Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM-strain): studies in vivo and in vitro.

The pathogenicity and cell tropism of mouse hepatitis virus (MHV-JHM-strain) in the developing mouse (Balb/c) and rat (Wistar and Lewis) brain were analysed. Intracranial infection of Balb/c mice at postnatal day 5 induced a lethal encephalitis in all animals. Of Wistar rats infected at day 2 or 5 after birth, 30 to 70%, respectively, survived. The distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus-infected cell type throughout the central nervous system. More than 75% of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. The cell tropism of MHV-JHM was examined further in virus-infected glial cell cultures derived from brains of rats or mice. In the glial cultures derived from Wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or Lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. Infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. Infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. These phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus-induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. The present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of MHV-JHM.

Cellular location of glutamine synthetase and lactate dehydrogenase in oligodendrocyte-enriched cultures from rat brain.

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.

Fatty acid acylation of viral proteins in murine hepatitis virus-infected cells. Brief report.

The fatty acid acylation of the cell-associated virus-specific proteins of mouse hepatitis virus (A 59-strain) was studied. 3H-palmitate label was associated with E2, one of the two virion glycoproteins and its intracellular precursor gp 150. A 110 K protein, the unglycosylated apoprotein of gp 150, accumulated by tunicamycin treatment, also incorporated radiolabeled palmitic acid. The addition of fatty acid to the MHV-A 59 E 2 protein is therefore not dependent on glycosylation.

The pestiviruses: where do they belong?

In this introduction to the meeting, the author specifies the place of pestiviruses among RNA viruses and briefly summarizes the main characteristics from this genus.

Equine arteritis virus-induced polypeptide synthesis.

Intracellular virus-specific proteins induced by equine arteritis virus (EAV) have been compared with in vitro translation products of virion and intracellular EAV RNAs. In infected BHK-21 cells, the two major virion proteins (C and E1) and polypeptides with mol. wt. of 60,000 (p60), 42,000 (p42) and 30,000 (p30) were found. There were no indications that the viral proteins were processed from a larger precursor as shown by pulse-chase, amino acid analogue and protease inhibitor experiments. The six polyadenylated RNAs that occur in EAV-infected cells were isolated and translated in an mRNA-dependent reticulocyte cell-free system. Translation of RNA6 resulted in the appearance of a product having the mol. wt. (14,000) of the nucleocapsid protein (C). EAV genomic RNA was translated into proteins of mol. wt. 30,000 and 200,000, while RNA1, the intracellular homologue of genomic RNA only encoded p30. The absence of large precursor molecules in infected cells and the results from the in vitro translation experiments both suggest that at least some of the proteins are primary translation products.

Intracellular equine arteritis virus (EAV)-specific RNAs contain common sequences.

Equine arteritis virus (EAV) is a nonarthropod-borne togavirus. Six virus-specific RNA species have been found in EAV-infected cells having the following molecular weights: 4.3 X 10(6) (RNA1), 1.3 X 10(6) (RNA2), 0.9 X 10(6) (RNA3), 0.7 X 10(6) (RNA4), 0.3 X 10(6) (RNA5), and 0.2 X 10(6) (RNA6). RNA1 comigrates with the viral genome (M. F. Van Berlo, M. C. Horzinek, and B. A. M. Van der Zeijst, 1982, Virology 118, 345-352). All RNAs hybridized with a radio-labeled cDNA probe representing RNA6, indicating that they contain common sequences. To study this homology in more detail, RNase T1 oligonucleotide fingerprinting of the RNAs was undertaken. This confirmed the presence of common sequences and showed more specifically that the intracellular viral RNAs form a nested set. The number of oligonucleotides in RNA1, however, is only one-third of the expected value. In all aspects studied the replication mechanism of EAV differs from that of other known positive-stranded RNA viruses.

Restricted replication of mouse hepatitis virus A59 in primary mouse brain astrocytes correlates with reduced pathogenicity.

Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) are drastically attenuated in their pathogenic properties. Intracerebral inoculation of mice with 10(5) PFU of mutant ts342 results in prolonged infection of the central nervous system, whereas 100 PFU of wild-type virus are lethal (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). In the Sac(-) cell line ts342 grows as well at 37 degrees C (the body temperature of mice) as at 31 degrees C (the permissive temperature). There is, however, a difference in primary cultures of mouse brain astrocytes. After infection with ts342, astrocytes produced low levels of infectious virus (5.2 +/- 3.7%) compared with virus yields after infection with wild-type virus. The fraction of wild-type virus- and ts342-infected cells was similar. Electron microscopy showed in wild-type virus-infected cells abundant virions in smooth vesicles usually closely associated with a well-developed Golgi apparatus. In mutant-infected cells no mature ts342 virus particles were found. There was no difference between ts342 and wild-type virus regarding the intracellular virus-specific RNAs. In ts342-infected cells the viral glycoproteins E2 and E1 were not detectable or were barely detectable. Either the mRNAs for the glycoproteins are not translated or the proteins are rapidly broken down. Revertants of ts342 were isolated. They grew as well as wild-type virus in astrocytes, indicating that they apparently produced sufficient amounts of E2 and E1, the ts defect itself rather than a second site mutation is responsible for the defect in replication, and the ts defect acts in unison with host-cell factors. The revertants also regained the lethal properties of wild-type virus.

Changes in the circadian rhythmicity of hydroxyindole-O-methyl transferase (HIOMT) activity in the synthesis of 5-methoxyindoles in the pineal gland of 28 day old male Wistar rats exposed to white, red and green light.

Hydroxyindole-O-methyl transferase activity for the synthesis of 5-methoxyindoles was investigated in the pineal gland of 28 day old male Wistar rats after exposure to white, red and green light for 12 hours daily. It could be demonstrated that, in comparison to white light, red light causes a shift of HIOMT activity for the synthesis of melatonin/5-methoxytryptophol towards an earlier period being highest between 12 and 16 hours. The synthesis of 5-methoxytryptamine and of 5-methoxytryptophan is shifted to 16 hours, while the synthesis of 5-methoxyindole-3-acetic acid, which normally peaks at 16 hours, is increased at this same hour. Green light shifts HIOMT activity for the synthesis of melatonin/5-method being highest between 12 and 16 hours. The synthesis of 5-methoxytryptamine and of 5-methoxytryptophan is shifted to 16 hours, while the synthesis of 5-methoxyindole-3-acetic acid, which normally peaks at 16 hours, is increased at this same hour. Green light shifts HIOMT activity for the synthesis of melatonin/5-method being highest between 12 and 16 hours. The synthesis of 5-methoxytryptamine and of 5-methoxytryptophan is shifted to 16 hours, while the synthesis of 5-methoxyindole-3-acetic acid, which normally peaks at 16 hours, is increased at this same hour. Green light shifts HIOMT activity for the synthesis of melatonin/5-methoxytryptophol to a later period, showing a peak at 24 hours. The synthesis of 5-methoxytryptamine is significantly increased at 24 hours. An increase is also observed in the synthesis of 5-methoxyindole-3-acetic acid at 12 and at 4 hours, times at which this synthesis is also maximal using white light, whereas the synthesis of 5-methoxytryptophan is generally decreased. A possible relationship between the present results and those obtained after exposure to different wavelengths of light on N-acetyltransferase activity, the effect of pterins on HIOMT activity and the effect of different wavelengths on gonadal growth are discussed.

Temperature-sensitive mutants of equine arteritis virus.

Seventeen temperature-sensitive mutants of equine arteritis virus, a nonarthropod-borne togavirus, have been isolated. 5-Fluorouracil, o-methylhydroxylamine and ethyl methanesulphonate were used as mutagens. The mutants were characterized by their ability to synthesize virus RNA and virus proteins at the permissive (35 degrees C) and restrictive temperature (40 degrees C) using autoradiography of cells labelled with 3H-uridine in the presence of actinomycin D and immunofluorescence respectively. Among the mutants, four were unable to synthesize virus RNA and virus proteins at 40 degrees C (RNA-/protein-). The other mutants were RNA-/protein+ (3); RNA +/-/protein- (2); RNA+/protein+ (6) and RNA+/protein- (1).

Estimation of the methylating capacity of the pineal gland. With special reference to indole metabolism.

In order to obtain more information on the methylating capacity of the pineal gland, a method determining the formation of different 5-methoxyindoles in the pineal gland was developed. The method depends on measuring the incorporation of labelled methyl groups into the various hydroxyindoles present in the pineal gland, after incorporation of pineal tissue with labelled S-adenosyl methionine. Hydroxyindoles were not added to the incubation medium. After incubation thin-layer chromatography was performed with pineal tissue together with the incubation medium; the spots were scraped and counted.