J C de la Torre

Astrocytes are the primary source of tissue factor in the murine central nervous system. A role for astrocytes in cerebral hemostasis.

Hemostasis in the brain is of paramount importance because bleeding into the neural parenchyma can result in paralysis, coma, and death. Consistent with this sensitivity to hemorrhage, the brain contains large amounts of tissue factor (TF), the major cellular initiator of the coagulation protease cascades. However, to date, the cellular source for TF in the central nervous system has not been identified. In this study, analysis of murine brain sections by in situ hybridization demonstrated high levels of TF mRNA in cells that expressed glial fibrillary acidic protein, a specific marker for astrocytes. Furthermore, primary mouse astrocyte cultures and astrocyte cell lines from mouse, rat, and human constitutively expressed TF mRNA and functional protein. These data indicated that astrocytes are the primary source of TF in the central nervous system. We propose that astrocytes forming the glia limitans around the neural vasculature and deep to the meninges are intimately involved in controlling hemorrhage in the brain. Finally, we observed an increase in TF mRNA expression in the brains of scrapie-infected mice. This modulation of TF expression in the absence of hemorrhage suggested that TF may function in processes other than hemostasis by altering protease generation in normal and diseased brain.

Arenaviruses: genomic RNAs, transcription, and replication.

The arenavirus genome comprises two single-stranded RNA molecules of negative polarity, designated L and S, that contain essentially nonoverlapping sequence information (for extensively referenced reviews see Buchmeier et al. 1980; Lehmann-Grube 1984; Howard 1986; Salvato 1993a; Southern 1996). There is some variability in the lengths of the genomic RNA segments for individual arenaviruses (L approximately 7,200 bases, S approximately 3,400 bases), but the general organization of the L and S genomic RNA species appears to be well conserved across the virus family. An extensive compilation of arenavirus genomic RNA sequence is now available, and this information has been used to derive phylogenetic relationships amongst the known arenaviruses (Bowen et al. 1997; Albarino et al. 1998). Sequence alignments have also facilitated the development of a generalized scheme to amplify arenavirus genomic RNAs by RT-PCR (Lozano et al. 1997) This innovative diagnostic resource, together with a specific RT-PCR assay for LCMV (Park et al. 1997), should have a significant positive impact on the rapid recognition and characterization of new arenavirus infections.

Contribution of LCMV Towards Deciphering Biology of Quasispecies In Vivo

Arenaviruses have often been viewed as relatively stable genetically with amino acid sequence homologies of 90%–95% among different strains of lymphocytic choriomeningitis virus (LCMV) and of 44%–63% for homologous proteins of different arenaviruses: LCMV, Pichinde, Junin, Machupo and Lassa (Southern and Bishop 1987; Southern and Oldstone 1988). Yet considerable variation in biological properties among LCMV strains soon became apparent (reviewed in Dutko and Oldstone 1983; Southern and Oldstone 1988). Hotchin already recognized the importance of passage history in determining the biological properties of LCMV (Hotchin 1962). He showed that early mouse brain passages of LCMV-induced immunologic tolerance in newborn mice and mortality was low. In contrast, late mouse brain passages of LCMV lost the tolerance-inducing capacity, and mortality was high. Neonatal infection of certain mouse strains with LCMV strains Armstrong (ARM) and E-350-induced growth hormone deficiency and severe hypoglycemia, which frequently resulted in the death of the infected mice, while strains WE and Traub did not cause this syndrome. This difference was associated with the ability of LCMV ARM and E-350, but not WE and Traub, to replicate at high levels in the GH-producing cells in the anterior pituitary (Oldstone et al. 1985). Other biological differences among LCMV strains include the capacity of the virus to invade s-cells in the islets of Langerhans, to cause alterations in glucose tolerance, or differences in the formation of immune complexes and lethality for adult guinea pigs (Southern and Oldstone 1988), as well as to induce generalized immunosuppression in adult mice. Selection of LCMV variants with distinguishable phenotypes occurs in different organs of infected mice persistently infected since birth with ARM tend to produce acute infection in adult mice, whereas isolates from the spleen of the same mice tend to persist (Ahmed and Oldstone 1988).

Human plasmacytoid dendritic cells sense lymphocytic choriomeningitis virus-infected cells in vitro.

ABSTRACT We previously reported that exosomal transfer of hepatitis C virus (HCV) positive-strand RNA from human Huh-7 hepatoma cells to human plasmacytoid dendritic cells (pDCs) triggers pDC alpha/beta interferon (IFN-α/β) production in a Toll-like receptor 7 (TLR7)-dependent, virus-independent manner. Here we show that human pDCs are also activated by a TLR7-dependent, virus-independent, exosomal RNA transfer mechanism by human and mouse hepatoma and nonhepatoma cells that replicate the negative-strand lymphocytic choriomeningitis virus (LCMV).

Molecular biology of Borna disease virus

Originally described in the early nineteenth century as a fatal encephalitis in horses, Borna disease (BD) has become an extraordinarily valuable model for the study of both molecular mechanisms and biological consequences of persistent virus infection in the CNS (Nicolau and Galloway 1928; Zwick 1939, this volume). BD is an immune-mediated neurologic syndrome characterized by behavioral abnormalities, meningeal and parenchymal inflammatory cell infiltrates in the brain, and the accumulation of disease-specific antigen in limbic system neurons (Joest and Degen 1911; Seifried and Spatz 1930; Ludwig et al. 1988; Richt et al. 1992). As a natural infection, BD has only been confirmed to occur in horses and sheep, but experimentally it can be transmitted to an extraordinary wide range of host species, including birds, rodents and nonhuman primates (Zwick et al. 1926; Nicolau and Galloway 1928; Zwick 1939; Matthias 1954; Nitzschke 1963; Heinig 1969; Anzil et al. 1973; Ludwig et al. 1973; Metzler et al. 1976; Ludwig and Thein 1977; Sprankel et al. 1978; Stitz et al. 1980; Hirano et al. 1983, Narayan et al. 1983; Gosztonyi and Ludwig 1984; Kao et al. 1984; Ludwig et al. 1985; Waelchli et al. 1985; Richt et al. 1992). Serological data indicate that the host range may even extend to humans, although no infectious material has been isolated from human subjects (Amsterdam et al. 1985; Rott et al. 1985, 1991 ; Bode et al. 1988, 1992).

Arenavirus Diversity and Evolution: Quasispecies In Vivo

Arenaviruses exist as viral quasispecies due to the high mutation rates of the low-fidelity viral RNA-dependent RNA polymerase (RdRp). This genomic heterogeneity is advantageous to the population, allowing for adaptation to rapidly changing environments that present varying types and degrees of selective pressure. The significant variation in biological properties observed among lymphocytic choriomeningitis virus (LCMV) strains, the prototypic arenavirus, indicates to what extent a quasispecies dynamics may play a role in arenavirus adaptability and pathogenesis. Several aspects of arenavirus variability and its contribution to pathogenesis will be discussed.

Reverse Genetics of Arenaviruses

Arenaviruses merit significant attention both as experimental models to study acute and persistent infections and as clinically important human pathogens, including hemorrhagic fever agents such as Lassa virus.

Lymphocytic Choriomeningitis Virus: Molecular Biology

Arenaviruses merit significant attention both as tractable model systems to study acute and persistent viral infections, and as clinically important human pathogens. Several arenaviruses, including Lassa fever, cause hemorrhagic fever (HF) disease in humans, and evidence indicates that the worldwide distributed prototypic arenavirus lymphocytic choriomeningitis virus is a neglected human pathogen. In the absence of immunization or effective treatment, these agents pose a public health concern, and thereby it is important to develop effective vaccines and better antiviral drugs to combat arenavirus infections. The development of reverse genetics systems for arenaviruses is allowing investigators to conduct a detailed characterization of the viral cis-acting signals and trans-acting factors that control each of the steps of the arenavirus life cycle, including RNA synthesis, packaging, and budding. This new knowledge will contribute to a better understanding of the arenavirus molecular and cell biology, and will facilitate the establishment of novel assays to identify and characterize novel antiviral compounds capable of interfering with specific steps of the arenavirus life cycle. Likewise, the ability to generate predetermined specific mutations within the arenavirus genome, and analyze their phenotypic expression, would significantly contribute to the elucidation of arenavirus–host interactions, including the bases arenavirus persistence and associated disease, as well as to cause severe HF disease in humans. These approaches could also lead to the development of novel potent and safe arenavirus vaccines.