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A neuroattenuated variant bunyavirus, designated RFC/25B.5 (B.5), was selected by serial passage of a reassortant clone (RFC virus) of a California serogroup virus in BHK-21 cells, followed by plaque purification of that passaged stock. Based on its virulence index (ratio of PFU/50% lethal dose), clone B5 was over 40,000-fold less virulent than its unpassaged RFC parent after intracerebral (i.c.) inoculation into adult mice. Clone B.5 also exhibited markedly reduced neuroinvasiveness after subcutaneous injection into neonatal mice, although it retained its ability to replicate and kill suckling mice after i.c. injection. A murine neuroblastoma line (NA cells) can be used as an in vitro surrogate for the adult mouse brain, since clone B.5 replicated to at least 1,000-fold-lower titers in NA cells than did several neurovirulent California serogroup viruses. Clone B.5 replicated in BHK-21 cells at 37 degrees C to titers similar to those achieved by other California serogroup viruses but was temperature sensitive (ts) since its replication was markedly restricted at 38.9 degrees C. Ten ts revertant clones of B.5 virus were selected at 38.9 degrees C, and all of them lost their ts phenotype and regained the ability to replicate to high titer in NA cells and to kill adult mice after i.c. injection. Clone B.5 is the first described California serogroup virus which is truly attenuated after i.c. inoculation, and its availability will permit genetic analysis of bunyavirus neurovirulence.

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChR) expressed in chick heart are pharmacologically, immunologically, and biochemically distinct from mAChR expressed in mammalian heart. A chicken genomic clone encoding a mAChR whose deduced amino acid sequence is most homologous to the mammalian m4 receptor has been isolated. Northern blot analysis demonstrated that this gene is expressed in both chick heart and brain. The receptor encoded by this gene was expressed in stably transfected Chinese hamster ovary (CHO) and Y1 adrenal carcinoma cells in order to examine its ligand binding and functional properties. The receptor expressed in CHO and Y1 cells exhibits high affinity binding for the muscarinic antagonists quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the cloned chick m4 receptor exhibits pharmacological properties similar to those previously reported for the chick cardiac receptor. This m4 receptor was able to mediate both agonist-dependent inhibition of forskolin-stimulated cAMP accumulation and agonist-dependent stimulation of phosphoinositide metabolism when expressed in CHO cells. In contrast, when expressed in Y1 cells, the chick m4 receptor mediated agonist-dependent inhibition of forskolin-stimulated cAMP accumulation, but not stimulation of phosphoinositide metabolism. Thus, as with the mammalian cardiac (m2) receptor, the functional specificity of the chick cardiac receptor appears to be dependent on the cell type in which it is expressed.

The thyrotropin-releasing hormone receptor from rat brain was solubilized in a stable unbound form. The natural glycoside digitonin was the only detergent of a variety tested capable of solubilizing the active receptor. The digitonin-solubilized receptor exhibited binding kinetics for 3-[3H]methylhistidine thyrotropin-releasing hormone virtually identical to membrane preparations and responded with a similar order of potency to a series of thyrotropin-releasing hormone analogues that inhibit binding of the labeled ligand. Gel filtration analysis and sucrose density gradient centrifugation indicated a Stokes radius of 5.2 nm, a sedimentation coefficient of 11.2 s, and a corresponding calculated molecular weight of 244,000 for the detergent-receptor complex. The soluble receptor shows a gradual loss of binding activity over 48 to 72 hr when kept at 4 degrees C. However, the preparation may be frozen at -20 degrees C with no significant loss of activity.

The tottering mouse (tg/tg) is a single-locus mutant, phenotypically characterized by the development of epilepsy associated with distinct electroencephalographic abnormalities. Because of reported alterations in muscarinic receptor (mAChR) number in various seizure states, mAChR density was examined in discrete brain regions of tottering (tg/tg) and coisogenic wild-type (+/+) mice. Saturation binding experiments revealed a widespread decrease in membrane mAChR density in the CNS of adult tottering (tg/tg) mice as compared with age-matched control wild-type (+/+) mice. The decrease was most pronounced in the hippocampus, where tg/tg mice exhibited a 40-60% reduction in mAChR density with no change in the affinity of the receptor for antagonists or agonists. At postnatal day 10, before the reported onset of electroencephalographic abnormalities, 114 and 65% increases in mAChR density were observed in the tg/tg hippocampus and cortex, respectively. Following the development of seizure activity at postnatal day 22, mAChR density in the tg/tg hippocampus was reduced by 29%. No change in brain mAChR density was seen in adult heterozygotes (+/tg), which do not develop electroencephalographic or seizure abnormalities. These results indicate that the development of reduced mAChR number in the CNS of the tg/tg mouse is secondary to abnormal neuronal activity, providing further support for the hypothesis that membrane depolarization can cause a decrease in neuronal mAChR density.

Prolonged treatment of chick embryos in vivo with the muscarinic agonists oxotremorine or carbachol leads to dose- and time-dependent decreases in the number of brain muscarinic acetylcholine receptors (mAChR) as measured by the specific binding of the potent muscarinic ligand L-[3H]quinuclidinyl benzilate to brain membranes. Maximal doses of agonists reduced the number of mAChR as much as 44%. Maximal loss of mAChR occurs 4 hr after treatment, but can be prevented or totally reversed within 24 hr by blockade of agonist-receptor interactions with muscarinic antagonists. After sustained in vivo oxotremorine treatment, brain mAChR show a decreased apparent affinity for agonists owing to a decrease in the affinities of both the high- and low-affinity agonist binding sites.

The ultrastructure of visna, a slowly progressive menigo-encephalomyelitis of sheep, was studied in animals sacrificed one month after intracerebral inoculation of visna virus. The major pathological changes, representative of those seen during the first year after infection, consist of inflammation and minor focal destructive lesions of grey and white matter. The inflammatory infiltrates, both subependymal and perivascular as well as of the choroid plexus, were composed mainly of lymphocytes and macrophages with varying numbers of plasma cells. The demyelination seen was of the secondary or Wallerian type. There was no evidence of primary demyelination. Visna virions were not seen in any of the CNS material studied. The ultrastructural findings are compatible with the view that lesions in visna may be induced by a cell-mediated immune response. However, changes characteristic of an autoimmune reaction to myelin antigens were not observed.

Two Icelandic sheep with clinical signs of visna appearing 6-7 years after intracerebral infection with visna virus were killed, fixed by perfusion and the central nervous system lesions examined by light and electron microscopy. Both sheep showed similar pathological changes. In the brain there was a severe periventricular inflammatory process with small foci of liquefaction necrosis and scattered small granulomas. In some areas of inflammation there was evidence of primary demyelination but it was not prominent. In the spinal cord there were focal plaques of primary demyelination. At the ultrastructural level the spinal cord lesions showed unambiguous primary demyelination with many naked axons; various stages of remyelination with peripheral type of myelin were also common. These observations indicate that the CNS lesions of visna, as seen in Icelandic sheep, fall into two categories: (a) an inflammatory process which often begins within weeks of infection and which occurs in the majority of infected animals in the absence of clinical paresis; and (b) focal demyelinating lesions of the spinal cord which are seen in sheep with clinical paresis but are uncommon in animals prior to onset of clinical signs. Both types of lesions may coexist.