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Alphavirus-based replicons are a promising nucleic acid vaccine platform characterized by robust gene expression and immune responses. To further explore their use in vaccination, replicons were engineered to allow conditional control over their gene expression. Riboswitches, comprising a ribozyme actuator and RNA aptamer sensor, were engineered into the replicon 3′ UTR. Binding of ligand to aptamer...
The immunogenicity of alphavirus replicon vaccines is determined by many factors including the level of antigen expression and induction of innate immune responses. Characterized attenuated alphavirus mutants contain changes to the genomic 5′ UTR and mutations that result in altered non-structural protein cleavage timing leading to altered levels of antigen expression and interferon (IFN) induction...
We previously described a single-cycle dengue vaccine (RepliVAX D2) engineered from a capsid (C) gene-deleted West Nile virus (WNV) expressing dengue virus serotype 2 (DENV2) prM/E genes in place of the corresponding WNV genes. That work demonstrated that adaptation of RepliVAX D2 to grow in WNV C-expressing cells resulted in acquisition of non-synonymous mutations in the DENV2 prM/E and WNV NS2A/NS3...
The lifecycle of intracellular pathogens, especially viruses, is intimately tied to the macromolecular synthetic processes of their host cell. In the case of positive-stranded RNA viruses, the ability to translate and, thus, replicate their infecting genome is dependent upon hijacking host proteins. To identify proteins that participate in West Nile virus (WNV) replication, we tested the ability of...
West Nile virus (WNV) infections in vertebrates are generally acute but persistent infections have been observed. To investigate the ability of WNV to produce persistent infections, we forced subgenomic WNV replicons to replicate within a cell without causing cell death. Detailed analyses of these cell-adapted genomes revealed mutations within the nonstructural protein genes NS2A (D73H, M108K), NS3...
To help understand the mechanism of pathogenesis of dengue virus (DV), we set out to create an infectious cDNA of the Brazilian prototype strain of DV serotype 1 (DV1-BR/90). PCR-amplified fragments of DV1-BR/90 cDNA were readily assembled into a subgenomic cDNA that could be used to produce replicating RNAs (replicons), lacking the structural protein-encoding regions of the genome. However, assembly...
Dengue viruses (DENV) are the most important arboviral pathogens in tropical and subtropical regions throughout the world. DENV transmission includes both a sylvatic, enzootic cycle between nonhuman primates and arboreal mosquitoes of the genus Aedes, and an urban, endemic/epidemic cycle between Aedes aegypti, a mosquito with larval development in peridomestic water containers, and human reservoir...
To develop new vaccine candidates for flavivirus infections, we have engineered two flaviviruses, yellow fever virus (YFV) and West Nile virus (WNV), that are deficient in replication. These defective pseudoinfectious viruses (PIVs) lack a functional copy of the capsid (C) gene in their genomes and are incapable of causing spreading infection upon infection of cells both in vivo and in vitro. However,...
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...
West Nile virus (WNV), the leading cause of viral encephalitis in the United States, is an arthropod-transmitted member of the family Flaviviridae. We have explored the interaction of this positive-strand RNA virus with signaling pathways involved in induction of the host's innate immune response. Phosphorylation of STAT-1 in response to interferon (IFN) treatment and the ability of IFN to establish...
Flaviviruses are emerging threats to public health worldwide. Recently, one flavivirus, West Nile virus (WNV), has caused the largest epidemic of viral encephalitis in US history. Like other flaviviruses, WNV is thought to cause a persistent infection in insect cells, but an acute cytopathic infection of mammalian cells. To study adaptation of WNV to persistently replicate in cell culture and generate...
Picornavirus infection induces the proliferation and rearrangement of intracellular membranes in response to the synthesis of nonstructural proteins, including 3A. We have previously shown that changes in 3A are associated with the inability of a Taiwanese strain of foot-and-mouth disease virus (FMDV) (OTai) to grow in bovine cells and cause disease in cattle, although the virus grows to high titers...
Swine are an important amplifier of Japanese encephalitis (JE) virus in the paradomestic environment. In this study, two JE DNA vaccine candidates were evaluated for immunogenicity in swine. Both vaccine plasmids encode a cassette consisting of the signal of premembrane (prM), prM, and envelope (E) coding regions of JE virus. One plasmid, designated pcJEME, is based on a commercial vector (pcDNA3),...
Recombinant Japanese encephalitis (JE) vaccine candidates based on a highly attenuated vaccinia virus (NYVAC-JEV) and a canarypox virus (ALVAC-JEV) were evaluated for their ability to induce specific antibodies and cytotoxic T lymphocytes (CTLs) in mice. Six- to eight-week-old male Balb/c mice that received one or two intraperitoneal inoculations with these JE vaccine candidates at a dose of 1 × 10...
To identify the active-site residues of the 3C proteinase of foot-and-mouth disease virus (FMDV), we introduced mutations into the 3C coding region and examined the activity of mutant enzymes on various substrates. Based on alignment of FMDV 3C with other picornavirus 3C proteinases and with the trypsin family of serine proteinases, mutations were introduced at residues presumed to be part of the...
A series of double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding the immunogenic proteins of Japanese encephalitis virus (JEV) [prM-E, prM-E-NS1, NS1-NS2A, 80%E (encodes the amino-terminal 80% part of E), and NS1] were constructed and analyzed for their ability to confer protective immunity in mice against lethal challenge with neurovirulent JEV. The cassettes were...
Double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding prM-E or a C-terminally truncated form of E of Japanese encephalitis virus (JEV) were constructed. The products were efficiently expressed in both mammalian and mosquito cell lines infected with the dsSIN recombinants. However, suppression of prM-E secretion from mammalian cells infected with dsSIN-prM-E recombinants...
We have previously reported that foot-and-mouth disease virus (FMDV) can enter an Fc receptor (FcR)-expressing cell line by antibody-dependent enhancement. Since FMDV can establish a persistent infection in animals in the presence of high levels of neutralizing antibodies (carrier state), we examined macrophages for their ability to be infected by the virus in the presence of antibody. The murine...
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