strains of Eastern Equine Encephalitis Virus

2005 ◽  
Vol 79 (17) ◽  
pp. 11300-11310 ◽  
Author(s):  
Patricia V. Aguilar ◽  
Slobodan Paessler ◽  
Anne-Sophie Carrara ◽  
Samuel Baron ◽  
Joyce Poast ◽  
...  
Keyword(s):  
Human Disease ◽  
Encephalitis Virus ◽  
Consistent Difference ◽  
Eastern Equine Encephalitis Virus ◽  
Wild Type ◽  
Survival Times ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Ifn Γ

ABSTRACT Eastern equine encephalitis virus (EEEV) causes human encephalitis in North America (NA), but in South America (SA) it has rarely been associated with human disease, suggesting that SA strains are less virulent. To evaluate the hypothesis that this virulence difference is due to a greater ability of NA strains to evade innate immunity, we compared replication of NA and SA strains in Vero cells pretreated with interferon (IFN). Human IFN-α, -β, and -γ generally exhibited less effect on replication of NA than SA strains, supporting this hypothesis. In the murine model, no consistent difference in IFN induction was observed between NA and SA strains. After infection with most EEEV strains, higher viremia levels and shorter survival times were observed in mice deficient in IFN-α/β receptors than in wild-type mice, suggesting that IFN-α/β is important in controlling replication. In contrast, IFN-γ receptor-deficient mice infected with NA and SA strains had similar viremia levels and mortality rates to those of wild-type mice, suggesting that IFN-γ does not play a major role in murine protection. Mice pretreated with poly(I-C), a nonspecific IFN inducer, exhibited dose-dependent protection against fatal eastern equine encephalitis, further evidence that IFN is important in controlling disease. Overall, our in vivo results did not support the hypothesis that NA strains are more virulent in humans due to their greater ability to counteract the IFN response. However, further studies using a better model of human disease are needed to confirm the results of differential human IFN sensitivity obtained in our in vitro experiments.

scholarly journals 49. Clinical Characteristics of the 2019 Eastern Equine Encephalitis Virus Outbreak in Michigan

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S154-S154
Author(s):  
Adam T Ladzinski ◽  
Matthew T Rumschlag ◽  
Aditya Mehta ◽  
Eric Edewaard ◽  
Pimpawan Boapimp ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
The United States ◽  
Encephalitis Virus ◽  
Eastern Equine Encephalitis Virus ◽  
Radiographic Images ◽  
Eastern Equine Encephalitis ◽  
Male Predominance ◽  
Equine Encephalitis Virus ◽  
Neuroinvasive Disease ◽  
Neurologic Sequelae

Abstract Background Eastern Equine Encephalitis Virus (EEEV) is a mosquito-borne alphavirus responsible for unpredictable outbreaks of severe neurologic disease in humans. While the vast majority of human EEEV infections are either asymptomatic or clinically nonspecific, a minority of patients develops neuroinvasive disease (EEE), which is a devastating illness with a mortality of at least 30%. No treatments are known to be effective. EEEV infection is relatively rare in the United States, with an annual average nationwide incidence of 7 cases between 2009 and 2018. However, 2019 was an exceptionally active year for human EEEV disease, yielding 38 nationwide confirmed cases, including 10 in Michigan, comprising the state’s largest outbreak to date. Methods EEE cases were identified by a regional network of physicians. Cases were defined by presentation with clinical symptoms of encephalitis, and by identification of EEEV IgM antibodies or RNA in cerebrospinal fluid (CSF), or EEEV-specific IgM in serum as confirmed by plaque reduction neutralization test. Radiographic images were evaluated and clinical data abstracted through chart review and clinical follow-up where possible. Results Records from 7 patients were identified and reviewed. The median age was 64, with a male predominance, and all presented in August. Notably, commercial arboviral CSF serology was uniformly negative on the initial CSF sample, and diagnosis was not made until a mean of 23 days (range: 12–38 days) after presentation. Testing in public health laboratories yielded the diagnosis in 5 out of 7 cases. Imaging findings were heterogeneous, but most patients exhibited abnormal findings in the thalamus and/or basal ganglia, and one patient displayed prominent pons and midbrain abnormalities. 4 patients died, while 2 patients survived with severe neurologic sequelae, and 1 patient recovered without sequelae. One patient underwent a limited postmortem examination, which revealed diffuse meningoencephalitis and focal vascular necrosis. Conclusion EEE is a frequently fatal condition whose diagnosis is often delayed, and for which no effective treatments are known. Improved diagnostics are needed to facilitate further clinical studies of EEE and encourage the development of potential therapies. Disclosures All Authors: No reported disclosures

scholarly journals Active Circulation of Madariaga Virus, a Member of the Eastern Equine Encephalitis Virus Complex, in Northeast Brazil

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 983
Author(s):  
Laura H. V. G. Gil ◽  
Tereza Magalhaes ◽  
Beatriz S. A. S. Santos ◽  
Livia V. Oliveira ◽  
Edmilson F. Oliveira-Filho ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Case Fatality ◽  
Febrile Illness ◽  
Encephalitis Virus ◽  
Northeast Brazil ◽  
Eastern Equine Encephalitis Virus ◽  
Eastern Equine Encephalitis ◽  
Vaccination Programs ◽  
Transmission Cycle ◽  
Equine Encephalitis Virus

Madariaga virus (MADV) is a member of the eastern equine encephalitis virus (EEEV) complex that circulates in Central and South America. It is a zoonotic, mosquito-borne pathogen, belonging to the family Togaviridae. Disturbances in the natural transmission cycle of this virus result in outbreaks in equines and humans, leading to high case fatality in the former and acute febrile illness or neurological disease in the latter. Although a considerable amount of knowledge exists on the eco-epidemiology of North American EEEV strains, little is known about MADV. In Brazil, the most recent isolations of MADV occurred in 2009 in the States of Paraíba and Ceará, northeast Brazil. Because of that, health authorities have recommended vaccination of animals in these regions. However, in 2019 an equine encephalitis outbreak was reported in a municipality in Ceará. Here, we present the isolation of MADV from two horses that died in this outbreak. The full-length genome of these viruses was sequenced, and phylogenetic analyses performed. Pathological findings from postmortem examination are also discussed. We conclude that MADV is actively circulating in northeast Brazil despite vaccination programs, and call attention to this arbovirus that likely represents an emerging pathogen in Latin America.

Mapping Eastern Equine Encephalitis Virus Risk on Equestrian Trails in Florida State Parks

2021 ◽  
Author(s):  
Joni Downs ◽  
Mehrdad Vaziri ◽  
Abigail Lavallin ◽  
Kristi Miley
Keyword(s):  
Risk Model ◽  
Risk Index ◽  
Management Strategies ◽  
Encephalitis Virus ◽  
Transmission Risk ◽  
State Parks ◽  
Eastern Equine Encephalitis Virus ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Index Model

Equestrian trail riding is a popular recreational activity but can expose horses and riders to arboviral diseases, such as Eastern Equine Encephalitis Virus (EEEV). This study uses a geographic information system (GIS) -based risk index model to measure and map risk associated with EEEV transmission to horses on equine trails in State Parks across Florida. The risk model assesses EEEV transmission risk of individual trail locations on a continuous scale of 0.0 (no measurable risk) to 1.0 (maximal risk). Risk is evaluated based on the composition and configuration of habitat types that support vectors and hosts of the virus. The results suggest that visitors using equestrian trails in the Florida State Park System may potentially be exposed to high levels of EEEV risk during times that the virus is actively circulating. Accordingly, park management strategies are recommended for reducing transmission risk to both horses and riders in areas where risk is high.

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