Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus.

Most previous studies of interferon-alpha/beta (IFN-α/ß) response antagonism by alphaviruses have focused upon interruption of IFN-α/ß induction and/or receptor signaling cascades. Infection of mice with Venezuelan equine encephalitis alphavirus (VEEV) or Sindbis virus (SINV) induces serum IFN-α/ß, that elicits a systemic antiviral state in uninfected cells successfully controlling SINV but not VEEV replication. Furthermore, VEEV replication is more resistant than that of SINV to a pre-existing antiviral state in vitro. While host macromolecular shutoff is proposed as a major antagonist of IFN-α/ß induction, the underlying mechanisms of alphavirus resistance to a pre-existing antiviral state are not fully defined, nor is the mechanism for the greater resistance of VEEV. Here, we have separated viral transcription and translation shutoff with multiple alphaviruses, identified the viral proteins that induce each activity, and demonstrated that VEEV nonstructural protein 2-induced translation shutoff is likely a critical factor in enhanced antiviral state resistance of this alphavirus.

Role of angiotensin II in experimental Venezuelan equine encephalitis in rats.

Venezuelan equine encephalitis (VEE) is a viral disease transmitted by mosquitoes. The inflammation induced by the VEE virus is associated with a high mortality rate in mice. Angiotensin II (Ang II), a pro-inflammatory molecule, is produced in the normal rat brain. There is no information about the role of this molecule in the inflammatory events occurring during VEE and the effect of inflammation on the mortality rate in VEE-virus-infected rats. This study was designed to determine the role of Ang II in VEE and to analyze the effect of inflammation on mortality in infected rats. Two groups of rats were studied: 1) Virus-infected animals and controls (n = 60) were treated with losartan (a blocker of the Ang II-AT1 receptor) or with pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB) or left untreated and analyzed for morbidity and mortality. 2) Animals treated using the same protocol (n = 30) were sacrificed at day 4 postinfection and analyzed by immunohistochemistry and histopathology and for cytokine production. Increased expression of Ang II, ICAM-1, ED-1 and cytokines (IL-1α, MCP-1, IL-6 and IL-10) in infected animals was observed. The main histopathology findings were dilated capillaries and capillaries with endothelial detachment. Losartan and PDTC reduced the expression of IL-1α, MCP-1, and IL-10, and the number of dilated capillaries and capillaries with endothelial detachment. Survival analysis showed that 100% mortality was reached earlier in infected rats treated with losartan (day 14) or PDTC (day 11) than in untreated animals (day 19). These findings suggest that Ang II plays a role in VEE and that brain inflammation is protective against viral infection.

Melatonin, minocycline and ascorbic acid reduce oxidative stress and viral titers and increase survival rate in experimental Venezuelan equine encephalitis.

Venezuelan equine encephalitis (VEE) virus causes an acute central nervous system infection in human and animals. Melatonin (MLT), minocycline (MIN) and ascorbic acid (AA) have been shown to have antiviral activities in experimental infections; however, the mechanisms involved are poorly studied. Therefore, the aim of this study was to determine the effects of those compounds on the viral titers, NO production and lipid peroxidation in the brain of mice and neuroblastoma cultures infected by VEE virus. Infected mouse (10 LD50) were treated with MLT (500 µg/kg bw), MIN (50mg/kg bw) or AA (50mg/kg bw). Infected neuroblastoma cultures (MOI: 1); MLT: 0.5, 1, 5mM, MIN: 0.1, 0.2, 2 µM or AA: 25, 50, 75 µM. Brains were obtained at days 1, 3 and 5. In addition, survival rate of infected treated mice was also analyzed. Viral replication was determined by the plaque formation technique. NO and lipid peroxidation were measured by Griess׳ reaction and thiobarbituric acid assay respectively. Increased viral replication, NO production and lipid peroxidation were observed in both, infected brain and neuroblastoma cell cultures compared with uninfected controls. Those effects were diminished by the studied treatments. In addition, increased survival rate (50%) in treated infected animals compared with untreated infected mice (0%) was found. MLT, MIN and AA have an antiviral effect involving their anti-oxidant properties, and suggesting a potential use of these compounds for human VEE virus infection.

Modulation of GSK-3ß activity in Venezuelan equine encephalitis virus infection.

Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3ß is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer's. Hence inhibition of GSK-3ß in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3ß inhibitors and GSK-3ß siRNA experiments indicated that GSK-3ß was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC(50) of ∼0.5 µM and a CC(50) of >100 µM. BIOder was a more potent inhibitor of GSK-3ß than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3ß is found in three distinct complexes in VEEV infected cells, whereas GSK-3ß is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3ß inhibitors for modulating VEEV infection.

Natural killer cell mediated pathogenesis determines outcome of central nervous system infection with Venezuelan equine encephalitis virus in C3H/HeN mice.

TC83 is a human vaccine with investigational new drug status and is used as a prototype Venezuelan equine encephalitis virus for pathogenesis and antiviral research. Differing from other experimental models, the virus causes high titer infection in the brain and 90-100% mortality in the C3H/HeN murine model. To better characterize the susceptibility to disease development in C3H/HeN mice, we have analyzed the gene transcriptomes and cytokine production in the brains of infected mice. Our analysis indicated the potential importance of natural killer cells in the encephalitic disease development. This paper describes for the first time a pathogenic role for natural killer cells in VEEV encephalitis.

Venezuelan equine encephalitis and 2 human deaths, Peru.

Studies have suggested that enzootic strains of Venezuelan equine encephalitis (VEE) subtype ID in the Amazon region, Peru, may be less pathogenic to humans than are epizootic variants. Deaths of 2 persons with evidence of acute VEE virus infection indicate that fatal VEEV infection in Peru is likely. Cases may remain underreported.

Analysis of Venezuelan equine encephalitis virus capsid protein function in the inhibition of cellular transcription.

The encephalitogenic New World alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses, constitute a continuing public health threat in the United States. They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that these viruses have developed the ability to interfere with cellular transcription and use it as a means of downregulating a cellular antiviral response. The results of the present study suggest that the N-terminal, approximately 35-amino-acid-long peptide of VEEV and EEEV capsid proteins plays the most critical role in the downregulation of cellular transcription and development of a cytopathic effect. The identified VEEV-specific peptide C(VEE)33-68 includes two domains with distinct functions: the alpha-helix domain, helix I, which is critically involved in supporting the balance between the presence of the protein in the cytoplasm and nucleus, and the downstream peptide, which might contain a functional nuclear localization signal(s). The integrity of both domains not only determines the intracellular distribution of the VEEV capsid but is also essential for direct capsid protein functioning in the inhibition of transcription. Our results suggest that the VEEV capsid protein interacts with the nuclear pore complex, and this interaction correlates with the protein's ability to cause transcriptional shutoff and, ultimately, cell death. The replacement of the N-terminal fragment of the VEEV capsid by its Sindbis virus-specific counterpart in the VEEV TC-83 genome does not affect virus replication in vitro but reduces cytopathogenicity and results in attenuation in vivo. These findings can be used in designing a new generation of live, attenuated, recombinant vaccines against the New World alphaviruses.

Tunicamycin enhances neuroinvasion and encephalitis in mice infected with Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis (VEE) viruses cause natural outbreaks in humans and horses and represent a significant biothreat agent. The effect of tunicamycin on the course of the disease in mice with VEE was investigated, and the combined effects of these agents was characterized. CD-1 mice given 2.5 microg of tunicamycin had >1,000-fold more virus in the brain 48 hours after infection with the virulent VEE strain V3000 and > or =100-fold of the attenuated strain V3034 at all tested times than did untreated mice, indicating enhanced neuroinvasion. Tunicamycin did not alter the viremia profiles of these viruses nor the replication of V3000 in the brain itself. Tunicamycin alone caused ultrastructural blood-brain barrier damage, yet neuroinvasion by V3000 in treated mice appeared to occur via the olfactory system rather than the blood-brain barrier. Tunicamycin-treated, V3000-infected mice also exhibited earlier and more severe weight loss, neurological signs, neuronal infection, neuronal necrosis and apoptosis, and inflammation than untreated, V3000-infected mice. The mean survival time of tunicamycin-treated, V3000-infected mice was 7.3 days versus 9.9 days for untreated, V3000-infected mice. These studies imply that animals that ingest toxins similar to tunicamycin, including the agent of annual ryegrass toxicity in livestock, are conceivably at greater risk from infections by encephalitis viruses and that humans and horses exposed to agents acting similar to tunicamycin may be more susceptible to encephalitis caused by VEE viruses. The exact mechanism of tunicamycin-enhanced neuroinvasion by VEE viruses requires further study.

Venezuelan equine encephalitis virus transmission and effect on pathogenesis.

Quantifying the dose of an arbovirus transmitted by mosquitoes is essential for designing pathogenesis studies simulating natural infection of vertebrates. Titration of saliva collected in vitro from infected mosquitoes may not accurately estimate titers transmitted during blood feeding, and infection by needle injection may affect vertebrate pathogenesis. We compared the amount of Venezuelan equine encephalitis virus collected from the saliva of Aedes taeniorhynchus to the amount injected into a mouse during blood feeding. Less virus was transmitted by mosquitoes in vivo (geometric mean 11 PFU) than was found for comparable times of salivation in vitro (mean saliva titer 74 PFU). We also observed slightly lower early and late viremia titers in mice that were needle injected with 8 PFU, which represents the low end of the in vivo transmission range. No differences in survival were detected, regardless of the dose or infection route.

Inflammation is a component of neurodegeneration in response to Venezuelan equine encephalitis virus infection in mice.

Infection with the mosquito-transmitted Venezuelan equine encephalitis virus (VEE) causes an acute systemic febrile illness followed by meningoencephalitis. In this communication we characterize the cytokine profile induced in the central nervous system (CNS) in response to virulent or attenuated strains of VEE using RNase Protection Assays. Virulent VEE causes an upregulation of multiple pro-inflammatory genes including inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-alpha). To determine if iNOS and TNF-alpha contribute to the neuropathogenesis of VEE infection, iNOS and TNF receptor knockout mice were used in VEE mortality studies and exhibited extended survival times. Finally, CNS tissue sections labeled for VEE antigen, and adjacent sections double-labeled for an astrocyte marker and apoptosis, revealed that apoptosis of neurons occurs not only in areas of the brain positive for VEE-antigen, but also in areas of astrogliosis. These findings suggest that the inflammatory response, which is in part mediated by iNOS and TNF-alpha, may contribute to neurodegeneration following encephalitic virus infection.

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

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.

[The efficacy of the therapeutic and prophylactic actions of immunomodulators in experimental infection due to the causative agent of Venezuelan equine encephalomyelitis]. / Effektivnost' lechebno-profilakticheskogo deistviia immunomoduliatorov pri éksperimental'noi infektsii, vyzvannoi vozbuditeliami venesuél'skogo éntsefalomielita loshadei.

The investigation of the prevention and treatment action of some immunomodulators (ridostin, reaferon and polyribonate) used alone and in combinations was conducted on laboratory animals infected aerogenically by Venezuelan equine encephalitis (VEE) virus. A lower death rate of the aerogenically infected mice (10-30 respiratory LD50) was observed after intramuscular injection of ridostin. The preventive affect of ridostin and ridostin + reaferon administered intranasally and intramuscularly was achieved in the aerogenically infected guinea pigs (10 respiratory LD50). The results of the study on the early virus reproduction in the animals were used for the choice of the treatment scheme. The immunomodulators had no effect when the treatment was started 1 day after the VEE virus infection.

[The antiviral activity of inhibitors of ADP ribosylation in experimental alpha- and bunyavirus infections]. / Protivovirusnaia aktivnost' nekotorykh ingibitorov ADF-ribozilirovaniia pri éksperimental'nykh al'fa- i bun'iavirusnykh infektsiiakh.

The prophylactic and therapeutic effects of ADP-ribosylation inhibitors, 3,N-acetylaminobenzamide and 3,N-butyrylaminobenzamide, were studied in mice inoculated with an alphavirus or a bunyavirus. Both drugs were shown to have high levels of antiviral activity when given as a single subcutaneous injection in a dose of 10 mg/kg while increasing the number of injections did not increase their efficacy or might even decrease it.

[The effect of the peroxidation of ribamidil-containing liposomes on the results of chemotherapy in experimental viral infections]. / Vliianie perekisnogo okisleniia ribamidilsoderzhashchikh liposom na rezul'taty khimioterapii nekotorykh éksperimental'nykh virusnykh infektsii.

A relationship between ribamydil concentration and the intensity of accumulation of peroxidation products was found while storing ribamydil-containing liposomes which indicated a significant pro-oxidant activity of ribamydil. Increasing lethality of guinea pigs and white mice infected with Venezuelan equine encephalomyelitis virus was shown while using liposomal ribamydil containing from 4.78 to 6.82 nmol. ml-1 malonic dialdehyde which appeared to be associated with disordered function of the antioxidant system of the experimental animals.

[The course of airborne infection in rabbits infected with the Venezuelan encephalomyelitis virus]. / Techenie infektsii u krolikov, aérogenno zarazhennykh virusom venesuél'skogo éntsefalomieltia loshadei,

The experimental data on aerosol challenge of rabbits with Venezuelan equine encephalomyelitis (VEE) virus are presented. Lethal infection of rabbits was followed by a rise in body temperature (greater than 0.5 degrees C) within 1-2 days after challenge. Two waves of lethality in aerosol infection experiments were recorded. Three variants of the infection outcome were observed after VEE virus aerosol challenge of rabbits. The causes of death of the infected rabbits are discussed.

[The antiviral efficacy of ribamidil in an experimental infection of animals with the Venezuelan equine encephalomyelitis virus]. / Protivovirusnaia éffektivnost' ribamidila pri éksperimental'noi infektsii zhivotnykh, zarazhennykh virusom venesuél'skogo éntsefalomielita loshadei.

Considerable differences in antiviral efficacy of ribamydil were demonstrated in various animals experimentally infected with Venezuelan equine encephalomyelitis virus. The infection in baboons was quite similar to the manifestations of this disease in man as reported in the literature. With ribamydil used prophylactically, manifestations of the infection in monkeys were less marked.

Venezuelan equine encephalitis virus: comparison of infectivity and virulence of strains V-38 and P676 in donkeys.

Two strains of Venezuelan equine encephalitis (VEE) virus were examined for the ability to replicate in, as well as to produce death among donkeys. One, a low passage strain known as strain P676 was originally isolated from mosquitos in Venezuela. The other, strain V-38 was isolated from a horse brain in 1938 and had undergone an unknown number of laboratory passages; it is used extensively for the preparation of inactivated VEE vaccine. Both strains were found to be approximately equal in their ability to infect donkeys. However, a quantity as small as 50% hamster intraperitoneal infectious units of strain V-38 resulted in fatal infection. On the other hand, as much as 631 million infectious units of strain P676 were nonfatal in one of two donkeys. It appears that strain V-38 is approximately 100 million times more virulent than strain P676 in equine species. One donkey which received strain P676 demonstrated a biphasic pattern of clinical illness and viremia, and there is suggestive evidence that another animal experienced a second and fatal clinical response 3 weeks after virus inoculation.

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

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.