Complementation analysis of measles virus mutants isolated from persistently infected lymphoblastoid cell lines.

Human lymphoblastoid cell lines persistently infected with measles virus release a heterogeneous population of virions. At least 80% of the infectious particles were temperature sensitive for plaque formation at 39 degrees C. Plaque-purified temperature-sensitive mutants from four persistently infected human lymphoblastoid cell lines were shown to be heterogeneous with respect to efficiency of plating at 31 and 39 degrees C, as well as to antigen and RNA production at 39 degrees C. The heterogeneity was confirmed by complementation analysis in which 21 temperature-sensitive isolates were found to represent at least four of the five previously described complementation groups of measles virus. Two isolates complemented four reference temperature-sensitive mutants. These isolates either represent new complementation groups or are members of the fifth complementation group, group E. The majority of isolates were found to have multiple mutations, and group B mutants (RNA-) predominated. Two temperature-sensitive isolates were able to interfere with production of parental measles virus at both permissive and nonpermissive temperatures.     

Clonal analysis of mammalian cell cultures persistently infected with Japanese encephalitis virus.

More than 200 cells were cloned from populations of mammalian cells persistently infected with Japanese encephalitis virus. Only four cloned cultures contained cells that had viral antigen measurable by immunofluorescence and that released infectious virus, yet all clones harbored virus-specific RNA. Superinfection of cloned cells with wild-type Japanese encephalitis virus did not produce cytopathic effects, but resulted in production of viral antigen and infectious virus in formerly nonproducing clones. Cocultivation of nonproducer clone cells with normally permissive cells did not induce virus production, nor did treatment of nonproducer clones with various inhibitors of DNA, RNA, or protein synthesis. It is suggested that the cloning procedure may have selected for a particular subpopulation of cells and that defective virus is also involved in establishment and maintenance of persistent infection.     

Cells persistently infected with newcastle disease virus: I. Properties of mutants isolated from persistently infected L cells

The strain of Newcastle disease virus (NDV_pi) present in persistently infected L cells differed markedly from the Herts strain (NDV₀) used to initiate the infection. NDV_pi produced small plaques (less than 1 mm) in chick embryo cell cultures, whereas the wild type (NDV₀) produced large plaques (2 to 3 mm). The two viruses differed in a number of additional properties. Whereas 80% of adsorbed NDV₀ eluted from chicken red blood cells at 37 C, only about 20% of NDV_pi was recovered under similar conditions. There was no significant difference in the neuraminidase content of the two viruses. The infectivity of NDV₀ was stable for 1 hr at 48 C, whereas 99.9% of the infectivity of NDV_pi was destroyed. The two viruses also differed in lethality for chick embryos; NDV_pi had significantly reduced lethality for 9-day-old chick embryos when compared to NDV₀. In contrast to NDV₀, which produced an abortive infection in L cells, NDV_pi not only replicated effectively and destroyed these cells, but also induced significantly higher quantities of interferon than did NDV₀. These data furnished additional evidence for the lack of relationship of interferon production to abortive infection of L cells with NDV₀. In contrast, interferon was found to play a significant role in the maintenance of persistent infection.     

HIV-1 replication in the central nervous system occurs in two distinct cell types

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can lead to the development of HIV-1-associated dementia (HAD). We examined the virological characteristics of HIV-1 in the cerebrospinal fluid (CSF) of HAD subjects to explore the association between independent viral replication in the CNS and the development of overt dementia. We found that genetically compartmentalized CCR5-tropic (R5) T cell-tropic and macrophage-tropic HIV-1 populations were independently detected in the CSF of subjects diagnosed with HIV-1-associated dementia. Macrophage-tropic HIV-1 populations were genetically diverse, representing established CNS infections, while R5 T cell-tropic HIV-1 populations were clonally amplified and associated with pleocytosis. R5 T cell-tropic viruses required high levels of surface CD4 to enter cells, and their presence was correlated with rapid decay of virus in the CSF with therapy initiation (similar to virus in the blood that is replicating in activated T cells). Macrophage-tropic viruses could enter cells with low levels of CD4, and their presence was correlated with slow decay of virus in the CSF, demonstrating a separate long-lived cell as the source of the virus. These studies demonstrate two distinct virological states inferred from the CSF virus in subjects diagnosed with HAD. Finally, macrophage-tropic viruses were largely restricted to the CNS/CSF compartment and not the blood, and in one case we were able to identify the macrophage-tropic lineage as a minor variant nearly two years before its expansion in the CNS. These results suggest that HIV-1 variants in CSF can provide information about viral replication and evolution in the CNS, events that are likely to play an important role in HIV-associated neurocognitive disorders.     

Cytotoxic T cells isolated from the central nervous systems of mice infected with Theiler''s virus.

Intracerebral inoculation of resistant mice (C57BL/10SNJ) with Theiler's murine encephalomyelitis virus (TMEV) results in acute encephalitis followed by subsequent clearance of virus from the central nervous system (CNS). In contrast, infection of susceptible mice (SJL/J) results in virus persistence and chronic immune-mediated demyelination. Both resistance and susceptibility to TMEV-induced disease appear to be immune mediated, since immunosuppression results in enhanced encephalitis in resistant mice but diminished demyelination in susceptible mice. The purpose of these experiments was to determine whether anti-TMEV cytotoxic T lymphocytes (CTLs) are generated during acute and chronic TMEV infection. Nonspecific lectin-dependent cellular cytotoxicity was used initially to detect the cytolytic potential of lymphocytes infiltrating the CNS irrespective of antigen specificity. Using TMEV-infected targets, H-2-restricted TMEV-specific CTLs of the CD8+ phenotype were demonstrated in lymphocytes from the CNS of susceptible and resistant mice, arguing against the hypothesis that the ability to generate CD8+ CTLs mediates resistance. In chronically infected SJL/J mice, TMEV-specific CTL activity was detected in the CNS as late as 226 days postinfection. These experiments demonstrate that virus-specific CTLs are present in the CNS during both acute and chronic TMEV infection. Anti-TMEV CTLs in the CNS of chronically infected SJL/J mice may play a role in demyelination through their ability to lyse TMEV-infected glial cells.     

Temperature-sensitive mutants isolated from hamster and canine cell lines persistently infected with Newcastle disease virus.

Evidence is presented which confirms that temperature-sensitive (ts) mutants with an RNA- phenotype are spontaneously selected in persistent infection of cell lines with Newcastle disease virus. Persistently infected BHK-21 cells, maintained since 1973, produce no interferon and are completely susceptible to vesicular stomatitis virus. Persistent infection of a canine kidney cell line (MDCK) terminated with destruction of all cells at about 100 days. Even under these conditions, a high proportion (33%) of RNA- temperature-sensitive mutants was present in the virus population 60 days after the infection was initiated.     

Effect of central nervous system acting drugs on brain cell replication in vitro

The role in the regulation of cell replication of the neurotransmitter compounds and the drugs which affect their balance was studied in vitro, using morphologically preserved brain slices. Compounds affecting noradrenergic, dopaminergic and serotoninergic neurotransmitter systems reduced the brain cell replication, measured in terms of the rate of [³H]thymidine incorporation into DNA. The reduction was dose dependent and half-maximal effects were obtained at about 1–5×10^–4 M concentrations. Although agonists and antagonists both showed similar inhibitory effect, the action of agonists was reversed by the appropriate antagonists. Also, the pharmacologically active isomers were several-fold more effective than the inactive isomers in forebrain slices, although with cerebellar slices the selectivity was less marked. Cyclic nucleotides and drugs affecting cholinergic neurotransmitter systems were apparently ineffective. Tese results indicate that monoamines may be involved in the regulation of cell replication in the developing brain. Furthermore, as some of the CNS acting drugs tested are suspected behavioural teratogens the present results suggest that the reported behavioural abnormalities in the offspring may be related, in part, to a chronologically determined interference with the formation of certain cell types.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures

Abnormal prion protein (PrP(Sc)) plays a central role in the transmission of prion diseases, but the molecular basis of prion strains with distinct biological characteristics remains to be elucidated. We analyzed the characteristics of prion disease by using mice inoculated with the Chandler and Fukuoka-1 strains propagated in a cultured mouse neuronal cell line, GT1-7, which is highly permissive to replication of the infectious agents. Strain-specific biological characteristics, including clinical manifestations, incubation period as related to the infectious unit, and pathological profiles, remained unchanged after passages in the cell cultures. We noted some differences in the biochemical aspects of PrP(Sc) between brain tissues and GT1-7 cells which were unlikely to affect the strain phenotypes. On the other hand, the proteinase K-resistant PrP core fragments derived from Fukuoka-1-infected tissues and cells were slightly larger than those from Chandler-infected versions. Moreover, Fukuoka-1 infection, but not Chandler infection, gave an extra fragment with a low molecular weight, approximately 13 kDa, in both brain tissues and GT1-7 cells. This cell culture model persistently infected with different strains will provide a new insight into the understanding of the molecular basis of prion diversity.     

Mechanisms involved in activity-dependent synapse formation in mammalian central nervous system cell cultures

Differences in neuronal activity produced by electrical stimulation lead to competition between synapses from sensory afferents converging on a common spinal cord neuron. Studies were performed on neurons dissociated from the mouse spinal cord and grown in culture dishes with three compartments. Synaptic efficacy from stimulated afferents was increased compared with unstimulated convergents, and the number of functional connections was increased by stimulation compared with control cultures. Blocking NMDA channel activation with 100 microM APV in medium containing 1.8 mM calcium inhibited this synaptic plasticity, but plasticity was not blocked by APV in medium in which the calcium concentration was elevated to 3 mM. These experiments support the view that electrical activity differentially influences processes that cause a persistent decrease in synaptic efficacy or lead to synapse elimination and those that increase synaptic strength or lead to synapse augmentation. We interpret our results in terms of a model in which these antagonistic mechanisms are both regulated via changes in calcium levels and second messengers that are modulated by electrical activity. A significant portion of the activity-related calcium influx relevant to synaptic plasticity passes through the NMDA channel, but other sources of calcium are involved. In particular, competitive elimination of synapses appears to occur during blockade of NMDA channels if the extracellular concentration of calcium is elevated moderately. The outcome of competition between the two calcium-dependent but antagonistic processes may depend either on their differential sensitivity to intracellular calcium concentration or separate specificities to NMDA and non-NMDA receptor-linked mechanisms.     

Characterization of mouse hepatitis virus-specific cytotoxic T cells derived from the central nervous system of mice infected with the JHM strain.

The cytotoxic T lymphocyte (CTL) activity of spleen cells from BALB/c (H-2d) mice immunized with the neurotropic JHM strain of mouse hepatitis virus (JHMV) was stimulated in vitro for 7 days. CTL were tested for recognition of target cells infected with either JHMV or vaccinia virus recombinants expressing the four virus structural proteins. Only target cells infected with either JHMV or the vaccinia virus recombinant expressing the JHMV nucleocapsid protein were recognized. Cytotoxic T cell lines were established by limiting dilution from the brains of mice undergoing acute demyelinating encephalomyelitis after infection with JHMV. Twenty of the 22 lines recognized JHMV-infected but not uninfected syngeneic target cells, indicating that they are specific for JHMV. All T-cell lines except one were CD8+. The specificity of the CTL lines was examined by using target cells infected with vaccinia virus recombinants expressing the JHMV nucleocapsid, spike, membrane, and hemagglutinin-esterase structural proteins. Seventeen lines recognized target cells expressing the nucleocapsid protein. Three of the JHMV-specific T-cell lines were unable to recognize target cells expressing any of the JHMV structural proteins, indicating that they are specific for an epitope of a nonstructural protein(s) of JHMV. These data indicate that the nucleocapsid protein induces an immunodominant CTL response. However, no CTL activity specific for the nucleocapsid protein could be detected in either the spleens or cervical lymph nodes of mice 4, 5, 6, or 7 days after intracranial infection, suggesting that the CTL response to JHMV infection within the central nervous system may be induced or expanded locally.     

Transgenic mouse model for central nervous system demyelination.

A common feature of demyelinating diseases such as multiple sclerosis in humans and experimental autoimmune encephalomyelitis in rodents is the marked elevation in the expression of the major histocompatibility complex (MHC) antigens in the involved sites. By specific targeting of a syngeneic MHC class I gene to oligodendrocytes, we have generated transgenic mice which not only exhibit severe involuntary tremors and develop tonic seizures but also show extensive demyelination in both the brain and the spinal cord. The fact that demyelination in these mice occurs in the absence of immune infiltration dismisses an autoimmune involvement but suggests that the MHC class I antigens play a direct role in inducing disease. Our findings lend support to the possibility that demyelinating diseases are induced by infectious agents such as viruses which can either directly activate MHC gene expression in oligodendroglia or indirectly activate expression through the release by reactive T cells of gamma interferon in the brain.     

Intracellular calcium recordings from isolated cells of the mammalian central nervous system

Measurements made with two different techniques of intracellular calcium levels from small isolated cells of the mammalian central nervous system are described and compared. Recordings in cultured mouse embryo spinal cord and dorsal root ganglion neurons, made with double-barrelled borosilicate Ca2+-selective microelectrodes yielded a mean Ca2+ level of 2.3 (SE +/- 0.54) microM for the lowest values recorded in 24 out of 46 cells. Intracellular Ca2+ dependence on membrane potential was apparent with levels of calcium greater than or equal to 4 microM (r = 0.371, n = 29). Both cyclic fluctuations induced by tetraethylammonium and an apparent increase in Ca2+ evoked by the depolarizing excitatory amino acid, L-aspartate, were observed. In contrast, estimates of intracellular Ca2+ obtained by spectrofluorimetry of suspensions of mouse embryo brain cells, loaded with the intracellular Ca-binding fluorescent probe, quin2 provided a approximately equal to 10-fold lower value, 152 (SE +/- 7) nM. This more closely resembles levels reported for large neurons where large-tip microelectrodes with greater sensitivity were used, and in spite of the heterogeneity of the cells this value is presumed to be a more accurate estimate of intraneuronal Ca2+ concentration. In these fluorescence studies KCl readily evoked increases in intracellular Ca2+ which could be blocked by verapamil and Cd2+ and were not induced in the absence of Ca2+. Increases were also produced by N-methyl-D-aspartate, but not by the kainate-like Lathyrus neurotoxin, L-3-oxalylamino-2-aminopropionic acid. These results provide preliminary evidence for both voltage-sensitive and receptor-activated Ca channels in embryonic brain cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-dependent poliovirus replication in the mouse central nervous system is determined by internal ribosome entry site-mediated translation

Mouse cells are not permissive for the replication of human rhinovirus type 2 (HRV2). To determine the role of the HRV2 internal ribosome entry site (IRES) in determining species specificity, a recombinant poliovirus (P1/HRV2) was constructed by substituting the poliovirus IRES with the IRES from HRV2. This recombinant virus replicated in all human and murine cell lines examined, demonstrating that the HRV2 IRES does not limit viral replication in transformed murine cells. P1/HRV2 replicated in the brain and spinal cord in neonatal but not adult mice transgenic for the poliovirus receptor, CD155. Passage of P1/HRV2 in mice led to selection of a virus that caused paralysis in neonatal mice. To determine the relationship between HRV2 IRES-mediated translation and replication of P1/HRV2 in mice, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The results demonstrate that the HRV2 IRES mediates translation in organs of neonatal but not adult mice. These findings show that HRV2 IRES-mediated translation is a determinant of virus replication in the murine brain and spinal cord and suggest that the IRES determines the species specificity of HRV2 infection.     

The development of isolated central nervous system preparations

1. Use of invertebrate isolated CNS preparations showed that identified neurons have distinct specific repeatable pharmacological properties. 2. Isolated CNS preparations have been developed from invertebrates to include fish, amphibian, reptile and mammal CNS preparations. 3. The isolated mammalian (mouse, hamster, rat, guinea pig) CNS preparation can remain alive for up to 72 hr and show cells with good resting potentials, action potentials; good functional pathways and tracts; and good responses to drug application. 4. Some possible future developments of isolated mammalian CNS preparations are described.