A multivariate statistical analysis to follow the course of disease after infection of cats with different strains of the feline immunodeficiency virus (FIV).

A descriptive multivariate assay is described which is suitable to analyze results of a biological experiment with small sample size but high qualitative and quantitative complexity of variables. This type of assay allows evaluation of multiple variables observed in the course of an experimental virus infection (e.g. viremia, nucleic acid detection, antibody titers, clinical parameters, anti-microbial treatments or vaccination) in a single graph. In our study, a multiple correspondence analysis (MCA) was used to correlate a total of 145 measurements from each of a dozen of variables measured in five groups of three cats infected by five isolates of feline immunodeficiency virus (FIV). Three groups of virus isolates with distinct virulence were defined and correlation between dynamics of lymphocyte subset counts and viral virulence was established. Comparison between the primary stages of illness and follow-up examinations were of prognostic value and are thus helpful for development and monitoring of therapeutic strategies.

Productive infection of primary cultures of endothelial cells from the cat liver sinusoid with the feline immunodeficiency virus.

Given the similarities between the two viruses, the feline immunodeficiency virus (FIV) is becoming an interesting animal model for human immunodeficiency virus (HIV) studies. To explore the still controversial role of the liver in the development of HIV infection, sinusoidal endothelial cells (SEC) were isolated, and primary cultures were infected with the FIV Villefranche IFFA strain. The isolated cells were characterized by their typical fenestrations, the presence of von Willebrand factor (vWf), and their ability to take up acetylated low-density lipoproteins and denatured collagen. Two weeks after infection, significant amounts of FIV p24 antigen were detected by immunofluorescence in both multinucleated giant and single cells and by enzyme-linked immunosorbent assay in the culture medium. High amounts of viral particles were observed together with different steps of budding at the plasma membrane or at the membrane of intracytoplasmic vacuoles. The released viral particles were shown to be infectious for a permissive cell line. During the first 3 weeks of infection, the only cytopathic effect of FIV was syncytia formation. No noticeable impairment of the pattern of fenestrations and the modulation of their number by a cytoskeleton-mediated process occurred. The productive infection of SEC may contribute to the progression of the infection.

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.

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.

[Response of splenic lymphocytes to phytohemagglutinin and bacterial lipopolysaccharide in mice, sensitive and resistant to mouse hepatitis virus 3 (MHV3)]. / Etude de la réponse des lymphocytes spléniques à la phytohémagglutinine et au lipopolysaccharide bactérien chez les Souris sensibles et résistantes au virus de l'hépatite murine 3 (MHV3).

The responsiveness of splenic cells to phytohemagglutinin but not to bacterial endotoxin was inhibited during MHV3 infection of both sensitive (C57Bl6) and resistant (A/J) Mice. This result could be interpreted as the consequence of an inhibition that selectively affects the T lymphocytes during infection. In C57Bl6 sensitive Mice the inhibition could be a consequence of the destruction of splenic T lymphocytes. In A/J resistant Mice, no splenic lesions have been observed; therefore another mechanism must be considered in order to explain the inhibition of the responsiveness of splenic cells to phytohemagglutinin.

Preparation and properties of an inhibitory extract from frog virus 3 particles.

The structural constituents of the frog virus 3 particle were solubilized by treatment with a nonionic detergent followed by the addition of a high salt concentration. This soluble viral extract (SVE) inhibits host nucleic acid synthesis. Its activity on RNA synthesis was studied in KB cells and found to be dependent on the presence of DEAE dextran. Inactivation of the inhibitory properties of SVE were obtained by trypsin digestion, treatment with urea, or heat denaturation. Neutralization of the activity of SVE was obtained by anti-frog virus 3 serum but not by anti-BHK serum. In vitro a complex may be formed between polynucleotides and the inhibitor indicating a possible mechanism for vivo inhibition.