Characterization of temperature-sensitive mutants mouse hepatitis virus type 3.
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Animals↗
Biomedical subjects
A Kirn
Publications and source records attributed to A Kirn.
Increased susceptibility of mice to MHV 3 infection induced by hypercholesterolemic diet: impairment of Kupffer cell function.
Nutritionally induced hypercholesterolemia in A/J mice causes susceptibility to Mouse Hepatitis type 3 (MHV 3), whereas normal A/J mice are fully resistant. A/J mice fed with a hypercholesterolemic diet for 15 to 60 days develop 5 to 7 days after MHV 3 infection an acute hepatitis which led to high levels of mortality. A direct relationship was found between the high levels of plasma and hepatic cholesterol and the mortality. In attempting to define the dietary-induced physiological changes which led to the loss of resistance, the Kupffer cells were shown to exhibit an impairment of functions in their ability to become activated by LPS in order to take up C3-coated IgM opsonized sheep red blood cells, C3(IgM)SRBC, or 3H-thymidine Escherichia coli, and the susceptibility to interferon (IFN) for the induction of an antiviral state. Peritoneal macrophages which were studied in comparison with the Kupffer cells showed no impaired functions. The findings presented here indicate an inhibition of host resistance, by nutritional hypercholesterolemia, of A/J mice to MHV 3 infection and that, at least one site of impairment occurs specifically at the stage of Kupffer cells function.
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The Kupffer cell is the first target in ricin-induced hepatitis.
The first target of ricin in the liver appears to be the Kupffer cells which are heavily damaged as early as four hours after the intravenous inoculation of 6 LD100 into mice. At that time, the only endothelial cell damage is constituted by more or less extended interruptions of the fenestrated cytoplasm. Hepatocyte injury affects the endoplasmic reticulum, the glycogen, the mitochondria as well as the plasmic membrane; however, it never results in cytolysis or complete necrosis. The intravascular coagulation observed may be induced either by the endotoxins, which are no longer cleared by the Kupffer cells, or as a result of the loss of the sinusoidal lining with the ensuing platelet activation.
Alanine Transaminase↗
Restriction of frog virus 3 polypeptide synthesis to immediate early and delayed early species by supraoptimal temperatures.
Multiplication of frog virus 3 (FV 3) occurs in mammalian cells provided they are incubated at temperatures lower than 33 degrees. The expression of the viral genome at supraoptimal temperatures was followed by analyzing the polypeptides produced in CHO-infected cells and comparing with those obtained under restrictive conditions provoked by amino acid analogs or metabolic inhibitors. Late polypeptides were not detected at 33 degrees and the number of delayed early species decreased gradually with increasing temperatures consequently the synthesis of all delayed early proteins was not turned on in response to a unique event. At 37 degrees the synthesis was limited to the immediate early species, i.e., the proteins synthesized after cycloheximide reversal. Temperature shift experiments suggested that delayed early genes remained untranscribed at 37 degrees. Thus, incubation of FV 3-infected mammalian cells at 37 degrees provides a unique way of limiting viral synthesis to immediate early proteins without the side-effect provoked by inhibitors.
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Thermosensitivity of frog virus 3 genome expression: defect in early transcription.
The influence of temperature on the transcription of the frog virus 3 genome was studied in CHO cells infected both at 29 and at 37 degrees, the nonpermissive temperature for virus multiplication. It was definitely established that late genes were not transcribed at 37 degrees. Although immediate early genes were expressed at 37 degrees, their transcription was altered but there was no sequestration of mRNAs in the nucleus which could impair their translation; these viral mRNAs were also efficiently translated in vitro. These results indicate that an immediate early viral protein involved in the transcription of delayed early genes is likely to be thermosensitive. Furthermore, one event taking place at the very beginning of the infection, possibly related to the activity of a viral structural component, facilitates the transcription of immediate early genes at 29 degrees and this step is partially impaired at 37 degrees.
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Interaction of mistletoe lectin I with Kupffer cells and endothelial cells of mouse liver.
The in vitro-formation of blebs by endothelial and Kupffer cells of the mouse liver after treatment with the toxic lectin I from mistletoe are demonstrated by means of scanning electron microscopy. The interaction of toxic lectins with nonparenchymal liver cells can be of importance concerning the elimination of these lectins in vivo.
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Localization and sequence of a vaccinia virus gene required for multiplication in human cells.
A vaccinia virus host range mutant (hr mutant) deleted of 18 kilobase pairs at the left end of the genome has been employed to precisely map a viral gene required for multiplication in human cells. DNA fragments from the wild-type virus were inserted into the thymidine kinase gene of mutant virus by means of in vivo homologous recombination, and the recombinants obtained were screened for their ability to multiply in human cells. A short sequence, 855 base pairs long, overlapping the HindIII M and K fragments was able to restore a normal host range on the mutant virus. A single long open reading frame that could encode a polypeptide of 32.5 kDa was found in the nucleotide sequence of the host range gene. The direction of transcription and the length of the open reading frame are in excellent agreement with previous mapping of mRNAs within this region of the genome. In vitro translation of infected cell early mRNA, selected by hybridization to the host range gene, yielded a prominent polypeptide product whose size (29 kDa) was close to that expected from the predicted amino acid sequence. The phenotype of the hr mutant suggests that the host range gene plays a role in maintaining a high level of protein synthesis in human cells. It may behave positively by complementing the lack of an analogous cellular activity or negatively by antagonizing a cell function that inhibits viral multiplication.
Amino Acid Sequence↗
C3-mediated phagocytosis induced in murine Kupffer cells by "in vitro" activation with endotoxin.
Binding and phagocytosis of red blood cells by Kupffer cells, mediated by C3 receptors, have been studied by incubating sheep red blood cells opsonized with C3, and Kupffer cells isolated from rat and mouse livers. Sheep red blood cell-binding was followed by internalization and phagocytosis only after preincubation of Kupffer cells with bacterial endotoxin. In both species of cells there was a direct relationship between the value of the phagocytic index and the amount of endotoxin needed to stimulate the cells. Increased phagocytosis was related to an increase in the number of phagocytosing cells; it did not depend on an increase in the number of red blood cells internalized per Kupffer cell. C3-mediated phagocytosis, together with the intrinsic and extrinsic antiviral activities, and the synthesis of interferon which take place in activated cells may play an important role in non-specific immunity.
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In vitro antiviral properties of endotoxin-activated rat Kupffer cells.
Kupffer cells isolated from the liver of normal rats were checked for their antiviral activities. The intrinsic antiviral effect against vaccinia virus was high whether the cells were activated in vitro with endotoxin or not. The expression of the extrinsic antiviral activity measured by mixing isolated Kupffer cells with vaccinia-virus-infected target cells was remarkably enhanced by prior treatment of the Kupffer cells with LPS. Interferon was shown to be not responsible for that inhibitory activity. Different experimental data suggest that the virus replication in the target cells is blocked at a late stage in the replication cycle.
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Rapid diagnosis of acute mumps infection by a direct immunoglobulin M antibody capture enzyme immunoassay with labeled antigen.
A new immunoglobulin M (IgM) antibody capture enzyme immunoassay with peroxidase-labeled mumps antigen (dMACEIA) is described, and its suitability for practical diagnosis of acute mumps infection is evaluated. All 54 patients with proven mumps infection that were tested showed mumps-specific IgM antibodies. On the other hand, no specific IgM antibodies were present in 16 cases of suspected mumps that could not be confirmed by classical complement fixation serology, and IgM mumps virus antibodies could be detected neither in the sera of 100 healthy individuals nor in those of 16 patients positive for rheumatoid factor. In all, 22 children with acute respiratory illness caused by parainfluenza virus and 44 patients with infections due to other viruses showed no IgM response in mumps dMACEIA. The particular characteristic in which complement fixation antibodies against mumps nucleocapsids appear before and disappear earlier than antibodies to the enveloped mumps virus could not be demonstrated in the dMACEIA. In an extensive epidemic of mumps virus infection, the dMACEIA gave a clear diagnosis of mumps infection in 200 out of 371 suspected cases. By day 2 of the illness, 71% of the patients had detectable IgM, and by day 3, all of them had detectable IgM. In 99% of the cases, dMACEIA gave a positive result in the first available serum specimens, most of which were negative for complement fixation antibodies. A positive but only moderate correlation was thus observed between the two serological procedures. IgM antibodies persisted for at least 6 weeks. The dMACEIA, performed in 3 h, offers a reliable, simple, and rapid alternative to routine methods for detection of acute mumps infection.
Adult↗
Inhibition of mouse hepatitis virus type 3 multiplication in activated Kupffer cells.
Mouse Hepatitis Virus type 3 (MHV3) multiplication in isolated murine Kupffer cells was partially inhibited by pretreatment of the cells with lipopolysaccharide (LPS). Supernatants of LPS-treated Kupffer cells contained large amounts of interferon. Inhibition of MHV3 multiplication was also observed when normal Kupffer cells were cultivated in a medium containing supernatants of LPS-treated Kupffer cells. In addition to the antiviral effect of the released interferon, there seems to be another effect of LPS, since Kupffer cells cultured in medium containing anti-interferon alpha beta antibodies were partially activated by LPS to inhibit MHV3 replication. The in vivo consequences of these effects for the local immunity of the liver against MHV3 infection are discussed.
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In vitro cytostatic properties of unactivated rat Kupffer cells.
Kupffer cells from the liver of normal rats were checked for their natural cytostatic capabilities using an in vitro target cell growth inhibition assay. A strong cytostatic effect was observed on an human tumor cell line and was shown to be exerted on various transformed or normal target cells with only small differences in their susceptibility. The inhibition of target cell proliferation was shown to depend on the effector/target cell ratio. Different experimental data suggest that an intimate membranal contact between Kupffer cells and target cells is required.
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Kupffer and endothelial liver cell damage renders A/J mice susceptible to mouse hepatitis virus type 3.
Damage to the Kupffer and endothelial cells of the liver sinusoids induced by the administration of sublethal doses of frog virus 3 (FV 3) renders A/J mice which are genetically resistant to mouse hepatitis virus type 3 (MHV 3) highly susceptible to this virus. Liver histopathology of these animals revealed typical necrotic foci containing MHV 3-specific antigens. FV 3-pretreated mice, after MHV 3 infection, showed higher levels of serum transaminase (GPT) than controls, and MHV 3 replicated more rapidly and to higher titres. Our results bear out the important role of the liver sinusoidal lining in protecting against hepatocyte infection and its direct involvement in the resistance of A/J mice to MHV 3 infection.
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Probable role of endogenous endotoxins in hepatocytolysis during murine hepatitis caused by frog virus 3.
Three new observations bear out the role of endogenous endotoxins in the pathogenesis of murine hepatitis caused by frog virus 3. First, the LD50 of endotoxin is 20 times lower in mice pretreated for 2.5 hr with a sublethal dose of frog virus 3 than in untreated mice. Animals inoculated with one sublethal dose of lipopolysaccharide 2.5 hr after injection of one sublethal dose of virus die, all having developed extensive hepatocellular necrosis. This hypersensitivity varies according to the intensity of virus-induced destruction of Kupffer cells, which are the intrahepatic target of the virus. Second, mortality is significantly lower and the interval between infection and death longer in axenic mice, which are largely protected from portal endotoxemia. Third, the impairment of some biologic activities of endotoxin (through treatment with polymyxin B or indomethacin, for example) protects mice against hepatic damage and death. Likewise, mice rendered tolerant to endotoxins, and C3H/HeJ mice, which are genetically resistant to endotoxins, survive challenge with frog virus 3 and are refractory with regard to hepatocytolysis . These findings suggest that, in hepatitis caused by frog virus 3, endogenous endotoxins are responsible for extensive hepatocytolysis since virus-induced damage to the hepatic reticuloendothelial system prevents their detoxification.
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Fate of frog virus 3 DNA replicated in the nucleus of arginine-deprived CHO cells.
In a productive infection, frog virus 3 (FV 3) DNA was synthesized in both the cell nucleus and cytoplasm. The infection was aborted in arginine-starved Chinese hamster ovary cells and viral DNA replication was then restricted to the nuclear compartment. It has been demonstrated that the newly synthesized FV 3 DNA present in the nucleus is of genomic size. After the addition of arginine, this DNA is transferred into the cytoplasm and can be encapsidated. The formation of infectious particles occurred even if an inhibitor of DNA synthesis was added simultaneously with arginine. Although the synthesis of early FV 3 polypeptides and their intracellular distribution were comparable in the presence or in the absence of arginine, the production of late species was greatly reduced by arginine deprivation; one of these late proteins must be involved in the passage from the nuclear to the cytoplasmic phase of FV 3 DNA replication. This system made it possible to carry out an independent analysis of the nuclear events that comprise the first stage of FV 3 multiplication.
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Interaction between mouse hepatitis viruses and primary cultures of Kupffer and endothelial liver cells from resistant and susceptible inbred mouse strains.
Infection with mouse hepatitis virus type 3 (MHV 3) of primary cultures of Kupffer and endothelial cells from the livers of resistant (A/J) and susceptible (BALB/c) mice was followed by the appearance of typical syncytia and comparable yields of virus. Using cells from A/J mice there was a delay of about 24 to 36 h in the appearance of the first particles detected by electron microscopy, the maximum viral titre and the number and size of syncytia. The partial resistance to MHV 3 multiplication expressed by cells from A/J mice was also observed when they infected with a strain of low virulence (JHM strain). The delay in MHV multiplication found in the sinusoidal liver cells, mainly in the endothelial cells, may be an important factor in their resistance, by allowing time for the local and systemic responses to clear the infective particles as well as in determining their degree of hepatotropism.
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Membrane lipids of hepatocytes, Kupffer cells and endothelial cells.
The membrane lipid composition of isolated hepatocytes, Kupffer cells and endothelial cells was determined. The hepatocytes are characterized by a lower quantity of gangliosides, cholesterol, sphingomyelin and a reduced cholesterol/phospholipid molar ratio when compared to the two other liver cell types. The main gangliosides of Kupffer and endothelial cells are the GM3 species, and those of hepatocytes are of the polysialogangliosides species.
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Sodium dodecyl sulfate-mediated transfer of electrophoretically separated DNA-binding proteins.
A modified procedure for the transfer of electrophoretically-separated proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels onto nitrocellulose filters has been developed. During the diffusion mediated transfer, the SDS-protein complexes were maintained and SDS was added to the buffer. This increases the number of polypeptide species bound to the filter thereby giving an accurate replica of the original gel pattern. The immobilization in the gel of certain polypeptides characterized as DNA-binding proteins, which is observed when SDS is eliminated prior to blotting is avoided. The molecules blotted in the presence of SDS remain immunoreactive and able to bind DNA.
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