Interplay of acute and persistent infections caused by Venezuelan equine encephalitis virus encoding mutated capsid protein.

Venezuelan equine encephalitis virus (VEEV) is a significant human and animal pathogen. The highlight of VEEV replication in vitro, in cells of vertebrate origin, is the rapid development of cytopathic effect (CPE), which is strongly dependent upon the expression of viral capsid protein. Besides being an integral part of virions, the latter protein is capable of (i) binding both the nuclear import and nuclear export receptors, (ii) accumulating in the nuclear pore complexes, (iii) inhibiting nucleocytoplasmic trafficking, and (iv) inhibiting transcription of cellular ribosomal and messenger RNAs. Using our knowledge of the mechanism of VEEV capsid protein function in these processes, we designed VEEV variants containing combinations of mutations in the capsid-coding sequences. These mutations made VEEV dramatically less cytopathic but had no effect on infectious virus production. In cell lines that have defects in type I interferon (IFN) signaling, the capsid mutants demonstrated very efficient persistent replication. In other cells, which have no defects in IFN production or signaling, the same mutants were capable of inducing a long-term antiviral state, downregulating virus replication to an almost undetectable level. However, ultimately, these cells also developed a persistent infection, characterized by continuous virus replication and beta IFN (IFN-beta) release. The results of this study demonstrate that the long-term cellular antiviral state is determined by the synergistic effects of type I IFN signaling and the antiviral reaction induced by replicating viral RNA and/or the expression of VEEV-specific proteins. The designed mutants represent an important model for studying the mechanisms of cell interference with VEEV replication and development of persistent infection.

Pro-inflammatory response resulting from sindbis virus infection of human macrophages: implications for the pathogenesis of viral arthritis.

Several viruses cause acute and chronic joint inflammation in humans, and among them, the alphaviruses are of special interest due to the increasing number of outbreaks in which they are the etiological factor. Sindbis virus (SinV), a member of the Alphavirus genus, is the most widely distributed of all known arboviruses. Although SinV causes arthritis in humans, the molecular and cellular factors that contribute to the pathogenesis of this disease are almost completely unknown. Despite the crucial role of macrophages in the development of arthritis, these cells have not been recognized as potential targets for viruses causing arthritis. In this study, replication of SinV in human macrophages was demonstrated. The infection promoted macrophage activation, leading to the release of macrophage migration inhibitor factor (MIF) from intracellular stores and inducing the expression and secretion of TNF-alpha, IL-1beta, and IL-6. Production of these cytokines was followed by the expression of matrix metalloproteinases (MMPs) 1 and 3, which could be involved in the articular damage that has been observed in disease induced by SinV. The use of different strategies to block MIF action, including an anti-MIF antibody, the MIF inhibitor ISO-1 and knockout mice for the MIF gene, showed that cytokine secretion and MMP expression during infection were regulated by MIF, suggesting that this cytokine acts in autocrine and paracrine manner upstream in the macrophage activation cascade. Thus, these are remarkable similarities between macrophage responses induced by SinV infection and those observed in rheumatoid arthritis, despite the different etiologies of infectious and autoimmune arthritides.

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic.

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.

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.