Venezuelan equine encephalitis: state-of-the-art

The paper provides the currently available data on the global prevalence of Venezuelan equine encephalitis (VEE), its epidemiology, clinical picture, and specific prevention in human beings. It also discussed the problem of potential use of the causative agent of VEE as a subject of bioterrorism.     

Venezuelan equine encephalitis. Review

The Venezuelan equine encephalomyelitis (VEE) is one the most serious viral infections of the nervous system. It has a wide geographic distribution and may give rise to sequela like mental retardation, amnesia, abortion, epilepsy and hidroanencephaly in infected humans and animals. The pathology of this infection is focused mainly in two tissues: lymphohematopoietic and nervous. The VEE virus has a special cytopathic activity on the nervous cells (glia and neurons) while the lesions produced in the myelin are probably a consequence of the immunological response of the host to the infection. The alterations produced by the VEE virus in different neuronal types can originate changes in the brain concentrations of several neurotransmitters and their receptors. Some biochemical modifications that have also been reported could be due to the cytopathic effect of the virus.     

Spread of Venezuelan equine encephalitis virus in mice olfactory tract

Summary Spread of Venezuelan equine encephalitis (VEE) virus and damage of the central nervous system (CNS) in mice infected by respiratory route was studied. Virus concentration in organs and blood, dose-effect relationships, and ultrastructural lesions in various tissues were examined in immune and normal mice. We showed, via three independent methods — characteristic curve investigations, tissue virus concentration dynamics, and ultrastructural methods — the spread of VEE virus through the olfactory tract into the brain of immune mice. From these experiments it was concluded that in case of respiratory challenge VEE virus can enter the CNS of normal mice by both vascular and olfactory pathways, while in immune mice the main route is olfactory.     

Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge

An anti-poxvirus vaccine based on replicon particles of Venezuelan equine encephalitis virus (VRP) is being developed. The cowpox virus genes encoding structural proteins corresponding to vaccinia virus proteins A33, B5, and A27 were each expressed from VRP. High serum IgG titers against these proteins were generated in BALB/c mice vaccinated with each of these VRP. VRP induced both IgG1 and IgG2a with a strong predominance of IgG2a production. The response is long-lasting, as evidenced by the retention of high anti-B5 serum IgG titers through at least 50 weeks after priming immunization. Mice vaccinated with B5-, A33- or A27-VRP individually or together survived intranasal challenge with cowpox virus, with the multivalent vaccine formulation providing more effective protection from weight loss and clinical signs of illness than the monovalent vaccines. These results demonstrate that VRP may provide an effective alternative to vaccinia virus vaccines against poxvirus infection.     

Virion envelope glycoproteins as epidermiological markers of Venezuelan encephalitis virus isolates.

Virion polypeptide compositions of 26 isolates of Venezuelan encephalitis virus were analyzed by a reproducible and comparative technique of discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Although the molecular weight of the core polypeptide for each isolate was 36,000, numbers and molecular weights of envelope glycoproteins were heterogeneous. Isolates associated with human, but not equine, disease usually had two glycoproteins of 50,000 to 51,000 and 51,000 to 55,000 molecular weight, whereas isolates associated with both human and equine disease usually had an additional, third polypeptide band of either 45,000 to 46,000 or 56,000 to 58,000 molecular weight. The former isolates were in hemagglutination inhibition subtypes I-D, I-E, III, or IV, and the latter were in subtypes I-A, I-B, I-C, or II. Thus virion envelope glycoproteins should be useful markers of Venezuelan encephalitis virus isolates in epidemiological investigations.     

Monoclonal antibodies cross-reacting with the tick-borne encephalitis virus and with the Venezuelan equine encephalomyelitis virus]

Employment of radioimmunoassay led to the demonstration of serological crossing between tick-borne encephalitis (TBE) virus and Venezuelan equine encephalomyelitis (VEE) virus. Using hybridoma technology, three hybridomas were produced secreting monoclonal antibodies (MAb) cross-reacting with these two viruses. With MAb, the epitope of binding of these antibodies was shown to be located on protein E of TBE virus and protein E1 of VEE virus. Despite the low percentage (14%) of homology of amino acid sequences of these proteins, 12 areas with homology from 24% to 63% were demonstrated. Considering conservative replacements, homology of these areas was 53%-75%. The assumed existence of some of these areas in alpha-helical conformation may explain the observed immunological crossing.     

Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.     

Human Venezuelan equine encephalitis virus infection and diabetes in Zulia State, Venezuela

Venezuelan equine encephalitis (VEE) virus has been implicated as producing alterations in glucose metabolism in animals. We performed oral glucose tolerance tests and measured serum immunoreactive insulin responses in 13 patients who were infected by VEE virus during an epidemic in 1969, in Zulia State, Venezuela. No significant alterations in the glucose tolerance test were found. Sera of 86 diabetic outpatients and 98 control individuals with normal glycemia at a local hospital were tested for antibodies to VEE virus by hemagglutination inhibition. No statistically significant difference was found between the two groups; 10.4% of the diabetic patients had detectable antibodies against VEE virus, compared to 7.1% of controls. Seventy-three percent of the diabetics with antibodies were individuals over 40 yr old, whose diabetes could be catalogued as insulin independent. The results of these studies indicate no relationship of VEE virus infection to subsequent diabetes.     

Biochemical and antigenic comparison of the envelope glycoproteins of Venezuelan equine encephalomyelitis virus strains.

Pulse-chase experiments after synchronous initiation of translation indicate that the larger Venzuelan equine encephalomyelitis (VEE) virus membrane glycoprotein E2, is derived by proteolytic cleavage of the precursor, PE2. The structural proteins of VEE virus strains representing each of the antigenic subtypes and varieties have been compared by discontinuous SDS-polyacrylamide gel electrophoresis. Nucleocapsid proteins of all isolates were similar in size (mol. wt. 35 to 36 X 10(3). The mol. wt. of E1 varied from 48 to 51 X 10(3) and the mol. wt. of E2 glycoproteins ranged from 53 to 59 X 10(3). Pixuna virus contained a third envelope glycoprotein of 59 X 10(3) mol. wt. in addition to the two major glycoproteins of mol. wt. 53 X 10(3) and 48 X 10(3) respectively. The isoelectric points (pI) of E1 and E2 for all VEE strains studied were approx. 7 and 9 respectively. Both glycoproteins of TC-83 virus induced precipitating antibodies which reacted only with the homologous purified E1 and E2 glycoproteins. Antibodies to E2 protein of each virus neutralized virus infectivity and inhibited the agglutination of goose erythrocytes by virions. Haemagglutination-inhibition tests using antisera to E2 glycoproteins of prototype viruses, representing each of the antigenic subtypes and varieties, differentiated the viruses into subtypes I, II, III and IV with subtype I divided into variants 1AB, 1C, 1D and 1E.     

Placental and fetal alterations due to Venezuelan equine encephalitis virus in rats.

Histopathological changes in the placentas, embryos, and fetuses of rats inoculated intraperitoneally with the virulent Guajira strain of Venezuelan equine encephalitis virus were studied by light microscopy and immunoperoxidase methods. Rats inoculated before day 15 of pregnancy showed necrosis and hemorrhages in the embryonic disks. Swelling of cytoplasm and nuclear pyknosis of cyto- and syncytotrophoblastic cells were noted as early as 2 days after inoculation. During weeks 1 and 2 of pregnancy, death of the embryos was always observed 3 to 4 days after Venezuelan equine encephalitis virus inoculation. Placental and fetal damage varied among the specimens. In rats 18 days pregnant and sacrificed 2 days after inoculation, there were some viable fetuses; the placentas showed inflammatory reactions in the mesometrial and decidual vessels. Other rats sacrificed at 3 to 4 days after inoculation showed large placental infarcts with fetal death. Viremia peaked during day 2 after inoculation. Immunoperoxidase stains demonstrated viral antigens present in the decidua, myometrium, and cyto- and syncytotrophoblastic cells. These experiments provide additional data regarding the pathogenesis and structural damage in the placental and fetal tissues caused by Venezuelan equine encephalitis virus.     

Laboratory studies of Venezuelan equine encephalitis virus in equines, Texas, 1971.

During the summer and fall of 1971, epizootic and epidemic Venezuelan equine encephalitis was detected in Texas. Isolates of epizootic (IB) and vaccine (TC-83) strains were distinguished by virulence of the former for guinea pigs. Vaccine virus was isolated from 1 to 14 days after vaccination and neutralization tests demonstrated the appearance of antibody about a week after vaccination. Viremia titers of subtype IB in horses ranged from 2.2 to 8.3 log10 suckling mouse intracranial 50% lethal doses per ml. Of 101 equines from which Venezuelan equine encephalitis virus (IB or TC-83) strains were isolated, 87 had no neutralizing antibody against Venezuelan, eastern or western equine encephalitis viruses.     

Development of a functional monoclonal single-chain variable fragment antibody against Venezuelan equine encephalitis virus

We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.