Objective: The purpose of this study was to characterize the innate or early immune responses of glial cells, specifically astrocytes and microglia, to Venezuelan equine encephalitis (VEE) virus infection. VEE is a viral pathogen endemic to a very restricted range in South America; however, this pathogen has a long history of research as a weapon of bioterrorism (bioweapon). Features that make VEE virus suitable as a bioweapon include: a capability of being produced in vast quantities, relatively stable yet highly infectious properties easily spread through aerosolization, and a lack of naturally acquired immunity held by most of the human population.
Design: The experimental design of this research included both in vitro and in vivo models. These models were then infected with a range of VEE phenotyes and utilized as a model system. Samples were then collected and processed by immunohistochemistry, mRNA characterization by RNase Protection Assays (RPAs), Polymerase Chain Reaction (PCR), or protein quantitation using Enzyme-linked immunosorbent assays (ELISAs).
Population, Sample, Setting, and Years: In vitro studies utilized primary cell cultures of rat astrocytes or microglia, or cell-lines that were immortalized from these cell types. For each virus phenotype, three cell cultures were infected. In vivo studies utilized an inbred mouse strain, as well as, transgenic mice that had mutations to eliminate specific immune responses. Mice were infected in groups of 10/1 virus phenotype, usually including a mock-infected control, and three virus phenotypes. VEE infection experiments were conducted in a Bio-Safety Laboratory level 3 (BSL-3) at the Uniformed Services University, Bethesda, MD, and the research protocol was approved the University's institutional animal care and use committee. Data were collected from 1998 to 2001.
Intervention and Outcome Variables: Primary astrocyte cell cultures were infected with one of three molecularly cloned strains of VEE that differed in virulence, and samples were collected over an experimental time course at 0, 2, 6, 12, 24, 48, and 72 hours post-infection. Mice were also infected with one of these VEE stains and their brains were harvested over an experimental time course of 0-7 days post-infection to quantitate viral load and characterize immune responses of the central nervous system (CNS) to VEE infection.
Methods: Characterization of cytopathic effects of cell cultures to VEE infection, or mortality rates, were determined. Samples of tissues were then harvested and sterilized using formaldehyde fixation, or phenol for mRNA extraction, and processed in standard laboratory facilities. Immunohistochemistry was utilized to demonstrate VEE infection in the CNS, as well as to quantitate the degree of gliosis, a characteristic of inflammation, and cell death. RPA, PCR and ELISA techniques were used to characterize the induction of pro-inflammatory genes.
Findings: Results demonstrate that VEE is capable of a productive infection in glial cells, and that the consequence of VEE infection is cell death. The rate of cell death and the pro-inflammatory responses mounted against VEE infection are directly related to the virulence of the VEE strain. Specifically, inducible nitric oxide synthase and tumor necrosis alpha were upregulated in response to VEE infection. Mortality rates and survival times for mice infected with VEE also demonstrated significant differences. Mice infected with a virulent strain of VEE survived only 8.1 ± 0.3 days with a 100% mortality rate, whereas mice infected with one of two attenuated strains of VEE had average survival times of 8.5 ± 1.0 and 10 ± 0.0 days, with mortality rates of 20 and 10 percent respectively (p < 0.05). Brain weights, a measure of cerebral edema, were also significantly different. Mice infected with the virulent strains of VEE had significantly more cerebral edema.
Conclusions: VEE infection in glial cells and in mice caused significant pathology. Glial cells and the CNS responded to this infection by the upregulation of pro-inflammatory genes. This pro-inflammatory response was directly correlated to the virulence of the VEE strain used for infection. It is unclear at this time if these pro-inflammatory responses are beneficial or detrimental to the host. Understanding the delicate balance of host-protection and disease will be crucial to the development of treatment strategies.
Implications: Due to the current high-threat potential for use of biological agents such as VEE, and the fact that nurses will most likely participate in first-responder teams, whether on the battlefield or the domestic front, it is important for nurses to understand the characteristics of these biological agents. Further, nurse scientists, working in collaboration with other researchers, are instrumental in bringing knowledge gained at the bench to the bedside.
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