Alphaviruses are a group of significant human and animal pathogens. The nearly 30 members of this genus are transmitted by mosquitoes to higher vertebrates that serve as amplifying hosts (54). Alphaviruses cause different diseases but have similar replication strategies and life cycles. In insect vectors, they cause persistent lifelong infections and viral replication does not critically affect the viability of the hosts (51). These viruses also often establish persistent infection in cultured mosquito cells. In contrast, vertebrate hosts usually develop acute infection that often results in disease prior to virus clearance by the immune system (27, 31). The infection of susceptible vertebrate cells typically leads to rapid cytopathic effect (CPE) (54) and cell death.Sindbis virus (SIN) is the prototype member of the Alphavirus genus. It can replicate productively in a wide variety of cell lines of insect and vertebrate origin and causes age-dependent encephalomyelitis in mice (28). As do all other alphaviruses, SIN enters the cells via receptor-mediated endocytosis. pHdependent fusion of viral and endosomal membranes leads to release of nucleocapsids into the cytoplasm (12) followed by nucleocapsid disassembly and genome replication (64).The SIN genome is a single RNA molecule of positive polarity, 11.7 kb in length. It contains a 5Ј methylguanylate cap and a 3Ј polyadenylate tract and is translated by host-cell machinery similar to cellular mRNAs (52). The 5Ј two-thirds of the genome encodes the nonstructural proteins (nsPs), which comprise the viral components of the RNA replicase/transcriptase required for replication of the viral genome and transcription of subgenomic RNA. The 4.1-kb subgenomic RNA corresponds to the 3Ј third of the genome and is translated into structural proteins that form virus particles. Replication of SIN is very rapid and leads to high-level accumulation of virusspecific RNAs and structural proteins. Finally, a large number of viral particles released by budding from the cell surface disseminate the infection. Viral replication profoundly affects cell metabolism, with downregulation of host cell protein synthesis playing the central role (31). Cells lose integrity and die within 24 to 48 h postinfection (p.i.). In many cell types, death is accompanied by apoptotic changes (37). Replication of viral RNA and/or accumulation of SIN nsPs is sufficient to cause translational shutoff and cell death. However, expression of viral structural proteins significantly accelerates the development of CPE (18).In recent studies, we selected SIN self-replicating RNAs (replicons) that were capable of persisting in some vertebrate cell lines for an unlimited number of passages without causing CPE (1, 16). These noncytopathic replicons replicated less efficiently than the parent and contained point mutations in the gene encoding one of the nsPs, nsP2. One adaptive mutation, P 726 3L, was at the same position as that found in the SIN-1 variant that also exhibits reduced cytopathogenicity (11,63).

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, ...

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.Venezuelan equine encephalitis virus (VEEV) is an enveloped virus with a nonsegmented, positive-sense RNA genome of approximately 11.4 kb and belongs to the Alphavirus genus in the Togaviridae family. The 5Ј two-thirds of the genome contains four nonstructural proteins (nsP1 to nsP4) that form an enzyme complex required for viral replication (46-48). After release of the viral genome into the cytoplasm, a nonstructural polyprotein is translated directly from this RNA and utilized in the production of a full-length, negative-sense replicative RNA intermediate (45). The full-length RNA then serves as a template for the synthesis of positive-sense genomic RNA and for transcription of a subgenomic 26S RNA (46). The approximately 4-kb-long, subgenomic RNA corresponds to the 3Ј onethird of the viral genome and is translated into a structural polyprotein that is proteolytically cleaved into the capsid and the envelope glycoproteins E2 and E1 (34). Two hundred forty copies of the capsid protein enclose the genomic viral RNA to form an icosahedral nucleocapsid that buds from the plasma membrane, acquiring a lipid envelope with embedded protein spikes formed by E1/E2 heterodimers (41, 48).Venezuelan equine encephalitis virus is a zoonotic pathogen...

No abstract

Both the 5 end of the Sindbis virus (SIN) genome and its complement in the 3 end of the minus-strand RNA synthesized during virus replication serve as parts of the promoters recognized by the enzymes that comprise the replication complex (RdRp). In addition to the 5 untranslated region (UTR), which was shown to be critical for the initiation of replication, another 5 sequence element, the 51-nucleotide (nt) conserved sequence element (CSE), was postulated to be important for virus replication. It is located in the nsP1-encoding sequence and is highly conserved among all members of the Alphavirus genus. Studies with viruses containing clustered mutations in this sequence demonstrated that this RNA element is dispensable for SIN replication in cells of vertebrate origin, but its integrity can enhance the replication of SIN-specific RNAs. However, we showed that the same mutations had a deleterious effect on virus replication in mosquito cells. SIN with a mutated 51-nt CSE rapidly accumulated adaptive mutations in the nonstructural proteins nsP2 and nsP3 and the 5 UTR. These mutations functioned synergistically in a cell-specific manner and had a stimulatory effect only on the replication of viruses with a mutated 51-nt CSE. Taken together, the results suggest the complex nature of interactions between nsP2, nsP3, the 5 UTR, and host-specific protein factors binding to the 51-nt CSE and involved in RdRp formation. The data also demonstrate an outstanding potential of alphaviruses for adaptation. Within one passage, SIN can adapt to replication in cells of a vertebrate or invertebrate origin.The Alphavirus genus of the Togaviridae family contains nearly 30 known members, including a number of human and animal pathogens (reviewed in reference 12). The majority of alphaviruses are transmitted by mosquitoes to higher vertebrates that serve as amplifying hosts. In insect vectors, alphaviruses cause persistent life-long infections and do not greatly affect the viability of their hosts (3). Accordingly, they establish persistent infections in cultured mosquito cells (2). In vertebrates, alphaviruses usually cause acute infections that often result in disease (14), and the infection of susceptible cultured vertebrate cells leads to a progressive cytopathic effect (CPE) and cell death (8,20).Sindbis virus (SIN) is a prototype member of the Alphavirus genus, and it has always been a highly valuable source of information about the mechanisms of alphavirus replication and virus-host cell interactions (reviewed in reference 44). This virus can productively replicate in a wide variety of cell lines of insect and vertebrate origins. Like other alphaviruses, SIN enters cells via receptor-mediated endocytosis (4). The fusion of viral and endosomal membranes leads to the release of nucleocapsids into the cytoplasm (10), and after their ribosome-mediated disassembly, viral genomes become capable of translation and replication (42,49).The SIN genome is a single-stranded RNA of positive polarity that is almost 12 kb long (43). It is ca...