Mechanisms involved in natural and experimental neuropathogenicity of influenza viruses: evidence and speculation.
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Animals↗
Biomedical subjects
G L Bradshaw
Publications and source records attributed to G L Bradshaw.
Encoding effects of remindings.
Understanding requires prior knowledge. Most theories and empirical work on understanding have focused on the use of general prior knowledge. In the experiments presented here, we examined the role of specific prior episodes in understanding. Subjects read ambiguous stories that had some superficial cue to make them think back to an earlier story. Experiment 1 shows that a superficial cue to an earlier story affects how the new story is interpreted. Experiment 2, in which a reading-time measure was used, provides evidence that this effect of the earlier story occurs during the initial understanding of the new story. We discuss the implications of these results for theories of understanding and the use of remindings in analogy.
Adult↗
Replication of H1N1 influenza viruses in cultured mouse embryo brain cells: virus strain and cell differentiation affect synthesis of proteins encoded in RNA segments 7 and 8 and efficiency of mRNA splicing.
The aims of these studies are (1) to determine whether, and by what mechanism(s), underexpression of M1 and/or NS1 protein restricts replication and cytopathogenicity in mouse brain cells of human influenza viruses which are closely related to the neurovirulent WSN variant but not selected for the neurovirulent phenotype; (2) to learn, ultimately, whether similarly restricted replication in natural infections might be enough to cause direct or indirect, immunologically mediated, neuropathology. On the basis of immunostaining, we have suggested that, in "aged" mouse embryo brain (MEB) cell cultures infected with A/PR/8/34 (PR8) or A/WS/33 (WS), M1 protein expression is restricted mainly in mature astrocytes (the dominant cell type in such cultures), but not in mature oligodendrocytes or neurons. Here we show that amounts of radiolabeled M1 protein in lysates of MEB cultures infected with PR8, WS, or WSN differ in proportion to previously reported single-cycle yields of trypsin-activated infectious virions. Low or undetectable cell-associated M1 does not reflect accelerated degradation, but tends to be accompanied by increased M2 protein (a product of spliced mRNA7). Radiolabeled NS1 is reduced, NS2 relatively increased, in "aged" MEB cultures infected at low m.o.i. with PR8, at high m.o.i. with WS as well, but not with WSN. In contrast, actively dividing and differentiating astrocyte-enriched or "young" MEB cultures tend to produce greatly increased amounts of NS2 even though NS1 may be at "normal" levels, both relative to those in similarly infected CEF cultures. We show, in extension of comparative studies by others on permissive and abortive FPV-infected cell systems, that virus strain-, cell type-, and perhaps differentiation-dependent variations in efficiency of mRNA 7 and 8 transcription and/or splicing are primary factors controlling variable expression of M and NS proteins in mouse brain cell cultures.
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Role of hemagglutinin cleavage and expression of M1 protein in replication of A/WS/33, A/PR/8/34, and WSN influenza viruses in mouse brain.
The combined presence of WSN gene segments 6 (neuraminidase), 7 (M1 and M2), and 8 (NS1 and NS2) in reassortants of WSN with A/Aichi/2/68 (H3N2) has been found by others to be necessary for full expression of neurovirulence in mice. We are examining the expression of the analogous three gene segments in brains of mice after intracerebral infection with non-neuroadapted strains A/WS/33 (WS) (from which WSN was derived) and A/PR/8/34 (PR8). Our aim is to determine possible mechanisms by which one or more of the five gene products may restrict replication of these strains in mouse brain cells to a single cycle, yielding noninfectious hemagglutinating particles (incomplete growth cycle). We found that minority subsets of such particles did produce plaques, provided they were activated by trypsin (analogous to other abortive systems producing virions with uncleaved HA), a step obviated for some WSN virions by indirect promotion of hemagglutinin cleavage by the neuraminidase of that strain. The percentage of such potentially infectious virions, relative to total hemagglutinating particles, was significantly lower in WS- or PR8-infected than in WSN-infected brains, suggesting possible defects in synthesis or function of M1 protein in the former. Cells in immunostained sections and appropriate bands in Western blots (immunoblots) of viral proteins electrophoretically separated from lysates of PR8-infected brains reacted with antibody to nucleoprotein but not to M1 protein. Either method revealed the presence of both proteins in WSN-infected brains. In contrast, Western blot analyses of particles concentrated from PR8-, WS-, or WSN-infected brains by hemadsorption, elution, and pelleting did reveal NP and M1 bands with comparable relative peroxidase-antiperoxidase staining intensities. The findings suggest that availability of M1 protein is a factor influencing the extent or rate of assembly of potentially infectious (i.e., trypsin-activated) progeny virions in mouse brains and that in this respect the two non-neurovirulent strains differ from WSN quantitatively rather than qualitatively.
Animals↗
Effects of cell differentiation on replication of A/WS/33, WSN, and A/PR/8/34 influenza viruses in mouse brain cell cultures: biological and immunological characterization of products.
The responses of mouse embryo brain (MEB) cell cultures and of Madin-Darby canine kidney cells and chicken embryo fibroblasts to infection with A/PR/8/34 (PR8), A/WS/33 (WS), or the neurovirulent WSN variant were compared in terms of (i) single-cycle yields of hemagglutinating and associated neuraminidase (NA) activities and plaque-forming particles, the latter with or without trypsin activation [PFU(TR++) or PFU(TR--), respectively], and (ii) expression of nucleoprotein (NP), M1, and NS1 protein, determined for specific cell types by immunostaining, for whole culture lysates by Western blot analysis of NP and M1. Primary MEB cultures grown in serum-enriched medium were infected after 6 days (young), when none of the cells reacted specifically and exclusively with any of the nerve cell marker antibodies used, or after greater than or equal to 21 days (aged), when astrocytes (the predominant cell type), neurons, and oligodendrocytes were morphologically and immunologically mature. Secondary astrocyte-enriched cultures were used when they contained 90 to 99% of their cells as astrocytes at an early stage of differentiation. By all criteria, young MEB cultures were only marginally less permissive for each of the three viruses than were chicken embryo fibroblasts or Madin-Darby canine kidney cells. Aged MEB cultures, by comparison, produced undiminished NP, hemagglutinin, and neuraminidase, but yields of PFU(TR++) and expression of M1 protein (relative to NP) were reduced for all three viruses, most for PR8 and least for WSN; relative reduction of NS1 protein was demonstrable only in PR8-infected aged cultures. Immunostaining revealed low levels of M1 and NS1 expression only in astrocytes, not in oligodendrocytes and neurons. In PR8-infected mature astrocytes, NP accumulated in the nucleus; it persisted in some cells for at least 8 weeks after infection. The presence of NP did not seem to interfere with cell division. Secondary MEB cultures containing 90 to 99% immature astrocytes were less restricted than were aged primary cultures. Thus, it appears that reduced permissivity of nerve cell cultures, as measured in this study, is most closely correlated with advancing differentiation and maturity of astroglial cells. Assembled virions, including those that score as PFU(TR++) in restricted cultures (e.g., PR8-infected aged MEB), may be mainly products of mature oligodendrocytes and neurons.
Animals↗
The growth requirements of BHK-21 cells in serum-free culture.
A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm2, this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.
Animals↗
Supplementary factors required for serum-free culture of rat kidney cells of line NRK-49F.
Factors required as supplements to basal tissue culture medium for the multiplication of cells of the cloned rat fibroblast line called normal rat kidney 49F (NRK-49F) were identified as epidermal growth factor, fibronectin, insulin, and retinoic acid. The requirement for fibronectin was manifested on a clean glass surface but not on the polystyrene plastic surface tested. This set of required factors differs substantially from the factor sets required by the Madin-Darby canine kidney (MDCK) and LLC-PK1 pig kidney lines of epithelial cells and the baby hamster kidney 21 (BHK-21) line of fibroblasts. The serum-free medium supplemented with the four factors supported rapid growth of NRK-49F cells when the initial cell population density was about 8,000 cells/cm2 or greater. At lower initial densities, cell multiplication was markedly increased by adding serum-free medium that had been conditioned by NRK-49F cells. Cell growth rate in the defined serum-free medium stayed high through two serial passages but declined in the third serial passage unless the cell-conditioned medium was added.
Animals↗
Polyoma virus transformation of rat kidney fibroblasts results in loss of requirement for insulin and retinoic acid.
Cloned cells of the rat kidney fibroblast line designated NRK-49F, which requires epidermal growth factor (EGF), fibronectin, insulin, and retinoic acid for rapid multiplication in serum-free culture, were transformed by polyoma virus. Cells from two independent transformation events were isolated and cloned, as were cells from two corresponding control untransformed cultures not treated with virus. Tests in serum-free culture showed that the two transformed subclones required EGF and fibronectin but not insulin or retinoic acid for rapid multiplication, whereas the two control subclones retained the requirements for all four factors. Although EGF at 10 to 50 ng/ml stimulated the multiplication of all four subclones, EGF at 500 ng/ml strongly inhibited multiplication of the two transformed subclones but not the two control subclones.
Animals↗
Inactivation of transforming DNA by a transient product of the interaction of Cu2+ and hydroquinone.
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Bacillus subtilis↗