Pathogenicity of an attenuated, temperature-sensitive mutant of western equine encephalitis virus induced by a chemical mutagen.

To know the pathogenicity of the chemically induced, temperature-sensitive (ts) mutant of western equine encephalitis virus, designated tsNG39, the lethality for mice injected with tsNG39, virus yield, interferon production, and histological changes in the brains of these mice were examined in parallel with those of mice inoculated with the parent strain (PS). All of the mice injected intracranially with PS died within 3.5 days after injection irrespective of the inoculum size of virus, whereas the lethality of the mice inoculated with tsNG39 varied from 94.3 to 65.5% among groups of mice and this variation seemed to be correlated with the inoculum size of virus rather than with the maximum virus titer in the brain. By histological examination, two types of changes in the brain were distinguished, inflammatory and degenerative ones. Inflammatory changes were more prominent in the brains injected with tsNG39 than in those receiving PS. Degenerative changes were dominant in the brains injected with PS, but they were slight in the earlier phase of infection by tsNG39 became prominent only later. The degree of degenerative change was well correlated with both the virus titer in the mouse brain and the death pattern of mice injected with PS or tsNG39. Since degenerative changes are thought to be caused by the direct effect of injected virus, these results indicated that the factor responsible for the low virulence of tsNG39 was the slow viral growth in the brain.     

Virulence of a temperature-sensitive mutant of western equine encephalitis virus

Summary Two temperature-sensitive mutants of western equine encephalitis virus were detected among 96 plaques derived from virus treated with N-methyl-N-nitro-N-nitrosoguanidine. One of the two, tsNG 39 mutant, was able to stimulate substantial ribonucleic acid synthesis at nonpermissive temperature of 42° C, though it did not produce infectious virus or hemagglutinin. At that temperature the wild-type and mutant virus particles had identical heat stability, while the mutant virus was found to be highly thermolabile at 50° C. In addition, the mutant exhibited low virulence for adult mice. Growth and back-mutation of the mutant in the brain of mice is discussed.     

Construction and characterization of monoclonal antibodies against western equine encephalitis virus

A repertoire of mouse monoclonal antibodies (MAbs) against western equine encephalitis virus (WEE) was constructed and characterized. Anti-WEE antibodies were expressed from hybridomas and purified by protein G chromatography. Each of the antibodies was functionally assessed by indirect enzyme-linked immunosorbent assays (ELISAs), Western blotting, and immunoprecipitations. All antibodies bound to WEE antigen in ELISAs, whereas only a subgroup of antibodies was found to be active in Western blotting and immunoprecipitations. A subset of antibodies was found to cross-react with other alphaviruses, such as Sindbis virus (SIN), Venezuelan equine encephalitis (VEE), and eastern equine encephalitis (EEE). Because many of the antibodies were highly reactive to WEE antigen in one or more of the assays, these antibodies are excellent candidates for immunodetection and immunotherapy studies.     

Isolation and preliminary characterization of temperature-sensitive mutants of western equine encephalitis virus

Twelve temperature-sensitive (ts) mutants were newly isolated from western equine encephalitis virus treated with nitrous acid or nitrosoguanidine. All these mutants were found to be thermostable and defective in viral RNA synthesis. Complementation assays were performed with these RNA-defective is mutants and one RNA⁺ is mutant reported previously. A total of three complementation groups has been identified among these is mutants.     

Effect of cordycepin on the replication of western equine encephalitis virus

Summary 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.With 3 Figures     

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

Summary 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.With 6 Figures     

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.     

Isolation of the structural proteins of western equine encephalitis virus by isoelectric focusing

Summary Western equine encephalitis virus was disrupted with Triton X-100 and subjected to isoelectric focusing in a sucrose or urea gradient. The two envelope proteins, E 1 and E 2 were not well separated in a sucrose gradient, while the E 1 and E 2 proteins were distinguished as two major peaks which focused in a urea gradient at about pH 7.5 and 10, respectively. Isolated E 1 protein refocused at pH 6.5 in a sucrose gradient isoelectric focusing column. When Western equine encephalitis virus was treated with Triton X-100 in 0.01 M phosphate buffer (pH 6), hemagglutinating E 1 protein was solubilized, which isoelectrofocused at pH 6.5.Purified nucleocapsids focused at pH 4 in a sucrose gradient on an isoelectric focusing column. After ribonuclease treatment of the purified nucleocapsid more than 95 per cent of the viral RNA was acid-soluble, and the nucleocapsid protein isoelectrofocused at about pH 4.With 6 Figures     

Studies with a pathogenic and an attenuated strain of Venezuelan equine encephalitis virus and aedes aegypti (L.) mosquitoes

Summary Venezuelan equine encephalitis virus was passaged in KB cell cultures. The virus lost its mouse pathogenicity following subcutaneous inoculation during KB cell passage; the attenuated strain also produced smaller plaques than the pathogenic strain. Both strains grew to the same extent inAedes aegypti (L.) mosquitoes after intrathoracic inoculation. If any reversions to pathogenicity occur during development of the attenuated virus in mosquitoes, then the mutation frequency per duplication per particle must be smaller than 3.5×10^–6.     

Effect of exogenous interferon and an interferon inducer on western equine encephalitis virus disease in a hamster model

Mice are used as models for western equine encephalitis virus (WEEV) infection, but high mortality is generally only seen with intracranial or intranasal challenge, while peripheral inoculation results in approximately 50% mortality and is not dose-dependent. Hamsters were therefore studied as a model for WEEV infection. Hamsters were highly sensitive to intraperitoneal (i.p.) infection with WEEV. Disease progression was rapid, and virus titers in serum, brain, liver, and kidney of infected hamsters peaked between 2 and 4 days post-virus inoculation (dpi). Foci of virus infection were detected in neurons of the cerebral cortex and midbrain. Pre-treatment i.p. with either interferon alfacon-1 (5 microg/kg/day) or with Ampligen (3.2 mg/kg/day) resulted in complete survival, reduced brain titers, and improved weight gain. This model of WEEV infection in hamsters appears to serve as a suitable model for the evaluation of potential therapeutic agents for the treatment of WEE disease.     

Interferon responses of various populations of lymphocytes of inbred mice with various degrees of sensitivity to the western equine encephalitis virus

The sensitivity of mice of three lines: CBA, BALB/C, and C57BL/6 (males aged greater than or equal to 4-5 and one week) to peripheral inoculation with Western equine encephalomyelitis (WEE) virus was studied. The C57BL/6 line was found to be the most and CBA the least sensitive. The first generation hybrids between these lines are intermediate in their sensitivity to WEE virus. A correlation was established between the increased sensitivity to WEE virus of C57BL/6 mice and the defect of the interferon system in these mice manifested by the extremely low capacity of their thymocytes for production of beta and alpha-interferons, as well as by a significant decrease of interferon responses of splenocytes to Newcastle disease virus, L-1 (alpha-interferon), lafarin, beta-interferon, concanaxalin-A (alpha-interferon) after immunization with antigens of different origin.     

Selection for accelerated penetration in cell culture coselects for attenuated mutants of Venezuelan equine encephalitis virus

Previous studies with Sindbis virus (SB) suggested that a single point mutation in glycoprotein E2 (serine 114 to arginine 114) conferred three phenotypic alterations: attenuation in neonatal mice, accelerated penetration of cultured cells, and efficient neutralization by two E2-specific monoclonal antibodies (Davis, Fuller, Dougherty, Olmsted, and Johnston (1986) Proc. Natl. Acad. Sci. USA 83, 6771-6775). Moreover, selection for rapidly penetrating mutants of SB coselected for attenuation in vivo, indicating that a domain of SB E2 which influences penetration in culture overlaps an E2 domain which influences pathogenesis (Olmsted, Meyer, and Johnston (1986) Virology 148, 245-254). To test the possibility that overlapping penetration and pathogenesis domains exist in other alphaviruses, the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus (TRD-VEE) was serially passed in baby hamster kidney (BHK) cells under a stringent selective pressure for accelerated penetration. Isolates were biologically cloned from the first through the fourth passages and were characterized as to penetration time course in BHK cells and virulence in adult mice following intraperitoneal inoculation. Twenty-two of the 27 isolates segregated into two major categories: slowly penetrating and virulent (like the TRD-VEE parent) and rapidly penetrating and avirulent. Mice which received the avirulent mutants were positive for anti-VEE neutralizing antibody and were refractory to challenge with TRD-VEE. Of the seven mouse avirulent mutants, two also were attenuated in hamsters, indicating the presence of at least two genetic loci at which mutations may influence both pathogenesis and penetration.     

Biochemical and biological characteristics of epitopes on the E1 glycoprotein of western equine encephalitis virus

Antigenic determinants identified by monoclonal antibodies (Mabs) on the E1 glycoprotein of western equine encephalitis (WEE) virus have been characterized by their serological activity, requirements for secondary structure, expression on the mature virion, and their role in protecting animals from WEE virus challenge. On the basis of a cross-reactivity enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition assay, eight antigenic determinants (epitopes) on the E1 glycoprotein have been identified, ranging in reactivity from WEE-specific to alphavirus group reactive. No neutralization of virus infectivity was demonstrable with any of the Mabs. An alphavirus group-reactive hemagglutination (HA) site, a WEE complex-reactive HA site, and a WEE virus-specific HA site were identified. Spatial arrangement of these epitopes was determined by a competitive binding ELISA. Four competition groups defining three distinct antigenic domains were identified. Antibodies directed against four E1 epitopes were capable of precipitating the E1/E2 heterodimer from infected cells or purified virus disrupted with nonionic detergents. These same antibodies precipitated only E1 in the presence of 0.1% SDS. That E1 conformation was important was shown by the inability of antibodies specific for seven of the epitopes to bind to virus denatured in 0.5% SDS. As determined by equilibrium gradient analysis of virus-antibody mixtures, four epitopes were found to be fully accessible on the mature virion, three epitopes were inaccessible, and one epitope was partially accessible to antibody binding. Antibodies specific for three epitopes were able to passively protect mice from WEE virus challenge.     

IRES-dependent replication of Venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells

The development of infectious cDNA for different alphaviruses opened an opportunity to explore their attenuation by extensively modifying the viral genomes, an approach that might minimize or exclude the reversion to the wild-type, pathogenic phenotype. Moreover, the genomes of such alphaviruses can be engineered to contain RNA elements that would be functional only in cells of vertebrate, but not insect, origin. In the present study, we developed a recombinant VEEV that is more attenuated than TC-83 and capable of replicating only in vertebrate cells. This phenotype was achieved by rendering the translation of the viral structural proteins, and ultimately viral replication, dependent on the internal ribosome entry site of encephalomyocarditis virus (EMCV IRES). This recombinant virus was viable, but required additional, adaptive mutations in nsP2 that strongly increased its replication rates. In spite of efficient replication in cultured vertebrate cells, the genetically modified VEEV demonstrated a highly attenuated phenotype in newborn mice, and yet induced protective immunity against VEEV infection.