Temperature sensitivity of two different steps in the viral life cycle of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) multiplication is totally blocked by incubation of infected cells at 41 degrees. This inhibition does not take place with a thermoresistant strain of FIV, designated m41, indicating the role played by the viral genome in temperature sensitivity. We have investigated the steps in the life cycle of wild-type FIV that are thermosensitive and found that they depend on the host cells infected. In CrFK cells, FIV replication was inhibited after the penetration step at 41 degrees. Synthesis of viral RNA and DNA was barely detectable and no viral antigen appeared in the extracellular medium. Nevertheless, viral multiplication resumed on incubation at 37 degrees, suggesting a state of latency at the elevated temperature. In peripheral blood mononuclear cells (PBMCs), the FIV cycle was inhibited at 41 degrees after the synthesis of viral DNA. Several viral mRNAs failed to appear as fully spliced products and no viral antigen was found in the extracellular medium. As in CrFK cells, viral multiplication occurred in PBMCs after a shift to the permissive temperature. These results suggest that at least two steps in the viral life cycle are sensitive to 41 degrees and that two different viral functions of the thermoresistant mutant m41 are modified to overcome temperature sensitivity.

A thermoresistant strain of feline immunodeficiency virus (FIV) with an altered cytopathic effect and a restricted cell tropism.

A thermoresistant strain, designated m41, of feline immunodeficiency virus (FIV) was selected after 31 successive passages of chronically infected IRC4 cells at 41 degrees C. The wild-type virus (wt) which served as a control was cultivated the same number of times at 37 degrees C. In Crandell feline kidney cells (CrFK), the replication of m41 was similar at 37 degrees C and 41 degrees C, whereas wt multiplied only at 37 degrees C. Furthermore, m41 was more resistant than the wt strain at temperatures ranging from 37 to 56 degrees C. Syncytia formation was observed with m41 when the CrFK were incubated at 41 degrees C whereas neither m41 nor wt produced syncytia at 37 degrees C. The level of replication of wt and m41 on feline lymphoid primary cells at 37 degrees C was similar. In contrast to wt, m41 was unable to infect bone marrow macrophages. Since one or several mutations in the envelope (env) gene could be involved in changes of cell fusion properties and of cellular tropism, the nucleotide sequence of the env gene derived from wt and m41 respectively was determined. Ten mutations were found in the env gene of m41, thus leading to 9 amino acid modifications in the envelope glycoproteins. These results suggest that structural modifications of the viral envelope proteins are prerequisites for the replication of a thermoresistant FIV strain at elevated temperature and are correlated with the newly acquired viral phenotype.

Evidence of feline immunodeficiency virus replication in cultured Kupffer cells.

OBJECTIVE: To determine if cultured feline Kupffer cells (KC) are as permissive for feline immunodeficiency virus (FIV) as cultured human liver macrophages are for HIV. Two types of infection likely to be relevant to the in vivo situation were used. KC were infected with either free virus or autologous infected peripheral blood mononuclear cells (PBMC). METHODS: Feline KC were isolated by centrifugal elutriation from collagenase-perfused liver; cultured cells were characterized by their morphological appearance and their erythrophagocytotic properties. After infection, viral replication was measured by enzyme-linked immunosorbent assay, reverse transcriptase activity, immunofluorescence assay, in situ hybridization and electron microscopic observations. RESULTS: Three days after isolation, 85% of cultured KC were able to internalize red blood cells; 45% were CD4-positive and 65% expressed a 24 kD protein thought to be a receptor for FIV (CD9). After the addition of autologous infected PBMC or cell-free supernatant of chronically infected IRC4 cells to KC cultures, a peak of viral replication was detected at day 28. Antigen revealed by immunofluorescence assay was present in only 0.4%, and viral RNA was detected by in situ hybridization in 2% of the infected cells. CONCLUSIONS: FIV can replicate in cultured feline KC without inducing any cytopathic effect, which suggests that these cells may play a role in the physiopathology of FIV infection.

Contrary results on mouse hepatitis virus type 3 susceptibility in A/J mouse hepatocytes of phosphatidylserine treatment and of a hypercholesterolaemic diet: no correlation with membrane fluidity levels.

The aim of this study was to examine whether or not membrane fluidity directly influences infection by enveloped viruses, and, more precisely here, the susceptibility of A/J mouse hepatocytes to Mouse Hepatitis Virus type 3 (MHV3). We therefore studied, in parallel, the effects on hepatocyte membrane fluidity and on intracellular viral titre of two treatments, i) a hypercholesterolaemic diet to increase the hepatocyte membrane cholesterol content, ii) direct phosphatidylserine incorporation into hepatocyte membrane. Membrane fluidity was monitored on isolated hepatocytes by fluorescence anisotropy with TMA-DPH, and the viral titre was determined by plaque assay. The results clearly demonstrate that membrane fluidity is not directly involved in viral infection mechanisms.

Pathogenicity of neutralization escape mutants of mouse hepatitis virus: correlation with T- and B-cell depletions.

Viral pathogenicity is a result of an imbalance between viral replication and the host's immune defences. When the virus is lymphotropic, understanding the pathogenic process of the viral disease becomes complicated because virus/lymphocyte interactions can alter the cell's integrity and subsequently induce immunodeficiency. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3). The use of attenuated escape mutants provides a tool to study the role of viral properties involved in its pathogenicity. We selected MHV3 mutants by virtue of their resistance to neutralization by monoclonal antibodies (mAb), in order to study their pathogenic properties. We reported that two MHV3 escape mutants were attenuated in their pathogenic properties according to inoculation site and with regard to survival time and ability to deplete T- and B-cell subpopulations in the spleen, thymus and bone marrow of susceptible Balb/c mice. The highly attenuated CL12 mutant could not induce depletion in T or B cells following intraperitoneal (i.p.) or intranasal (i.n.) inoculations, at three days postinfection. The less attenuated 51.6 mutant, however, maintained the ability to deplete T and B cells following i.p. inoculation, as described with the pathogenic MHV3. In contrast, no depletion of T cells following i.n. inoculation was induced with this mutant, although B lineage cells decreased. The use of such mutants enabled us to examine the role of each compartment of the immune system, since the highly attenuated CL12 mutant induced no immunodeficiency, as defined by immune cell depletion, whereas the less attenuated 51.6 mutant maintained its ability to decrease only the B-cell compartment after i.n. inoculation. Results are discussed with regard to the virus/lymphocyte interactions during the pathogenic process.

Creatine kinase-BB: a marker of liver sinusoidal damage in ischemia-reperfusion.

Cell damage within the sinusoidal lining of human liver grafts during transplantation is an early event that is critical in ischemia-reperfusion injury and probably plays a key role in primary liver dysfunction after transplantation. No simple biochemical marker for sinusoidal injury is currently available. Because creatine kinase activity has been described in heart endothelial cells, we hypothesized that release of this enzyme might serve as an index of sinusoidal injury. To test this hypothesis, we used several in vivo and in vitro experimental models. Occlusion of the rat hepatic pedicle in situ for 60 min (normothermic ischemia) induced a significant increase in serum creatine kinase levels relative to those in laparotomized controls (2,530 +/- 530 vs. 389 +/- 64 IU/L, mean +/- SEM; p < 0.005). In the isolated perfused rat liver, 60-min ischemia induced early (< or = 3 min) creatine kinase and AST release (0.87 +/- 0.14 vs. 0.08 +/- 0.01 IU/min/gm liver, respectively). A similar phenomenon was observed after 24-hr or 48-hr hypothermic conservation in University of Wisconsin solution. Electrophoretic analysis and immunoinhibition studies showed that creatine kinase activity comprised creatine kinase-BB (approximately 50%) and mitochondrial creatine kinase. Trypan blue infusion showed a loss of viability in sinusoidal cells, whereas hepatocytes were relatively spared. Finally, murine sinusoidal cells were isolated, cultured and then lysed by a freeze-thaw cycle and sonication. Creatine kinase activity was found in endothelial cells (creatine kinase-BB), Kupffer cells (creatine kinase-BB) and Ito cells (creatine kinase-MM). Creatine kinase-BB was not found in hepatocytes, but mitochondrial creatine kinase was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased susceptibility to mouse hepatitis virus type 3 (MHV3) infection induced by a hypercholesterolaemic diet with increased adsorption of MHV3 to primary hepatocyte cultures.

The administration of a hypercholesterolaemic (HC) diet rendered genetically resistant A/J mice susceptible to mouse hepatitis virus 3 (MHV3) infection. The animals died of acute hepatitis with high viral titres in the liver accompanied by many necrotic foci and high serum transaminase levels. Resistance to virus was re-established by refeeding HC mice with a normal diet for 2 weeks. This modification of pathogenesis was accompanied by an increase in the susceptibility of hepatocyte cultures from HC mice to MHV3 and could be explained by an enhancement in virus adsorption. We hypothesize that the incorporation of cholesterol into the plasma membranes of hepatocytes of HC mice, thereby decreasing the membrane fluidity, may lead to an increase in the availability of virus receptors.

Inhibition in fat-storing cell multiplication by a factor produced by normal cultured murine hepatocytes.

In this study we demonstrate that hepatocytes isolated from normal mice may efficiently inhibit the multiplication of fat-storing cells (FSC) in culture, either in a coculture system where both cell types are separated by a filter of 0.45 microns pore size or via their conditioned medium. The inhibition may be completely reversed when the hepatocytes are removed and the culture medium is renewed. The inhibitory factor appears as early as 8 h in the medium with an almost maximum effect being reached after 24 h, as long as protein synthesis is allowed. It rapidly loses its efficiency through dilution. The inhibitory capacity of the conditioned medium is maintained after heating at 56 degrees C, dialysis of 100,000 x g centrifugation, but reduced after trypsin treatment. The infection of the hepatocytes by ectromelia virus causes an almost total suppression of the synthesis of the inhibitory factor. This latter result suggests that the multiplication of FSC, which may be inhibited by normal hepatocytes, would no longer be hindered in case of disregulation.

[Effects of estrogen treatment on the antiviral properties of murine Kupffer cells]. / Répercussions d'un traitement aux oestrogènes sur les propriétés antivirales des cellules de Kupffer murines.

Adult female mice were administered 17 beta-oestradiol at pharmacological dosages by subcutaneous injections. Histopathological examination revealed an increase in cells of the mononuclear lineage infiltrating the sinusoids of the liver. In vitro culture of Kupffer cells demonstrated that the treatment did not alter their antiviral properties but did rather induce an activation of their non-specific immune properties. This activation might be beneficial or deleterious for the infected host and must be discussed in each particular pathological process.

Ultrastructural and biochemical evidence of the trimeric nature of frog virus 3 (FV3) six-coordinated capsomers.

Image analysis of freeze-etch replicas of cylindrical aberrant forms of FV3 provided evidence for three morphological subunits protruding from the six-coordinated capsomers. Negatively stained capsomers displayed both triangular and hexagonal profiles which suggests that their innermost portion is pseudohexagonal. Images from underfocused micrographs of capsomers are indicative of a central channel. The trimeric nature of the capsomer has been established by electrophoresis in the presence of Triton X-100, which showed that the molecular weight of the nondissociated capsomer is about 140,000 whereas that of the polypeptide itself is 48,000. This trimeric association does not occur via disulfide bonds, and inside the capsomers there are no free amino groups accessible to the usual bifunctional reagents. Thus, the chemical nature of the interpolypeptide bonds inside the trimers is still unknown. We have previously estimated the triangulation number (T) of FV3 to be 147 or 133 (Darcy-Tripier et al., 1984). The present study, using optical diffraction of the facets of FV3, allowed a better determination of the angle of skewness and is in favor of T = 133 (h = 9, k = 4, 18 degrees).

Ultrastructural and biochemical study of frog virus 3 uptake by BHK-21 cells.

Ultrastructural studies of the uptake of enveloped and naked frog virus 3 (FV 3) particles by BHK-21 cells have shown that enveloped viruses are internalized by adsorptive endocytosis via coated pits. The enveloped particles then appear to move through endosomes and finally lysosomes. Naked viruses may also follow the same pathway but only rarely. Their more frequent mode of entry is by fusion between the virus shell and the cellular membranes, thus allowing the virus to shed its nucleoprotein content directly into the cytoplasm. This difference in the mechanism of penetration has been confirmed by the use of lysosomotropic agents: the inhibition of viral growth being far more drastic for enveloped FV 3 than for naked virus implies that a lysosomal step is required for the multiplication of enveloped viral particles.

The organization of frog virus 3 as revealed by freeze-etching.

A variety of freeze-fracture techniques has been employed in this study with the aim of dissecting the frog virus 3 virion and obtaining further information about its architecture. The icosahedral capsid has a skew symmetry with a triangulation number of 133 or 147. The capsomers are closely packed with a center-to-center spacing of 72 A. The inner membrane contains transmembrane proteins which appear as intra-membranous particles on both fracture faces. Rod-like structures (about 100 A in diameter) are present in the virus interior suggesting that the DNA-protein complex is highly organized.

Ultrasonic absorption evidence for structural fluctuations in frog virus 3 and its subparticles.

The structural fluctuations specific to self-assemblies of biological molecules have been investigated further with ultrasonic techniques by using frog virus 3 (FV3). We compared the ultrasonic properties of complete FV3 virions and of several subparticles that may be obtained from this DNA virus: (i) the central nucleoprotein core versus its component DNA and proteins in a dissociated state; (ii) the core versus the capsidless subparticle, consisting of the core surrounded by the lipid membrane; and (iii) the complete virus versus the capsidless subparticle. The ultrasonic absorption by the core particle was quite large compared with the absorption by other nucleoprotein assemblies, suggesting that the core contains some organized structure. Both the core and the complete virus absorbed ultrasound more than did the capsidless subparticle. The difference spectrum for the virion relative to the capsidless subparticle may represent a single relaxation and is analyzed, by using a recent model, in terms of volume fluctuations due to radial movements in the virion. These fluctuations are much smaller than can be detected in virus crystals with present-day x-ray techniques.

Induction of intranuclear microtubules in chick embryo fibroblasts by frog virus 3.

Intranuclear microtubules appear in chick embryo fibroblasts upon infection with Frog Virus 3 (FV 3). Both the diameter and the annular shape of the microtubule profiles, established from electron microscopic observations using a goniometer, suggest that they are identical to naturally occurring cytoplasmic microtubules. Furthermore, the use of vinblastine allowed demonstration of the tubulin composition of the intranuclear microtubules.

Frog virus 3 morphogenesis: effect of temperature and metabolic inhibitors.

The different stages of frog virus 3 (FV 3) morphogenesis have been investigated in chick embryo fibroblasts infected at an optimal temperature for virus growth (29 degrees C). The metabolic requirements for morphogenesis were determined by adding inhibitors of macromolecular synthesis at different periods in the virus growth cycle. The effect of a non-permissive temperature for FV 3 replication (37 degrees C) was also studied in shift up experiments. The following results were obtained: (I) when DNA replication was inhibited, neither immature nor mature virus particles appeared; (2) continuous protein synthesis was required for every stage of virus morphogenesis. However, the assembly of virions into paracrystalline arrays seemed to be a passive phenomenon. (3) Continuous mRNA transcription was not necessary for assembly of capsid constituents, although most of these capsids appeared empty; there was also a striking increase in the number of aberrant virus structures. (4) If infected cells were shifted to a non-permissive temperature, virus maturation was completely inhibited.

Hypersensitivity to procarbazine associated with angioedema, urticaria, and low serum complement activity.

Hypersensitivity to procarbazine associated with urticaria, angioedema, and painful joint swelling was found in a 20-year-old student being treated for Hodgkin's disease. A marked fall in complement component activity occurred simultaneously with the development of symtoms. It is suggested that generation of products of complement component activation could be important in the pathogenesis of hypersensitivity to some drugs.

[New data on the structure and the budding of FV3 (frog virus 3) (author's transl)]. / Données nouvelles sur la structure et le bourgeonnement du FV3 (frog virus 3)

The freeze-etching technique when applied to FV3 suspensions revealed the perfectly icosahedral structure of the viral nucleocapsids. This allowed a fine substructure suggesting the existence of numerous subunits of small dimensions to be detected at their surface. Observations of the replicas of infected cells obtained by freeze-etching revealed modifications of the external face of the cytoplasmic membrane occurring at the level of the budding viral particles; in these zones parallel stripes appeared, whose elementary structure is constant (13,5 nm). Observations of the infected cells with the scanning microscope enabled the spreading of the budding process on all the cells of a same culture to be studied. By counting the number of budding particles per mum2, it was possible to demonstrate the heterogeneity of the budding process at the surface of the cells.