Isolation and characterization of infectious full-length DNA clones of chimpanzee foamy viruses SFV6 and SFV7: evidence for a Taf-dependent internal promoter.

We have cloned complete viral genomes directly from Hirt supernatant DNAs of simian foamy virus types 6 and 7 (SFV6 and SFV7) -infected cells. These clones were shown to be infectious by transfection into cells and subsequent infection of susceptible cells either by cocultivation or by passage of cell-free supernatants. The presence of virus particles, suggested by a typical cytopathic effect, was confirmed by electron microscopy. These viruses were characterized at different levels of the replication cycle. The proviral genomes revealed a taf deletion comparable to that previously described in the human foamy virus (HFV) bel1 gene. Analysis of viral RNAs revealed similar patterns of transcripts for SFV6- and SFV7-infected cells, with predominant expression of accessory genes. Characteristic major viral polypeptides were identified by radioimmunoprecipitation for both isolates. Sequences homologous to the gene encoding Taf and to a potential internal promoter were identified in the infectious clones and subcloned into expression vectors. Their functional properties were tested by transfection assays, which provided evidence for the presence of a Taf-dependent internal promoter in both SFV6 and SFV7 isolates.

In vitro studies on interferon-inducing capacity and sensitivity to IFN of human foamy virus.

We demonstrate in this article that human foamy virus (HFV) fails to induce interferon (IFN) production in two different human tissue culture cell lines: U373-MG and AV3. We also show the effect of human alpha-, beta- and gamma IFN on the multiplication cycle of HFV. Treatment of cells with 100 IU/ml of any IFN led to strong inhibition of an HFV-induced cytopathic effect. This effect was associated with a significant diminution of reverse transcriptase activity in supernatant fluids of IFN-treated infected cultures, and a substantial decrease in viral particle production, as detected by electron microscopy. All these effects were accompanied by strong inhibition of both viral proteins and RNA synthesis, as well as almost total disappearance of free and integrated proviral DNA. In light of our data, human IFN action on HFV seems to be mediated by a mechanism which differs from that observed in the case of other retroviruses (type C and D for instance); however, it evokes that described for HIV.

Unintegrated HTLV-I proviral DNA in cell lines and uncultured peripheral blood mononuclear cells from tropical spastic paraparesis (TSP/HAM) and adult T-cell leukemia (ATL) patients.

Ultrastructural studies on cell cultures derived from TSP/HAM and ATL patients, show the presence of large quantities of HTLV-I viral particles in extracellular spaces and budding at the cytoplasmic membrane. In addition, mature enveloped particles and images of endopinocytosis of virions are seen in the cytoplasm vacuoles suggesting the existence of a reinfection phenomenon. In this context, we decided to investigate some features of the replicative cycle, in particular the synthesis of unintegrated proviral forms. To increase the sensitivity of detection, we applied a procedure which combines the electrophoretic separation of closed circular forms and PCR amplification. By this procedure we produced evidence for the existence of supercoiled HTLV-I DNA in established cell lines from TSP/HAM and ATL and in patients peripheral blood mononuclear cells. These HTLV-I unintegrated proviral forms may play an important role in the physiopathology of HTLV-I associated diseases. Preliminary results of AZT/interferon treatment in ALT patients are largely superior to chemotherapy. The therapeutic effect of AZT, it known inhibitor of reverse transcriptase, may be through its inhibition of the synthesis of HTLV-I unintegrated proviral DNA.

HTLV-1-like particles and HTLV-1-related DNA sequences in an unambiguous case of Sèzary syndrome.

An unambiguous case of Sèzary syndrome associated with the presence of unusual retroviral infection markers is described. The blood smear showed 15% typical Sèzary cells but also rare atypical lymphocytes with convoluted nuclei, evocative of characteristic adult T-cell leukemia (ATL) flower cells. However, the patient did not present any clinical or biological manifestations of ATL, and human T-cell leukemia virus type 1 (HTLV-1) serology was consistently negative. After being cultured for 4 months, peripheral blood mononuclear cells (PBMC) produced typical type C retrovirus-like particles with budding forms strongly resembling HTLV-1 virions. The producer cells did not express HTLV-1-specific antigens detectable by indirect immunofluorescence (IIF). Southern blotting of uncultured PBMC DNA, submitted to digestion with the restriction enzymes PstI and SacI, and hybridized with a full genomic HTLV-1 probe, showed the presence of specific homologous sequences, absent in all of the healthy donor control PBMC DNAs. These HTLV-1-like sequences presented a restriction enzyme pattern distinct from that of the HTLV-1 prototype genome and of other HTLV-1 proviruses studied up to now. Polymerase chain reaction (PCR) with highly conserved HTLV-1 derived pol and env primers was consistently negative with the patient's DNA. All these results taken together suggest that our patient carries a retroviral agent partially homologous to, but probably different from HTLV-1. The possibility is discussed that this type of retroviral agent might be associated with a subtype of cutaneous T-cell lymphoma (CTCL) represented by a typical Sèzary syndrome with a very low percentage of ATL-like flower cells in the blood smear.

Transient immunosuppressive effect induced in rabbits and mice by the human spumaretrovirus prototype HFV (human foamy virus).

Spumaviruses (foamy viruses) constitute one of the three retroviral genera isolated from man. Although spumaviruses have not been clearly linked to a given pathology in humans and other infected species, it is well established that they lead in vivo to chronic infections without detectable viral expression. We thought it of interest to investigate certain aspects of the pathology induced in laboratory animals by human foamy virus (HFV). In this work, we demonstrate that HFV infection of rabbits and mice gives rise to a transient immunosuppressive effect, as evaluated in vitro by lymphocyte transformation tests. This phenomenon occurs shortly after viral inoculation, at around 4-5 days, and regresses within thirty days.

The effects of poly(I).poly(C12U) and interferon on the multiplication of a mammalian type C retrovirus in human cells.

Poly(I).poly(C12U) or interferon treatment inhibited multiplication of the xenotropic baboon type C endogenous retrovirus M7 in chronically infected human AV3-M7 cells, as determined by a reverse transcriptase (RT) assay and electron microscopy. Furthermore, this polynucleotide induced 2'5' oligoadenylate (2'5'A) synthetase activity. In contrast to interferon (IFN), poly(I).poly(C12U) did not give rise to the appearance of a trapping phenomenon observable by electron microscopy. When AV3-M7 cells were treated simultaneously with poly(I).poly(C12U) and anti-IFN-beta/alpha antibodies, the induction of 2'5'A synthetase was abolished without any alteration of the inhibitory effect of RT activity. Taken together, these results suggest that different mechanisms are used by poly(I).poly(C12U) and IFN in blocking type C retrovirus multiplication.

First heterotransplantation of a human carcinoid tumor into nude mice.

The first successful heterotransplantation of a human carcinoid tumor into nude mice is reported. CSH, a voluminous hepatic metastasis of a primary bronchial carcinoid tumor (CSB) was resected and transplanted into three irradiated nude (Swiss-nu/nu) mice both by subcutaneous (SC) and intramuscular (IM) routes; the success rate was five of six. Heterotransplanted tumors took 4 to 5 months to appear in the mice and 1 month to attain a width of 0.5 cm. Both human and mouse tumors (named CSH-SC and CSH-IM) were studied by light and electron microscopy. They were Grimelius-positive, neuron-specific enolase-positive, and bombesin-negative by immunocytochemistry. Furthermore, CSH-SC cells presented characteristic (pear-shaped, rod-shaped, or tadpole-shaped) neurosecretory granules. Although CSB and CSH were slightly serotonin positive by immunocytochemistry, only a few serotonin-positive cells were found in CSH-SC and none in CSH-IM, suggesting partial loss of differentiation or an increase in serotonin catabolism during transplantation.

No evidence for HTLV-I infection in 24 cases of French and Portuguese mycosis fungoïdes and Sezary syndrome (as seen in France).

A survey in search of evidence for HTLV-I infection was conducted on French and Portuguese patients residing in France with a diagnosis of mycosis fungoïdes or Sezary syndrome. Methods used in this investigation included serological assays (ELISA, Western blot, particle agglutination, indirect immunofluorescence) and DNA molecular studies (Southern blot and polymerase chain reaction). Cultures of peripheral blood mononuclear cells were performed and checked by electron microscopy and reverse transcriptase assay. The results indicate that neither HTLV-I nor a closely related retrovirus are associated with mycosis fungoïde or Sezary syndrome in the cases studied.

Paradoxical effects of recombinant mouse beta-interferon on different expression levels of intracisternal A particle genes.

We have studied the effect of mouse recombinant beta-Interferon on the expression of the intracisternal A particle (IAP) genes in a transformed mouse fibroblast cell line. Northern and immunoprecipitation analysis showed an increase in specific transcripts and polypeptides. Run-on experiments on isolated nuclei and transfection assays with an IAP long terminal repeat construct, coupled with the chloramphenicol acetyl transferase gene, showed no enhancement of the transcription rate following beta-IFN treatment. This suggests that the increase in IAP expression depends on a post-transcriptional event. Electron microscopic analysis revealed that in spite of the higher levels of IAP related polypeptides, the number of particles was significantly reduced by interferon.

Cell surface phenotype and human T lymphotropic virus type 1 antigen expression in 12 T cell lines derived from peripheral blood and cerebrospinal fluid of West Indian, Guyanese and African patients with tropical spastic paraparesis.

Twelve long-term cell lines were established from peripheral blood mononuclear cells (PBMC) or cerebrospinal fluid cells of patients with human T lymphotropic virus type I (HTLV-1) seropositive tropical spastic paraparesis (TSP) originating from the French West Indies, French Guyana or the Central African Republic. Most of these long-term interleukin-2-dependent cell lines exhibited a pattern characteristic of CD4(+)-activated T cells with high expression of CD2, CD3 and CD4 antigens, associated with a strong density of TAC and DR molecules. Nevertheless, in five cases CD8 expression was present at a significant level. HTLV-I antigens were never detected in uncultured PBMC, but they were expressed in a few cells after short-term culture and after 4 months the majority of the cells were HTLV-I positive, as demonstrated by indirect immunofluorescence (IF) using polyclonal or monoclonal anti-p19 and anti-p24 antibodies. Low and variable levels of reverse transcriptase activity were detected in supernatant fluids of these cell lines only after 4 months of culture, when at least 50% of the cells exhibited HTLV-I antigens by IF. However, numerous type C HTLV-I-like viral particles were detected, mostly in the extracellular spaces, with rare budding particles. Similar findings were found in three T cell lines derived from West Indian and African patients with adult T-cell leukaemia/lymphoma (ATLL). Differences in high Mr polypeptides were detected by Western blot in cell lysates when comparing TSP- or ATLL-derived T cell lines. Thus a signal of 62K was easily detectable in all the TSP lines, but not in the ATLL lines. In all cell lines bands corresponding to p53, p24 and p19 viral core polypeptides were present, as was the env gene-coded protein p46.

Effects of human recombinant alpha and gamma and of highly purified natural beta interferons on simian Spumavirinae prototype (simian foamy virus 1) multiplication in human cells.

The present study demonstrates the inhibitory effect of human recombinant interferons (r-Hu-IFN) alpha and gamma, and that of highly purified natural human interferon beta on the replication of simian foamy virus type 1 (SFV1) in human AV3-cell cultures. All IFN led to strong inhibition of the SFV1 cytopathic effect. Electron microscopy showed a 70 to 95% decrease in viral particles. Significant inhibition of virus-associated reverse transcriptase activity was found in supernatant fluids of infected IFN-treated cultures. Metabolic labelling of the virus confirmed the inhibition of extracellular release of SFV1. PAGE analysis of immunoprecipitates indicated a reduction in viral-specific protein bands. Altogether, these results indicate that the mechanism of inhibition of Spumavirinae infection by interferon differs from that described for the other Retroviridae, and particularly for types B, C and D viruses. Our data is of therapeutic interest since Spumavirinae have been linked to pathological processes such as de Quervain thyroiditis.

In vitro induction of early mouse embryo intracisternal particles (epsilon particles) in cultured cell lines.

The experimental induction of epsilon particles, retrovirus-like structures corresponding to the small IA particles of the mouse, was studied by electron microscopy in rodent-cultured cell lines. Among the chemicals tested, only IdUr was shown to be an effective inducer, but not cycloheximide, puromycin , deoxy-fluorouracil or 5-azacytidine. However, only two mouse-derived cell lines: Ki-BALB and FG 10, among 27 cell lines of mouse, rat and mink origins tested, expressed epsilon particles upon IdUr treatment. Epsilon particles thus respond to chemical inducers very differently in comparison with large IAP. Moreover, the addition of interferon previously shown to attenuate IAP production, had no effect on that of epsilon particles.

Effect of recombinant human alpha A interferon on Visna virus multiplication in acutely infected ovine cells.

We have studied the effects of recombinant human alpha A interferon (IFN) on the multiplication of Visna retrovirus in ovine cells. Pretreatment with IFN followed by continuous IFN treatment, drastically blocked the Visna virus induced cytopathogenic effect and the emergency of neoformed virions. Thus, virus yield and virus dependent reverse transcriptase activity were highly reduced in supernatant fluids. All these effects were accompanied by a strong inhibition of viral protein synthesis. In the light of these data, human IFN action on Visna retrovirus seems to be determined by a mechanism of action distinct from that described in the case of other Retroviridae.

Chronic myelopathies associated with human T-lymphotropic virus type I. A clinical, serologic, and immunovirologic study of ten patients in France.

Chronic myelopathy associated with human T-lymphotropic virus type I (HTLV-I) has been described in HTLV-I endemic areas. In Paris, 167 neurologic patients were screened for HTLV-I by enzyme-linked immunosorbent, indirect immunofluorescent, and Western blot assays. Ten of the 11 patients with positive results had a chronic spastic paraparesis with IgG oligoclonal bands and elevated HTLV-I antibody index. Two of them had been born and were living in France, without HTLV-I risk factors. Evoked potentials were abnormal in the nine tested patients and brain magnetic resonance images in three of seven patients. No improvement was observed with steroid treatment. A retrovirus similar to HTLV-I was isolated in five cases at different periods of the disease. Hypotheses of limited endemic areas in western countries are discussed. Early presence and persistence of HTLV-I suggest that it is the etiologic agent.

Characterization of HTLV-I isolates and T lymphoid cell lines derived from French West Indian patients with tropical spastic paraparesis.

Lymphoid cell lines derived from the peripheral blood of French West Indian patients with HTLV-I sero-positive Tropical Spastic Paraparesis and HTLV-I isolates were characterized. While patients' peripheral blood lymphocytes did not express detectable HTLV-I antigens when uncultured, they did so after short-term culture. Established cell lines were of T-cell lineage: CD2+, CD3+, CD4+, CD7+, WT31+ with activated T-cell markers CD25+, DR+ and a clonal rearrangement of the beta and gamma genes of the T-cell receptor. HTLV-I antigens were detected in cell lines by indirect immunofluorescence, Western blot and radio-immunoprecipitation assays. After 4 months in culture, low levels of Mg2+ dependent reverse transcriptase activity were detected and electron microscopy revealed numerous type-C retroviral particles similar to HTLV-I virions. Western blot and radio-immunoprecipitation analysis of purified viruses revealed gp46, p24, p19 and Pr53gag proteins similar to those detected in HUT 102 and MT2 cell lines. Deep analysis of env-coded precursor of one TSP versus ATL isolates revealed minor differences in their molecular weights. Southern blot analysis using 32P HTLV-I env gene as a probe showed the presence of HTLV-I proviral fragments clonally integrated into the genome of the cell lines. Our data suggest that HTLV-I isolated from Tropical Spastic Paraparesis does not differ significantly from the leukemogenic prototypes. Does HTLV-I induce either acute lymphoproliferative diseases or chronic neuromyelopathies depending upon as yet unknown co-factors? This question remains to be determined.

Electron microscopic characterisation of retrovirus and retrovirus-like particles induced by demethylating agents (5-azacytidine and 5-azadeoxycytidine) in Syrian hamster (Mesocricetus auratus) cells.

In a framework of a study on retrovirus expression in Syrian hamster (mesocricetus auratus), three cell lines were examined by electron microscopy. As we had previously demonstrated the efficacy of demethylating drug in activating the formation of retroviral particles, we used 5-azacytidine to activate endogenous retrovirus expression. Proceeding to a more detailed survey we were able to observe various types of retrovirions. All of them were present in the different activated cell lines. They were: intracisternal R-type particles (IRP), intracisternal A-type particles (IAP), intracytoplasmic A-type particle (ICAP), intramitochondrial A-type particle (IMAP) and M-432 virus-like particles. Some of these particles have never been described before in Syrian hamster cells. The significance of their presence is discussed here.

Detection of retrovirus particles and reverse transcriptase activity in mid-term cultured peripheral blood and lymph node cells from a French woman with Sezary syndrome.

Retrovirus particles, with an ultrastructure of type C-virus similar to HTLV-I were observed in several mid-term cultures of leukemic cells derived from a woman with a well characterised Sezary syndrome who had always resided in France. Reverse transcriptase activity was detected in supernatant fluids from day 6 to day 40 of culture. However, negative anti HTLV-I serology and the absence of specific molecular hybridization between leukemic cell DNA and two HTLV-I derived probes, argue against a HTLV-I virus.

[Large-scale production of amastigotes by a human monoblastoid cell line]. / Production massive d'amastigotes par une lignée de cellules monoblastoides humanines.

In this study, a human monoblastoid cell line (TPH-1) was tested in vitro for the production of Leishmania amastigotes. The number of TPH1 cells increased with time and 6 days after promastigote infection the percentage of infected cells was around 45%. Pre-treatment of TPH1 cells with retinoic acid induced the cells to differentiate into unreplicating macrophage-like cells. Ninety per cent was parasitized 6 days after promastigote infection; the number of amastigotes quintuplied during this period of time; this result was irrespective of the Leishmania species used for experiments. Viable and infective parasites were obtained from treated and nontreated cells. TPH1 cells merit further consideration for research concerning new molecules active against Leishmania.

Dexamethasone stimulates expression of transposable type A intracisternal retroviruslike genes in mouse (Mus musculus) cells.

Dexamethasone treatment enhances expression of transposable intracisternal type A particles (IAP) at RNA and proteins levels in a murine retrovirus-transformed cell line (Ki-BALB). This effect was ascertained by electron microscopic numeration of IAP. By sequence comparison, we located glucocorticoid-responsive elements in IAP long terminal repeats. Their regulatory potential was tested on the promoter activity of an IAP long terminal repeat construct coupled with the chloramphenicol acetyltransferase gene. Our findings suggest that the IAP activation by dexamethasone occurs at the level of transcription.

[Establishment of a T lymphoid cell line producing a retrovirus of HTLV-I type, from peripheral blood of a patient with tropical spastic paraparesis]. / Etablissement d'une lignée lymphoïde T productrice d'un rétrovirus de type HTLV-I à partir du sang périphérique d'un patient atteint de paraparésie spastique tropicale.

HTLV-I, the causative agent of Adult T cell Leukemia, has recently been found to be associated with chronic neuromyelopathies common in tropical areas and in Japan. We report here the establishment of a lymphoid T cell line from peripheral blood lymphocytes of a patient with Tropical Spastic Paraparesis. This cell line produces a retrovirus whose morphologic, antigenic and genetic characteristics show no detectable difference from the leukemogenic prototype of HTLV-I.