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 humanpathogenic 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.Venezuelan equine encephalitis virus (VEEV) is a member of the Alphavirus genus in the Togaviridae family. VEEV is an enveloped virus with a nonsegmented, positive-sense RNA genome of approximately 11.5 kb. The 5Ј two-thirds of the genome encodes four nonstructural proteins (nsP1 to nsP4) that form an enzyme complex required for viral replication (45). After viral RNA entry into the cytoplasm, a nonstructural polyprotein is translated directly from the viral genome and utilized in the production of a full-length, negative-sense replicative RNA intermediate. This RNA is then used as a template for synthesis of positive-sense genomic RNA and for transcription of a subgenomic 26S RNA. The ca. 4-kb subgenomic RNA corresponds to the 3Ј one-third of the viral genome and is translated into a structural polyprotein that is proteolytically cleaved into the capsid and envelope glycoproteins E2 and E1 (39). Two hundred forty copies of the capsid protein combine with the genomic viral RNA to form an icosahedral nucleocapsid. Finally, the nucleocapsid buds from the plasma membrane to acquire a lipid envelope with embedded protein spikes containing E1-E2 heterodimers (42,45).VEEV was a significant human and equine pathogen for much of the past century, and recent epidemics (40, 50) indicate that VEEV still represents a serious public health threat. Furthermore, ...

RNA viruses are notorious for their genetic plasticity and propensity to exploit new host-range opportunities, which can lead to the emergence of human disease epidemics such as severe acute respiratory syndrome, AIDS, dengue, and influenza. However, the mechanisms of host-range change involved in most of these viral emergences, particularly the genetic mechanisms of adaptation to new hosts, remain poorly understood. We studied the emergence of Venezuelan equine encephalitis virus (VEEV), an alphavirus pathogen of people and equines that has had severe health and economic effects in the Americas since the early 20th century. Between epidemics, VEE disappears for periods up to decades, and the viral source of outbreaks has remained enigmatic. Combined with phylogenetic analyses to predict mutations associated with a 1992-1993 epidemic, we used reverse genetic studies to identify an envelope glycoprotein gene mutation that mediated emergence. This mutation allowed an enzootic, equine-avirulent VEEV strain, which circulates among rodents in nearby forests to adapt for equine amplification. RNA viruses including alphaviruses exhibit high mutation frequencies. Therefore, ecological and epidemiological factors probably constrain the frequency of VEE epidemics more than the generation, via mutation, of amplification-competent (high equine viremia) virus strains. These results underscore the ability of RNA viruses to alter their host range, virulence, and epidemic potential via minor genetic changes. VEE also demonstrates the unpredictable risks to human health of anthropogenic changes such as the introduction of equines and humans into habitats that harbor zoonotic RNA viruses.alphavirus ͉ arbovirus ͉ equine ͉ evolution

Mosquito-borne alphaviruses, which replicate alternately and obligately in mosquitoes and vertebrates, appear to experience lower rates of evolution than do many RNA viruses that replicate solely in vertebrates. This genetic stability is hypothesized to result from the alternating host cycle, which constrains evolution by imposing compromise fitness solutions in each host. To test this hypothesis, Sindbis virus was passaged serially, either in one cell type to eliminate host alteration or alternately between vertebrate (BHK) and mosquito (C6/36) cells. Following 20 to 50 serial passages, mutations were identified and changes in fitness were assessed using competition assays against genetically marked, surrogate parent viruses. Specialized viruses passaged in a single cell exhibited more mutations and amino acid changes per passage than those passaged alternately. Single host-adapted viruses exhibited fitness gains in the cells in which they specialized but fitness losses in the bypassed cell type. Most but not all viruses passaged alternately experienced lesser fitness gains than specialized viruses, with fewer mutations per passage. Clonal populations derived from alternately passaged viruses also exhibited adaptation to both cell lines, indicating that polymorphic populations are not required for simultaneous fitness gains in vertebrate and mosquito cells. Nearly all passaged viruses acquired Arg or Lys substitutions in the E2 envelope glycoprotein, but enhanced binding was only detected for BHK cells. These results support the hypothesis that arbovirus evolution may be constrained by alternating host transmission cycles, but they indicate a surprising ability for simultaneous adaptation to highly divergent cell types by combinations of mutations in single genomes.Sindbis virus (SINV) is the type species of the genus Alphavirus, a group of RNA viruses with nonsegmented singlestranded genomes of approximately 11.7 kb (23, 25). The alphavirus genome is capped at the 5Ј end and contains a 3Ј poly(A) tail. The 5Ј two-thirds of the genome encode the nonstructural proteins 1 to 4, which are necessary for viral replication. The structural proteins, capsid and E1 and E2 envelope glycoproteins, are translated from a subgenomic mRNA (26S) which is identical in sequence to the 3Ј one-third of the genome (Fig. 1). The Sindbis virion contains an icosahedral nucleocapsid that consists of 240 copies of the capsid protein surrounded by a lipid envelope derived from the plasma membrane of infected cells, into which the glycoprotein E1/E2 heterodimers are embedded (4).Nearly all alphaviruses rely on horizontal mosquito-borne transmission among vertebrate hosts, requiring alternating replication in highly divergent hosts and cell types (7,34). Alphaviruses and other arthropod-borne viruses also appear to undergo lower rates of evolution than many other animal RNA viruses that replicate solely in vertebrates (6,33,35). Rates of SINV nucleotide substitution, deduced from oligonucleotide fingerprinting, are approximately 4 ϫ 10 Ϫ4 ...

Since Venezuelan equine encephalitis virus (VEEV) was isolated in Peru in 1942, >70 isolates have been obtained from mosquitoes, humans, and sylvatic mammals primarily in the Amazon region. To investigate genetic relationships among the Peru VEEV isolates and between the Peru isolates and other VEEV strains, a fragment of the PE2 gene was amplified and analyzed by single-stranded conformation polymorphism. Representatives of seven genotypes underwent sequencing and phylogenetic analysis. The results identified four VEE complex lineages that cocirculate in the Amazon region: subtypes ID (Panama and Colombia/Venezuela genotypes), IIIC, and a new, proposed subtype IIID, which was isolated from a febrile human, mosquitoes, and spiny rats. Both ID lineages and the IIID subtype are associated with febrile human illness. Most of the subtype ID isolates belonged to the Panama genotype, but the Colombia/Venezuela genotype, which is phylogenetically related to epizootic strains, also continues to circulate in the Amazon basin.

Significance Mosquito-borne alphaviruses are important causes of epidemic encephalomyelitis. The immune response plays an important role in disease; however, immune-mediated mechanisms of pathogenesis and regulation are not understood. In this study, we determined that a pathogenic Th17 response occurs during fatal alphavirus encephalitis. Furthermore, the regulatory cytokine, interleukin 10, plays an important role in modulating the pathogenic Th17 response. In the absence of interleukin 10, the Th17 response is increased in magnitude and displays a more pathogenic phenotype, resulting in accelerated disease progression. These findings are important for understanding the pathogenesis of virus infections in the central nervous system and the identification of therapeutic interventions that focus on immune modulation in the central nervous system.