A framework for automated scalable designation of viral pathogen lineages from genomic data.

Pathogen lineage nomenclature systems are a key component of effective communication and collaboration for researchers and public health workers. Since February 2021, the Pango dynamic lineage nomenclature for SARS-CoV-2 has been sustained by crowdsourced lineage proposals as new isolates were sequenced. This approach is vulnerable to time-critical delays as well as regional and personal bias. Here we developed a simple heuristic approach for dividing phylogenetic trees into lineages, including the prioritization of key mutations or genes. Our implementation is efficient on extremely large phylogenetic trees consisting of millions of sequences and produces similar results to existing manually curated lineage designations when applied to SARS-CoV-2 and other viruses including chikungunya virus, Venezuelan equine encephalitis virus complex and Zika virus. This method offers a simple, automated and consistent approach to pathogen nomenclature that can assist researchers in developing and maintaining phylogeny-based classifications in the face of ever-increasing genomic datasets.

Post-exposure intranasal IFNα suppresses replication and neuroinvasion of Venezuelan Equine Encephalitis virus within olfactory sensory neurons.

BACKGROUND: Venezuelan Equine Encephalitis virus (VEEV) may enter the central nervous system (CNS) within olfactory sensory neurons (OSN) that originate in the nasal cavity after intranasal exposure. While it is known that VEEV has evolved several mechanisms to inhibit type I interferon (IFN) signaling within infected cells, whether this inhibits virologic control during neuroinvasion along OSN has not been studied. METHODS: We utilized an established murine model of intranasal infection with VEEV and a repository of scRNAseq data from IFN-treated OSN to assess the cellular targets and IFN signaling responses after VEEV exposure. RESULTS: We found that immature OSN, which express higher levels of the VEEV receptor LDLRAD3 than mature OSN, are the first cells infected by VEEV. Despite rapid VEEV neuroinvasion after intranasal exposure, olfactory neuroepithelium (ONE) and olfactory bulb (OB) IFN responses, as assessed by evaluation of expression of interferon signaling genes (ISG), are delayed for up to 48 h during VEEV neuroinvasion, representing a potential therapeutic window. Indeed, a single intranasal dose of recombinant IFNα triggers early ISG expression in both the nasal cavity and OB. When administered at the time of or early after infection, IFNα treatment delayed onset of sequelae associated with encephalitis and extended survival by several days. VEEV replication after IFN treatment was also transiently suppressed in the ONE, which inhibited subsequent invasion into the CNS. CONCLUSIONS: Our results demonstrate a critical and promising first evaluation of intranasal IFNα for the treatment of human encephalitic alphavirus exposures.

Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function.

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

Everglades virus: an underrecognized disease-causing subtype of Venezuelan equine encephalitis virus endemic to Florida, USA.

Everglades virus (EVEV) is subtype II of the Venezuelan equine encephalitis virus (VEEV) complex (Togaviridae: Alphavirus), endemic to Florida, USA. EVEV belongs to a clade that includes both enzootic and epizootic/epidemic VEEV subtypes. Like other enzootic VEEV subtypes, muroid rodents are important vertebrate hosts for EVEV and certain mosquitoes are important vectors. The hispid cotton rat Sigmodon hispidus and cotton mouse Peromyscus gossypinus are important EVEV hosts, based on natural infection (virus isolation and high seropositivity), host competence (experimental infections), and frequency of contact with the vector. The mosquito Culex (Melanoconion) cecedei is the only confirmed vector of EVEV based upon high natural infection rates, efficient vector competence, and frequent feeding upon muroid rodents. Human disease attributed to EVEV is considered rare. However, cases of meningitis and encephalitis are recorded from multiple sites, separated by 250 km or more. Phylogenetic analyses indicate that EVEV is evolving, possibly due to changes in the mammal community. Mutations in the EVEV genome are of concern, given that epidemic strains of VEEV (subtypes IAB and IC) are derived from enzootic subtype ID, the closest genetic relative of EVEV. Should epizootic mutations arise in EVEV, the abundance of Aedes taeniorhynchus and other epizootic VEEV vectors in southern Florida provides a conducive environment for widespread transmission. Other factors that will likely influence the distribution and frequency of EVEV transmission include the establishment of Culex panocossa in Florida, Everglades restoration, mammal community decline due to the Burmese python, land use alteration by humans, and climate change.

Deep spatial profiling of Venezuelan equine encephalitis virus reveals increased genetic diversity amidst neuroinflammation and cell death during brain infection.

Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host's response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5-6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure. IMPORTANCE Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance.

Development of novel SARS-CoV-2 viral vectors.

The authentic SARS-CoV-2 requires to be handled in Biosafety Level 3 laboratories, which restrains investigation by the broader scientific community. Here, we report the development of a novel SARS-CoV-2 viral vector composed of all 4 SARS-CoV-2 structural proteins, the packaging signal sequence of SARS-CoV-2, a reporter gene, and an RNA amplification component of Venezuelan equine encephalitis virus (VEEV). This VEE-SARS-CoV-2 viral vector transduces target cells in an ACE2-dependent manner, and all 4 structural proteins of SARS-CoV-2 are indispensable for its transduction activity. Comparative studies show that the incorporation of the VEEV self-amplification mechanism increases the gene expression level by ~ 65-fold and extends the transgene expression up to 11 days in transduced cells. Additionally, we demonstrated the significant applications of this new VEE-SARS-CoV-2 vector for neutralizing antibody quantification and antiviral drug testing. The VEE-SARS-CoV-2 vectors developed will be an important and versatile tool for investigating SARS-CoV-2 molecular virology, developing antiviral agents targeting receptor binding, and studying RNA genome packaging and function of the essential but not well studied structural proteins of SARS-CoV-2.

Vaccine elicitation and structural basis for antibody protection against alphaviruses.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.

Mutations in the non-structural protein coding region regulate gene expression from replicon RNAs derived from Venezuelan equine encephalitis virus.

Self-replicating RNA (repRNA) derived from Venezuelan equine encephalitis (VEE) virus is a promising platform for gene therapy and confers prolonged gene expression due to its self-replicating capability, but repRNA suffers from a suboptimal transgene expression level due to its induction of intracellular innate response which may result in inhibition of translation. To improve transgene expression of repRNA, we introduced point mutations in the non-structural protein 1-4 (nsP1-4) coding region of VEE replicon vectors. As a proof of concept, inflammatory cytokines served as genes of interest and were cloned in their wild type and several mutant replicon vectors, followed by transfection in mammalian cells. Our data show that VEE replicons bearing nsP1GGAC-nsP2T or nsP1GGAC-nsP2AT mutations in the nsP1-4 coding region could significantly reduce the recognition by innate immunity as evidenced by the decreased production of type I interferon, and enhance transgene expression in host cells. Thus, the newly discovered mutant VEE replicon vectors could serve as promising gene expression platforms to advance VEE-derived repRNA-based gene therapies.

A roadmap for developing Venezuelan equine encephalitis virus (VEEV) vaccines: Lessons from the past, strategies for the future.

Venezuelan equine encephalitis (VEE) is a zoonotic infectious disease caused by the Venezuelan equine encephalitis virus (VEEV), which can lead to severe central nervous system infections in both humans and animals. At present, the medical community does not possess a viable means of addressing VEE, rendering the prevention of the virus a matter of paramount importance. Regarding the prevention and control of VEEV, the implementation of a vaccination program has been recognized as the most efficient strategy. Nevertheless, there are currently no licensed vaccines or drugs available for human use against VEEV. This imperative has led to a surge of interest in vaccine research, with VEEV being a prime focus for researchers in the field. In this paper, we initially present a comprehensive overview of the current taxonomic classification of VEEV and the cellular infection mechanism of the virus. Subsequently, we provide a detailed introduction of the prominent VEEV vaccine types presently available, including inactivated vaccines, live attenuated vaccines, nucleic acid, and virus-like particle vaccines. Moreover, we emphasize the challenges that current VEEV vaccine development faces and suggest urgent measures that must be taken to overcome these obstacles. Notably, based on our latest research, we propose the feasibility of incorporation codon usage bias strategies to create the novel VEEV vaccine. Finally, we prose several areas that future VEEV vaccine development should focus on. Our objective is to encourage collaboration between the medical and veterinary communities, expedite the translation of existing vaccines from laboratory to clinical applications, while also preparing for future outbreaks of new VEEV variants.

Clinical trials of self-replicating RNA-based cancer vaccines.

Therapeutic cancer vaccines, designed to activate immune effectors against tumor antigens, utilize a number of different platforms for antigen delivery. Among these are messenger RNAs (mRNA), successfully deployed in some prophylactic SARS-CoV2 vaccines. To enhance the immunogenicity of mRNA-delivered epitopes, self-replicating RNAs (srRNA) that markedly increase epitope expression have been developed. These vectors are derived from positive-strand RNA viruses in which the structural protein genes have been replaced with heterologous genes of interest, and the structural proteins are provided in trans to create single cycle viral replicon particles (VRPs). Clinical stage srRNA vectors have been derived from alphaviruses, including Venezuelan Equine Encephalitis (VEE), Sindbis, and Semliki Forest virus (SFV) and have encoded the tumor antigens carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), prostate specific membrane antigen (PSMA), and human papilloma virus (HPV) antigens E6 and E7. Adverse events have mainly been grade 1 toxicities and minimal injection site reactions. We review here the clinical experience with these vaccines and our recent safety data from a study combining a VRP encoding HER2 plus an anti-PD1 monoclonal antibody (pembrolizumab). This experience with VRP-based srRNA supports recent development of fully synthetic srRNA technologies, where the viral structural proteins are replaced with protective lipid nanoparticles (LNP), cationic nanoemulsions or polymers.

Codon usage bias of Venezuelan equine encephalitis virus and its host adaption.

Venezuelan equine encephalitis virus (VEEV) is an emerging zoonotic virus in the alphavirus genus. It can be transmitted to humans due to spillover from equid-mosquito cycles. The symptoms caused by VEEV include fever, headache, myalgia, nausea, and vomiting. It can also cause encephalitis in severe cases. The evolutionary features of VEEV are largely unknown. In this study, we comprehensively analyzed the codon usage pattern of VEEV by computing a variety of indicators, such as effective number of codons (ENc), codon adaptation index (CAI), relative synonymous codon usage (RSCU), on 130 VEEV coding sequences retrieved from GenBank. The results showed that the codon usage bias of VEEV is relatively low. ENc-GC3s plot, neutrality plot, and CAI-ENc correlation analyses supported that translational selection plays an important role in shaping the codon usage pattern of VEEV whereas the mutation pressure has a minor influence. Analysis of RSCU values showed that most of the preferred codons in VEEV are C/G-ended. Analysis of dinucleotide composition found that all CG- and UA-containing codons are not preferentially used. Phylogenetic analysis showed that VEEV isolates can be clustered into three genera and evolutionary force affects the codon usage pattern. Furthermore, a correspondence analysis (COA) showed that aromaticity and hydrophobicity as well as geographical distribution also have certain effects on the codon usage variation of VEEV, suggesting the possible involvement of translational selection. Overall, the codon usage of VEEV is comparatively slight and translational selection might be the main factor that shapes the codon usage pattern of VEEV. This study will promote our understanding about the evolution of VEEV and its host adaption, and might provide some clues for preventing the cross-species transmission of VEEV.

Serological and Molecular Evidence of the Circulation of the Venezuelan Equine Encephalitis Virus Subtype IIIA in Humans, Wild Vertebrates and Mosquitos in the Brazilian Amazon.

Understanding the interaction between viruses and ecosystems in areas with or without anthropic interference can contribute to the organization of public health services, as well as prevention and disease control. An arbovirus survey was conducted at Caxiuanã National Forest, Pará, Brazil, where 632 local residents, 338 vertebrates and 15,774 pools of hematophagous arthropods were investigated. Neutralization antibodies of the Venezuelan Equine Encephalitis virus, subtype IIIA, Mucambo virus (MUCV) were detected in 57.3% and 61.5% of humans and wild vertebrates, respectively; in addition, genomic fragments of MUCV were detected in pool of Uranotaenia (Ura.) geometrica. The obtained data suggest an enzootic circulation of MUCV in the area. Understanding the circulation of endemic and neglected arboviruses, such as MUCV, represents an important health problem for the local residents and for the people living in the nearby urban centers.

Efficacy of Western, Eastern, and Venezuelan Equine Encephalitis (WEVEE) Virus-Replicon Particle (VRP) Vaccine against WEEV in a Non-Human Primate Animal Model.

The purpose of this study was to evaluate the effects of the route of administration on the immunogenicity and efficacy of a combined western, eastern, and Venezuelan equine encephalitis (WEVEE) virus-like replicon particle (VRP) vaccine in cynomolgus macaques. The vaccine consisted of equal amounts of WEEV, EEEV, and VEEV VRPs. Thirty-three animals were randomly assigned to five treatment or control groups. Animals were vaccinated with two doses of WEVEE VRPs or the control 28 days apart. Blood was collected 28 days following primary vaccination and 21 days following boost vaccination for analysis of the immune response to the WEVEE VRP vaccine. NHPs were challenged by aerosol 28 or 29 days following second vaccination with WEEV CBA87. Vaccination with two doses of WEVEE VRP was immunogenic and resulted in neutralizing antibody responses specific for VEEV, EEEV and WEEV. None of the vaccinated animals met euthanasia criteria following aerosol exposure to WEEV CBA87. However, one NHP control (total of 11 controls) met euthanasia criteria after infection with WEEV CBA87. Statistically significant differences in median fever hours were noted in control NHPs compared to vaccinated NHPs, providing a quantitative measure of infection and efficacy of the vaccine against a WEEV challenge. Alterations in lymphocytes, monocytes, and neutrophils were observed. Lymphopenia was observed in control NHPs.

Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the central nervous system (CNS), were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, VEEV genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

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

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.

Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods.

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.

Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.

Identification of Quinolinones as Antivirals against Venezuelan Equine Encephalitis Virus.

Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.

Structure and Sequence Requirements for RNA Capping at the Venezuelan Equine Encephalitis Virus RNA 5' End.

Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5' end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.

The transcriptional landscape of Venezuelan equine encephalitis virus (TC-83) infection.

Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions.