The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants. (9/310)

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.  (+info)

A single-site mutant and revertants arising in vivo define early steps in the pathogenesis of Venezuelan equine encephalitis virus. (10/310)

The early stages of Venezuelan equine encephalitis virus (VEE) pathogenesis in the mouse model have been examined using a genetic approach. Disease progression of a molecularly cloned single-site mutant was compared with that of the parental virus to determine the step in the VEE pathogenetic sequence at which the mutant was blocked. Assuming that such a block constitutes a genetic screen, isolates from different tissues thought to be distal to the block in the VEE pathogenetic sequence were analyzed to determine the pathogenetic step at which revertants of the mutant were selected. Directed mutation and analysis of reversion in vivo provide two powerful genetic tools for the dissection of the wild-type VEE pathogenetic sequence. Virus from the parental virulent clone, V3000, first replicated in the draining lymph node after subcutaneous inoculation in the left rear footpad. Movement of a cloned avirulent mutant, V3010 (E2 76 Glu to Lys), to the draining lymph node was impaired, replication in the node was delayed, and spread beyond the draining lymph node was sporadic. Serum, contralateral lymph node, spleen, and brain isolates from V3010 inoculated animals were invariably revertant with respect to sequence at E2 76 and/or virulence in mice. Revertants isolated from serum and contralateral lymph node retained the V3010 E2 Lys 76 mutation but also contained a second-site mutation, Glu to Lys at E2 116. Modification of the V3010 clone by addition of the second-site mutation at E2 116 produced a virus that bypassed the V3010 block at the draining lymph node but that did not possess full wild-type capacity for replication in the central nervous system or for induction of mortality. A control construct containing only the E2 116 reverting mutation on the V3000 background was identical to V3000 in terms of early pathogenetic steps and virulence. Therefore, analysis of mutant replication and reversion in vivo suggested (1) that the earliest steps in VEE pathogenesis are transit to the draining lymph node and replication at that site, (2) that the mutation in V3010 impairs transit to the draining lymph node and blocks dissemination to other tissues, and (3) that reversion can overcome the block without restoring full virulence.  (+info)

Pegylated alpha interferon is an effective treatment for virulent venezuelan equine encephalitis virus and has profound effects on the host immune response to infection. (11/310)

Venezuelan equine encephalitis virus (VEEV) is a highly infectious alphavirus endemic in parts of Central and South America. The disease is transmitted by mosquitoes, and the natural reservoir is the small rodent population, with epidemics occurring in horses and occasionally humans. Following infection, VEEV replicates in lymphoid tissues prior to invasion of the central nervous system. Treatment of VEEV-infected BALB/c mice with polyethylene glycol-conjugated alpha interferon (PEG IFN-alpha) results in a greatly enhanced survival from either a subcutaneous or an aerosol infection. Virus is undetectable within PEG IFN-alpha-treated individuals by day 30 postinfection (p.i.). Treatment results in a number of changes to the immune response characteristics normally associated with VEEV infection. Increased macrophage activation occurs in PEG IFN-alpha-treated BALB/c mice infected with VEEV. The rapid activation of splenic CD4, CD8, and B cells by day 2 p.i. normally associated with VEEV infection is absent in PEG IFN-alpha-treated mice. The high tumor necrosis factor alpha production by macrophages from untreated mice is greatly diminished in PEG IFN-alpha-treated mice. These results suggest key immunological mechanisms targeted by this lethal alphavirus that can be modulated by prolonged exposure to IFN-alpha.  (+info)

Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice. (12/310)

The arbovirus, Venezuelan equine encephalitis virus (VEE), causes disease in humans and equines during periodic outbreaks. A murine model, which closely mimics the encephalitic form of the disease, was used to study mechanisms of attenuation. Molecularly cloned VEE viruses were used: a virulent, epizootic, parental virus and eight site-specific glycoprotein mutants derived from the parental virus. Four of these mutants were selected in vitro for rapid binding and penetration, resulting in positive charge changes in the E2 glycoprotein from glutamic acid or threonine to lysine (N. L. Davis, N. Powell, G. F. Greenwald, L. V. Willis, B. J. Johnson, J. F. Smith, and R. E. Johnston, Virology 183, 20-31, 1991). Tissue culture adaptation also selected for the ability to bind heparan sulfate as evidenced by inhibition of plaque formation by heparin, decreased infectivity for CHO cells deficient for heparan sulfate, and tight binding to heparin-agarose beads. In contrast, the parental virus and three other mutants did not use heparan sulfate as a receptor. All eight mutants were partially or completely attenuated with respect to mortality in adult mice after a subcutaneous inoculation, and the five mutants that interacted with heparan sulfate in vitro had low morbidity (0-50%). These same five mutants were cleared rapidly from the blood after an intravenous inoculation. In contrast, the parental virus and the other three mutants were cleared very slowly. In summary, the five VEE viruses that contain tissue-culture-selected mutations interacted with cell surface heparan sulfate, and this interaction correlated with low morbidity and rapid clearance from the blood. We propose that one mechanism of attenuation is rapid viral clearance in vivo due to binding of the virus to ubiquitous heparan sulfate.  (+info)

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles. (13/310)

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.  (+info)

Extension of the mean time to death of mice with a lethal infection of Venezuelan equine encephalomyelitis virus by antithymocyte serum treatment. (14/310)

The mean time to death of mice infected with Venezuelan equine encephalomyelitis (VEE) virus was increased 2 days by antithymocyte serum (ATS) treatment given 1 day before and 1 day after virus inoculation. Virus assays of blood, brain, and spleen indicated that VEE virus replication was delayed by ATS. Additionally, mice treated with ATS exhibited neurological signs later than untreated mice. During the infection, the percentage of splenic B lymphocytes as determined by surface immunoglobulin staining increased. ATS treatment caused a further elevation of the percentage of splenic B lymphocytes. These results show a selective depletion of the non-immunoglobulin-bearing lymphocyte population during VEE virus infection and support the hypothesis that ATS destroys or alters an important population of cells associated with the normal course of pathogenesis and the replication of VEE virus to high titers in the mouse.  (+info)

Adjuvant effects of low doses of a nuclease-resistant derivative of polyinosinic acid . polycytidylic acid on antibody responses of monkeys to inactivated Venezuelan equine encephalomyelitis virus vaccine. (15/310)

Polyriboinosinic.polyribocytidylic acid [poly(I).poly(C)] stabilized with poly-l-lysine and carboxymethylcellulose [poly(ICLC)] has been previously shown to be a compound with marked adjuvant activity when given in high doses with inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccine. This study investigated the effects of much lower doses of poly(ICLC) on the magnitude and kinetics of the primary and secondary humoral antibody responses of rhesus monkeys to inactivated VEE virus vaccine. Monkeys given a single injection of vaccine developed very low neutralizing antibody titers, whereas those given adjuvant plus vaccine had 30- to 100-fold-higher titers which remained elevated for longer than 6 months. Low doses of poly(ICLC) given with VEE virus vaccine resulted in a profound but transient increase in priming of secondary antibody responses to the antigen. In contrast, the administration of poly-l-lysine and carboxymethylcellulose alone without the poly(I).poly(C) component of the complex had no adjuvant effect on antibody responses of monkeys to VEE virus vaccine. The temporal development of antibody by class (immunoglobulin M-immunoglobulin G) in monkeys given two injections of adjuvant-vaccine was not different from that with vaccine alone. Serial hematological and clinical chemistry determinations on monkeys given single or multiple doses of poly(ICLC) with vaccine were not different from values in monkeys given vaccine alone.  (+info)

Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon system protects chickens against lethal infection with Hong Kong-origin H5N1 viruses. (16/310)

Venezuelan equine encephalitis virus replicon particles (VRP) containing the gene expressing hemagglutinin (HA) from the human Hong Kong Influenza A isolate (A/HK/156/97) were evaluated as vaccines in chicken embryos and young chicks. Expressed HA was readily detected in bird-tissue staining with anti-H5 HA antibody and in chicken cells infected with the replicon preparations following immunoprecipitation with monoclonal antibody. Birds challenged with a dose of the lethal parent virus were protected to different extents depending on the age of the bird. In ovo and 1-day-old inoculated animals that received no boost with the VRP were partially protected; birds 2 weeks of age were completely protected with a single dose of VRP.  (+info)