Clinicopathologic features of viral agents of potential use by bioterrorists.

Concerns regarding the possible use of viral agents as weapons of mass destruction have heightened our need to recognize disease syndromes caused by these pathogens and to increase our understanding of potential countermeasures. This article reviews the clinical and pathologic features of various viruses that are generally thought to be potential biowarfare threats, and other related agents of topical interest. The epidemiologic and clinical aspects of recent natural outbreaks of disease caused by exotic viral agents are briefly described. Viral tissue targets, immune responses to these agents, relevant animal models, and diagnostic and potential therapeutic modalities also are discussed.

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.

Early expression of IFN-alpha/beta and iNOS in the brains of Venezuelan equine encephalitis virus-infected mice.

To investigate the roles of type I interferon (IFN-alpha/beta) and other mediators of innate immune responses (e.g., inducible nitric oxide synthase [iNOS]) in early dissemination of Venezuelan equine encephalitis virus (VEE) infection, we used mice with targeted deletions in either their IFN-alpha/beta-receptor (IFNAR-1-/-) or interferon regulatory factor 2 (IRF-2-/-) genes. Following footpad infection, both IFNAR-1-/- and IRF-2-/- mice were more susceptible than control mice to VEE. The IFNAR-1-/- mice also exhibit accelerated VEE dissemination to serum, spleen, and brain, and compared with control mice, they evidenced faster kinetics in the upregulation of proinflammatory genes. In contrast, in IRF-2-/- mice, iNOS gene induction was completely absent following peripheral virulent VEE infection. In evaluating the role of cells involved in iNOS production, primary microglial cell cultures were found to be highly permissive to VEE infection. Moreover, VEE infection increased levels of nitric oxide (NO) in resting microglial cultures but decreased NO production in IFN-gamma-stimulated microglia. Thus, these findings suggest that reactive nitrogen species play an important contributory role in VEE dissemination and survival of the host. Our results further suggest the necessity for a carefully balanced host response that follows a middle course between immunopathology and insufficient inflammatory response to VEE infection.

Astrocytes as targets for Venezuelan equine encephalitis virus infection.

Venezuelan equine encephalitis virus (VEE) produces an acute infection in humans and induces a well-characterized cytopathic effect in neurons of the central nervous system (CNS). However, little is known about the role of glial cells in response to VEE infection of the CNS. Our results demonstrate that VEE is capable of a productive infection in primary astrocyte cultures and that this infection is cytotoxic. Further, there were significant differences in the growth kinetics comparing virulent and attenuated strains of VEE. Additionally, VEE infection of astrocyte cultures induced gene expression of two neuro-immune modulators, tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). Assays for TNF-alpha protein and nitric oxide (NO) demonstrated high levels of TNF-alpha protein and low levels of NO in response to VEE infection of astrocytes. These observations suggest an important role of astrocytes in this virus-induced encephalitis, and that interactions between astrocytes, other glial cells, and neurons may be important in VEE pathogenesis. Such interactions, which could impact neuronal survival, may include loss of functional changes in astrocytes or, alternatively, their production of neurotoxic molecules.

Role of interferon and interferon regulatory factors in early protection against Venezuelan equine encephalitis virus infection.

To investigate the role of type I interferon (IFN) and its regulatory transacting proteins, interferon regulatory factors (IRF-1 and IRF-2), in early protection against infection with virulent Venezuelan equine encephalitis virus (VEE), we utilized mice with targeted mutations in the IFN-alpha/beta receptor, IRF-1, or IRF-2 genes. IFN-alpha/beta-receptor knockout mice are highly susceptible to peripheral infection with virulent or attenuated VEE, resulting in their death within 24 and 48 h, respectively. Treatment of normal macrophages with anti-IFN-alpha/beta antibody prior to and during infection with molecularly cloned virulent VEE resulted in increased VEE replication. However, treatment with high doses of IFN or IFN-inducing agents failed to alter percentage mortality or average survival times in mice challenged with a low dose of virulent VEE. In IRF-1 and IRF-2 knockout mice (IRF-1(-/-) and IRF-2(-/-)), the 100% protection against virulent VEE that is conferred by attenuated VEE within 24 h in control C57BL/6 mice was completely absent in IRF-2(-/-) mice, whereas 50% of IRF-1(-/-) mice were protected. IRF-2(-/-) mice were deficient in clearing VEE virus from the spleen and the brain compared to the heterozygous IRF-2(+/-) knockout or C57BL/6 (+/+) mice. Furthermore, a distinct pattern of histopathological changes was observed in brains of IRF-2(-/-) mice after VEE exposure. Taken together, these findings imply that the altered immune response in IRF-1 and IRF-2 knockout mice results in altered virus dissemination, altered virus clearance, and altered virus-induced pathology. Thus, type I interferon, as well as IRF-1 and IRF-2, appears to play an important and necessary role in the pathogenesis of, and protection against, VEE infection.

Kinetics of cytokine expression and regulation of host protection following infection with molecularly cloned Venezuelan equine encephalitis virus.

In the mouse model, the arbovirus Venezuelan equine encephalitis virus (VEE) replicates in lymphoid tissues prior to either inducing protective immunity (attenuated VEE mutant) or progressing to lethal encephalitis (virulent parent VEE). To investigate the mechanism of the protective response, cytokine gene expression was examined during the course of the primary in vivo immune response to molecularly cloned, virulent VEE and a single-site attenuated VEE mutant, using a quantitative reverse transcriptase-polymerase chain reaction assay. VEE-induced cytokine gene expression was 100-fold elevated over that of untreated controls for IFN-gamma and IL-6 and 10-fold increased for IL-12, IL-10, and TNF-alpha. There was no qualitative difference in cytokine gene induction comparing mice infected with the attenuated and the virulent VEE; however, there were significant differences in the cytokine gene expression kinetics. In mice infected with the attenuated VEE, elevated cytokine gene expression was delayed 24 hr when compared to mice infected with the virulent parent VEE clone at the same dose. Further, IFN-gamma protein secretion by cells from the draining lymph node mimicked the pattern of IFN-gamma gene induction by cells harvested from the same site. IFN-gamma gene expression was elevated at an earlier time point in mice given virulent V3000 24 hr after attenuated V3032 injection compared to mice infected with virulent V3000 alone. The combined V3000/V3032 infection resulted in host protection. Treatment of mice with IL-12 prior to infection with virulent VEE failed to reduce the severity of infection, while anti-IL-12 antibody did not prevent the early protective effect of attenuated virus. In contrast, administration of anti-IFN-alpha/beta antibody prior to VEE infection worsened virulent VEE disease. These results indicate that the attenuated VEE strain elicits a similar but delayed cytokine response compared to the virulent strain, suggesting that the kinetics of cytokine expression and the particular cytokine produced may influence the development of a host protective response. Furthermore, IFN-alpha/beta, but not IL-12, seems to be a major factor in the induction of early protection against VEE infection and disease.

Virulent and attenuated mutant Venezuelan equine encephalitis virus show marked differences in replication in infection in murine macrophages.

One of the first target cells at the site of inoculation with an alphavirus may be monocytes or macrophages. The replication kinetics of virulent and attenuated molecularly cloned Venezuelan equine encephalitis virus (VEE) in murine macrophages were therefore compared. Infection of both quiescent and activated mouse primary peritoneal macrophages with a molecularly cloned, virulent VEE termed V3000 resulted in peak virus titres of 10(4) plaque forming units (PFU)/ml supernatant by 24 h post-infection (pi), followed by rapidly decreasing virus titres. In contrast, a molecularly cloned attenuated VEE mutant, V3032, that differs from V3000 by a single amino acid at E2 glycoprotein position 209 (glu-->lys) replicated more slowly and to higher titres (10(8) PFU/ml supernatant) that peaked at 72 h pi. Replication of V3032, but not V3000, was sharply restricted by prior activation of macrophages with lipopolysaccharide or interferon-gamma. These results indicate that virulent V3000 and attenuated V3032 differ in their growth kinetics in both quiescent and activated macrophages. Thus, macrophages, and their specific activation state, may play a major role in virulent and attenuated VEE replication and pathogenesis.

Specific restrictions in the progression of Venezuelan equine encephalitis virus-induced disease resulting from single amino acid changes in the glycoproteins.

The pathogenesis of Venezuelan equine encephalitis virus (VEE) was examined in the mouse model using V3000, a virus derived from a molecular clone of the Trinidad donkey strain of VEE. These results were compared in parallel experiments with avirulent mutants of VEE derived by site-directed mutagenesis of the clone. Adult mice, inoculated subcutaneously in their left rear footpad with V3000, were followed in a time course study for 6 days in which 15 organs were tested for histopathological changes, for the presence of viral antigen by immunohistochemical staining, for the presence of viral nucleic acid by in situ hybridization analysis, and for content of viable virus. Virus was detected in the footpad inoculation site, but until 12 hr postinoculation (pi), the level of virus did not suggest early viral replication. By 4 hr pi, however, replication of V3000 was evident in the draining popliteal lymph node. At this early time point, no virus could be isolated from any other organ examined. At 12 hr, a significant serum viremia was observed, and virus was detected at a low level in a number of well vascularized organs, including spleen, heart, lung, liver, kidney, and adrenal gland. By 18 hr, high virus titers were present in serum and all the lymphoid organs examined, and these tissues appeared to be the major peripheral sites of V3000 replication. Virus in serum and peripheral organs was cleared by 3-4 days pi. In a second phase of the infection, V3000 invaded the central nervous system (CNS), replicated predominantly in neurons, and persisted in the brain until death by encephalitis. Pathologic findings as well as the results of immunocytochemical and in situ hybridization examination were generally coordinate with virus titration. A site-directed mutant of V3000, V3010, contained a mutation in the gene for the E2 glycoprotein at codon 76 (Glu to Lys) which rendered it avirulent after footpad inoculation. Detection of V3010 replication in the draining lymph node was sporadic and was sometimes delayed to as long as 3 days pi. Infrequent and/or delayed virus spread to other sites also was observed. Analogous experiments were performed with other mutants which were avirulent by the footpad inoculation route: V3014, a mutant differing from V3000 at three loci (E2 Lys 209, E1 Thr 272, and E2 Asn 239), as well as single-site mutants V3032 (E2 Lys 209) and V3034 (E1 Thr 272).(ABSTRACT TRUNCATED AT 400 WORDS)

A molecular genetic approach to the study of Venezuelan equine encephalitis virus pathogenesis.

Viral pathogenesis can be described as a series of steps, analogous to a biochemical pathway, whose endpoint is disease of the infected host. Distinct viral functions may be critical at each required step. Our genetic approach is to use Venezuelan equine encephalitis virus (VEE) mutants blocked at different steps to delineate the process of pathogenesis. A full-length cDNA clone of a virulent strain of VEE was used as a template for in vitro mutagenesis to produce attenuated single-site mutants. The spread of molecularly cloned parent or mutant viruses in the mouse was monitored by infectivity, immunocytochemistry, in situ hybridization and histopathology. Virulent VEE spread through the lymphatic system, produced viremia and replicated in several visceral organs. As virus was being cleared from these sites, it began to appear in the brain, frequently beginning in the olfactory tracts. A single-site mutant in the E2 glycoprotein appeared to block pathogenesis at a very early step, and required a reversion mutation to spread beyond the site of inoculation. The feasibility of combining attenuating mutations to produce a stable VEE vaccine strain has been demonstrated using three E2 mutations.

Host-dependent variation of bluetongue virus neutralization.

To determine if neutralizing epitopes of Bluetongue virus (BTV) 17 are host dependent, e.g., that monoclonal antibodies (mAb) to Bluetongue virus 17 (BTV 17) differ in their ability to neutralize BTV infectivity in insect versus mammalian cells, a panel of neutralizing mAb was developed. The relative neutralizing titer of eight mAb for BTV 17 infectivity in mammalian versus insect target cells was determined. Four mAb differed in their relative neutralization titer when assayed on mammalian target cells as compared to insect target cells. These findings suggest that different epitopes involved in neutralization might be important in virus infectivity and neutralization in mammalian versus insect target cells. To determine which viral protein(s) these mAb bind, the specificity of the mAb was determined by radioimmunoprecipitations. Five BTV 17 neutralizing mAb bound to the major outer coat protein P2, a 98-kDa protein, whereas the BTV protein(s) bound by the other three neutralizing mAb could not be determined. The potential role of the two BTV outer coat proteins in infection of mammalian and insect host cells is discussed.

Antigenic topography of bluetongue virus 17.

Neutralizing monoclonal antibodies (mAbs) have been produced and used to map the topographical relationship of the surface antigenic determinants of bluetongue virus (BTV) 17 that mediate neutralization. Eight monoclonal antibodies, at least five of which were directed to the major outer coat protein of BTV 17, P2, were studied in neutralization assays using variant BTV 17 and in competition binding experiments. Five different epitopes were identified that are involved in neutralization of viral infectivity. Three of the five epitopes are clearly associated with P2, while the location of the other two epitopes is not known. The potential association of these two epitopes with one or both outer coat proteins of BTV is discussed.

Antiidiotypic antibody mimicry of a bluetongue virus neutralizing antigen.

Syngeneic antiidiotypic (Anti-Id) mAb were produced to the Bluetongue virus (BTV) serotype 17 neutralizing mAb. Three anti-Id mAb had the characteristics of an internal image of the Id as demonstrated by 1) the internal image anti-Id mAb were capable of mimicking BTV by blocking the neutralizing activity of the idiotypic neutralizing mAb and 2) the anti-Id mAb bound specifically to the surface of BTV susceptible cells in indirect binding experiments as determined by immunocytochemistry and flow cytometric analysis. The potential application of these internal image anti-Id mAb in this arbovirus system is discussed.

Conformationally dependent epitopes of bluetongue virus neutralizing antigen.

The neutralizing epitopes of Bluetongue virus serotype 17 (BTV 17) were evaluated by virus neutralization assay, flow cytometry, radioimmuno-precipitation, and Western blot. We showed that neutralizing monoclonal antibodies (MABs) raised to BTV 17 bound to viral proteins on intact virus when examined by virus neutralization and by an indirect binding assay analyzed by flow cytometry. In contrast, when the viral proteins were solubilized, a loss in reactivity with some of the neutralizing MABs was observed. Additionally, when the viral proteins were subjected to denaturing conditions, none of the neutralizing MABs reacted with the viral proteins. The results indicate that these neutralizing MABs bind conformationally dependent epitopes.

Immunobiology of bluetongue virus.

Following BTV infection or vaccination, sheep develop both anti-virus antibody (which may include neutralizing antibody) and a cellular immune response. Yet, it still is unclear what aspects of the response are most critical in preventing infection and disease from this virus. This is, in part, the result of a lack of knowledge of all of the viral virulence factors. However, with the current work involving subunit vaccines and the efforts to more carefully characterize virulence factors and tissue tropism, there will be a more thorough understanding of the immunobiology of the Bluetongue virus.