Endogenous LINE-1 (Long Interspersed Nuclear Element-1) Reverse Transcriptase Activity in Platelets Controls Translational Events Through RNA-DNA Hybrids.

OBJECTIVE: One source of endogenous reverse transcriptase (eRT) activity in nucleated cells is the LINE-1/L1 (long interspersed nuclear element-1), a non-LTR retrotransposon that is implicated in the regulation of gene expression. Nevertheless, the presence and function of eRT activity and LINE-1 in human platelets, an anucleate cell, has not previously been determined. APPROACH AND RESULTS: We demonstrate that human and murine platelets possess robust eRT activity and identify the source as being LINE-1 ribonucleoprotein particles. Inhibition of eRT in vitro in isolated platelets from healthy individuals or in people with HIV treated with RT inhibitors enhanced global protein synthesis and platelet activation. If HIV patients were treated with reverse transcriptase inhibitor, we found that platelets from these patients had increased basal activation. We next discovered that eRT activity in platelets controlled the generation of RNA-DNA hybrids, which serve as translational repressors. Inhibition of platelet eRT lifted this RNA-DNA hybrid-induced translational block and was sufficient to increase protein expression of target RNAs identified by RNA-DNA hybrid immunoprecipitation. CONCLUSIONS: Thus, we provide the first evidence that platelets possess L1-encoded eRT activity. We also demonstrate that platelet eRT activity regulates platelet hyperreactivity and thrombosis and controls RNA-DNA hybrid formation and identify that RNA-DNA hybrids function as a novel translational control mechanism in human platelets.

Genetic variability in coding regions of the surface antigen and reverse transcriptase domain of hepatitis B virus polymerase, Colombia, 2002-2014

Introduction: Despite the availability of an effective vaccine and treatment to reduce the viral load and progressive hepatocellular injury, approximately 240 million people worldwide are chronically infected with the hepatitis B virus (HBV). In Colombia, the circulation of different viral genotypes has been confirmed. Mutations in the genome have been associated to antiviral therapy resistance, viral escape to neutralizing antibodies, occult infection and progression to hepatocellular carcinoma. Objective: To identify the genotypes and the presence of mutations in the coding region of the surface (S) antigen and the reverse transcriptase (RT) domain of the polymerase of HBV obtained from serum samples for hepatitis B diagnosis received by the Instituto Nacional de Salud during the period 2002-2014. Materials and methods: A total of 495 serum samples with previous HBsAg reactive result were used for molecular detection. A fragment of 1,591 nucleotides was sequenced, and the corresponding phylogenetic analysis was performed. Results: We detected the viral genome of HBV in 66 samples and 28 were successfully sequenced. The phylogenetic analysis allowed the identification of subgenotypes F3 and A2. The L180M and M204V resistance mutations were simultaneously identified in one sample, while the I169L resistance mutation was identified in another one. A single escape mutation, P120Q, was identified in one more. Two samples showed a deletion of 105 nucleotides in the preS1-preS2 region. Conclusions: The circulation of genotypes/subgenotypes F3 and A2 of HBV in Colombia was corroborated, as well as the presence of some resistance and escape mutations. The present study constitutes a contribution to the molecular epidemiology of HBV in Colombia.

Reverse transcriptase viral load correlates with RNA in SIV/SHIV-infected macaques.

Monitoring of viral load in macaques has usually been carried out using in-house PCR-based methods. A novel viral load (VL) kit (ExaVir Load) based on the measurement of lentivirus reverse transcriptase (RT) activity provides a potential alternative to methods that measure plasma viral RNA. RT is a fundamental and conserved activity of all retroviruses and the method should theoretically detect RT from all lentiviruses. To test this we compared VL measured by a commercially available RT kit with an in-house QC RT-PCR in macaques infected with SIV and SHIV. Both RT and RNA levels were measured over time in both sets of macaques. Results indicated that the relationship between both tests was strong for SIV and SHIV (r = 0.95 and r = 0.92, p < 0.0001, respectively). The VL trends also followed each other, indicating that both techniques measured the same process of viral replication. Furthermore, the RT load obtained using standardized control plasma samples supplied by NIBSC gave values close to the designated VL. However, when comparing RT load with QC RT-PCR a consistently three to five time higher level was obtained with the RT assay, highlighting potential differences in assay calibration. Even so, the data suggest that the RT assay is both sensitive and robust for use in the SIV/SHIV macaque model, particularly where molecular-based assays for SIV VL determinations are not easily available. The assay is also a commercially available kit and hence has the potential to reduce the variability seen between laboratories using in-house PCR.

No indication for activation of exogenous retroviruses in patients with renal allograft rejection.

AIM: Reactivation of latent BK virus in kidney-transplanted patients results in severe graft dysfunction. The role of retroviruses infecting also latently target cells is not investigated so far in this setting. We determined the presence or induction of retroviruses in sera of immunosuppressed patients with renal allografts at the timepoint of organ rejection or ongoing polyomavirus nephropathy. PATIENTS AND METHODS: Sera of patients with acute kidney rejection or polyomavirus nephropathy (n=25) and controls (n=8) were tested for reverse transcriptase activity by the ultrasensitive product enhanced reverse transcriptase (PERT) assay. In parallel, kidney biopsies were investigated for histological signs of kidney rejection or polyomavirus nephropathy confirmed by either immunofluorescence or immunohistochemistry. RESULTS: None of the investigated sera, specifically those of patients with ongoing BK virus nephropathy, indicated reverse transcriptase activity. CONCLUSION: Our results do not support the idea of the induction of known or unknown retroviruses in patients with kidney transplantation, even under highly immunosuppressive therapies.

Cytotoxicity of human endogenous retrovirus K-specific T cells toward autologous ovarian cancer cells.

PURPOSE: To determine whether HERV-K envelope (ENV) protein could function as a tumor-associated antigen and elicit specific T-cell responses against autologous ovarian cancer cells. EXPERIMENTAL DESIGN: The expression of HERV-K transcripts and ENV protein, the presence of serum antibodies against HERV-K, reverse transcriptase (RT) activities, and cellular immune responses in primary ovarian cancer tissues and patient blood samples were analyzed and compared with samples from patients with benign ovarian diseases and normal female donors. RESULTS: Ovarian cancer cells in primary tumors and ascites expressed markers of cancer stem cells and markers of both mesenchymal and epithelial cells. Expression of HERV transcripts and HERV-K ENV protein and reverse transcriptase activities were higher in ovarian cancer compared with adjacent normal and benign tissues. The ovarian cancer patient plasma also had high reverse transcriptase activities and the ovarian cancer patient sera contained HERV-K immunoreactive antibodies. HERV-K-specific T cells generated from autologous dendritic cells pulsed with HERV-K ENV antigens exhibited phenotypes and functions consistent with a cellular immune response including T-cell proliferation, IFNγ production, and HERV-K-specific cytotoxic T lymphocyte (CTL) activity. Significantly higher CTL lysis of autologous tumor cells than of uninvolved normal cells was demonstrated in patients with ovarian cancer than patients with benign diseases and further enhanced lysis was observed if T regulatory cells were depleted. CONCLUSION: Endogenous retroviral gene products in ovarian cancer may represent a potentially valuable new pool of tumor-associated antigens for targeting of therapeutic vaccines to ovarian cancer. Clin Cancer Res; 21(2); 471-83. ©2014 AACR.

A new quantitative HIV load assay based on plasma virion reverse transcriptase activity for the different types, groups and subtypes.

BACKGROUND: Plasma viral load monitoring is an integral part of the standard of care for HIV-infected patients in industrialized countries. In developing countries, viral load assay is either unaffordable or hindered by on-site maintenance and/or technical problems. OBJECTIVES: To evaluate a new and simple quantitative assay for plasma HIV reverse transcriptase (RT) activity; and to compare RT activity-based and RNA-based quantification in plasma samples from patients infected by different subtypes of HIV-1 group-M, HIV-1 group-O and HIV-2. METHODS: The RT-based viral load assay involves separation of the virion-protected RT and quantification of its activity with an enzyme immunoassay. Plasma viraemia was quantified both by RT activity and by RNA copies in 322 samples from 236 HIV-1 group M-infected patients, including serial samples from 54 patients. Samples from 49 patients infected by HIV-1 group O or HIV-2 were also tested. RESULTS: RT activity and RNA copies were detected in 70% of plasma samples; respectively 25% and 1% of samples contained detectable RNA copies or RT activity alone. Measured RT activity corresponded to 48%, 96% and 100% of samples with 1.7-4.0 log(10), 4.1-4.8 log(10) and 4.9-6.7 log(10) RNA copies/ml, respectively. The values of the two assays correlated independently of the HIV subtype (P < 0.0001) and group/type (P < 0.03). Patient follow-up showed a similar pattern of viraemia with the two assays. CONCLUSION: Plasma RT activity assay is a simple, cheap and reliable alternative for HIV viral load determination. As such, it could be particularly valuable for diagnosis and treatment monitoring in developing countries.

Retroviral expression in mononuclear blood cells isolated from a patient with osteopetrosis (Albers-Schönberg disease).

We report the presence of reverse transcriptase activity in the supernatant of long-term culture of mononuclear blood cells (monocytes and lymphocytes) isolated from a 27-year-old patient suffering from benign osteopetrosis. The enzyme was purified to homogeneity according to the technique of Chandra and Steel, by chromatography, first on DEAE-cellulose (DE 52) and then on phosphocellulose (P11). After purification, the enzyme was characterized biochemically for its template specificity and ionic requirements. The purified enzyme was able to transcribe poly(rA).(dT)12-18 and poly(rC).(dG)12-18 very efficiently and had a marked preference for Mg2+ ions over Mn2+ ions. The pattern of ionic dependency for this enzyme is similar to that of reverse transcriptases purified from human lymphotropic viruses. The patient was tested and found sero-negative for HIV-1, HIV-2, and HTLV-I and seropositive (immunoglobulin G) for cytomegalovirus. Epstein-Barr virus nuclear antigens (EBNA) were detected in the patient's B lymphocytes. Since reverse transcriptase is the hallmark of retroviruses, we suggest that a retrovirus may be involved in the etiology of osteopetrosis.

Lack of evidence for a role of T-cell-associated retroviruses as an etiology of schizophrenia.

The evidence that schizophrenia may involve infection by a virus (or viruses) has been indirect. The recent discovery, however, of the human retroviruses--human T-cell lymphoma-leukemia virus-I, and II (HTLV-I, -II) and human immunodeficiency virus (HIV)--now also known to affect the central nervous system (CNS), together with the development of new techniques in retrovirology, have made it possible to investigate more directly the role of this class of viruses as an etiology of schizophrenia. In our first effort to screen for the presence of a T-cell lymphotropic virus in schizophrenia, short-term tissue cultures of peripheral lymphocytes from 17 chronic schizophrenic patients and 10 normal controls were established. The cells were cultured in the presence of T-cell growth factor (TCGF, IL-2), and the culture supernatants were tested for the presence of the retroviral enzyme reverse transcriptase. No T-cell-associated reverse transcriptase activity was detected in cultures from patients or normal controls. Therefore, the data do not provide evidence for a role for T-cell lymphotropic retroviruses as an etiology of schizophrenia.

A rapid phenotypic assay for detecting multiple nucleoside analogue reverse transcriptase inhibitor-resistant HIV-1 in plasma.

Zidovudine and other nucleoside analogue reverse transcriptase inhibitors (NRTIs), like zalcitabine and didanosine used for treatment of individuals infected with HIV-1, can select for viruses with Q151M and other associated mutations (for example, A62V, S68G, V751, F77L, F116Y) in the reverse transcriptase (RT) enzyme. These mutations confer resistance to multiple nucleoside analogues, and thereby compromise the efficacy of this class of drugs. Presently available phenotypic assays for detection of multiple nucleoside analogue resistant (MNR) HIV-1 require testing for each NRTI individually. Here we report an enzymatic RT assay that uses resistance to zidovudine triphosphate (zidovudine-TP) as a diagnostic biochemical marker of MNR HIV-1. This assay exploits the different biochemical mechanisms for zidovudine-resistance conferred by either Q151 M or T215Y/F mutations and the inability of conventional RT assays to detect T215Y/F-associated zidovudine resistance. The assay detects RT activity directly in plasma by using Amp-RT, an ultra-sensitive PCR-based RT assay. We show that enzymatic resistance to zidovudine-TP is specific to MNR RT and is distinguishable from both wild-type (WT) and RT containing classical zidovudine-resistant mutations (D67N, K70R, T215Y/F, K219Q). Compared to WT, MNR HIV-1 RT had 5- to 36-fold increases in the concentration of drug required to inhibit 50% (IC50) of RT activity, depending on the presence of Q151 M alone or with additional MNR mutations. A screening assay utilizing 1 microM zidovudine-TP was developed and validated on 14 reference isolates, 37 plasma specimens, and seven patient-derived viruses. Twenty-three specimens were found to have reduced susceptibility to zidovudine-TP, and all had Q151 M. In contrast, 21 specimens were sensitive to zidovudine-TP, of which 12 had WT genotypes, four had T215Y/F, and five had T69S-insertions along with T215Y/F mutations. This RT-based phenotypic assay provides a specific and rapid tool for the direct identification and monitoring of Q151M-associated MNR HIV-1 in plasma.

Correlation of a specific chromosomal marker, 21q-, and retroviral indicators in patients with thrombocythemia.

A chromosomal anomaly, 21q-, has been found in association with retroviral indicators in patients with myeloproliferative disorders (MPD) including polycythemia vera (PV), essential thrombocythemia (ET), chronic myelocytic leukemia (CML) and acute non-lymphocytic leukemia (ANLL). The viral indicators are found in platelet homogenates of thrombocythemic patients. Evidence is presented from 2 laboratories (Philadelphia, USA and Bologna, Italy) for the 21q- deletion in MPD patients. Thirty patients evaluated for the presence of both viral and chromosomal markers in Philadelphia showed positive correlations.

Platelet DNA polymerase levels in CML: implications for oncogenesis.

Partially purified platelet DNA polymerase (PDP) was able to synthesize DNA transcripts of platelet polyadenylated RNA. PDP was elevated in the earliest stages of CML and PV. In PV, successful chemotherapy resulted in rapid return of PDP to normal levels while in CML this was not the case. An hypothesis is presented proposing that PDP contributes to oncogenesis by regulating the expression of oncogenes.

Effects of SKF 108922, an HIV-1 protease inhibitor, on retrovirus replication in mice.

Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polypeptides in cultures of human T lymphocytes infected with human immunodeficiency virus type 1 (HIV-1) and therefore suppress the infectivity of HIV-1 in vitro. We have previously reported the antiviral activity in vitro of HIV-1 protease inhibitors against the C-type retrovirus Rauscher murine leukemia virus (RMuLV) and the lentivirus simian immunodeficiency virus (SIV). The same compounds which blocked the infectivity of HIV-1 also inhibited the infectivity of RMuLV and SIV in vitro. This report extends these findings by testing the antiviral activity of HIV-1 protease inhibitors in vivo in the RMuLV model. RMuLV-infected mice were treated twice a day (bid) with either an active (SKF 108922) or inactive (SKF 109273) compound for fourteen days by the intraperitoneal (i.p.) route. Compared with excipient control, SKF 108922, formulated with hydroxypropyl-beta-cyclodextrin (HPB), reduced virus-induced splenomegaly, viremia, and serum reverse transcriptase (RT) levels, while SKF 109273 was inactive. The HPB vehicle by itself enhanced replication of RMuLV. The effects of changing the formulation and the route of administration were examined. SKF 108922, formulated in HPB, had similar antiviral activity when administered by the i.p. or subcutaneous (SC) routes. However, SKF 108922 administered as a colloidal suspension in cholesterol sulfate (CS) had no detectable antiviral effect. Measurements of the circulating levels of the protease inhibitor in plasma explained this result. Plasma concentrations of SKF 108922 exceeded 1000 nM within 10 min after SC administration of the compound solubilized in HPB, but SKF 108922 was not detected in plasma after SC administration of the same dose formulated with CS. Information on optimal conditions for administering these agents should prove useful in guiding their clinical application Therefore, RMuLV should provide a good model for the preclinical evaluation and development of this class of agents for the treatment of HIV.

An in-vivo infectivity assay for cloned retroviruses lacking a susceptible cell culture.

The lymphoproliferative disease virus (LPDV) of turkeys is the retroviral agent of etiology of a rapidly developing, naturally occurring, lymphoproliferative process. Recently we have molecularly cloned the viral genome. The lack of a susceptible cell culture which can sustain LPDV replication hampered the analysis of the infectious capability of the cloned genome. Based on the efficient in-vivo replication of LPDV we have developed a sensitive in-vivo approach aimed at establishing the infectious capability of the cloned provirus. According to this approach, peripheral leukocytes withdrawn from 3-week-old turkeys were transfected with the cloned DNA and the transfected leukocytes were re-injected into the turkey from which they had been obtained. The injected leukocytes enabled the efficient expression of the viral genome and the release into the blood stream of LPDV virions, which thereafter could travel to their appropriate in-vivo target lymphoid cells and start multiple replication cycles, resulting in the development of a detectable viremia. The applicability of this in-vivo assay for other cloned viral genomes is discussed.

Reactivity of five anti-HIV-1 subtype O specimens with six different anti-HIV screening ELISAs and three immunoblots.

Five anti-subtype O specimens were tested by anti-HIV-1/2 screening and confirmatory assays. They can be divided into three specimens, reactive with all ELISAs, independent of the nature of the antigen (recombinant proteins or peptides) and test configuration (indirect ELISA or double antigen/sandwich ELISA). One specimen was not detected by one peptide based ELISA. One specimen was only recognized by two ELISAs and should be considered as a marker sample for the weakness of currently used ELISAs with anti-subtype O. Three different immunoblot assays available commercially detected two of the specimens with a major binding of gp160 and other viral bands, especially the integrase and reverse transcriptase. Another two specimens lacked reactivity with glycoproteins almost completely, but showed some staining with the enzymes of HIV, and would most probably be interpreted as indeterminate. The fifth specimen, which was also missed by most of the ELISAs, had very faint staining of the gp160 and a very weak staining of p24, and would most probably be interpreted as negative. Adaption of currently available tests to anti-subtype O is needed for the future reliability of anti-HIV diagnostic reagents.

Development of an assay that detects transcriptionally competent human immunodeficiency virus type one particles.

To study the functional properties of HIV-1 reverse transcriptase (RT) from intact viral particles without the requirement for tissue culture expansion, a method that couples HIV-1 reverse transcription utilizing its endogenous RT (ERT) with polymerase chain reaction amplification (PCR) was developed. Detection of endogenous reverse transcripts from HIV particles by ERT-PCR was compared to HIV RNA PCR detection using avian myeloblastosis virus (AMV) RT from plasma samples from 45 HIV-1 infected patients. The HIV ERT-PCR method was capable of detecting plasma viremia with the same efficiency (29/29 patients) as the AMV RT HIV RNA PCR in patients with CD4 cell counts of less than 500/mm3. The determination of HIV-RT drug sensitivities using four well-characterized HIV-1 lab strains was assessed. The ERT-PCR method detected reduced sensitivity to TIBO R82150 (10 microM) in a TIBO resistant strain but not in the TIBO sensitive HTLV-IIIB viral mixture or an HTLV-IIIB clone. In summary, the HIV ERT-PCR method provides a useful approach for the detection of HIV and the characterization of RT sensitivities among HIV-1 strains.

Reverse transcriptase activity (RTA) in lymphocyte cultures of HIV-infected patients receiving short treatments of HPA 23. A biological evaluation.

Ammonium tungsto antimoniate (HPA 23) is a potent inhibitor of nucleic acid polymerases and reverse transcriptases of retroviruses. Its in vivo activity as an HIV inhibitor was previously published. However, its clinical use is limited by pharmacological parameters (short half-life and intravenous administration) and significant side effects (thrombocytopenia). In order to evaluate the place of this drug in the therapeutic strategy of HIV-infected patients, we administered 1.5 mg/kg of HPA 23 in 15 infected patients at various stages of the disease twice a day during 14 days. A significant decrease of reverse transcriptase activity (less than 15% of the initial value) was noticed in 13 patients. This activity remained low at least 6 weeks after the end of the treatment in 8 patients. Thrombocytopenia was the only significant side effect reported and was always transient. This study suggests that HPA 23 can be used as an induction treatment in patients infected by HIV. A maintenance treatment has to be defined, as well as the association to other drugs.

Busulphan: effect on platelet RNA dependent DNA polymerase--implications in the treatment of polycythemia vera, thrombosis and atherosclerosis.

Analysis of controlled studies performed by the Polycythemia Vera Study Group (P.V.S.G.) and the European Organization for Research in Treatment of Cancer (E.O.R.T.C.) indicate that busulphan (Myleran) (BU) is the treatment of choice for polycythemia vera (PV). BU is particularly effective as compared to aspirin and dipyridamole (Persantine) or radioactive phosphorus (32P) in preventing the thrombotic and atherosclerotic complications of PV. In contradistinction to chlorambucil (CM), BU is not associated with an unacceptable increase in the incidence of leukemia. The pharmacology of BU remains unclear, but certainly it cannot be considered a classic alkylating agent. BU suppresses the activity of the reverse transcriptase-like RNA dependent DNA polymerase in the platelets of these patients. A clearer understanding of the role of BU in the treatment of the myeloproliferative disorders will provide important insights into the etiology and pathogenesis not only of preneoplastic states, but also thrombosis and atherosclerosis.

Preliminary analysis of the polypeptides of the salmon leukemia virus (SLV) and evidence for development of a bimodal viremia following SLV infection.

A retrovirus, known as salmon leukemia virus (SLV), was purified from farm-reared chinook salmon (Oncorhynchus tshawytscha) with plasmacytoid leukemia (PL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified SLV revealed the presence of 9 virus-associated polypeptides with molecular weights from 82 kDa to 15 kDa. Endoglycosidase digestion and alcian blue staining of viral polypeptides separated by SDS-PAGE, and immunoprecipitation experiments using hyperimmune antisera suggest that the non-glycosylated 27 kDa polypeptide may represent a capsid-associated protein and the 82 kDa glycoprotein may represent an envelope-associated protein, which appears to be composed of a 67 kDa protein moiety. Fish injected with PL-positive tissue homgenate developed a bimodal viremia, as indicated by the presence of cell-free, virus-associated reverse transcriptase activity and SLV in serum of fish from 1 to 3 wk post-injection and again from 7 wk on through the rest of the study. If horizontal transmission of SLV and PL occurs in infected chinook salmon, it is most likely to occur after the second viremic period begins.

Effect of lymphoproliferative disease virus and of niridazole on the in vitro blastogenic response of peripheral blood lymphocytes of turkeys.

Turkeys inoculated at 5 weeks of age with lymphoproliferative disease (LPD) virus developed typical lesions in the spleen, thymus, and pancreas. The in vitro blastogenic response of peripheral blood lymphocytes to the mitogens phytohemagglutinin and concanavalin A was drastically (up to 90%) suppressed in the inoculated turkeys 1 to 4 weeks postinoculation compared with uninoculated controls, and even at 11 weeks the response was about 50% inhibited. A lethal (about LD33) dose of antihelminthic drug niridazole, 100 mg/kg given each day for 3 days to 4-week-old turkeys, caused a transient inhibition of the blastogenic response within 32 days of treatment, which was less pronounced than that observed in turkeys inoculated with LPD virus, whether pretreated with niridazole or not. Virus-associated reverse transcriptase activity in the plasma was significantly higher in the turkeys pretreated with niridazole, and LPD lesions developed to the same extent in the untreated and treated groups, as determined 9 weeks post virus inoculation. A sublethal dose of niridazole, 50 mg/kg given each day for 4 days, did not suppress the blastogenic response to mitogens at any time determined (starting 10 days post-treatment) and did not affect the pathogenesis of LPD and the viremia. Body weights were significantly decreased by virus infection and by treatment with lethal doses of niridazole.

Retrovirus-like activity in an immunosuppressed dog: pathological and immunological findings.

A putative retrovirus was isolated from a dog with a severe, acquired immunodeficiency-like syndrome. The haematological abnormalities and immunological deficiencies included anaemia, leucopenia (lymphopenia and neutropenia), thrombocytopenia, decreased humoral immunity, and ineffective T-cell responses in-vitro. The necropsy findings included generalized lymphoid depletion, severe bone marrow hypoplasia, plasmacytic infiltrates in lymphoid and non-lymphoid organs, and severe secondary infections. Supernates of peripheral blood mononuclear cell cultures from the affected dog contained an agent with manganese-dependent reverse transcriptase (RT) activity that sedimented at a density of 1.122 g/ml. RT activity was also found post-mortem in extracts prepared from the bone marrow, lymph nodes, and small intestine. The lymph nodes and small intestine expressed a 3.8 kb mRNA that was recognized by a bovine leukaemia virus (BLV) pol DNA probe by Northern blotting. DNA isolated from the lymph nodes and small intestine from the affected dog showed distinct band patterns by Southern analysis, suggesting an exogenous retrovirus. The retrovirus could be propagated in normal canine peripheral blood mononuclear cells or short-term canine lymphocyte cell lines in-vitro, and was cytopathogenic for cells of canine, but not human, origin. These results suggest the existence of a pathogenic canine retrovirus capable of producing disease of the type associated with retroviruses in other species.