Structural and functional basis of VLDLR usage by Eastern equine encephalitis virus.

The very-low-density lipoprotein receptor (VLDLR) comprises eight LDLR type A (LA) domains and supports entry of distantly related alphaviruses, including Eastern equine encephalitis virus (EEEV) and Semliki Forest virus (SFV). Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage more than one LA domain simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection. Whereas all EEEV strains show conservation of two VLDLR-binding sites, the EEEV PE-6 strain and a few other EEE complex members feature a single amino acid substitution that enables binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.

Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

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.

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.

Human eastern equine encephalitis: immunohistochemistry and ultrastructure.

The brain of a 7-year-old boy who died of eastern equine encephalitis (EEE) was examined by immunohistochemical and ultrastructural techniques to detect the presence and distribution of viral antigen. A mouse polyclonal antibody was most effective for demonstrating the presence of antigen previously unreported in this disease in humans. Antigen was localized to the perikaryon and dendrites of neurons; little was detected in glial cells. Cell death by apoptosis was conspicuous, but it was primarily identified in glial and inflammatory cells. Neuronal death was most commonly marked by cytoplasmic swelling or eosinophilia and nuclear pyknosis. A disassociation between the degree of inflammation and the presence of antigen was noted, especially in cerebral cortex and spinal cord, presumably where infected cells already had been cleared. Ultrastructurally, rare mature viral particles were seen in extracellular spaces.

Patterns of eastern equine encephalomyelitis virus infection in Culiseta melanura (Diptera: Culicidae).

Infection of the mosquito Culiseta melanura (Coquillett) by eastern equine encephalomyelitis virus was examined using transmission electron microscopy (TEM) of whole tagmata and fluorescent antibody assay (FA) and infectious assay (IA) of dissected tissues. Following infectious blood meals from chicks circulating different virus titers, mosquitoes were examined after extrinsic incubation intervals of 1-22 d. Virus was first detected by FA and IA in midguts and nonalimentary tissues 24 h after infection. Nascent virus was first visualized by TEM in several tissues, including midgut, fat body, and salivary glands, of high-titer-infected mosquitoes 48 h after they engorged. Moderate- and low-titer blood meals resulted in slightly slower appearance and dissemination of virus. Results were consistent with dissemination of virus from the posterior midgut to salivary glands via the hemolymph. Neural tissues contained little or no virus, whereas fat body appeared to be an important organ for virus replication and dissemination. Dissemination barriers did not accompany mosquito infections.

Human eastern equine encephalitis. Electron microscopic study of a brain biopsy.

An electron microscopic study was done on brain biopsy tissue from an eight-month-old female with acute eastern equine encephalitis diagnosed by indirect immunofluorescence. Rare clusters of round virions were found almost exclusively in the extracellular space. All virions observed had spiked envelopes, and their sizes averaged approximately 55 nm. Also found were rare enveloped virions along with degenerate organelles in a membrane-bound structure in the cytoplasm of macrophages. Intracytoplasmic development of virions was not found. Tubuloreticular complexes were seen in the endothelial cells and macrophages. This is the first report of an electron microscopic study on biopsy material from a case of human eastern equine encephalitis. It will extend the usefulness of brain biopsy in the diagnosis of acute encephalitis.

Rapid infection of salivary glands in Culiseta melanura with eastern equine encephalitis virus: an electron microscopic study.

Transmission electron microscopy was used to determine if eastern equine encephalitis (EEE) virus infects and replicates in the salivary glands of Culiseta melanura after 3 days of extrinsic incubation (EI). The Cs. melanura studied were from a colony strain, were orally infected, and had EI periods of 55-69 hours. Both naked nucleocapsids and enveloped virions were present in aggregates, suggestive of viral replication, within salivary gland acinar cells. Nucleocapsids were present in the cytoplasm below the plasma membrane that lined apical cavities. Enveloped virions occurred in the salivary matrix within apical cavities. Some nucleocapsids appeared to be budding through the plasma membrane around apical cavities and maturing into infectious virions. These results suggest that Cs. melanura is capable of biological transmission of EEE virus after less than or equal to 3 days of EI.

The distribution and development of eastern equine encephalitis virus in its enzootic mosquito vector, Culiseta melanura.

The timing and sequence of eastern equine encephalitis (EEE) virus replication was studied in the organs from a colony strain of orally infected Culiseta melanura. Three methods of virus assay were used: fluorescent antibody (FA) staining of dissected organs; virus titration in cell culture of whole mosquitoes, dissected organs, hemolymph, and egg rafts; and transmission electron microscopy (TEM) of infected hindguts. EEE virus replicated rapidly in Cs. melanura, first in the posterior midgut, after which it disseminated into the hemocoel where hemolymph transported virus to other organs causing a systemic infection that eventually involved all organs examined, except ovarioles. No initial decrease in virus titer of whole mosquitoes or dissected organs was observed when mosquitoes were collected at daily intervals. Muscle tissue contained the greatest amount of specific fluorescence and the largest aggregates of virus that were visible by TEM. Dissemination of virus occurred rapidly, in some mosquitoes after less than or equal to 17 hours of extrinsic incubation (EI). All infected mosquitoes had disseminated infections after 3 days of EI. Maximum amounts of virus were obtained from whole mosquitoes on the 7th day of EI. FA staining of hindguts was determined to be a rapid and reliable method for detection of EEE virus dissemination and replication in Cs. melanura.

Eastern equine encephalomyelitis. Histopathologic and ultrastructural changes with isolation of the virus in a human case.

Enveloped Toga virus particles were demonstrated by means of an electron microscopy in the brain tissues of a 3-year-old girl with acute encephalitis. Areas of demyelinization and necrosis throughout the white matter and brainstem were revealed by light microscopy. These viral particles were identified as eastern equine encephalomyelitis virus in postmortem isolation of the virus utilizing young mice and complement-fixation studies. To the authors' knowledge, this is the first demonstration of eastern equine encephalomyelitis virus particles in human tissues by electron microscopy.