Shifts in receptors during submergence of an encephalitic arbovirus.

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

Astrocyte inflammatory signaling mediates α-synuclein aggregation and dopaminergic neuronal loss following viral encephalitis.

Viral infection of the central nervous system (CNS) can cause lasting neurological decline in surviving patients and can present with symptoms resembling Parkinson's disease (PD). The mechanisms underlying postencephalitic parkinsonism remain unclear but are thought to involve increased innate inflammatory signaling in glial cells, resulting in persistent neuroinflammation. We therefore studied the role of glial cells in regulating neuropathology in postencephalitic parkinsonism by studying the involvement of astrocytes in loss of dopaminergic neurons and aggregation of α-synuclein protein following infection with western equine encephalitis virus (WEEV). Infections were conducted in both wildtype mice and in transgenic mice lacking NFκB inflammatory signaling in astrocytes. For 2 months following WEEV infection, we analyzed glial activation, neuronal loss and protein aggregation across multiple brain regions, including the substantia nigra pars compacta (SNpc). These data revealed that WEEV induces loss of SNpc dopaminergic neurons, persistent activation of microglia and astrocytes that precipitates widespread aggregation of α-synuclein in the brain of C57BL/6 mice. Microgliosis and macrophage infiltration occurred prior to activation of astrocytes and was followed by opsonization of ⍺-synuclein protein aggregates in the cortex, hippocampus and midbrain by the complement protein, C3. Astrocyte-specific NFκB knockout mice had reduced gliosis, α-synuclein aggregate formation and neuronal loss. These data suggest that astrocytes play a critical role in initiating PD-like pathology following encephalitic infection with WEEV through innate immune inflammatory pathways that damage dopaminergic neurons, possibly by hindering clearance of ⍺-synuclein aggregates. Inhibiting glial inflammatory responses could therefore represent a potential therapy strategy for viral parkinsonism.

Functional pseudotyping of human immunodeficiency virus type 1 vectors by Western equine encephalitis virus envelope glycoprotein.

We investigated the ability of western equine encephalitis virus envelope glycoproteins (WEEV GP) to pseudotype lentiviral vectors. The titers of WEEV GP-pseudotyped human immunodeficiency virus type 1 (HIV) ranged as high as 8.0 x 10(4) IU/ml on permissive cells. Sera from WEEV-infected mice specifically neutralized these pseudotypes; cell transduction was also sensitive to changes in pH. The host range of the pseudotyped particles in vitro was somewhat limited, which is atypical for most alphaviruses. HIV vectors pseudotyped by WEEV GP may be a useful tool for characterizing WEEV cell binding and entry and screening for small-molecule inhibitors.

Dissemination of western equine encephalomyelitis virus in the potential vector, Culex pipiens pallens.

Two western equine encephalomyelitis virus (WEEV) strains have been isolated in China. Our previous studies have verified that the mosquito Culex pipiens pallens Coquillett (Diptera: Culicidae) infected with WEEV was capable of transmitting this arbovirus, but it was not clear how the sequential multiplication and spread of virus occurred within the mosquito. In this study, we observed the distribution of WEEV antigen in orally-infected Cx. p. pallens by immunohistochemistry in order to better understand the initial infection, dissemination, and transmission of WEEV in the potential vector. Orally-infected WEEV dissemination varied within the different tissues of Cx. p. pallens, with virus antigen consistently observed in the salivary glands, foregut, midgut epithelial cells, Malpighian tubules, hindgut, and ovarian follicles of some individuals after various days of extrinsic incubation. We suggest that Cx. p. pallens, the potential vector of WEEV, has the ability to harbor the virus through the alimentary system, and the midgut epithelial cell may be the initial site of WEEV replication after ingestion of a viremic blood meal.

Properties of monoclonal antibodies against glycoproteins of western equine encephalitis virus.

To analyze the biological activities of the alphavirus glycoproteins, eight different monoclonal antibodies against the two glycoproteins of western equine encephalitis virus were isolated. Five of the eight monoclonal antibodies were shown to be specific for E1 and three for E2 protein by an enzyme-linked immunosorbent assay and by radioimmunoprecipitation. Three of the five anti-E1 and all of the anti-E2 monoclonal antibodies inhibited hemagglutination by purified virions. One anti-E1 and two anti-E2 monoclonal antibodies possessed high virus-neutralizing activity.

Maturation defect of a temperature-sensitive mutant of western equine encephalitis virus.

The defective step of a temperature-sensitive mutant of western equine encephalitis virus, which synthesize viral RNA but not mature virus at the restrictive temperature, was studied. Cells infected with the mutant virus at the restrictive temperature synthesized the same intracellular viral RNA as that in wild type infection. Cells infected with the mutant at the restrictive temperature formed three proteins (E1, E2 and C) which migrated to positions identical with those of purified virions and a precursor protein of E2 (PE2). The mutant virus was also able to form cytoplasmic nucleocapsids sedimenting at 140S as in the case of wild type infection. On the other hand, cells infected with the mutant could not induce a significant amount of hemadsorbing ability and the ability induced at the permissive temperature disappeared immediately after shifting up to the restrictive temperature. These results suggested that the mutant virus produced a defective envelope protein responsible for hemagglutination at the restrictive temperature. Owing to the incompleteness of the modification of the cell plasma membrane by the envelope proteins, viral nucleocapsids in the mutant infected cells could not bind to the plasma membrane.

Evidence for the presence of the minor capsid protein of Western equine encephalitis virus.

A minor capsid protein was found in Western equine encephalitis virus. The minor capsid protein appeared to be produced by proteolytic cleavage of part of the newly synthesized capsid protein in infected cells and to be incorporated into nucleocapsids.

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus.

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus were studied. Electron micrographs of the infected cells showed that most of mutant virions bud from the plasma or vacuolar membrane as a multiploid particle containing a various number of nucleocapsids enclosed with a defined common envelope. The mutant virions contained three polypeptides which migrated to the position identical with those of wild type on discontinuous acrylamide gels. Cells infected with the mutant virus synthesized the same intracellular viral RNA species as was made after infection of wild type. Cytoplasmic nucleocapsids of the mutant sedimented at 140S and contained 42S virion RNA as those of wild type; they were indistinguishable from those of wild type in an electron microscope examination. On the other hand, mutant nucleocapsids isolated from extracellular virions sedimented as heterogeneous particles larger thant 140S and were shown to be pleomorphic and aggregate in electron micrographs. The budding process of this mutant seemed to be modified, so that it might form the multiploid with the alteration of its nucleocapsids.

A variant of western equine encephalitis virus with nonglycosylated E3 protein.

A variant clone (At/A125) of western equine encephalitis virus was isolated from a line of mosquito cells persistently infected with a temperature-sensitive parent strain, A125. Variant-infected cells produced an altered form of PE2 protein which migrated with a higher electrophoretic mobility than wild type or A125 PE2. The altered PE2, like PE2 of wild type, was precipitated by anti-envelope proteins serum but not by anti-E1 serum. In pulse-chase experiments the altered PE2 protein was shown to yield E2 of normal electrophoretic mobility in SDS-polyacrylamide gel electrophoresis. The unglycosylated form of the altered PE2 synthesized in the presence of tunicamycin migrated at the same position as the unglycosylated PE2 obtained from tunicamycin-treated, parent strain-infected cells. This suggested that migration difference might be ascribable to incomplete glycosylation of PE2, possibly of its E3 component. E3 is released into culture fluid of wild-type-infected cells as an approximately 11-kd glycoprotein, while variant-infected culture fluid yielded a smaller, apparently virus-specific protein. The protein could not be labeled with [3H]mannose, suggesting that the polypeptide moiety of E3 in the variant infected cells failed to be glycosylated. The parent strain, A125, and a revertant of the variant, At/A125/rev, did not synthesize such altered PE2 and E3 proteins. The growth of At/A125 in mosquito cells was similar to that of parent or wild type but depressed in vertebrate cells.

In vitro mechanisms of monoclonal antibody neutralization of alphaviruses.

We have previously identified at least eight epitopes on the E2 glycoprotein of Venezuelan equine encephalomyelitis (VEE) virus vaccine strain TC-83 by using monoclonal antibodies (MAbs). Several of these antibodies identified a critical neutralization (N) domain in competitive binding assays. Passive transfer of these MAbs protected animals from a lethal virus challenge. Using radioactive, purified virus as a marker, we have demonstrated that antibody-mediated virus N, preattachment, can be effected by one of three mechanisms. Interaction of antibody can block virus attachment to susceptible Vero or human embryonic lung cells. The MAbs that were most efficient at blocking attachment were those that defined epitopes spatially proximal to the E2c epitope. The E2c MAbs were, however, the most efficient antibodies for neutralizing virus postattachment. Other E2 MAbs were unable to efficiently block virus attachment to cells; however, resulting replication as monitored by plaque assay or intracellular viral RNA synthesis could not be detected. One novel MAb that defined the E2f epitope appeared to enhance virus attachment to Vero cells, but not BHK-21 or LLC-MK2 cells, by stabilizing virus-cell interaction. This antibody did, however, efficiently neutralize virus infectivity. Once virus had attached to cells, the ability of most MAbs to neutralize infectivity was diminished, except for E2c MAbs. On a molar basis antibody Fab fragments were less efficient than intact antibody at blocking virus attachment.

Effect of cordycepin on the replication of western equine encephalitis virus.

Cordycepin (3'-deoxyadenosine) inhibited viral RNA synthesis in the replication of western equine encephalitis virus, thereby causing a reduction of virus production. The rate of inhibition of viral RNA synthesis was dependent on drug concentration and the period of treatment with the drug. These results suggest that the virus RNA synthesizing system is sensitive to the drug.

Correlation between virus-cell receptor properties of alphaviruses in vitro and virulence in vivo.

Virulent and avirulent clones of Venezuelan, Western, and Eastern equine encephalitis viruses were examined for their in vitro attachment characteristics to the surface of cultured cell monolayers. These attachment characteristics were correlated with in vivo plasma clearance rates and virulence. For the clones investigated, avirulence correlated in vitro with attachment pH optima close to physiologic pH and in vivo with a rapid clearance from plasma. Conversely, virulent clones had lower in vitro attachment pH optima and low plasma clearances in vivo.

Spontaneous and induced mutagenesis in Western Equine encephalomyelitis virus in chick embryo cells with different repair activity.

Evidence was obtained indicating differences in the survival rate of Western equine encephalomyelitis virus after exposure to ultraviolet radiation and methyl methanesulfonate in commercial and leukosisfree chick embryo cells that differed in repair activity. The levels of spontaneous mutagenesis (on the basis of the yield of small palque variants of the encephalomyelitis virus) did not essentially change when the virus was passage in leukosis-free chick embryo cells, whereas an increase in the number of small palque variants was observed in the cells of commercial chick embryos. A 10-fold increase in the number of induced virus variants was observed in commercial chick embryo cells in experiments with methyl methanesulfonate as compared with the contorl, whereas the induction of virus variants was not noted in leukosis-free cells.

Photochemical inactivation of DNA and RNA viruses by psoralen derivatives.

Western equine encephalitis virus, and RNA virus, and herpes simplex virus type I, a DNA virus, were efficiently inactivated in less than I min by exposure to long-wave ultraviolet light (320 to 380 nm) in the presence of several psoralen derivatives. The psoralen photochemical reaction was chosen for study due to its known specificity for nucleic acids. Neither the light nor any of the drugs alone caused appreciable inactivation. The inactivation kinetics and dependence on light intensity and on different derivatives of psoralen were studied. The high solubility of a new aminomethyl psoralen derivative was found to be advantageous in the photochemical inactivation of the RNA virus, but was not in the case of the more easily inactivated DNA virus. Within its limited solubility range trimethylpsoralen was superior to its aminomethyl derivative on a molar basis for the inactivation of both types of viruses under most of the conditions studied.

A heterologous coiled coil can substitute for helix I of the Sindbis virus capsid protein.

Alphavirus core assembly proceeds along an assembly pathway involving a dimeric assembly intermediate. Several regions of the alphavirus capsid protein have been implicated in promoting and stabilizing this dimerization, including a putative heptad repeat sequence named helix I. This sequence, which spans residues 38 to 55 of the Sindbis virus capsid protein, was implicated in stabilizing dimeric contacts initiated through the C-terminal two-thirds of the capsid protein and nucleic acid. The studies presented here demonstrate that helix I can be functionally replaced by the corresponding sequence of a related alphavirus, western equine encephalitis virus, and also by an unrelated sequence from the yeast transcription activator, GCN4, that was previously shown to form a dimeric coiled coil. Replacing helix I with the entire leucine zipper domain of GCN4 (residues 250 to 281) produced a virus with the wild-type phenotype as determined by plaque assay and one-step growth analysis. However, replacement of helix I with a GCN4 sequence that favored trimer formation produced a virus that exhibited approximately 40-fold reduction in virus replication compared to the wild-type Sindbis virus. Changing residues within the Sindbis virus helix I sequence to favor trimer formation also produced a virus with reduced replication. Peptides corresponding to helix I inhibited core-like particle assembly in vitro. On the basis of these studies, it is proposed that helix I favors capsid protein-capsid protein interactions through the formation of dimeric coiled-coil interactions and may stabilize assembly intermediates in the alphavirus nucleocapsid core assembly pathway.