Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE-and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5 untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.

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...

We developed chimeric Sindbis (SINV)/Eastern equine encephalitis (EEEV) viruses and investigated their potential for use as live virus vaccines against EEEV. One vaccine candidate contained structural protein genes from a typical North American EEEV strain, while the other had structural proteins from a naturally attenuated Brazilian isolate. Both chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in mice. Vaccinated mice did not develop detectable disease or viremia, but developed high titers of neutralizing antibodies. Upon challenge with EEEV, mice vaccinated with >10 4 PFU of the chimeric viruses were completely protected from disease. These findings support the potential use of these SIN/EEEV chimeras as safe and effective vaccines.

A stable cell system for high-efficiency packaging of West Nile virus (WNV) subgenomic replicons into virus-like particles (VLPs) was developed. VLPs could be propagated on these packaging cells and produced infectious foci similar to foci produced by WNV. Focus size correlated with the replicative capacity of WNV replicons, indicating that genome copy number, rather than amount of trans-complementing structural proteins, was rate-limiting in packaging of VLPs. Comparison of VLP production from replicon genomes encoding partial or complete C genes indicated that portions of C downstream of the cyclization sequence could improve genome replication or that cis expression of C could enhance packaging. Interestingly, a rapid loss of replicon-encoded reporter gene activity was detected within two serial passages of reporter gene-containing VLPs. The loss of reporter activity correlated with gene deletion and better VLP growth, indicating a powerful selection pressure for WNV genomes lacking reporter genes.

During the last decade, alphaviruses became widely used for expression of heterologous genetic information and development of recombinant vaccines against a variety of human and animal pathogens. In this study, we compared a number of vectors based on the genome of Sindbis (SINV) and Venezuelan equine encephalitis (VEEV) viruses for their ability to express the Rift Valley fever virus (RVFV) envelope glycoprotein Gn and induce a protective immune response against RVFV infection. Our results suggest that (i) application of VEEV-based expression systems appears to be advantageous, when compared to similar systems designed on the basis of the SINV genome. (ii) Alphavirus-specific E3 and E2 proteins and furin-specific cleavage sites can be used for engineering secreted forms of the proteins. (iii) Alphaviruses can be modified for expression of the large fragments of heterologous proteins on the surface of chimeric, infectious viral particles. Thus, alphavirus-based expression systems may have the potential for a broader application beyond their current use as replicons or double-subgenomic vectors.

We established a system for propagation of Sindbis virus (SIN)-based replicons in tissue culture in the form of a tricomponent genome virus. Three RNA fragments containing complementing genetic information required for virus replication are packaged into separate viral particles, and each cell produces at least 1,000 packaged replicons and the number of packaged helpers sufficient to perform the next passage. This system can be used to generate large stocks of packaged replicons. The formation of infectious recombinant SIN virus was not detected in any experiments. These features make multicomponent genome SIN an attractive system for a variety of research and biotechnology applications.During the last decade, significant progress in understanding the mechanisms of replication and virus-host interactions has led to the wide use of viruses with RNA genomes for the delivery and expression of heterologous genes in vivo and in vitro (2,7,12,13,(18)(19)(20). Alphaviruses were among the first to be adapted for application as vector systems (16,28), and presently they are widely used in recombinant vaccine development and large-scale protein production (23,29).The cDNAs encoding infectious genomes have been developed for a number of alphaviruses, including Sindbis virus (SIN) (24), Semliki Forest virus (SFV) (17), Venezuelan equine encephalitis (VEE) (3), eastern equine encephalitis (EEE) (26), and others. Cloning of these genomes facilitated in vitro manipulations of viral genetic material and reverse genetics experiments. These studies strongly promoted the application of alphaviruses as gene delivery vehicles.The alphavirus genome consists of a single-stranded RNA of positive polarity that is about 12 kb in length (27). It is capped at the 5Ј end and contains a 3Ј-terminal poly(A) tail. The genome serves as a template for synthesis of viral nonstructural proteins (nsPs) forming the replicase-transcriptase complex (RdRp) required for synthesis of a genome-length, minusstrand intermediate that is a template for transcription of both genome-length, plus-strand RNA and the subgenomic 26S RNA. The latter RNA, encoded by the 3Ј one-third of the genome, accumulates in infected cells to high levels and is translated into viral structural proteins. The structural proteins are dispensable for viral RNA replication and transcription, and their coding sequence can be replaced by heterologous genes (11,28). The deletion of the structural genes makes these RNAs (replicons) unable to spread, but they can be packaged into viral particles by cotransfecting the cells with in vitro-synthesized replicon RNAs and one or two helper RNAs (1, 5, 11, 16). These helpers contain 5Ј and 3Ј cis-acting RNA elements that serve as replication promoters utilized by the viral replicative enzymes provided by replicons in trans and the subgenomic promoters that drive the expression of viral structural proteins. The required structural proteins can be expressed as a single polyprotein, or the capsid-and glycoproteins-coding genes can be separated betwe...

Replication of alphaviruses strongly depends on the promoters located in the plus- and minus-strands of virus-specific RNAs. The most sophisticated promoter is encoded by the 5' end of the viral genome. This RNA sequence is involved in the initiation of translation of viral nsPs, and synthesis of both minus- and plus-strands of the viral genome. Part of the promoter, the 51-nt conserved sequence element (CSE), is located in the nsP1-coding sequence, and this limits the spectrum of possible mutations that can be performed. We designed a recombinant Venezuelan equine encephalitis virus genome, in which the promoter and nsP1-coding sequences are separated. This modification has allowed us to perform a wide variety of genetic manipulations, without affecting the amino acid sequence of the nsPs, and to further investigate 51-nt CSE functioning. The results of this study suggest a direct interaction of the amino terminal domain of nsP2 with the 5' end of the viral genome.