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20 pages, 5052 KiB

Open AccessArticle

In Silico Screening of Inhibitors of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease

by Xin Hu, Elaine Morazzani, Jaimee R. Compton, Moeshia Harmon, Veronica Soloveva, Pamela J. Glass, Andres Dulcey Garcia, Juan J. Marugan and Patricia M. Legler

Viruses 2023, 15(7), 1503; https://doi.org/10.3390/v15071503 - 4 Jul 2023

Cited by 3 | Viewed by 2397

Abstract

The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.B79) is essential for viral replication. High throughput in silico/in vitro screening using a focused set of known cysteine protease inhibitors identified two epoxysuccinyl prodrugs, E64d and CA074 methyl ester [...] Read more.

The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.B79) is essential for viral replication. High throughput in silico/in vitro screening using a focused set of known cysteine protease inhibitors identified two epoxysuccinyl prodrugs, E64d and CA074 methyl ester (CA074me) and a reversible oxindole inhibitor. Here, we determined the X-ray crystal structure of the CA074-inhibited nsP2 protease and compared it with our E64d-inhibited structure. We found that the two inhibitors occupy different locations in the protease. We designed hybrid inhibitors with improved potency. Virus yield reduction assays confirmed that the viral titer was reduced by >5 logs with CA074me. Cell-based assays showed reductions in viral replication for CHIKV, VEEV, and WEEV, and weaker inhibition of EEEV by the hybrid inhibitors. The most potent was NCGC00488909-01 which had an EC₅₀ of 1.76 µM in VEEV-Trd-infected cells; the second most potent was NCGC00484087 with an EC₅₀ = 7.90 µM. Other compounds from the NCATS libraries such as the H1 antihistamine oxatomide (>5-log reduction), emetine, amsacrine an intercalator (NCGC0015113), MLS003116111-01, NCGC00247785-13, and MLS00699295-01 were found to effectively reduce VEEV viral replication in plaque assays. Kinetic methods demonstrated time-dependent inhibition by the hybrid inhibitors of the protease with NCGC00488909-01 (K_i = 3 µM) and NCGC00484087 (K_i = 5 µM). Rates of inactivation by CA074 in the presence of 6 mM CaCl₂, MnCl₂, or MgCl₂ were measured with varying concentrations of inhibitor, Mg²⁺ and Mn²⁺ slightly enhanced inhibitor binding (3 to 6-fold). CA074 inhibited not only the VEEV nsP2 protease but also that of CHIKV and WEEV. Full article

(This article belongs to the Special Issue Alphaviruses)

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21 pages, 6161 KiB

Open AccessArticle

Recombinant Virus Quantification Using Single-Cell Droplet Digital PCR: A Method for Infectious Titer Quantification

by Ksenija Korotkaja and Anna Zajakina

Viruses 2023, 15(5), 1060; https://doi.org/10.3390/v15051060 - 26 Apr 2023

Cited by 3 | Viewed by 3656

Abstract

The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop [...] Read more.

The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and β-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications. Full article

(This article belongs to the Special Issue Alphaviruses)

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18 pages, 2631 KiB

Open AccessArticle

Effect of Viral Strain and Host Age on Clinical Disease and Viral Replication in Immunocompetent Mouse Models of Chikungunya Encephalomyelitis

by Elizabeth J. Anderson, Audrey C. Knight, Mark T. Heise and Victoria K. Baxter

Viruses 2023, 15(5), 1057; https://doi.org/10.3390/v15051057 - 26 Apr 2023

Cited by 5 | Viewed by 2351

Abstract

The alphavirus chikungunya virus (CHIKV) represents a reemerging public health threat as mosquito vectors spread and viruses acquire advantageous mutations. Although primarily arthritogenic in nature, CHIKV can produce neurological disease with long-lasting sequelae that are difficult to study in humans. We therefore evaluated [...] Read more.

The alphavirus chikungunya virus (CHIKV) represents a reemerging public health threat as mosquito vectors spread and viruses acquire advantageous mutations. Although primarily arthritogenic in nature, CHIKV can produce neurological disease with long-lasting sequelae that are difficult to study in humans. We therefore evaluated immunocompetent mouse strains/stocks for their susceptibility to intracranial infection with three different CHIKV strains, the East/Central/South African (ECSA) lineage strain SL15649 and Asian lineage strains AF15561 and SM2013. In CD-1 mice, neurovirulence was age- and CHIKV strain-specific, with SM2013 inducing less severe disease than SL15649 and AF15561. In 4–6-week-old C57BL/6J mice, SL15649 induced more severe disease and increased viral brain and spinal cord titers compared to Asian lineage strains, further indicating that neurological disease severity is CHIKV-strain-dependent. Proinflammatory cytokine gene expression and CD4+ T cell infiltration in the brain were also increased with SL15649 infection, suggesting that like other encephalitic alphaviruses and with CHIKV-induced arthritis, the immune response contributes to CHIKV-induced neurological disease. Finally, this study helps overcome a current barrier in the alphavirus field by identifying both 4–6-week-old CD-1 and C57BL/6J mice as immunocompetent, neurodevelopmentally appropriate mouse models that can be used to examine CHIKV neuropathogenesis and immunopathogenesis following direct brain infection. Full article

(This article belongs to the Special Issue Alphaviruses)

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14 pages, 1833 KiB

Open AccessArticle

Chikungunya Virus Infection and Gonotrophic Cycle Shape Aedes aegypti Oviposition Behavior and Preferences

by Margaux Mulatier, Antoine Boullis, Christelle Dollin, Gerardo Cebrián-Torrejón and Anubis Vega-Rúa

Viruses 2023, 15(5), 1043; https://doi.org/10.3390/v15051043 - 25 Apr 2023

Cited by 2 | Viewed by 1944

Abstract

Targeting gravid females through chemical lures is a promising strategy in vector control; however, it requires the understanding of the factors susceptible to alter female oviposition behavior. Here, we evaluated the effect of infection with chikungunya virus (CHIKV) and the number of gonotrophic [...] Read more.

Targeting gravid females through chemical lures is a promising strategy in vector control; however, it requires the understanding of the factors susceptible to alter female oviposition behavior. Here, we evaluated the effect of infection with chikungunya virus (CHIKV) and the number of gonotrophic cycles (GCs) on oviposition activity in A. aegypti. Dual choice oviposition assays were performed, where dodecanoic acid, pentadecanoic acid, n-heneicosane and a Sargasssum fluitans (Børgesen) Børgesen extract were tested in uninfected females and females infected with CHIKV, at the 1st and 2nd GC. Infected females displayed a lower percentage of oviposition and a higher number of eggs laid at the 1st GC. Then, the combined effects of GC and CHIKV were observed on oviposition preferences, with a chemical-dependent effect. For instance, the deterrent effect of n-heneicosane and pentadecanoic acid increased at the 2nd GC in infected females. These results allow for a deeper understanding of the mechanisms involved in oviposition site selection and highlight the need for taking into account physiological stage changes to increase the control programs’ efficacy. Full article

(This article belongs to the Special Issue Alphaviruses)

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15 pages, 2679 KiB

Open AccessArticle

Pathways Activated by Infected and Bystander Chondrocytes in Response to Ross River Virus Infection

by Elisa X. Y. Lim, Julie A. Webster, Penny A. Rudd and Lara J. Herrero

Viruses 2023, 15(1), 136; https://doi.org/10.3390/v15010136 - 31 Dec 2022

Cited by 2 | Viewed by 2216

Abstract

Old world alphaviruses, such as Ross River virus (RRV), cause debilitating arthralgia during acute and chronic stages of the disease. RRV-induced cartilage degradation has been implicated as a cause of joint pain felt by RRV patients. Chondrocytes are a major cell type of [...] Read more.

Old world alphaviruses, such as Ross River virus (RRV), cause debilitating arthralgia during acute and chronic stages of the disease. RRV-induced cartilage degradation has been implicated as a cause of joint pain felt by RRV patients. Chondrocytes are a major cell type of cartilage and are involved in the production and maintenance of the cartilage matrix. It is thought that these cells may play a vital role in RRV disease pathogenesis. In this study, we used RNA-sequencing (RNA-Seq) to examine the transcriptomes of RRV-infected and bystander chondrocytes in the same environment. RRV containing green fluorescent protein (GFP) allowed for the separation of RRV-infected (GFP+) and bystander uninfected cells (GFP−). We found that whereas GFP+ and GFP− populations commonly presented similar gene expression profiles during infection, there were also unique signatures. For example, RIMS2 and FOXJ1 were unique to GFP+ cells, whilst Aim2 and CCL8 were only found in bystander chondrocytes. This indicates that careful selection of potential therapeutic targets is important to minimise adverse effects to the neighbouring uninfected cell populations. Our study serves as a resource to provide more information about the pathways and responses elicited by RRV in cells which are both infected and stimulated because of neighbouring infected cells. Full article

(This article belongs to the Special Issue Alphaviruses)

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19 pages, 3398 KiB

Open AccessArticle

Application of a Human Blood Brain Barrier Organ-on-a-Chip Model to Evaluate Small Molecule Effectiveness against Venezuelan Equine Encephalitis Virus

by Niloufar A. Boghdeh, Kenneth H. Risner, Michael D. Barrera, Clayton M. Britt, David K. Schaffer, Farhang Alem, Jacquelyn A. Brown, John P. Wikswo and Aarthi Narayanan

Viruses 2022, 14(12), 2799; https://doi.org/10.3390/v14122799 - 15 Dec 2022

Cited by 15 | Viewed by 2967

Abstract

The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity [...] Read more.

The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load. Full article

(This article belongs to the Special Issue Alphaviruses)

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16 pages, 21085 KiB

Open AccessArticle

Effect of IL-10 Deficiency on TGFβ Expression during Fatal Alphavirus Encephalomyelitis in C57Bl/6 Mice

by Nina M. Martin and Diane E. Griffin

Viruses 2022, 14(8), 1791; https://doi.org/10.3390/v14081791 - 16 Aug 2022

Cited by 2 | Viewed by 2283

Abstract

Sindbis virus (SINV) causes viral encephalitis in mice with strain-dependent virulence. Fatal encephalomyelitis in C57Bl/6 mice infected with a neuroadapted strain of SINV (NSV) is an immunopathogenic process that involves Th17 cells modulated by the regulatory cytokine IL-10. To further characterize the pathogenic [...] Read more.

Sindbis virus (SINV) causes viral encephalitis in mice with strain-dependent virulence. Fatal encephalomyelitis in C57Bl/6 mice infected with a neuroadapted strain of SINV (NSV) is an immunopathogenic process that involves Th17 cells modulated by the regulatory cytokine IL-10. To further characterize the pathogenic immune response to NSV, we analyzed the regulation of transforming growth factor (TGF)-b in both wild-type (WT) and IL-10-deficient mice. NSV infection upregulated the expression of TGFb1 and TGFb3 in the central nervous system (CNS). In the absence of IL-10, levels of brain Tgfb1 mRNA and brain and spinal cord mature active TGFβ1 and TGFβ3 proteins were higher than in WT mice. Compared to WT mice, IL-10-deficient mice had more TGFβ1-expressing type 3 innate lymphoid cells (ILC3s) and CD4⁺ T cells infiltrating the CNS, but similar numbers in the cervical lymph nodes. Expression of glycoprotein A repetitions predominant protein (GARP) that binds pro-TGFb on the surface of regulatory T cells was decreased on CNS cells from IL-10-deficient mice. Higher CNS TGFb was accompanied by more expression of TGFbRII receptor, activation of SMAD transcription factors, increased PCKα mRNA, and more RORγt-positive and IL-17A-expressing cells. These results suggest a compensatory role for TGFβ in the absence of IL-10 that fosters Th17-related immunopathology and more rapid death after NSV infection. Full article

(This article belongs to the Special Issue Alphaviruses)

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13 pages, 1860 KiB

Open AccessArticle

Inhibition of Venezuelan Equine Encephalitis Virus Using Small Interfering RNAs

by Amrita Haikerwal, Michael D. Barrera, Nishank Bhalla, Weidong Zhou, Niloufar Boghdeh, Carol Anderson, Farhang Alem and Aarthi Narayanan

Viruses 2022, 14(8), 1628; https://doi.org/10.3390/v14081628 - 26 Jul 2022

Cited by 2 | Viewed by 2239

Abstract

Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to [...] Read more.

Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV genome were designed and evaluated for antiviral activity in mammalian cells in the context of VEEV infection. The data demonstrate that vsiRNAs were able to effectively decrease the infectious virus titer at earlier time points post infection in the context of the attenuated TC-83 strain and the virulent Trinidad Donkey strain, while the inhibition was overcome at later time points. Depletion of Argonaute 2 protein (Ago2), the catalytic component of the RISC complex, negated the inhibitory effect of the vsiRNAs, underscoring the involvement of the siRNA pathway in the inhibition process. Depletion of the RNAi pathway proteins Dicer, MOV10, TRBP2 and Matrin 3 decreased viral load in infected cells, alluding to an impact of the RNAi pathway in the establishment of a productive infection. Additional studies focused on rational combinations of effective vsiRNAs and delivery strategies to confer better in vivo bioavailability and distribution to key target tissues such as the brain can provide effective solutions to treat encephalitic diseases resulting from alphavirus infections. Full article

(This article belongs to the Special Issue Alphaviruses)

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23 pages, 3502 KiB

Open AccessArticle

EGR1 Upregulation during Encephalitic Viral Infections Contributes to Inflammation and Cell Death

by Caitlin W. Lehman, Amy Smith, Jamie Kelly, Jonathan L. Jacobs, Jonathan D. Dinman and Kylene Kehn-Hall

Viruses 2022, 14(6), 1210; https://doi.org/10.3390/v14061210 - 2 Jun 2022

Cited by 15 | Viewed by 3662

Abstract

Early growth response 1 (EGR1) is an immediate early gene and transcription factor previously found to be significantly upregulated in human astrocytoma cells infected with Venezuelan equine encephalitis virus (VEEV). The loss of EGR1 resulted in decreased cell death but had no significant [...] Read more.

Early growth response 1 (EGR1) is an immediate early gene and transcription factor previously found to be significantly upregulated in human astrocytoma cells infected with Venezuelan equine encephalitis virus (VEEV). The loss of EGR1 resulted in decreased cell death but had no significant impact on viral replication. Here, we extend these studies to determine the impacts of EGR1 on gene expression following viral infection. Inflammatory genes CXCL3, CXCL8, CXCL10, TNF, and PTGS2 were upregulated in VEEV-infected cells, which was partially dependent on EGR1. Additionally, transcription factors, including EGR1 itself, as well as ATF3, FOS, JUN, KLF4, EGR2, and EGR4 were found to be partially transcriptionally dependent on EGR1. We also examined the role of EGR1 and the changes in gene expression in response to infection with other alphaviruses, including eastern equine encephalitis virus (EEEV), Sindbis virus (SINV), and chikungunya virus (CHIKV), as well as Zika virus (ZIKV) and Rift Valley fever virus (RVFV), members of the Flaviviridae and Phenuiviridae families, respectively. EGR1 was significantly upregulated to varying degrees in EEEV-, CHIKV-, RVFV-, SINV-, and ZIKV-infected astrocytoma cells. Genes that were identified as being partially transcriptionally dependent on EGR1 in infected cells included ATF3 (EEEV, CHIKV, ZIKV), JUN (EEEV), KLF4 (SINV, ZIKV, RVFV), CXCL3 (EEEV, CHIKV, ZIKV), CXCL8 (EEEV, CHIKV, ZIKV, RVFV), CXCL10 (EEEV, RVFV), TNF-α (EEEV, ZIKV, RVFV), and PTGS2 (EEEV, CHIKV, ZIKV). Additionally, inhibition of the inflammatory gene PTGS2 with Celecoxib, a small molecule inhibitor, rescued astrocytoma cells from VEEV-induced cell death but had no impact on viral titers. Collectively, these results suggest that EGR1 induction following viral infection stimulates multiple inflammatory mediators. Managing inflammation and cell death in response to viral infection is of utmost importance, especially during VEEV infection where survivors are at-risk for neurological sequalae. Full article

(This article belongs to the Special Issue Alphaviruses)

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Review

Jump to: Research, Other

36 pages, 2812 KiB

Open AccessReview

Advances in the Development of Small Molecule Antivirals against Equine Encephalitic Viruses

by Tyler J. Ogorek and Jennifer E. Golden

Viruses 2023, 15(2), 413; https://doi.org/10.3390/v15020413 - 1 Feb 2023

Cited by 4 | Viewed by 3902

Abstract

Venezuelan, western, and eastern equine encephalitic alphaviruses (VEEV, WEEV, and EEEV, respectively) are arboviruses that are highly pathogenic to equines and cause significant harm to infected humans. Currently, human alphavirus infection and the resulting diseases caused by them are unmitigated due to the [...] Read more.

Venezuelan, western, and eastern equine encephalitic alphaviruses (VEEV, WEEV, and EEEV, respectively) are arboviruses that are highly pathogenic to equines and cause significant harm to infected humans. Currently, human alphavirus infection and the resulting diseases caused by them are unmitigated due to the absence of approved vaccines or therapeutics for general use. These circumstances, combined with the unpredictability of outbreaks—as exemplified by a 2019 EEE surge in the United States that claimed 19 patient lives—emphasize the risks posed by these viruses, especially for aerosolized VEEV and EEEV which are potential biothreats. Herein, small molecule inhibitors of VEEV, WEEV, and EEEV are reviewed that have been identified or advanced in the last five years since a comprehensive review was last performed. We organize structures according to host- versus virus-targeted mechanisms, highlight cellular and animal data that are milestones in the development pipeline, and provide a perspective on key considerations for the progression of compounds at early and later stages of advancement. Full article

(This article belongs to the Special Issue Alphaviruses)

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13 pages, 327 KiB

Open AccessReview

Sequelae and Animal Modeling of Encephalitic Alphavirus Infections

by Rachel A. Reyna and Scott C. Weaver

Viruses 2023, 15(2), 382; https://doi.org/10.3390/v15020382 - 28 Jan 2023

Cited by 3 | Viewed by 2387

Abstract

Eastern (EEEV), Venezuelan (VEEV), and western equine encephalitis viruses (WEEV) are members of the genus Alphavirus, family Togaviridae. Typically spread by mosquitoes, EEEV, VEEV, and WEEV induce febrile illness that may develop into more severe encephalitic disease, resulting in myriad severe [...] Read more.

Eastern (EEEV), Venezuelan (VEEV), and western equine encephalitis viruses (WEEV) are members of the genus Alphavirus, family Togaviridae. Typically spread by mosquitoes, EEEV, VEEV, and WEEV induce febrile illness that may develop into more severe encephalitic disease, resulting in myriad severe neurologic sequelae for which there are no vaccines or therapeutics. Here, we summarize the clinical neurologic findings and sequelae induced by these three encephalitic viruses and describe the various animal models available to study them. We emphasize the crucial need for the development of advanced animal modeling combined with the use of telemetry, behavioral testing, and neuroimaging to facilitate a detailed mechanistic understanding of these encephalitic signs and sequelae. Through the use of these systems, much-needed therapeutics and vaccines can be developed. Full article

(This article belongs to the Special Issue Alphaviruses)

Other

Jump to: Research, Review

10 pages, 2056 KiB

Open AccessBrief Report

Suppressor of Cytokine Signalling 5 (SOCS5) Modulates Inflammatory Responses during Alphavirus Infection

by Lukasz Kedzierski, Abigail Er Qi Tan, Isabelle Jia Hui Foo, Sandra E. Nicholson and John K. Fazakerley

Viruses 2022, 14(11), 2476; https://doi.org/10.3390/v14112476 - 9 Nov 2022

Cited by 5 | Viewed by 1434

Abstract

CNS viral infections are one of the major causes of morbidity and mortality worldwide and a significant global public health concern. Uncontrolled inflammation and immune responses in the brain, despite their protective roles, can also be harmful. The suppressor of cytokine signalling (SOCS) [...] Read more.

CNS viral infections are one of the major causes of morbidity and mortality worldwide and a significant global public health concern. Uncontrolled inflammation and immune responses in the brain, despite their protective roles, can also be harmful. The suppressor of cytokine signalling (SOCS) proteins is one of the key mechanisms controlling inflammatory and immune responses across all tissues including the brain. SOCS5 is highly expressed in the brain but there is little understanding of its role in the CNS. Using a mouse model of encephalitis, we demonstrate that lack of SOCS5 results in changes in the pathogenesis and clinical outcome of a neurotropic virus infection. Relative to wild-type mice, SOCS5-deficient mice had greater weight loss, dysregulated cytokine production and increased neuroinflammatory infiltrates composed predominantly of CD11b⁺ cells. We conclude that in the brain, SOCS5 is a vital regulator of anti-viral immunity that mediates the critical balance between immunopathology and virus persistence. Full article

(This article belongs to the Special Issue Alphaviruses)

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