Phagocyte-expressed glycosaminoglycans promote capture of alphaviruses from the blood circulation in a host species-specific manner.

The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya, eastern- (EEEV), and Venezuelan- (VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology.

Influencing Factors of Recurrence of Lumbar Disc Herniation After Percutaneous Transforaminal Endoscopic Discectomy: A Meta-Analysis.

Background: Lumbar disc herniation (LDH) remains one of the extremely common diseases in the elderly population, and despite the fact that percutaneous transforaminal endoscopic discectomy (PTED) can be an effective treatment for LDH, prognostic recurrence of the patients is still a clinical problem that needs to be addressed. Objective: To perform a meta-analysis of the influencing factors of disease recurrence after PTED for LDH to provide evidence for clinical practice. Methods: By screening the PubMed, EMbase, and Cochrane Library databases for relevant studies on disease recurrence after PTED for LDH, we extracted the authors, publication time, outcome measures, and other indicators were extracted for meta-analyses using RevMan 5.3 software. Results: The online retrieval and rigorous screening returned 8 valid articles for analysis, all with high reference value, as their Newcastle Ottawa Scale (NOS) scores were above 6. According to meta-analyses, there were no differences in gender and LDH type and location among patients with LDH recurrence after PTED treatment (P > .05); however, statistical significance was present in Pfirrmann grading, incomplete nucleus pulposus removal during surgery, and Modic changes (P < .05), indicating that these indexes were the influencing factors of LDH recurrence. Conclusions: Pfirrmann grading, incomplete nucleus pulposus removal during surgery, and Modic changes are related factors affecting LDH recurrence after PTED.

Vertebrate-class-specific binding modes of the alphavirus receptor MXRA8.

MXRA8 is a receptor for chikungunya (CHIKV) and other arthritogenic alphaviruses with mammalian hosts. However, mammalian MXRA8 does not bind to alphaviruses that infect humans and have avian reservoirs. Here, we show that avian, but not mammalian, MXRA8 can act as a receptor for Sindbis, western equine encephalitis (WEEV), and related alphaviruses with avian reservoirs. Structural analysis of duck MXRA8 complexed with WEEV reveals an inverted binding mode compared with mammalian MXRA8 bound to CHIKV. Whereas both domains of mammalian MXRA8 bind CHIKV E1 and E2, only domain 1 of avian MXRA8 engages WEEV E1, and no appreciable contacts are made with WEEV E2. Using these results, we generated a chimeric avian-mammalian MXRA8 decoy-receptor that neutralizes infection of multiple alphaviruses from distinct antigenic groups in vitro and in vivo. Thus, different alphaviruses can bind MXRA8 encoded by different vertebrate classes with distinct engagement modes, which enables development of broad-spectrum inhibitors.

Phagocyte-expressed glycosaminoglycans promote capture of alphaviruses from the blood circulation in a host species-specific manner.

The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya (CHIKV), eastern-(EEEV), and Venezuelan-(VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate (HS) impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology. Significance Statement: Previously, evidence of arbovirus-GAG interactions in vivo has been limited to associations between viral residues shown to promote enhanced GAG-binding phenotypes in vitro and in vivo phenotypes of viral dissemination and pathogenesis. By directly manipulating host GAG expression, we identified virion-GAG interactions in vivo and discovered a role for phagocyte-expressed GAGs in viral vascular clearance. Moreover, we observe species-specific differences in viral vascular clearance of enhanced GAG-binding virions between murine and avian hosts. These data suggest species-specific variation in GAG structure is a mechanism to distinguish amplifying from dead-end hosts for arbovirus transmission.

Cryo-EM structures of alphavirus conformational intermediates in low pH-triggered prefusion states.

Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development.

In Vitro and In Vivo Phenotypes of Venezuelan, Eastern and Western Equine Encephalitis Viruses Derived from cDNA Clones of Human Isolates.

The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA "Animal Rule" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other "wild type" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.

LDLRAD3 is a receptor for Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans1. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.

Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKß Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKß inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKß and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKß kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKß can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients.

SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Cryo-EM structure of eastern equine encephalitis virus in complex with heparan sulfate analogues.

Eastern equine encephalitis virus (EEEV), a mosquito-borne icosahedral alphavirus found mainly in North America, causes human and equine neurotropic infections. EEEV neurovirulence is influenced by the interaction of the viral envelope protein E2 with heparan sulfate (HS) proteoglycans from the host's plasma membrane during virus entry. Here, we present a 5.8-Å cryoelectron microscopy (cryo-EM) structure of EEEV complexed with the HS analog heparin. "Peripheral" HS binding sites were found to be associated with the base of each of the E2 glycoproteins that form the 60 quasi-threefold spikes (q3) and the 20 sites associated with the icosahedral threefold axes (i3). In addition, there is one HS site at the vertex of each q3 and i3 spike (the "axial" sites). Both the axial and peripheral sites are surrounded by basic residues, suggesting an electrostatic mechanism for HS binding. These residues are highly conserved among EEEV strains, and therefore a change in these residues might be linked to EEEV neurovirulence.

Encephalitic Alphaviruses Exploit Caveola-Mediated Transcytosis at the Blood-Brain Barrier for Central Nervous System Entry.

Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB.IMPORTANCE VEEV, WEEV, and eastern equine encephalitis virus (EEEV) are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the bloodstream; however, the underlying molecular mechanisms have remained largely unknown. Here, we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPases within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens in the brain than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.

Macromolecular Synthesis Shutoff Resistance by Myeloid Cells Is Critical to IRF7-Dependent Systemic Interferon Alpha/Beta Induction after Alphavirus Infection.

Alphavirus infection of fibroblastic cell types in vitro inhibits host cell translation and transcription, leading to suppression of interferon alpha/beta (IFN-α/ß) production. However, the effect of infection upon myeloid cells, which are often the first cells encountered by alphaviruses in vivo, is unclear. Previous studies demonstrated an association of systemic IFN-α/ß production with myeloid cell infection efficiency. Murine infection with wild-type Venezuelan equine encephalitis virus (VEEV), a highly myeloid-cell-tropic alphavirus, results in secretion of very high systemic levels of IFN-α/ß, suggesting that stress responses in responding cells are active. Here, we infected myeloid cell cultures with VEEV to identify the cellular source of IFN-α/ß, the timing and extent of translation and/or transcription inhibition in infected cells, and the transcription factors responsible for IFN-α/ß induction. In contrast to fibroblast infection, myeloid cell cultures infected with VEEV secreted IFN-α/ß that increased until cell death was observed. VEEV inhibited translation in most cells early after infection (<6 h postinfection [p.i.]), while transcription inhibition occurred later (>6 h p.i.). Furthermore, the interferon regulatory factor 7 (IRF7), but not IRF3, transcription factor was critical for IFN-α/ß induction in vitro and in sera of mice. We identified a subset of infected Raw 264.7 myeloid cells that resisted VEEV-induced translation inhibition and secreted IFN-α/ß despite virus infection. However, in the absence of IFN receptor signaling, the size of this cell population was diminished. These results indicate that IFN-α/ß induction in vivo is IRF7 dependent and arises in part from a subset of myeloid cells that are resistant, in an IFN-α/ß-dependent manner, to VEEV-induced macromolecular synthesis inhibition.IMPORTANCE Most previous research exploring the interaction of alphaviruses with host cell antiviral responses has been conducted using fibroblast lineage cell lines. Previous studies have led to the discovery of virus-mediated activities that antagonize host cell antiviral defense pathways, such as host cell translation and transcription inhibition and suppression of STAT1 signaling. However, their relevance and impact upon myeloid lineage cell types, which are key responders during the initial stages of alphavirus infection in vivo, have not been well studied. Here, we demonstrate the different abilities of myeloid cells to resist VEEV infection compared to nonmyeloid cell types and begin to elucidate the mechanisms by which host antiviral responses are upregulated in myeloid cells despite the actions of virus-encoded antagonists.

Cooperativity between the 3' untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus.

Eastern 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 7 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 the 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.

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

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

Protective antibodies against Eastern equine encephalitis virus bind to epitopes in domains A and B of the E2 glycoprotein.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have 'elite' activity with 50 and 99% effective inhibitory concentrations (EC50 and EC99) of less than 10 and 100 ng ml-1, respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design.

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

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

The Interferon-Induced Exonuclease ISG20 Exerts Antiviral Activity through Upregulation of Type I Interferon Response Proteins.

Type I interferon (IFN)-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding the molecular basis of ISG restriction, the antiviral mechanisms of many remain unclear. The 20-kDa ISG ISG20 is a nuclear 3'-5' exonuclease with preference for single-stranded RNA (ssRNA) and has been implicated in the IFN-mediated restriction of several RNA viruses. Although the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, evidence has yet to be presented that virus inhibition in cells requires this activity. Here, we utilized a combination of an inducible, ectopic expression system and newly generated Isg20-/- mice to investigate mechanisms and consequences of ISG20-mediated restriction. Ectopically expressed ISG20 localized primarily to Cajal bodies in the nucleus and restricted replication of chikungunya and Venezuelan equine encephalitis viruses. Although restriction by ISG20 was associated with inhibition of translation of infecting genomic RNA, degradation of viral RNAs was not observed. Instead, translation inhibition of viral RNA was associated with ISG20-induced upregulation of over 100 other genes, many of which encode known antiviral effectors. ISG20 modulated the production of IFIT1, an ISG that suppresses translation of alphavirus RNAs. Consistent with this observation, the pathogenicity of IFIT1-sensitive alphaviruses was increased in Isg20-/- mice compared to that of wild-type viruses but not in cells ectopically expressing ISG20. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by regulating expression of other ISGs that inhibit translation and possibly other activities in the replication cycle.IMPORTANCE The host immune responses to infection lead to the production of type I interferon (IFN), and the upregulation of interferon-stimulated genes (ISGs) reduces virus replication and virus dissemination within a host. Ectopic expression of the interferon-induced 20-kDa exonuclease ISG20 suppressed replication of chikungunya virus and Venezuelan equine encephalitis virus, two mosquito-vectored RNA alphaviruses. Since the replication of alphavirus genomes occurs exclusively in the cytoplasm, the mechanism of nucleus-localized ISG20 inhibition of replication is unclear. In this study, we determined that ISG20 acts as a master regulator of over 100 genes, many of which are ISGs. Specifically, ISG20 upregulated IFIT1 genes and inhibited translation of the alphavirus genome. Furthermore, IFIT1-sensitive alphavirus replication was increased in Isg20-/- mice compared to the replication of wild-type viruses but not in cells ectopically expressing ISG20. We propose that ISG20 acts as an indirect regulator of RNA virus replication in the cytoplasm through the upregulation of many other ISGs.

Ribbon scanning confocal for high-speed high-resolution volume imaging of brain.

Whole-brain imaging is becoming a fundamental means of experimental insight; however, achieving subcellular resolution imagery in a reasonable time window has not been possible. We describe the first application of multicolor ribbon scanning confocal methods to collect high-resolution volume images of chemically cleared brains. We demonstrate that ribbon scanning collects images over ten times faster than conventional high speed confocal systems but with equivalent spectral and spatial resolution. Further, using this technology, we reconstruct large volumes of mouse brain infected with encephalitic alphaviruses and demonstrate that regions of the brain with abundant viral replication were inaccessible to vascular perfusion. This reveals that the destruction or collapse of large regions of brain micro vasculature may contribute to the severe disease caused by Venezuelan equine encephalitis virus. Visualization of this fundamental impact of infection would not be possible without sampling at subcellular resolution within large brain volumes.

Antibody Preparations from Human Transchromosomic Cows Exhibit Prophylactic and Therapeutic Efficacy against Venezuelan Equine Encephalitis Virus.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus that causes low mortality but high morbidity rates in humans. In addition to natural outbreaks, there is the potential for exposure to VEEV via aerosolized virus particles. There are currently no FDA-licensed vaccines or antiviral therapies for VEEV. Passive immunotherapy is an approved method used to protect individuals against several pathogens and toxins. Human polyclonal antibodies (PAbs) are ideal, but this is dependent upon serum from convalescent human donors, which is in limited supply. Non-human-derived PAbs can have serious immunoreactivity complications, and when "humanized," these antibodies may exhibit reduced neutralization efficiency. To address these issues, transchromosomic (Tc) bovines have been created, which can produce potent neutralizing human antibodies in response to hyperimmunization. In these studies, we have immunized these bovines with different VEEV immunogens and evaluated the protective efficacy of purified preparations of the resultant human polyclonal antisera against low- and high-dose VEEV challenges. These studies demonstrate that prophylactic or therapeutic administration of the polyclonal antibody preparations (TcPAbs) can protect mice against lethal subcutaneous or aerosol challenge with VEEV. Furthermore, significant protection against unrelated coinfecting viral pathogens can be conferred by combining individual virus-specific TcPAb preparations.IMPORTANCE With the globalization and spread or potential aerosol release of emerging infectious diseases, it will be critical to develop platforms that are able to produce therapeutics in a short time frame. By using a transchromosomic (Tc) bovine platform, it is theoretically possible to produce antigen-specific highly neutralizing therapeutic polyclonal human antibody (TcPAb) preparations in 6 months or less. In this study, we demonstrate that Tc bovine-derived Venezuelan equine encephalitis virus (VEEV)-specific TcPAbs are highly effective against VEEV infection that mimics not only the natural route of infection but also infection via aerosol exposure. Additionally, we show that combinatorial TcPAb preparations can be used to treat coinfections with divergent pathogens, demonstrating that the Tc bovine platform could be beneficial in areas where multiple infectious diseases occur contemporaneously or in the case of multipathogen release.

Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus.

Most previous studies of interferon-alpha/beta (IFN-α/ß) response antagonism by alphaviruses have focused upon interruption of IFN-α/ß induction and/or receptor signaling cascades. Infection of mice with Venezuelan equine encephalitis alphavirus (VEEV) or Sindbis virus (SINV) induces serum IFN-α/ß, that elicits a systemic antiviral state in uninfected cells successfully controlling SINV but not VEEV replication. Furthermore, VEEV replication is more resistant than that of SINV to a pre-existing antiviral state in vitro. While host macromolecular shutoff is proposed as a major antagonist of IFN-α/ß induction, the underlying mechanisms of alphavirus resistance to a pre-existing antiviral state are not fully defined, nor is the mechanism for the greater resistance of VEEV. Here, we have separated viral transcription and translation shutoff with multiple alphaviruses, identified the viral proteins that induce each activity, and demonstrated that VEEV nonstructural protein 2-induced translation shutoff is likely a critical factor in enhanced antiviral state resistance of this alphavirus.