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Lymphocytic choriomeningitis virus (LCMV) Armstrong strain selectively and persistently infects the majority of growth hormone (GH) producing cells in the anterior lobe of pituitary glands of C3H/St mice but negligibly infects GH producing cells of BALB/WEHI mice (Oldstone et al., Virology 142, 175--182, 1985; Oldstone et al., Science 218, 1125--1127, 1982). Although infected GH cells remain free of structural damage, disrupted initiation of GH transcription (Klavinskis and Oldstone, J. Gen. Virol. 68, 1867--1873, 1989; Valsamakis et al., Virology 156, 214--220, 1987) occurs with a resultant decrease in the synthesis of GH, leading to a failure of growth and development (Oldstone et al., Science 218, 1125--1127, 1982). Microsatellite mapping of DNA obtained from 101 individual C3H/St x BALB/WEHI F1 x F1 mice shows that the growth failure correlates with host genes linked (P value 0.0008) on chromosome 17 just outside of the H-2D MHC site between D17 Mit24 and D17 Mit51, a distance of 2.5 cM. The genetic mapping done here excludes alpha-dystroglycan (alpha-DG), a known receptor for LCMV (Cao et al., Science 282, 2079--2081, 1998) in pathogenesis of GH disease, as alpha-DG is encoded in the mouse by a gene residing on chromosome 9 (Yotsumoto et al., Hum. Mol. Genet. 5, 1259--1267, 1996)., (Copyright 2001 Academic Press.)

Recently, we reported that in vitro and in vivo persistent infection of neurons by lymphocytic choriomeningitis virus (LCMV) downregulated GAP43 expression, a protein involved in neuronal plasticity associated with learning and memory. Here, we investigated the transcriptional and posttranscriptional events involved. Persistent LCMV infection of PC12 cells (PC12Pi) caused reduced levels of GAP43 steady-state mRNA when compared to uninfected PC12 cells. In addition, an increase in the steady-state levels of GAP43 mRNA observed in PC12 cells in response to nerve growth factor (NGF) was abrogated in PC12Pi cells. Nuclear run-on analysis revealed that the rate of GAP43 transcription was reduced threefold in PC12Pi cells compared to uninfected PC12 cells. Moreover, analysis of the half-life of GAP43 mRNA indicated that NGF-mediated stabilization of GAP43 transcripts was significantly diminished in PC12Pi cells. Treatment of PC12Pi cells with basic fibroblast growth factor, dibutyryl cyclic AMP, and 12-o-tetradecanoyl-phorbol-13-acetate, a potent activator of protein kinase C, did not increase the GAP43 mRNA steady-state level, suggesting that LCMV infection interferes with a step downstream from protein kinases A and C in the NGF signal transduction pathway.

Borna disease virus (BDV) causes a central nervous system disease in several vertebrate species which is characterized by behavioral disturbances. Seroepidemiological data indicate an association of BDV infection with certain human mental disorders. Sclerosis of the hippocampus and astrocytosis constitute histopathological hallmarks of BDV infection in animals. Therefore, we searched for human brain autopsy cases with such histopathological features. Five of 600 cases examined were identified as having hippocampus sclerosis and astrocytosis. Using immunocytochemistry, RT-PCR, and in situ hybridization, we detected both BDV antigen and RNA in autopsy brain samples from 4 of these 5 patients, who presented with a clinical history of mental disorders involving memory loss and depression. This is the first demonstration that BDV can infect human brain tissue, possibly contributing to the pathophysiology of specific human neuropsychiatric disorders.

Populations of RNA viruses consist of heterogeneous mixtures of related genomes (quasispecies). Isolation of variants present at low levels within a population can result in clonal virus populations which display markedly different phenotypes upon infection of the host. The mechanisms by which these variants are maintained within the original quasispecies are not understood. Certain strains of lymphocytic choriomeningitis virus (LCMV) cause a growth hormone deficiency syndrome (GHDS) when inoculated into newborn C3H/St mice while others do not. We have previously described the isolation of virus clones from the GHDS-negative WE strain of LCMV which differ in their ability to cause GHDS. To investigate how disease-positive clones can remain hidden within a disease-negative parental population, we examined whether infection with mixtures of the GHDS-negative (WE c54) and GHDS-positive (WE c2.5) clones could cause GHDS. Neonatal C3H/ST mice infected with 100:1 or 10:1 ratios of WE c54 to WE c2.5 did not develop the syndrome, while animals infected with 1:1 or lower ratios did. Development of GHDS correlated with the extent to which the GH-producing cells of the anterior pituitary were infected. These data indicate that a large excess of disease-negative clones can restrict the replication of disease-positive clones in GH-producing cells, thus preventing the onset of GHDS. In addition, our results indicate that a threshold for phenotypic dominance exists. Interestingly, WE c54 did not entirely outcompete WE c2.5 in mice infected with the 100:1 ratio, suggesting a mechanism whereby pathogenic viruses can be maintained within a nonpathogenic viral population.

Neurons have a restricted expression of MHC heavy chain molecules which prevents presentation of antigens of infecting viruses. As a result, such infected cells escape immune surveillance and allow the establishment of noncytolytic persistent infection. Here we show that a chronic noncytolytic viral infection both in vitro and in vivo selectively perturbed the expression of GAP-43, a protein that plays a central role in neuronal plasticity processes accompanying learning and memory. GAP-43 expression was greatly decreased in the hippocampus, an area of heightened viral replication, while synaptic density was preserved. Concurrently, the ability to learn tasks was significantly impaired in these persistently infected mice. Yet, infected neurons remained free from structural injury.

Lymphocytic choriomeningitis virus (LCMV) is a nonlytic murine virus that provides a valuable model system for studying the behavioral correlates of CNS viral infection. Newborn or immunosuppressed mice infected with LCMV develop a persistent tolerant infection characterized by continuous viral production. Virus can be found in various body organs including lung, liver, kidney, and brain. In brain, neurons are the predominant CNS cells infected and the greatest number of persistently infected neurons are found in the cerebral cortex, hippocampus, other limbic structures and parts of the hypothalamus. Despite continuous infection throughout the animal's life, neurons show no structural injury or dropout. Mice from the DBA/2J strain were infected with LCMV (1000 plaque-forming units) within 18 h of birth and tested for behavioral function as adults. Plaque assays indicated persistent infection in virus-injected mice. Mice were tested for their ability to learn a Y-maze spatial discrimination to avoid the onset of a mild footshock (0.43 mA). The number of correct avoidance responses made during training was taken as a measure of acquisition performance. The virus-infected mice showed a deficit in acquisition of the Y-maze discrimination compared to that seen in vehicle-injected and noninjected controls. Following additional training to reach control levels of performance, the infected mice and the controls were injected with the cholinergic antagonist scopolamine. Scopolamine (2.0 mg/kg) disrupted the performance of the infected mice significantly more than control performance, suggesting that a cholinergic dysfunction accounted for some of the learning deficit. A separate group of virus-infected mice exhibited hypoactivity during the first exposure to a locomotor testing apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocytes and/or monocytes/macrophages carry viral genetic information in most, if not all, persistent and latent viral infections, and serve as potential reservoirs for maintaining or reintroducing the infection. Similarly, neurons can be persistently infected by several DNA and RNA viruses whose continued presence can alter the physiologic function of these cells, leading to disorders in neurotransmitters and disease. Here, we document that adoptive transfer of virus-specific cytotoxic T lymphocytes clears virus and viral nucleic acid sequences, in vivo, from individually infected lymphocytes, macrophages, and neurons. By plaquing, infectious center, Northern blot, and in situ hybridization at the single cell level, virus was efficiently removed from these cells.

Borna disease (BD) is a neurologic syndrome characterized by profound disturbances in behavior and the accumulation of specific antigens in limbic system neurons. The potency of brain homogenates from animals with BD to cause disease in normal animals is reduced by exposure to detergents. We have recently described isolation and characterization of clones derived from the BD agent. Here we present evidence that suggests that the BD agent is a negative-sense, single-strand RNA virus. The 8.5-kb genome of this virus appears to be associated with nuclei and encodes two major RNA transcripts of 2.1 and 0.8 kb.

Rat spinal cords were subjected to a 200 g/cm force acceleration injury at T10. Ten days later, the cords were totally transected at T10 and the rats separated into two groups: group C (controls) had the spinal cord realigned end-to-end; group X had 3 mm trimmed from proximal and distal cord stumps and a semifluid collagen matrix (CM) bioimplant was inserted in the gap. The CM polymerized to a firm gel at body temperature within 2 h. All rats were maintained 90 days posttransection (dpt). At 90 dpt, they were examined for local spinal cord blood flows, somatosensory evoked potentials, and a neurological evaluation. After killing, the cords were processed for electron and light microscopy and monoamine histofluorescence. The results indicated that CM can support the growth of central neurites, fibroblasts, and an adequate anastomotic network of blood vessels. Control scar tissue does not promote the presence of nerve fibers and blood vessels to the extent observed in the CM. Somatosensory evoked potential early waveforms were present in CM-bioimplanted rats but not in controls. No rat regained walking ability at 90 dpt but muscle tone and strength appeared better in CM-implanted than in control rats. We conclude that a CM bridge can provide a well vascularized, relatively nonhostile environment for central neurites and catecholaminergic axons extending from the proximal spinal cord tissue across the CM bridge and into the distal stump.

Cell lines persistently infected with foot-and-mouth disease virus (FMDV) have been established by growth of BHK-21 (c-13) or IBRS-2 (c-26) that survived standard cytolytic infections with FMDV. They maintain cytoplasmic FMDV RNA sequences, as shown by dot blot hybridization tests, using cloned FMDV cDNA as probes. Cell line C1-BHK-Rc1 was derived by infection of cloned BHK-21 c1 cells and plaque-purified FMDV C-S8 c1. Indirect immunofluorescence assays indicated the presence of FMDV antigens. It was resistant to superinfection by FMDV C-S8 c1, O-S7, or A5, but not by encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or Semliki forest virus (SFV). Infectious FMDV was detected in the culture medium only up to cell passage 65. The virus isolated from C1-BHK-Rc1 cells showed decreased plaque size and diminished yield in infections at 42 degrees. Multiple mutations in the intracellular FMDV RNA have been detected by T1 oligonucleotide fingerprinting of genomic RNA segments hybridized to FMDV cDNA fragments. At late cell passages, when no infectious FMDV is detected, cells continue to express viral antigens and FMDV RNAs with deletions of up to 3 kb have been identified by Northern blot analysis. We conclude that persistent infections of cell cultures with FMDV are readily established and that multiple genetic and phenotypic variations occur in the virus during persistence.

The regulatory region of the tetracycline resistance determinant from transposon Tn10 has been used to construct plasmid vectors for gene expression regulated by tetracycline. Plasmids pRS tetBam-8 and pRS tetBam-16 include the tet regulatory region, the segment coding for the first four amino acids of the tetracycline resistance protein (tetA protein), and a linker region with SalI, HpaII, and BamHI restriction sites for gene fusions. Plasmid pTB-1, a derivative of pRS tetBam-8 and of the beta-galactosidase gene-containing plasmid pMC1403, constitutively expresses a tetA fragment-beta-galactosidase fusion protein. If a multicopy runaway replication plasmid, pMOBglII-16 that includes a 2.7-kb BglII DNA fragment from Tnl10 that provides tetR protein is present along with pTB-1, the expression of beta-galactosidase is reduced eightfold. Tetracycline acts as an inducer of the system and restores the level of beta-galactosidase activity measured in transformants containing pTB-1 alone. Plasmid mutants unable to produce active tetR protein are ineffective in reducing expression. Escherichia coli carrying plasmids that express both tetA protein and tetR protein show an increase in the tetracycline resistance level after incubation with the drug. The observations are consistent with the previously proposed mechanism of regulation of tetracycline resistance in Tn10.