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.

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.

Neutralizing antibodies protect mice against Venezuelan equine encephalitis virus aerosol challenge.

Venezuelan equine encephalitis virus (VEEV) remains a risk for epidemic emergence or use as an aerosolized bioweapon. To develop possible countermeasures, we isolated VEEV-specific neutralizing monoclonal antibodies (mAbs) from mice and a human immunized with attenuated VEEV strains. Functional assays and epitope mapping established that potently inhibitory anti-VEEV mAbs bind distinct antigenic sites in the A or B domains of the E2 glycoprotein and block multiple steps in the viral replication cycle including attachment, fusion, and egress. A 3.2-Å cryo-electron microscopy reconstruction of VEEV virus-like particles bound by a human Fab suggests that antibody engagement of the B domain may result in cross-linking of neighboring spikes to prevent conformational requirements for viral fusion. Prophylaxis or postexposure therapy with these mAbs protected mice against lethal aerosol challenge with VEEV. Our study defines functional and structural mechanisms of mAb protection and suggests that multiple antigenic determinants on VEEV can be targeted for vaccine or antibody-based therapeutic 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.

Approach to Strain Selection and the Propagation of Viral Stocks for Venezuelan Equine Encephalitis Virus Vaccine Efficacy Testing under the Animal Rule.

Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical. An approach to selecting VEEV challenge strains for use under the Animal Rule was developed, taking into account Department of Defense (DOD) vaccine requirements, FDA Animal Rule guidelines, strain availability, and lessons learned from the generation of filovirus challenge agents within the Filovirus Animal Nonclinical Group (FANG). Initial down-selection to VEEV IAB and IC epizootic varieties was based on the DOD objective for vaccine protection in a bioterrorism event. The subsequent down-selection of VEEV IAB and IC isolates was based on isolate availability, origin, virulence, culture and animal passage history, known disease progression in animal models, relevancy to human disease, and ability to generate sufficient challenge material. Methods for the propagation of viral stocks (use of uncloned (wild-type), plaque-cloned, versus cDNA-cloned virus) to minimize variability in the potency of the resulting challenge materials were also reviewed. The presented processes for VEEV strain selection and the propagation of viral stocks may serve as a template for animal model development product testing under the Animal Rule to other viral vaccine programs. This manuscript is based on the culmination of work presented at the "Alphavirus Workshop" organized and hosted by the Joint Vaccine Acquisition Program (JVAP) on 15 December 2014 at Fort Detrick, Maryland, USA.

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.

Similarities and differences in antagonism of neuron alpha/beta interferon responses by Venezuelan equine encephalitis and Sindbis alphaviruses.

Venezuelan equine encephalitis virus (VEEV) is highly virulent in adult laboratory mice, while Sindbis virus (SINV) is avirulent regardless of dose or inoculation route, dependent upon functioning alpha/beta interferon (IFN-alpha/beta) responses. We have examined each virus' resistance to and/or antagonism of IFN-alpha/beta responses in neurons, a cell type targeted by both viruses in mice, by infecting IFN-alpha/beta-treated or untreated primary cultures with viruses or virus-derived replicons that lacked the structural proteins. Priming with IFN-alpha/beta prior to infection revealed that VEEV replication and progeny virion production were resistant to an established antiviral state while those of SINV were more sensitive. Postinfection IFN-alpha/beta treatment revealed that phosphorylation of STAT1 and STAT2 was partially blocked by infection with either virus, dependent upon expression of nonstructural proteins (nsP), but not structural proteins (sP). However, inhibition of STAT phosphorylation by VEEV replicons was not correlated with inhibition of IFN-stimulated gene (ISG) mRNA induction, yet ISG induction was inhibited when sP were present. Host translation was inhibited by VEEV nsP even when cells were pretreated with IFN-alpha/beta. SINV blocked ISG induction and translation, associated with nsP-mediated shutoff of macromolecular synthesis, but both activities were sensitive to IFN-alpha/beta pretreatment. We conclude that both VEEV and SINV limit ISG induction in infected neurons through shutoff of host transcription and translation but that inhibition by VEEV is more resistant to IFN-alpha/beta priming. Likewise, both viruses inhibit IFN receptor-initiated signaling, although the effect upon host responses is not clear. Finally, VEEV appears to be more resistant to effectors of the preestablished antiviral state.

Eastern and Venezuelan equine encephalitis viruses differ in their ability to infect dendritic cells and macrophages: impact of altered cell tropism on pathogenesis.

Eastern and Venezuelan equine encephalitis viruses (EEEV and VEEV, respectively) cause severe morbidity and mortality in equines and humans. Like other mosquito-borne viruses, VEEV infects dendritic cells (DCs) and macrophages in lymphoid tissues, fueling a serum viremia and facilitating neuroinvasion. In contrast, EEEV replicates poorly in lymphoid tissues, preferentially infecting osteoblasts. Here, we demonstrate that infectivity of EEEV for myeloid lineage cells including DCs and macrophages was dramatically reduced compared to that of VEEV, whereas both viruses replicated efficiently in mesenchymal lineage cells such as osteoblasts and fibroblasts. We determined that EEEV infection of myeloid lineage cells was restricted after attachment, entry, and uncoating of the genome. Using replicon particles and translation reporter RNAs, we found that translation of incoming EEEV genomes was almost completely inhibited in myeloid, but not mesenchymal, lineage cells. Alpha/beta interferon (IFN-alpha/beta) responses did not mediate the restriction, as infectivity was not restored in the absence of double-stranded RNA-dependent protein kinase, RNase L, or IFN-alpha/beta receptor-mediated signaling. We confirmed these observations in vivo, demonstrating that EEEV is compromised in its ability to replicate within lymphoid tissues, whereas VEEV does so efficiently. The altered tropism of EEEV correlated with an almost complete avoidance of serum IFN-alpha/beta induction in vivo, which may allow EEEV to evade the host's innate immune responses and thereby enhance neurovirulence. Taken together, our data indicate that inhibition of genome translation restricts EEEV infectivity for myeloid but not mesenchymal lineage cells in vitro and in vivo. In this regard, the tropisms of EEEV and VEEV differ dramatically, likely contributing to observed differences in disease etiology.