Research in Veterinary Science 1992, 52, 250-255

Isolation and preliminary characterisation of a novel retrovirus isolated from a leukaemic dog N. SAFRAN, K. PERK*, O. EYAL, Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot Campus, POB 12, Rehovot, Israel J. E. DAHLBERG, Advanced

Biotechnologies lnc, Rivers Park 1I, 9108 Guilford Road, Columbia, Maryland 21046, USA

and in dogs with lymphosarcoma and of viruslike particles in tumour specimens (Onions 1980, Strandstrrm and Bowen 1982, Tomley et al 1983, Sykes et al 1985). A feature common to many exogenous retroviruses, particularly all the known lentiviruses including the human immunodeficiency virus HIV-1, is the propensity to infect latently cells of the monocyte/macrophage lineage (Narayan and Zink 1988). In the case of lentivirus-infected animals, immature infected monocytes do not express significant levels of viral proteins, while upon maturation to macrophages they may synthesise high levels of virus particles (Narayan and Zink 1988). A programme has been initiated in which fresh mononuclear cells from normal and tumour-bearing animals which are seen in this clinic are screened for the presence of retroviruses. The characterisation and propagation of a novel retrovirus, canine retrovirus, isolated from a leukaemic dog is reported here.

A novel canine retrovirus was isolated from mononuelear cells of the peripheral blood of a leukaemie dog. The main clinical and pathological findings in this dog were lethargy, anorexia, weakness, dyspnoea, severe anaemia, thombocytopenia and a high white blood cell count, practically all of which were lymphoblasts. The virus was isolated from mononuclear cells obtained from the blood, cocultivated with indicator cells. The virus particles encode a reverse transcriptase with Mg ++ preference, have a density in sucrose gradients of 1"16 g ml-t, and induce syncytia in permissive cell cultures such as Himalayan tahr ovary and canine fetal thymus lines. This agent replicates to high titres. The virus exhibits a morphogenesis and morphology typical of lentiviruses. Immunoblotting and competitive radioimmunoassays failed to detect immunological crossreactivity with other representative lentiviruses and oncoviruses of the retrovirus family.

IN most domestic animals and in man, endogenous and, or, exogenous retroviruses have been isolated and frequently shown to be associated with severe disease (Gross 1983, Theilen and Madewell 1987, Varmus 1988). Furthermore, interest in retrovirus research has dramatically accelerated in the past decade, with the discoveries of human retroviruses that cause adult T cell leukaemia (Poiesz et al 1980) and acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al 1983, Gallo et al 1984, Levy et al 1984). However, despite a great deal of effort devoted to searching for a retrovirus in the dog, no verifiable isolate has yet been reported (Strandstrrm et al 1990), although there have been reports of reverse transcriptase activity in some normal dogs *Corresponding author

Materials and methods

The virus was isolated from a dog which was included in our screening programme for retroviruses of animals brought to the authors' veterinary teaching hospital.

Clinical, haematological and biochemicalfindings The dog was an eight-year-old female German shepherd. The main clinical signs were anorexia, weakness, lethargy, dyspnoea, pale mucous membranes and rapid pulse. The main haematological parameters were: red blood cells 1.74 × 106 mm-3; haemoglobin 5.3 g

250

Canine retrovirus dl-1; packed cell volume 14-7 per cent; white blood cells 49,000 mm -3, platelets 99,000 mm-3 mean corpuscular volume 84 #1. The blood smear showed that almost all white blood cells were immature lymphoblasts, indicating acute lymphoblastic leukaemia, Biochemical abnormalities included: lactic dehydrogenase 515/tg litre-1; aspartate aminotransferase 86/.tg litre-1; alanine aminotransferase 73 /zg litre-1; urea 89 mg dl-1; alkaline phosphatase 824/zg litre-1; creatine phosphokinase 132/.tg litre-1; albumin 1-9 g d1-1. Isolation of retroviruses

251

10 minutes at 4°C. The supernatants were pelleted over 20 per cent glycerol for one hour at 100,000 g. The pellet was resuspended to 50 #1 in 0.01 M Tris-HCl, pH 8.1, containing 0.2 per cent Nonidet P-40. After an incubation period of 10 minutes at 0°C, 10 ~1 dT:rA (poly(rA):oligo(dT)l 2. 18), 20/A BM/6/60 including 30 mM MgC12, 240 mM KC1, 2 mM DTT in 250 mM Tris-HC1, pH 8.1, and 20/zl 2 dNTP including 0.01 [tmol of 3HdTTP (50 Ci mmo1-1) and 0.2/zmol each of dGTP, dCTP and dATP were added to each sample. The samples were incubated for 30 minutes at 37°C, following which the reaction was stopped by the addition of 0-5 ml TCA mix. The precipitated nucleic acids were collected and filtered on nitrocellulose filters and washed with cold 5 per cent TCA. The filters were dried and the radioactivity was counted.

Blood was collected from the jugular vein in a vacutainer containing 143 usP units of sodium heparin per 10 ml tube. The blood was centrifuged for 20 minutes at 830 g. The buffy coat layer was collected and diluted to a final volume of 4 ml with phosphate buffered saline (I'Bs) contain- Density gradient ing 100 in ml-1 penicillin and 100/zg m1-1 strepThe tissue culture medium from infected cells tomycin. The diluted sample was carefully layered on an equal volume ofFicoll-Paque (Pharmacia), was centrifuged at 3000 g and 10,000 g for 10 and centrifuged for 20 minutes at 830 g. Cells minutes at 0°C. The supernatant fluid was layered were harvested from the interface, washed and on a 10 ml column of 20 per cent glycerol in 100 pelleted three times for 10 minutes at 150 g with mM NaC1, 10 mM tris, 1 mM EDTA (TNE) and Dulbecco's minimal essential medium (DMEM) spun at 100,000 g for one hour at 4°C. The resultcontaining antibiotics as above. The cells were ing pellet was resuspended in 3 ml TNE, layered finally resuspended in DMEM containing 20 per on a linear gradient of 20 to 50 per cent in the cent heat-inactivated fetal calf serum, and seeded SW27 rotor at 4°C for 16 hours. The gradient in 10 cm tissue culture dishes. Monocytes were was collected from the bottom of the tube in 30 left to adhere for 16 hours, then lymphocytes equal fractions. Twenty/11 of each fraction was and other non-adherent cells were removed, and measured for sucrose density with a refractomefresh medium was added to the plates. In some ter. Two gl of 5 per cent NP-40 detergent was cultures, 1-5 x 106 indicator cells (Himalayan added to 28/.tl of each fraction and incubated tahr ovary, [HTO] cells [HJ1 ov, ATCC CRL427] for 10 minutes at 0°C, and the reverse transcripcanine fetal thymus cells [Cf2Th3 ATCCCRL 1430] tase activity was measured by the method were added to enable any viruses present in the described above. monocytes to grow in the heterologous cell line. All cultures were monitored by microscopic examination and supernatant reverse transcrip- Mg 2+ and Mn 2+ assay tase activity. Mg 2+ and Mn2+-dependent reverse transcriptase activity was detected in purified density Reverse transcriptase activity assay sucrose gradient fractions, containing the canine Reverse transcriptase activity was measured retrovirus. The reverse transcriptase activity was in uninfected HTO cells, infected HTO cells, unin- measured with poly(rA):oligo(dT)12_18 template fected canine fetal thymus cells, and in infected primer, four different deoxyribonucleoside Cf2Th cells. Samples of 2 ml of supernatant fluids triphosphates and 240 mM KC1 for Mg 2+ and collected at the indicated times were clarified by 60 mM NaC1 for Mn 2+ assays (Colcher and centrifugation at 3000 g and then 10,000 g for Schlom 1980).

252 Electron microscopy

N. Safran, K. Perk, O. Eyal, J. E. Dahlberg

free supernatant fluids from this culture were collected, filtered through a 0.2/an filter, and Cell culture samples were fixed in situ with 3 added to fresh cultures of HTO. They were found per cent glutaraldehyde, scraped and pelleted and to be infectious. Syncytium induction was post fixed with 1 per cent osmium tetroxide. Thin observed within three days in the culture which sections were stained with uranyl acetate and received filtered medium obtained from the origlead citrate. inal coculture, but not in the control flask. Subsequent efforts to transmit this infectious agent were successful, and transmission has been Immunoblot analysis observed even in cultures which received superTwo hundred ~tg each of purified virus (canine natant fluids diluted by a factor of 1 × 105. The retrovirus, C-type cat endogenous virus, murine ability of this agent to replicate to high titre has mammary tumour virus, squirrel monkey retro- been verified by large scale production, in which virus, Mason-Pfizer monkey virus, and caprine yields of approximately 0-5 mg of purified virus arthritis encephalitis virus) were electrophoresed are obtained per litre of culture medium. It was on separate 10 per cent polyacrylamide gels. A subsequently found that canine retrovirus could comb was not used, so that the sample formed also be transmitted to canine fetal thymus cells. a continuous band across the gel. After elec- It is interesting to note that despite repeated trophoretic separation, the viral proteins were efforts, feline immunodeficiency virus did not transblotted on to nitrocellulose sheets (in 25 replicate in Cf2TH cells nor in IqTO cells. mM Tris, 192 mM glycine, 20 per cent methanol, Culture supernatants from infected HTO and pH 8.3), using a current of 100 mA for 16 hours. Cf2TH cells were analysed for reverse transcripThe blots were soaked in PBS containing 5 per tase activity. Optimal activity was obtained seven cent non-fat dried milk (NFDM) for two hours, days after infection using a poly(rA):oligo washed in deionised water (three times, 10 min- (dT)12.18 template primer (Fig 1), in excellent utes each) and dried at room temperature before correlation with maximum syncytia formation. being cut into 4 mm strips. Immunoblot analysis Fig 2 summarises data indicating that the canine was carried out by exposing strips to sera diluted retrovirus reverse transcriptase prefers magnein PBSwith NFDM overnight at room temperature, sium to manganese as its divalent cation. The followed by washing (PBS with 0.05 per cent virus particles have a density in sucrose gradients Tween 20), and exposure to the appropriate anti- of 1.16 g ml-1, as determined by measurieg reverse species IgG conjugated to horseradish peroxidase transcriptase activity on gradient fractions (Perk (one hour at room temperature). After additional et al 1974). washing, the strips were exposed to 4-chloro-1Electron microscopic examination of HTO culnaphthol. The strips were washed with water to tures exhibiting maximal syncytium formation halt the reaction (Towbin et al 1979, Bers and revealed a high proportion of the cells to be synGrafin 1985). thesising retroviral particles (Fig 3). As seen in the figures, budding (Fig 3a), immature and mature extracellular virions (Fig 3c) are readily Results and discussion apparent. Also seen are intracytoplasmic typeBuffy coat cells were obtained from a leukaemic A particles (Fig 3b). The morphology and mordog and cultured as described above. The mono- phogenesis of this canine retrov.irus bears striking cyte culture which was established began to similarities to the lentivirus subfamily. The virions degenerate after several days in culture and a bud at the cell plasma membrane with a crescentnumber of cell lines were cocultivated with these shaped nucleoid consisting of a thin outer unit membrane with a closely opposed wider electron monocytes. The coculture, containing HTO cells added to dense crescent membrane modelling it. The extraone of the monocyte-macrophage cultures, exhib- cellular mature virus particles usually contain a ited widespread small (three or four nuclei) syn- single electron-dense core. Occasionally, barcytia which continued to increase in size after shaped cores were seen. The intracytoplasmic four days. Within an additional six to nine days, type-A particles, which typically appear in large the monolayer was completely destroyed. Cell clusters, consist of two layers surrounding an

Canine retrovirus 14

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FIG 2: Mg2+and Mn2+- dependent reverse transcriptase (RT)activity in purified density sucrose gradient fractions, containing the canine retrovirus. The RT activity was measured with poly(rA):oligo(dT)12_18 template primer, 2dNTP and 240 mM KCI for Mg2+ and 60 mM NaCI for Mn2+ assays. • Mn+÷, [ ] Mg++

electron-lucent centre. Such particles, known to be equivalent to the core of type B retroviruses (for example, murine mammary tumour virus) (Dalton and Haguenau 1973, Dahlberg et al 1974, Fine and Schochetman 1978) or type D retroviruses (for example, Mason-Pfizer monkey virus) (Fine and Schochetman 1978), were also repeatedly reported in lentivirus infected cultures (Coward et al 1970, Bouthe and Van der Maaten 1974, Dahlberg et al 1981, Perk 1990). The intracytoplasmic particles were frequently aberrant in both size and shape and many were multilaminated. Such pleomorphism is also typical of the intracytoplasmic particles seen in cultures infected with other lentiviruses. The canine retrovirus was further characterised by testing for immunological relatedness to other retroviruses. Mason-Pfizer monkey virus, squirrel

C FIG 3: Electron micrographs of HTO-canine retrovirus infected culture, on day 7 after infection, showing (a) virions budding, with crescentshaped nucleoids from cell membrane; (b) intracytoplasmic type-A particles; (c) extracellular mature particles, a and b approximately × 80,500 and c approximately × 67,000

254

N. Safran, K. Perk, O. Eyal, J. E. Dahlberg

monkey retrovirus (as representatives of primate type D viruses), cat endogenous virus (as a representative type C virus), murine mammary tumour virus (as a representative type B virus), and caprine arthritis encephalitis virus (as a representative lentivirus), were tested in addition to canine retrovirus. Two hundred/.tg each of the purified viruses were elect'rophoresced on 10 per cent polyacrylamide gels and transferred to nitrocellulose. Strips of each blot were probed with antisera to each of these viruses except the canine retrovirus to determine if immunological crossreactivity to canine retrovirus could be detected. As shown in Fig 4, when canine retrovirus strips were analysed, the viral proteins were not identified by heterologous anti-retroviral sera. However, there were some background bands, possibly representing contaminating immunoglobulin heavy and light chains present in the virus preparation which reacted with the conjugate. On the other hand, heterologous crossreactivity was readily demonstrated by this method between CaRV 234

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squirrel monkey retrovirus and Mason-Pfizer monkey virus (as expected) (Colcher et al 1978, Devare 1978) and, more surprisingly, although nonreciprocally, between murine mammary tumour virus and squirrel monkey retrovirus (lane 15). These blots also detected crossreactivity between the major gag proteins of murine mammary tumour virus (p28, lane 16) and RD114 (p30, lane 19). Additionally, several sera identified the small highly conserved region of envelope glycoprotein (lane 20) shared by the primate type D viruses (squirrel monkey retrovirus and MasonPfizer monkey virus) and by both baboon endogenous virus and cat endogenous virus (which are closely related). These results indicate that Western blot analysis is a highly suitable technique for detecting immunological crossreactivity between quite distantly related retroviruses. In addition, several competitive radioimmunoassays capable of detecting nanogram levels of human immunodeficiency virus (HIV-1) p24, equine infectious anemia virus p 14, caprine arthritis encephalitis virus p27, and ovine progressive ~TV

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FIG 4: Immunoblot analysis of representative retroviruses. Except for the strips illustrated, all results using heterologous sera were negative for crossreactivity. The sera used are as follows: CaRV lane 1 negative control, 2 anti-(suRv) (goat), 3 anti RDl14 (goat), 4 anti-(Maw) (rabbit), 5 anti-HIV-1 (human), 6 anti-(cAEv) (rabbit); MPMV=7 negative control, 8 anti MPMV(goat), 9 anti-sMnv (rabbit); SMRV:10 negative control, 11 anti-sMRV (rabbit), 12 anti-aPMV (goat); MMTV:13 negative control, 14 anti-MaTV (rabbit), 15 anti-sMRV (goat), 16 anti-RD114 (rabbit); RD114:17 negative control, 18 anti-RD114 (rabbit), 19 antiMMTV(rabbit), 20 anti-MPMV(goat). The major gag proteins for each virus, as well as the major glycopretein for RD114, are indicated with arrows. CAEVCaprine arthritis encephalitis virus, CARVCanine retrevirus, MMTVMurine mammary tumou r virus, MPMVMason-Pfizer monkey virus, RD114 C-type cat endogenous virus, SMRVSquirrel monkey retrovirus

Canine retrovirus

pneumonia virus p28 wereused to test whether canine retrovirus was closely enough related to other animal and human lentiviruses to compete in the assays. Concentrated and purified canine retrovirus failed to compete in any of these radioimmunoassays (data not shown). The canine retrovirus which the authors have isolated has a number of morphological, physical and biological properties, (that is, syncytium induction, magnesium-dependent reverse transcriptase activity and morphogenesis), which suggests that it be classified as a new lentivirus. This novel naturally occurring canine retrovirus-lentivirus extends the range of retrovirus infection to the canine species. As a lentivirus, in addition to its relevance to veterinary medicine, it may provide a useful model for HIV and related research. Acknowledgement The authors thank Dr Niels Pedersen, School of Veterinary Medicine, Davis, California, USA, for the gift of feline immunodeficiency virus. References BARRt~-SINOUSSI,F., CHERMANN, J. C., REY, F., NUGEYRE, M. T., CHAMARET, S., GRUEST, J., DAUGUET, C., AXLERBLIN, C., VEZINET-BRUN, F., ROUZIOUX, C., ROZENBAUM, W. & MONTAGNIER, L. (1983) Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).Science 220, 868-871 BERS, G. & GRAFIN, D. (1985) Protein and nucleic acid blotting and immunobinchemical detection. BioTechniques 3, 276-288 BOUTHE, A. D. & VAN DER MAATEN, M. J. (1974) Ultrastructural studies of a visna-like syncytia-producing virus from cattle with lymphocytosis. Journal of Virology 13, 197-204 COLCHER, D. & SCHLOM, J. (1980) Purification and characterization of the RNA-directed DNA polymerase of a primate type-D retrovirus: Mason Pfizer virus. Biochimica et Biophysica Acta 607, 445-456 COLCHER, D., TERAMOTO, Y. A. & SCHLOM, J. (1978) Immunological and structural relationships between Langur virus and other primate type-D retroviruses. Virology 88, 384-388 COWARD, J. E., HARTER, D. H. & MORGAN, C. (1970) Electron microscopic observations of visna virus infected cells. Virology 40, 1030-1038 DAHLBERG, J. E., GASKIN, G. M. & PERK, K. (1981) Morphological and immunological comparison of caprine arthritis encephalitis and ovine progressive pneumonia viruses. Journal of Virology 39, 914-919 DAHLBERG, J. E., PERK, K. & DALTON, A. J. (1974) Viruslike particles induced in guinea pig ceils by 5-bromo-2'-deoxyuridine are morphologically similar to murine B-type virus. Nature 249, 828-830

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