Explore the vast range of titles available.

Encephalitogenic alphaviruses are mosquito-borne viruses that can cause fatal disease in humans and equines. Currently, there are no licensed vaccines or antiviral treatments for these infections. Western equine encephalitis virus (WEEV) is a member of this group that had not produced a human infection in over a decade. However, an outbreak of WEEV encephalitis in humans and equines was reported recently in South America, indicating a need for additional countermeasures. Blind passage approaches to generation of RNA virus live attenuated vaccines (LAVs) frequently result in acquisition of positively charged amino acid mutations that confer heparan sulfate (HS) binding and that are attenuating factors in resultant LAVs. To develop an informed approach for creation of alphavirus LAVs, we have utilized the WEEV McMillan (McM) strain as an HS weak/non-binding platform into which we have placed positively charged amino acid substitution mutations at positions in the E2 glycoprotein previously shown to confer HS-dependent infection upon other alphaviruses. This approach yielded four mutants with high efficiency HS binding and avirulence in mice, which were further subjected to yield optimization by in vitro selection of second-site mutations. Interestingly, the original mutations concomitantly increased HS interactions and reduced infection promoted by VLDLR and PCDH10 protein receptors, while the second site mutations improved infectivity mediated by VLDLR. Further, we report a newly generated 4.1Å cryo-EM reconstruction of WEEV McM strain into which we have mapped the mutations to provide an E2 glycoprotein domain-based representation of receptor binding site location.

Naturally circulating strains of eastern equine encephalitis virus (EEEV) bind heparan sulfate (HS) receptors and this interaction has been linked to neurovirulence. Previous studies associated EEEV-HS interactions with three positively charged amino acid clusters on the E2 glycoprotein. One of these sites has recently been reported to be critical for binding EEEV to the very-low-density lipoprotein receptor (VLDLR), an EEEV receptor protein. The proteins apolipoprotein E receptor 2 (ApoER2) isoforms 1 and 2, and LDLR have also been shown to function as EEEV receptors. Herein, we investigate the individual contribution of each HS interaction site to EEEV HS- and protein receptor-dependent infection in vitro and EEEV replication in animals. We show that each site contributes to both EEEV-HS and EEEV-protein receptor interactions, providing evidence that altering these interactions can affect disease in mice and eliminate mosquito infectivity. Thus, multiple HS-binding sites exist in EEEV E2, and these sites overlap functionally with protein receptor interaction sites, with each type of interaction contributing to tissue infectivity and disease phenotypes. Mutation of positively charged binding sites on the eastern equine encephalitis virus (EEEV) E2 glycoprotein affects both heparan sulfate and protein receptor binding, identifying an unexpected linkage between engagement of these two receptor types.

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.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus that can cause severe encephalitis in humans and horses with a high case fatality rate. There are no licensed EEEV vaccines or therapeutics for human use, warranting the need to better understand the human immune response against EEEV. Here we present a cryo-EM reconstruction of the chimeric virus, Sindbis (SINV)/EEEV, in complex with a potently neutralizing and efficacious intact human IgG1 antibody in a mouse model of infection and disease. This antibody requires bivalency to recognize a quaternary epitope on the E2 glycoprotein and cross-links two virus spikes across the icosahedral two-fold axis through a unique binding mode. Kinetic analysis of the binding interaction provides insights into this distinguishing feature. Mechanistically, the antibody inhibits viral entry into cells through blockade of receptor binding and early fusion events but does not block egress, thereby, exclusively targeting an epitope found on intact virions. The discovery of the quaternary epitope and unique binding mode recognized by this antibody together advance our understanding of the complexity of antibody-antigen interactions and can aid in vaccine design to elicit recognition of distinct epitopes of clinically relevant alphaviruses. Structural analyses of antibody-virus complexes offer critical insights into immune recognition mechanisms. In this report, we present how a patient-derived IgG recognizes a quaternary epitope on EEEV particles through strictly bivalent interactions.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans 1 . 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. LDLRAD3 is a receptor for infection with Venezuelan equine encephalitis virus, and in mouse models deletion of Ldlrad3 or treatment with a soluble LDLRAD3 decoy molecule abrogates infection and disease caused by this virus.

Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-dependent enhancement (ADE) of disease as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral/ocular) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7–10 days post-infection. To evaluate the potential for ADE, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.

Aerosol exposure to eastern equine encephalitis virus (EEEV) can trigger a lethal viral encephalitis in cynomolgus macaques which resembles severe human disease. Biomarkers indicative of central nervous system (CNS) infection by the virus and lethal outcome of disease would be useful in evaluating potential medical countermeasures, especially for therapeutic compounds. To meet requirements of the Animal Rule, a better understanding of the pathophysiology of EEEV-mediated disease in cynomolgus macaques is needed. In this study, macaques given a lethal dose of clone-derived EEEV strain V105 developed a fever between 2–3 days post infection (dpi) and succumbed to the disease by 6 dpi. At the peak of the febrile phase, there was a significant increase in the delta electroencephalography (EEG) power band associated with deep sleep as well as a sharp rise in intracranial pressure (ICP). Viremia peaked early after infection and was largely absent by the onset of fever. Granulocytosis and elevated plasma levels of IP-10 were found early after infection. At necropsy, there was a one hundred- to one thousand-fold increase in expression of traumatic brain injury genes (LIF, MMP-9) as well as inflammatory cytokines and chemokines (IFN-γ, IP-10, MCP-1, IL-8, IL-6) in the brain tissues. Phenotypic analysis of leukocytes entering the brain identified cells as primarily lymphoid (T, B, NK cells) with lower levels of infiltrating macrophages and activated microglia. Massive amounts of infectious virus were found in the brains of lethally-infected macaques. While no infectious virus was found in surviving macaques, quantitative PCR did find evidence of viral genomes in the brains of several survivors. These data are consistent with an overwhelming viral infection in the CNS coupled with a tremendous inflammatory response to the infection that may contribute to the disease outcome. Physiological monitoring of EEG and ICP represent novel methods for assessing efficacy of vaccines or therapeutics in the cynomolgus macaque model of EEEV encephalitis.

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.

Dengue, one of the most widespread infectious diseases, has affected U.S. military readiness throughout history. We explored the dengue diagnosis capability gap by circulating a questionnaire among military end users to determine in what capacity diagnostic test results are needed and how these results would be used at various roles of care in the Military Health System. Results were used to generate target product profiles for potential diagnostic tests. We determined that at far-forward locations, diagnostic tests need to be rugged and easy to use and are primarily needed to inform medical evacuation decisions. In mobile or fixed hospitals, diagnostics can be less portable but must be accurate enough to inform patient care decisions reliably. We then evaluated the suitability of using rapid diagnostic tests and enzyme-linked immunosorbent assays based on published performance characteristics, and we used a model to determine positive and negative predictive values in certain simulated deployments. In far-forward settings, a rapid diagnostic test comprising both antigen- and antibody-based detection can fulfill the capability gap with reasonable accuracy, whereas at higher roles of care immunoglobulin M-enzyme-linked immunosorbent assay was determined to be the most suitable option.

Naturally circulating strains of eastern equine encephalitis virus (EEEV) bind heparan sulfate (HS) receptors and this interaction has been linked to its neurovirulence. Previous studies associated EEEV-HS interactions with three positively charged amino acid clusters on the E2 glycoprotein. One of these sites has recently been reported to be critical for binding EEEV to very-low-density lipoprotein receptor (VLDLR), an EEEV receptor protein. The proteins apolipoprotein E receptor 2 (ApoER2) isoforms 1 and 2, and LDLR have also been shown to function as EEEV receptors. Herein, we investigate the individual contribution of each HS interaction site to EEEV HS- and protein receptor-dependent infection and EEEV replication in animals. We show that each site contributes to both EEEV-HS and EEEV-protein receptor interactions, providing evidence that altering these interactions can affect disease in mice and eliminate mosquito infectivity. Thus, multiple HS-binding sites exist in EEEV E2, and these sites overlap functionally with protein receptor interaction sites, with each type of interaction contributing to tissue infectivity and disease phenotypes.