Examination of Antiviral Resistance in Venezuelan Equine Encephalitis Virus

Encephalitis Virus ◽

Antiviral Resistance ◽

Nucleotide Polymorphisms ◽

Evolutionary Trajectory ◽

Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that causes Venezuelan equine encephalitis (VEE), which is characterized by a febrile illness that can progress to neurological disease and death. While no major outbreaks of VEE have occurred since 1995, VEEV is a virus of concern as, in addition to its spread through mosquitos, it can be aerosolized and used as a bioweapon. Unfortunately, there are currently no FDA-approved vaccines or antivirals against VEEV. Efforts have been made to discover small molecules with an inhibitory effect on VEEV, but the potential for emergence of antiviral resistance to these compounds will remain a concern because VEEV is an RNA virus with a high mutation rate and grows to high titers. To examine the evolutionary trajectory of antiviral resistance in VEEV, we developed a next-generation sequencing pipeline to examine single-nucleotide polymorphisms that emerged after repeated passaging of the virus with increasing concentrations of antiviral compounds. In addition, we examined the effect of the microenvironment on the evolution of antiviral resistance, both in cell culture and mouse models. We found that VEEV evolves resistance to the compound ML336 and its derivatives through mutations in the nsP2 and nsP4 genes, but the number, timing of emergence, and the extent of penetrance of these SNPs depend on the compound. These mutations emerged more slowly when infecting an astrocyte cell line. We also found that neurons in the mouse brain did not impose a selective pressure on VEEV during an infection. These results demonstrate how the population dynamics of RNA viruses can be tracked over time and the extent to which they are affected by selective pressures, as well as opening questions about how viruses can mutate and adapt at the molecular level.

Structure of Isolated Nucleocapsids from Venezuelan Equine Encephalitis Virus and Implications for Assembly and Disassembly of Enveloped Virus

Journal of Virology ◽

10.1128/jvi.77.1.659-664.2003 ◽

2003 ◽

Vol 77 (1) ◽

pp. 659-664 ◽

Cited By ~ 24

Author(s):

Angel Paredes ◽

Kathy Alwell-Warda ◽

Scott C. Weaver ◽

Wah Chiu ◽

Stanley J. Watowich

Keyword(s):

Conformational Changes ◽

Rna Virus ◽

Three Dimensional ◽

Encephalitis Virus ◽

Dimensional Structure ◽

Icosahedral Symmetry ◽

Enveloped Virus

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is ∼11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation.

Ablation of Programmed −1 Ribosomal Frameshifting in Venezuelan Equine Encephalitis Virus Results in Attenuated Neuropathogenicity

Journal of Virology ◽

10.1128/jvi.01766-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 16

Author(s):

Joseph A. Kendra ◽

Cynthia de la Fuente ◽

Ashwini Brahms ◽

Caitlin Woodson ◽

Todd M. Bell ◽

...

Keyword(s):

Cultured Cells ◽

Rna Virus ◽

Encephalitis Virus ◽

Ribosomal Frameshifting ◽

Positive Strand ◽

Live Attenuated Vaccines ◽

Positive Strand Rna

ABSTRACT The alphaviruses Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV) are arthropod-borne positive-strand RNA viruses that are capable of causing acute and fatal encephalitis in many mammals, including humans. VEEV was weaponized during the Cold War and is recognized as a select agent. Currently, there are no FDA-approved vaccines or therapeutics for these viruses. The spread of VEEV and other members of this family due to climate change-mediated vector range expansion underscores the need for research aimed at developing medical countermeasures. These viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to synthesize the viral trans-frame (TF) protein, which has previously been shown to be important for neuropathogenesis in the related Sindbis virus. Here, the alphavirus −1 PRF signals were characterized, revealing novel −1 PRF stimulatory structures. −1 PRF attenuation mildly affected the kinetics of VEEV accumulation in cultured cells but strongly inhibited its pathogenesis in an aerosol infection mouse model. Importantly, the decreased viral titers in the brains of mice infected with the mutant virus suggest that the alphavirus TF protein is important for passage through the blood-brain barrier and/or for neuroinvasiveness. These findings suggest a novel approach to the development of safe and effective live attenuated vaccines directed against VEEV and perhaps other closely related −1 PRF-utilizing viruses. IMPORTANCE Venezuelan equine encephalitis virus (VEEV) is a select agent that has been weaponized. This arthropod-borne positive-strand RNA virus causes acute and fatal encephalitis in many mammals, including humans. There is no vaccine or other approved therapeutic. VEEV and related alphaviruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to synthesize the viral trans-frame (TF) protein, which is important for neuropathogenesis. −1 PRF attenuation strongly inhibited VEEV pathogenesis in mice, and viral replication analyses suggest that the TF protein is critical for neurological disease. These findings suggest a new approach to the development of safe and effective live attenuated vaccines directed against VEEV and other related viruses.

Early activation of the host complement system is required to restrict central nervous system invasion and limit neuropathology during Venezuelan equine encephalitis virus infection

Journal of General Virology ◽

10.1099/vir.0.038281-0 ◽

2012 ◽

Vol 93 (4) ◽

pp. 797-806 ◽

Cited By ~ 13

Author(s):

Christopher B. Brooke ◽

Alexandra Schäfer ◽

Glenn K. Matsushima ◽

Laura J. White ◽

Robert E. Johnston

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Complement System ◽

Rna Virus ◽

Encephalitis Virus ◽

Central Nervous System Invasion ◽

Immunocompetent Mice

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genus Alphavirus, family Togaviridae, that is responsible for sporadic outbreaks in human and equid populations in Central and South America. In order to ascertain the role that complement plays in resolving VEEV-induced disease, complement-deficient C3−/− mice were infected with a VEEV mutant (V3533) that caused mild, transient disease in immunocompetent mice. In the absence of a functional complement system, peripheral inoculation with V3533 induced much more severe encephalitis. This enhanced pathology was associated with a delay in clearance of infectious virus from the serum and more rapid invasion of the central nervous system in C3−/− mice. If V3533 was inoculated directly into the brain, however, disease outcome in C3−/− and wild-type mice was identical. These findings indicate that complement-dependent enhancement of peripheral virus clearance is critical for protecting against the development of severe VEEV-induced encephalitis.

Emergence and Magnitude of ML336 Resistance in Venezuelan Equine Encephalitis Virus Depend on the Microenvironment

Journal of Virology ◽

10.1128/jvi.00317-20 ◽

2020 ◽

Vol 94 (22) ◽

Cited By ~ 2

Author(s):

Jasper Lee ◽

Jyothi Parvathareddy ◽

Dong Yang ◽

Shruti Bansal ◽

Kathryn O’Connell ◽

...

Keyword(s):

Population Level ◽

Encephalitis Virus ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Antiviral Compounds ◽

Generation Sequencing ◽

Over Time

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that can cause neurological disease and death in humans and equines following transmission from infected mosquitoes. Despite the continued epidemic threat of VEEV, and its potential use as a bioterrorism agent, there are no FDA-approved antivirals or vaccines for treatment or prevention. Previously, we reported the discovery of a small molecule, ML336, with potent antiviral activity against VEEV. To further explore the population-level resistance profiles of ML336, we developed a whole-genome next-generation sequencing (NGS) approach to examine single nucleotide polymorphisms (SNPs) from virus passaged in dose escalation studies in a nonhuman primate kidney epithelial and a human astrocyte cell line, Vero 76 and SVGA, respectively. We passaged VEEV TC-83 in these two cell lines over seven concentrations of ML336, starting at 50 nM. NGS revealed several prominent mutations in the nonstructural protein (nsP) 3 and nsP4 genes that emerged consistently in these two distinct in vitro environments—notably, a mutation at Q210 in nsP4. Several of these mutations were stable following passaging in the absence of ML336 in Vero 76 cells. Network analyses showed that the trajectory of resistance differed between Vero and SVGA. Moreover, the penetration of SNPs was lower in SVGA. In conclusion, we show that the microenvironment influenced the SNP profile of VEEV TC-83. Understanding the dynamics of resistance in VEEV against newly developed antiviral compounds will guide the design of optimal drug candidates and dosing regimens for minimizing the emergence of resistant viruses. IMPORTANCE RNA viruses, including Venezuelan equine encephalitis virus (VEEV), have high mutation rates that allow for rapid adaptation to selective pressures in their environment. Antiviral compounds exert one such pressure on virus populations during infections. Next-generation sequencing allows for examination of viruses at the population level, which enables tracking of low levels of single-nucleotide polymorphisms in the population over time. Therefore, the timing and extent of the emergence of resistance to antivirals can be tracked and assessed. We show here that in VEEV, the trajectory and penetration of antiviral resistance reflected the microenvironment in which the virus population replicates. In summary, we show the diversity of VEEV within a single population under antiviral pressure and two distinct cell types, and we show that population dynamics in these viruses can be examined to better understand how they evolve over time.

A Novel Compound, ML336, Inhibits VEEV Replication by Interfering with Viral RNA Synthesis

10.1101/343228 ◽

2018 ◽

Author(s):

Andrew M. Skidmore ◽

Robert S. Adcock ◽

Jasper Lee ◽

Colleen B. Jonsson ◽

Jennifer E. Golden ◽

...

Keyword(s):

Mechanism Of Action ◽

Rna Synthesis ◽

Rna Virus ◽

Unmet Need ◽

Encephalitis Virus ◽

Viral Rna ◽

Metabolic Labelling

AbstractVenezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to Central and South America. VEEV is known to cause periodic outbreaks of encephalitis in both humans and equids. There are currently no treatments or preventatives for VEEV disease. Our group has previously reported on the development of a novel VEEV inhibitor, ML336, which showed a potent antiviral effect in cell culture models. However, the mechanism of action had yet to be elucidated. Based on the discovery of mutations conferring resistance within nonstructural proteins, we hypothesized that ML336 inhibits viral RNA synthesis. We found that ML336 was able to inhibit VEEV RNA synthesis with an IC50value of 1.1 nM in a metabolic labelling assay. ML336 marginally affected cellular transcription at levels 20,000-fold above the IC50, and did not show any cytotoxicity up to 50 µM. Using a combination of fluorography, strand-specific qRT-PCR, and a metabolic labelling assay, we found that ML336 inhibits the synthesis of all forms of VEEV RNA. Structural analogues of ML336 showed a correlation between their RNA synthesis inhibitory activity and their antiviral activity in cells, leading us to propose that the primary mechanism of action of this class of compounds is viral RNA synthesis inhibition. The activities of ML336 were highly specific to VEEV, without measurable activity against Chikungunya virus. ML336 was efficacious even in a cell-free viral RNA synthesis assay, suggesting a direct interaction with viral proteins.ImportanceVenezuelan equine encephalitis virus (VEEV) is a pathogenic alphavirus that circulates in the Americas which can cause a lethal encephalitis in humans and equids. There are currently no licensed treatments or vaccines for VEEV. Due to the high potential for aerosol infection and severe outcomes, it is classified as an NIAID Category B agent. To address the unmet need for VEEV antivirals, we continue to advance a novel amidine compound, ML336, through medicinal chemistry and mechanism of action (MOA) studies. Here, we present the molecular MOA by which ML336 inhibits VEEV replication using cellular and biochemical approaches. Our data suggest that ML336 is a direct-acting antiviral that inhibits viral RNA synthesis by interfering with the viral replicase complex. Our studies provide new insights into approaches for the development of novel RNA virus replication inhibitors and the molecular mechanism of alphavirus RNA synthesis.

T Cells Facilitate Recovery from Venezuelan Equine Encephalitis Virus-Induced Encephalomyelitis in the Absence of Antibody

Journal of Virology ◽

10.1128/jvi.02545-09 ◽

2010 ◽

Vol 84 (9) ◽

pp. 4556-4568 ◽

Cited By ~ 27

Author(s):

Christopher B. Brooke ◽

Damon J. Deming ◽

Alan C. Whitmore ◽

Laura J. White ◽

Robert E. Johnston

Keyword(s):

T Cells ◽

T Cell ◽

Rna Virus ◽

Clinical Signs ◽

Encephalitis Virus ◽

Cell Subpopulations ◽

T Cell Subpopulations

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genus Alphavirus that is responsible for a significant disease burden in Central and South America through sporadic outbreaks into human and equid populations. For humans, 2 to 4% of cases are associated with encephalitis, and there is an overall case mortality rate of approximately 1%. In mice, replication of the virus within neurons of the central nervous system (CNS) leads to paralyzing, invariably lethal encephalomyelitis. However, mice infected with certain attenuated mutants of the virus are able to control the infection within the CNS and recover. To better define what role T cell responses might be playing in this process, we infected B cell-deficient μMT mice with a VEEV mutant that induces mild, sublethal illness in immune competent mice. Infected μMT mice rapidly developed the clinical signs of severe paralyzing encephalomyelitis but were eventually able to control the infection and recover fully from clinical illness. Recovery in this system was T cell dependent and associated with a dramatic reduction in viral titers within the CNS, followed by viral persistence in the brain. Further comparison of the relative roles of T cell subpopulations within this system revealed that CD4+ T cells were better producers of gamma interferon (IFN-γ) than CD8+ T cells and were more effective at controlling VEEV within the CNS. Overall, these results suggest that T cells, especially CD4+ T cells, can successfully control VEEV infection within the CNS and facilitate recovery from a severe viral encephalomyelitis.

Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Plants ◽

10.3390/plants10020346 ◽

2021 ◽

Vol 10 (2) ◽

pp. 346

Author(s):

Caitlin W. Lehman ◽

Kylene Kehn-Hall ◽

Megha Aggarwal ◽

Nicole R. Bracci ◽

Han-Chi Pan ◽

...

Keyword(s):

Antiviral Activity ◽

Fluorescent Protein ◽

Glycogen Synthase ◽

Encephalitis Virus ◽

Host Cells ◽

Dynamics Simulation ◽

Luciferase Reporter ◽

Equine Encephalitis Virus

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.