In vivo infection of ramified microglia from adult cat central nervous system by feline immunodeficiency virus.

Infection of microglial cells by the human immunodeficiency virus (HIV) is supposed to play an important role in the pathogenesis of AIDS-related central nervous system (CNS) complications. So far, however, experimental data about interactions between HIV and ramified microglia from the adult CNS were only occasionally reported, making it difficult to understand the exact nature of pathogenic events contributing to HIV-encephalopathy. Therefore, we used the animal model of feline immunodeficiency virus (FIV) infection of domestic cats to establish an experimental system which is suitable for studying the relationships between an immunodeficiency virus and the mature ramified microglia of the central nervous system. By means of density gradient centrifugation approximately 95% pure microglial cells could be isolated from adult feline brain that were characterized by their CD45(low) phenotype. Resident microglia extracted from the CNS of experimentally infected cats harbored FIV-specific DNA and cocultivation with mitogen-activated, but uninfected peripheral blood mononuclear cells (PBMC) resulted in recovery of high-titered infectious virus. Double labeling of brain cell monocultures explanted from persistently infected animals for both microglia and FIV markers disclosed less than 1% of viral antigen expressing microglial cells. This suggests that during the subclinical phase of the infection only a small number of brain-resident macrophages are productively infected. However, interaction of FIV-infected microglia and inflammatory lymphocytes may promote viral replication, thus supporting viral spread in brain tissue.

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.

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.

Mouse hepatitis virus type 3 infection provokes a decrease in the number of sinusoidal endothelial cell fenestrae both in vivo and in vitro.

Fenestrations of hepatic endothelial cells play an active role as a sieving barrier allowing extensive exchange between the blood and liver parenchyma. Alteration of these structures may be induced in the course of various pathological events and provoke important perturbations of liver function. We demonstrate here that sinusoidal endothelial cells are permissive for mouse hepatitis virus 3 (MHV3) in vivo and in vitro and that this infection leads to a striking decrease in the number of fenestrae. The disappearance of these structures observed under scanning electron microscopy or in cryofracture preparations in vivo and in vitro cannot be reversed by the action of cytochalasin B on the microfilament network. The decrease in the porosity seems to be related directly to the productive infection of the endothelial cells, because it was not observed in A/J mice resistant to the virus and in susceptible BALB/c mice immunized with a thermosensitive mutant in which no viral replication occurs. In conclusion, a viral infection of liver endothelial cells may cause extensive loss of the fenestrations and thus lead to important functional pertubations.

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.

Feline immunodeficiency virus can productively infect cultured endothelial cells from cat brain microvessels.

Feline immunodeficiency virus (FIV) provokes a disease in cats characterized by histopathological lesions similar to those observed in AIDS patients. In order to determine whether endothelial cells from brain microvessels are involved in the central nervous system disease to the same extent as macrophages and microglia, cells were isolated from healthy cat brains, cultured and infected in vitro with the FIV Villefranche IFFA 1/88 strain. The isolated cells displayed typical endothelial cell ultrastructural features and were characterized further by von Willebrand factor-labelling and the binding of specific lectins such as Ulex europaeus lectin on their membrane. They were also able to take up acetylated low density lipoproteins. Two weeks after infection, significant amounts of FIV p24 antigen were detected by indirect immunofluorescence in syncytia and single cells. Concomitantly, the same antigen could be detected in the culture medium of the infected cells by an ELISA technique. Numerous viral particles as well as different steps in the process of viral budding were observed under transmission electron microscopy. The synthesis of FIV p24 antigens still occurred in cells in which replication was blocked in the G2 phase with taxol. Our results suggest the possibility of a productive infection of brain microvascular endothelial cells by FIV in vivo, which could lead to important perturbations in the functions of the blood-brain barrier.

Altered pathogenicity in the liver induced by a mouse hepatitis virus type 3 thermosensitive mutant.

Intraperitoneal inoculation into sensitive BALB/c mice of D85, a thermosensitive (ts) mutant, provokes acute hepatitis followed by recovery of the mice. The ts mutant was able to replicate in the liver. However, the maximal viral titre was obtained 2 days later than was the case with the wild-type (wt) MHV 3 infection; the viral antigens remained localized within small foci and no invasion of the entire liver was observed. The hepatocytes infected with D85 showed strong steatosis similar to that induced by wt virus, but the other lesions induced by MHV 3 (closing of endothelial cell fenestrae and hepatocytolysis) were not seen. An important feature noticed with the D85 mutant concerned the establishment, in the surviving animals, of persistent infection: this phenomenon was demonstrated by the decrease of viral titre in the liver, viral RNA detection, and the fact that viral antigens gradually decreased until the 3rd month post-infection.

Temperature-sensitive mutants of mouse hepatitis virus type 3 (MHV-3): isolation, biochemical and genetic characterization.

Mouse hepatitis virus 3 (MHV-3) is highly hepatotropic in sensitive mice. Temperature-sensitive mutants (ts mutants) induced by N-methyl-N'-nitrosoguanidine and 5-fluorouracil were isolated. Twelve mutants which were able to induce the formation of syncytia at 33 degrees C but not at the restrictive temperature (39.5 degrees C) were selected for detailed study. No viral RNA synthesis was detected after infection at the restrictive temperature with six of the mutants (RNA-) whereas six others were RNA+, although they displayed RNA synthesis which was generally reduced. No differences have been detected in the size of the genome or the viral-intracellular RNA species found in wild type virus or ts mutant infected cells at permissive temperature. The pattern of virus-induced proteins analyzed after immunoprecipitation by SDS-PAGE was similar in wild type virus and RNA+ mutant infected cells at 39.5 degrees C. Complementation experiments between ts mutants enabled us to distinguish five groups. Three of the groups contained RNA- mutants and two of them RNA+. Plaques made by mutants in one group displayed characteristic features that distinguished them from the wild type.

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.

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.