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Epithelial cells from goat oviduct are highly permissive for productive infection with caprine arthritis–encephalitis virus (CAEV)
Virus Research 87 (2002) 69 – 77 www.elsevier.com/locate/virusres
Epithelial cells from goat oviduct are highly permissive for productive infection with caprine arthritis–encephalitis virus (CAEV) A. Lamara a,*, F. Fieni a, L. Mselli-Lakhal b, D. Tainturier a, Y. Chebloune b a
Laboratoire d’Etude des Risques Sanitaires lie´s aux Biotechnologies de la Reproduction, Ecole Nationale Ve´te´rinaire de Nantes, Atlanpole-La Chantrerie, BP 40706, 44307 Nantes Cedex, France b UMR 754 INRA/ENVL/UCBL Virologie Cellulaire, Mole´culaire et Maladies Emergentes, Ecole Nationale Ve´te´rinaire de Lyon, 1, A6. Bourgelat, B.P. 83, 69280 Marcy l’Etoile, France Received 25 October 2001; received in revised form 23 April 2002; accepted 23 April 2002
Abstract Caprine oviduct epithelial cells (COEC) are commonly used in in vitro goat embryo production protocols to stimulate early embryonic development. These feeder cells are usually collected from slaughterhouses from unknown serological status animals for caprine arthritis–encephalitis virus (CAEV) infection which is frequent in many regions of the world. Tissues derived from this source may be contaminated with CAEV and the use of such material in in vitro fertilisation systems may contribute to transmission of this pathogen to the cultured embryos and dissemination via embryo transfer (ET). The aim of this study was to determine the permissiveness of COEC to CAEV replication in vitro. Cells were isolated from goats from certified CAEV-free herds and then were inoculated with two CAEV strains: the molecularly-cloned isolate of CAEV (CAEV-pBSCA) and the French field isolate (CAEV-3112). Cytopathic effects (CPE) were observed on cell culture monolayers inoculated with both CAEV strains. Expression of CAEV proteins was shown both by immunocytochemistry using anti-p24 gag specific antibodies and by immunoprecipitation using a hyperimmune serum. The CAEV proteins were correctly and properly processed by artificially-infected COEC and the titers of virus released into the supernatant reached 106 TCID50/ml 6 days post-inoculation. Although the macrophage lineage cells are the main centre of infection in the virus-positive animal, these findings suggest that epithelial cells may be important in the viral life cycle probably as a reservoir allowing the viral persistence, dissemination and pathogenesis. These results suggest also that the use in in vitro fertilisation systems of co-culture feeder cells that support efficient replication of CAEV to high titers could represent a serious risk for permanent transmission of virus to the cultured embryos and to the surrogate dam involved. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Caprine oviduct epithelial cells; CAEV; Replication; Cytopathic effect
* Corresponding author. Tel.: + 33-24068-7714; fax: + 33-24068-7748 E-mail address: [email protected] (A. Lamara). 0168-1702/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 2 ) 0 0 0 8 2 - 5
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1. Introduction Artificial reproductive techniques are increasingly used for the expansion of stocks of highvalue goats from genetically selected parents. In view of the widespread prevalence of the caprine arthritis–encephalitis virus (CAEV) in goats in most regions of the world (Adams et al., 1984; Robinson and Ellis, 1986; Hanel, 1991), it is desirable to avoid any associated propagation of this economically damaging virus. CAEV is a lentivirus which chiefly targets cells of the monocyte/macrophage lineage in the infected animal (Narayan et al., 1982, 1983), and which is transmitted to the offspring through colostrum and milk (Kennedy-Stoskopf et al., 1985; McGuire, 1987). Infection is lifelong, and disease can affect multiple organs resulting in disability or even death (Narayan and Clements, 1989). CAEV, like other lentiviruses, has been shown to be capable of infecting epithelial cells from several organs, and viral transcripts can be found in kidney or small intestine (Zink et al., 1990; Brodie et al., 1995) or mammary gland (Mselli-Lakhal et al., 1999) epithelia from infected animals. Infection of epithelial cells by HIV-1 may contribute to milk transmission (Toniolo et al., 1995) or to primary infection of the reproductive tract (Howell et al., 1997). Infection of epithelial cells has also been observed with feline immunodeficiency virus (Park et al., 1995), bovine immunodeficiency virus (Zhang et al., 1997 and equine infectious anaemia virus (Carpenter and Chesebro, 1989). While CAEV screening of the parental donors and the surrogate dams for artificial goat reproduction is possible and desirable, it may not be sufficient to exclude the risk of virus transmission. Several potentially infectable epithelial cell tissues are employed in the artificial preparation of embryos for implantation. We have previously shown that granulosa cells, which are necessary for the efficient pre-fertilisation maturation of the oocytes, and which are often derived from untested slaughterhouse animals, can sustain infection by CAEV (Lamara et al., 2001). For the post-fertilisation development of the embryo, a feeder culture (Prichard et al., 1992; Crozet et
al., 1995; Martino et al., 1995) of caprine oviduct epithelial cells (COEC) is used to provide essential support for its early development (Galli and Moor, 1991; Crozet, 1991). In the present paper, we investigate the susceptibility of COEC to infection by CAEV. Control of cultures used as feeders in artificial reproduction of goats has not been specifically addressed, although the International Embryo Transfer Society (IETS, 1990) and the Office Internationale des Epizooties (Singh, 1985) have produced recommendations for the certification of donors, stringent washing procedures and rejection of embryos with a damaged zona pellucida (ZP). Some control may be desirable, because bovine viral diarrhoea virus (Booth et al., 1995) has been shown to be transmitted from such cells to the embryo during culture (Stringfellow et al., 2000).
2. Materials and methods
2.1. Animals and cells Oviducts from healthy 1–3-year-old Alpine or Saanen goats from certified CAEV-free herds in the Deux Se`vres region of France were removed surgically, rinsed and trimmed free of fat and connective tissue in modified phosphate buffered saline (PBS, Eurobio, France). Each oviduct was closed with a clip at one end and filled with a solution of trypsin (0.25%) in calcium and magnesium-free PBS supplemented with 0.02% EDTA (Gibco-BRL, France). The distal end was also sealed and the filled oviduct was incubated at 37 °C for 15 min. The liberated mucosal tissue was dissociated in 10 ml of minimal essential medium (MEM, Gibco-BRL) in a conical tube by repeated gentle syringing through an 18 g needle. Cells were harvested by centrifugation at 660 g for 5 min, washed twice with MEM and re-suspended in 10 ml of R10 medium, consisting of RPMI (Gibco-BRL) supplemented with 10% heat inactivated (56 °C for 30 min) foetal bovine serum (FBS, Sigma, France). The COEC were seeded at 106 cells per 25 ml culture flask
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(Nunc, Polylabo, France) and incubated at 37 °C in 5% CO2 in a moist atmosphere for 24 h, when non-adherent cells were removed and fresh R10 medium was added. Thereafter, medium was replaced at 3-day intervals until a near-confluent cell monolayer developed. GSM cells, originally derived from the explanted carpal synovial membrane of a colostrumdeprived newborn goat (Narayan and Clements, 1989), were cultured in MEM supplemented with 10% FBS for the production of virus.
2.2. Viruses CAEV-pBSCA was produced as described previously (Mselli-Lakhal et al., 1999), by transfecting GSM cells with the pBSCA plasmid, harvesting the medium and titrating the released virus on fresh GSM cells. CAEV-3112 is a field isolate obtained by culture of inflammatory cells aspirated from the synovium of a swollen carpus of a naturally-infected arthritic goat from the Lyon region of France (Blondin et al., 1989). CAEV-3112 produced in GSM cells regularly titres at 106 TCID50/ml on either GSM cells or caprine macrophages.
2.3. COEC infection and 6irus titration COEC were seeded at 105 cells per well in 2 ml R10 + 10% FBS in six-well plates (Polylabo, France), and incubated at 37 °C in 5% CO2 until they reached sub-confluence, when they were infected with either CAEV-pBSCA or CAEV-3112 at a multiplicity of infection (MOI) of 1. After 6 days further incubation, they were fixed in 10% formalin, stained with May-Gru¨ nwald-Giemsa stain and microscopically examined for the presence of multinucleated giant cells, the typical cytopathic effect (CPE) indicating CAEV infection. Virus was titrated in serial tenfold dilutions of supernatants from COEC cultures after filtration through 0.45 mm membranes (Polylabo, France) on GSM cells in 24-well plates (Polylabo, France). After 6 days, CPE was detected as above, and the titre calculated as TCID50/ml following Reed and Muench (1938).
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2.4. Immunocytochemistry Epithelial cell markers and CAEV antigens were detected by incubating infected or control COEC, cultured to sub-confluence in eight-chamber slides (Lab-Tek, France) and fixed in cold acetone, with specific antisera for 1 h. Cytokeratin was detected using a 1:100 dilution of K813 mouse monoclonal antibody (Sigma) and CAEVp25 using the 30-5A1 mouse monoclonal antibody (VMRD, France) at a dilution of 1:500 in PBS containing 1% bovine serum albumin (BSA). After washing, antibody uptake was revealed by incubation with a 0.5% solution of biotinylated goat anti mouse IgG, then with streptavidin peroxidase (Kit K0377, Dako, France) in PBS with 1% BSA at room temperature for 30 min. After washing, the slides were stained with diaminobenzidine for 8 min, counterstained with hematoxylin and mounted (Mselli-Lakhal et al., 1999). Antibody negative controls were unrelated mouse antisera of the same IgG subclass, and appropriate epithelial and infected cells served for positive controls.
2.5. Immunoprecipitation of 6iral proteins Cells were seeded into six-well plates at a density of 5× 105/well, and then were infected at a MOI of 1 with either CAEV-pBSCA or CAEV 3112 24 h later. After culture for 6 days, the medium was replaced by MEM lacking methionine and cysteine and 100 mCi of [35S]methionine/ cysteine (Amersham, France). After overnight culture, the supernatants were recovered and the cells were lysed in RIPA buffer. Viral proteins were detected by overnight incubation with 10 ml of undiluted polyclonal serum from a goat that had received multiple injections of a mixture of three different CAEV isolates and K1514 MaediVisna virus (G9615), and recovered using protein A-sepharose (Sigma). Bands separated by SDSPAGE were visualised by autoradiography.
2.6. PCR amplification of pro6iral DNA Freshly prepared or infected COEC (105 cells) were lysed as previously described (Chebloune et
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al., 1996) and CAEV proviral DNA was investigated by nested PCR (Guiguen et al., 2000). Sequences from the gag region were amplified using primers GEX5 (5%-GAAGTGTTGCTGCGAGAGGTGTTG-3% and GEX3 (5%-TGGCTGATCCATGTTAGCTTGTGC-3%), corresponding to bases 393–416 and the complement of bases 1268–1291 of CAEV-CO (Saltarelli et al., 1990). An initial denaturation at 94 °C for 3 min was followed by 35 rounds of amplification (denaturation: 92 °C for 1 min, annealing: 56 °C for 90 s and extension at 70 °C for 3 min). Five microliters of the product of this reaction was used as template for a second round of amplifications using the internal primers GIN5 (5%GATAGAGACATGGCGAGGCAAGT-3%) and GIN3 (5%-GAGGCCATGCTGCATTGCTACTGT-3%), situated at positions 524– 546 and 1013– 1036 in CAEV-CO. Sample DNA integrity was controlled by amplifying the b-actin gene using primers based on the human sequence (Joag et al., 1994). Amplified bands were visualised by ethidium bromide staining after electrophoresis through 1.5% agarose gel. This technique has been shown to be capable of detecting less than ten infected cell samples containing 106 or 107 cells (Chebloune et al., 1996).
3. Results
3.1. Morphology and properties of cultured COEC When seeded at a density of 106 cells in a 25 cm2 culture flask, the majority of oviduct epithelial cells attach to the plastic during the initial 24 h of culture. They form discrete disseminated foci of fibro-epithelial or typically epithelial (polygonal) cells then grow out to confluence around the 7th day. The initial monolayers are typically strewn with sparse scattered macrophages, which only disappear after the second or third passage. Cultures of COEC remained vigorous and healthy even after numerous passages. Cultured COEC retain their epithelial charac-
teristics as exemplified by cytokeratin, which is constitutively expressed by 100% of the cells in the cultures, irrespective of morphology (Fig. 1a, b). In contrast, cells from CAEV-seronegative donor animals were never recognised by a monoclonal antiserum specific for CAEV-p25 antigen. In addition, PCR amplification of DNA recovered from any of these cell cultures never produced a band corresponding to the CAEV gag sequences targeted, although actin gene amplification confirmed that the DNA samples were of adequate quality (see Section 3.2). Taken together, these results suggest that COEC taken from seronegative nannies from a certified CAEV-free flock are indeed negative for the presence of the virus.
3.2. Susceptibility of COEC to infection by CAEV Typical development of multinucleated giant cells occurs some 6 days after inoculation of COEC cultures with either CAEV-pBSCA or CAEV-3112, but the extent of CPE is consistently greater after infection with CAEV-3112 (Fig. 2). The cells show specific staining by the mouse monoclonal antibody directed towards CAEV p25 antigen (Fig. 1c). PCR analysis detects proviral sequences in the DNA from infected cells only (Fig. 3, lane 3 vs. lane 1), whereas actin-specific sequences are easily visible in both samples. No bands are visible in the DNA-free control (Fig. 3, lane 2). The normal set of viral proteins appear to be produced in cells infected by either of the two virus strains tested, as demonstrated by the presence of all the expected precursor and mature protein bands on radioimmunoprecipitation with polyvalent goat serum (Fig. 4). The mature components were also released into the supernatant medium (Fig. 4). These results suggest that COEC are competent for CAEV replication and this was confirmed by productive infection of GSM cells with the supernatants from infected COEC cultures. Titres of 105 and 106 TCID50/ml were obtained for CAEV-pBSCA and CAEV-3112 infections, respectively.
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Fig. 1. Immunocytochemical detection of cellular and viral proteins. Expression of cytokeratin as detected by the K813 monoclonal antibody on cultured COEC: (a) PBS negative control ( × 100), (b) K813 antibody ( × 200). Expression of CAEV antigens on cultured COEC: (c) Presence of positive cells after reaction with anti p25 Gag monoclonal antibody ( ×100).
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Fig. 2. Multinucleated giant cells induced by CAEV infection of COEC. Uninfected controls. CAEV-infected CEOC.
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4. Discussion Artificial reproduction of goats involves a series of cellular manipulations to achieve a viable embryo for implantation into a surrogate dam. First, the recovered oocytes require a feeder layer of granulosa cells to allow their proper maturation before in vitro fertilisation. We have already shown that these granulosa cells, which are often obtained from uncontrolled slaughterhouse nannies, can be productively infected by CAEV (Lamara et al., 2001). After fertilisation, the zygote again needs a feeder layer, this time of
Fig. 4. Viral proteins produced in COEC after infection by two CAEV strains. Autoradiographic visualisation of proteins immunoprecipitated by G9615 serum from the supernatant (SN) or cellular lysate (C) of cultured COEC, either mock infected (1), or infected with CAEV-pBSCA (2) or CAEV-3112 (3). Arrows mark the major viral Gag (p25) and Env (gp175; gp135) proteins; the molecular weight ladder is on the left.
Fig. 3. Presence of CAEV proviral DNA in infected COEC. DNA fragments amplified by nested PCR from (1) uninfected COEC from healthy does, (2) control lacking template DNA, and (3) cultured COEC 6 days after infection with CAEV. Bands were separated by electrophoresis through 1.5% agarose and stained with ethidium bromide.
oviduct epithelial cells, to supply the factors required for early embryonic development. These cells are, again, often obtained from slaughterhouse animals and represent a further potential source of infection for the high-value embryos produced by in vitro methods. CAEV occurs in a range of natural variants, which may have different biological properties, so we tested two different isolates for their capacity to infect COEC. Both the infectious molecular clone CAEV-pBSCA and a laboratory-maintained field isolate CAEV-3112 proved easily able to infect COEC, with later release of high titres of infectious virus.
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A crucial question is whether these accessory cells are, in fact, infected in CAEV-positive animals. At present it is impossible to provide a formal demonstration because, as we show here, the primary cultures always contain cells of the monocyte/macrophage lineage, and mature macrophages are regularly seen on the cultures at early passages. Macrophage lineage cells are the main centre of infection in the virus-positive animal (Narayan et al., 1983; Gorell et al., 1992; Singh et al., 1999), and so the presence of virus in the early cultures would not determine the cellular source. Although the macrophages disappear on continued passage, CAEV is produced efficiently by maturing macrophages, and we have shown that in vitro cultures of COEC are highly sensitive to infection. Presence of virus in cultures of this nature from CAEV-infected animals is almost inevitable and would not determine the in vivo source of infection. Nevertheless, many lentiviruses have some capacity to infect epithelial cells in vivo (Carpenter and Chesebro, 1989; Park et al., 1995; Zhang et al., 1997; Mselli-Lakhal et al., 1999), and the present demonstration that the epithelial cells of the proximal goat reproductive tract are easily infected raises concerns about their potential activity as a viral reservoir. Any infection present would undoubtedly be amplified greatly under the culture conditions of artificial reproduction. Such infections with bovine viral diarrhoea virus have been observed (Booth et al., 1995) and may cause infection of the embryo (Bielanski et al., 1998; Stringfellow et al., 2000). In goat artificial reproduction, the developing embryo could be directly infected, or adherent virus could infect the surrogate dam, and perhaps later the late stage foetus or the newborn kid. It is not known whether the early goat embryo can be infected with CAEV, although cells from 8– 16 cell embryos were shown to be able to transmit the CAEV in vitro, whereas intact embryos with an unruptured ZP failed to transmit the virus (data not shown). The ZP frequently acts as a protection against infectious agents (Philipott, 1993), although some enveloped viruses may penetrate it, or adhere strongly enough to resist washing (Shisong and Warthall, 1989). Rupture of the ZP does not stop embryo
development in vitro, and indeed may be deliberately performed to facilitate hatching, thereby opening a breach for potential infection. While the present study cannot demonstrate conclusively whether COEC are actively infected in the CAEV-positive goat, their susceptibility to infection in vitro and their capacity for viral amplification suggests caution in choosing their origins. Goats produced using in vitro fertilisation methods will mainly be high-value animals for improved agronomic use, or genetically modified animals, perhaps destined to produce pharmaceutical products. Secretion of biologically active molecules in the milk of a modified animal is an attractive way to harvest such products, and so any risk of contamination by a lentivirus naturally transmitted through milk should be avoided. In the absence of specific recommendations, and in view of the efficient CAEV multiplication in COEC that we show here, it would be prudent to use only COEC from CAEV-certified donors for the in vitro reproduction of goats.
References Adams, D.S., Oliver, R.E., Ameghino, E., De Martini, J.C., Verwoerd, D.W., Houwers, D.J., Waghela, S., Gorham, J.R., Hyllseth, B., Dawson, M., Trigo, F.J., McGuire, T.C., 1984. Global survey of serological evidence of caprine arthritis-encephalitis virus infection. Veterinary Res. 115, 493 – 495. Bielanski, A., Sapp, T., Lutze-Wallace, C., 1998. Association of bovine embryos produced by in vitro fertilization with a noncytopathic strain bovine viral diarrhea virus type II. Therio 49, 1231 – 1238. Blondin, I., Grillet, C., Thiogane, Y., 1989. Syncitia formation in cultures and analysis of the protein composition of various strains of caprine arthritis encephalitis virus (CAEV). Ann. Rech. Ve´ te´ rinaires 20, 153 – 158. Booth, P.J., Stevens, D.A., Collins, M.E., Brownlie, J., 1995. Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle. J. Reprod. Fertility 105, 17 –24. Brodie, S.J., Pearson, L.D., Zink, M.C., Bickle, H.M., Anderson, B.C., Marcom, K.A., DeMartini, J.C., 1995. Ovine lentivirus expression and disease virus replication, but not entry, is restricted to macrophages of specific tissues. Am. J. Pathol. 146, 250 – 263. Carpenter, S., Chesebro, B., 1989. Change in host cell tropisme associated with in vitro replication of equine infectious anemia virus. J. Virol. 63, 2492 – 2496.
A. Lamara et al. / Virus Research 87 (2002) 69–77 Chebloune, Y., Sheffer, D., Karr, B.M., Stephens, E., Narayan, O., 1996. Restrictive type of replication of ovine/ caprine lentiviruses in ovine fibroblast cell cultures. Virology 222, 21 – 30. Crozet, N., 1991. Manipulation of oocytes and in vitro fertilization. J. Reprod. Fertility Suppl. 43, 235 –243. Crozet, N., Ahmed-Ali, M., Dubos, M.P., 1995. Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J. Reprod. Fertility 103, 293 –298. Galli, C., Moor, R.M., 1991. Developmental of immature bovine oocytes into viable embryos in vitro. Bull. Assoc. Anatomistes 75, 67 – 71. Gorell, M.D., Brandon, M.R., Sheffer, D., Adams, R.J., Narayan, O., 1992. Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes. J. Virol. 66, 2679 – 2688. Guiguen, F., Mselli-Lakhal, L., Durand, J., Du, J., Favier, C., Fornazero, C., Grezel, D., Balleydier, S., Hausmann, E., Chebloune, Y., 2000. Experimental infection of Mouflondomestic sheep hybrids with caprine arthritis –encephalitis virus. Am. J. Vet. Res. 61 (4), 456 –461. Hanel, A., 1991. The serological diagnosis of caprine arthritis encephalitis in goats and visna-maedi in sheep. Tierarztliche Umschau. 46, 665 –673. Howell, A.L., Edkins, R.D., Rier, S.E., Yeaman, G.R., Stern, J.E., Franger, M.W., Wira, C.R., 1997. Human immunodeficiency virus type 1 infection of cells and tissues from the upper and lower human female reproductive tract. J. Virol. 71, 3498 –3506. IETS, Stringfellow, D.A., Saidel, S.M. (Eds.), 1990. Manual of the International Embryo Transfer Society, second ed. IETS, Champaign, IL. Joag, S.V., Stephens, E.B., Adams, R.J., Foresman, L., Narayan, O., 1994. Pathogenesis of SIV mac infection in Chinese and Indian rhesus macaques: effects of splenectomy on virus burden. Virology 200, 436 –446. Kennedy-Stoskopf, S., Narayan, O., Strandberg, J.D., 1985. The mammary gland as a target organ for infection with caprine arthritis encephalitis virus. J. Comp. Pathol. 95, 609 – 617. Lamara, A., Fieni, F., Mselli-Lakhal, L., Tainturier, D., Chebloune, Y., 2001. Efficient replication of caprine arthritis– encephalitis virus in goat granulosa cells. Virus Res. 79, 165 – 172. Martino, A., Mogas, T., Palomo, M.J., Paramio, M.T., 1995. In vitro maturation and fertilization of prepubertal goat oocytes. Therio 43, 473 –485. McGuire, T.C., 1987. The immune response to viral antigens as determinant of arthritis in caprine arthritis – encephalitis virus infection. Vet. Immunol. Immunopathol. 17, 465 – 470. Mselli-Lakhal, L., Guiguen, F., Fornazero, C., Jian, D., Favier, C., Durand, J., Grezel, D., Balleydier, S., Mornex, J.F., Chebloune, Y., 1999. Goat milk epithelial cells are highly permissive to CAEV infection in vitro. Virology 259, 67 – 73.
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Narayan, O., Clements, J.E., 1989. Biology and pathogenesis of lentiviruses. J. Gen. Virol. 70, 1617 – 1639. Narayan, O., Wolinsky, J.S., Clements, J.E., Strandberg, J.D., Griffin, D.E., Cork, L.C., 1982. Slow virus replication: the role of macrophages in the persistence and expression of Visna viruses of sheep and goats. J. Gen. Virol. 59, 345 – 356. Narayan, O., Kennedy-Stoskopf, S., Sheffer, D., Griffin, D.E., Clements, J.E., 1983. Activation of caprine arthritis – encephalitis virus expression during maturation of monocytes to macrophages. Infect. Immunol. 41, 67 – 73. Park, H.S., Kyaw-Tanner, M., Thomas, J., Robinson, W.F., 1995. Feline immunodeficiency virus replicates in salivary gland ductual epithelium during the initial phase of infection. Vet. Microbiol. 46, 257 – 267. Philipott, M., 1993. The danger of disease transmission by artificial insemination and embryo transfer. Br. Vet. J. 149, 339 – 369. Prichard, J.F., Thibodeaux, J.K., Pool, S.H., Blakewood, E.G., Me´ ne´ zo, Y., Godke, R.A., 1992. In vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells. Human Reproduction 7, 553 – 557. Reed, L., Muench, H., 1938. A simple method for estimating fifty per cent points. Am. J. Hygien. 27, 413 – 497. Robinson, W.F., Ellis, T.M., 1986. Caprine arthritis – encephalitis virus infection: from recognition to eradication. Austr.Vet. J. 5, 399 –406. Saltarelli, M., Querat, G., Konings, D.A., Vigne, R., Clements, J.E., 1990. Nucleotide sequence and transcriptional analysis of molecular clones of CAEV which generate infectious virus. Virology 179, 347 – 364. Shisong, C., Warthall, A.E., 1989. The importance of the zona pellucida for diseases control in livestock by embryo transfer. Br. Vet. J. 145, 129 – 140. Singh, E.L., 1985. Disease control: procedures for handling embryos. Rev. Sci. Technol. l’Office Int. Epizooties 4, 867 – 872. Singh, D., Chebloune, Y., Mselli-Lkhal, L., Karr, B., Narayan, O., 1999. Ovine lentivirus-infected macrophages mediate productive infection in cell types that are not susceptible to infection with cell-free virus. J. Gen. Virol. 80, 1437 – 1444. Stringfellow, D.A., Ridell, K.P., Galik, P.K., Damiani, P., Bishop, M.D., Wright, J.C., 2000. Quality controls for bovine viral diarrhea virus-free IVF embryos. Therio 53 (3), 827 – 839. Toniolo, A., Serra, C., Conaldi, P.G., Basolo, F.V., Dolei, A., 1995. Productive HIV-1 infection of normal human mammary epithelial cells. AIDS 9, 859 – 866. Zhang, S., Troyer, D.L., Kapil, S., Zheng, L., Kennedy, G., Weiss, M., Xue, X., Wood, C., Minocha, H.C., 1997. Detection of proviral DNA of bovine immunodeficiency virus in bovine tissues by polymerase chain reaction (PCR) and PCR in situ hybridization. Virology 236 (2), 249 – 257. Zink, M.C., Yager, J.A., Myers, J.D., 1990. Pathogenesis of caprine arthritis – encephalitis virus. Cellular localization of viral transcripts in tissues of infected goats. Am. J. Pathol. 136, 843 – 854.
Epithelial cells from goat oviduct are highly permissive for productive infection with caprine arthritis–encephalitis virus (CAEV) A. Lamara a,*, F. Fieni a, L. Mselli-Lakhal b, D. Tainturier a, Y. Chebloune b a
Laboratoire d’Etude des Risques Sanitaires lie´s aux Biotechnologies de la Reproduction, Ecole Nationale Ve´te´rinaire de Nantes, Atlanpole-La Chantrerie, BP 40706, 44307 Nantes Cedex, France b UMR 754 INRA/ENVL/UCBL Virologie Cellulaire, Mole´culaire et Maladies Emergentes, Ecole Nationale Ve´te´rinaire de Lyon, 1, A6. Bourgelat, B.P. 83, 69280 Marcy l’Etoile, France Received 25 October 2001; received in revised form 23 April 2002; accepted 23 April 2002
Abstract Caprine oviduct epithelial cells (COEC) are commonly used in in vitro goat embryo production protocols to stimulate early embryonic development. These feeder cells are usually collected from slaughterhouses from unknown serological status animals for caprine arthritis–encephalitis virus (CAEV) infection which is frequent in many regions of the world. Tissues derived from this source may be contaminated with CAEV and the use of such material in in vitro fertilisation systems may contribute to transmission of this pathogen to the cultured embryos and dissemination via embryo transfer (ET). The aim of this study was to determine the permissiveness of COEC to CAEV replication in vitro. Cells were isolated from goats from certified CAEV-free herds and then were inoculated with two CAEV strains: the molecularly-cloned isolate of CAEV (CAEV-pBSCA) and the French field isolate (CAEV-3112). Cytopathic effects (CPE) were observed on cell culture monolayers inoculated with both CAEV strains. Expression of CAEV proteins was shown both by immunocytochemistry using anti-p24 gag specific antibodies and by immunoprecipitation using a hyperimmune serum. The CAEV proteins were correctly and properly processed by artificially-infected COEC and the titers of virus released into the supernatant reached 106 TCID50/ml 6 days post-inoculation. Although the macrophage lineage cells are the main centre of infection in the virus-positive animal, these findings suggest that epithelial cells may be important in the viral life cycle probably as a reservoir allowing the viral persistence, dissemination and pathogenesis. These results suggest also that the use in in vitro fertilisation systems of co-culture feeder cells that support efficient replication of CAEV to high titers could represent a serious risk for permanent transmission of virus to the cultured embryos and to the surrogate dam involved. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Caprine oviduct epithelial cells; CAEV; Replication; Cytopathic effect
* Corresponding author. Tel.: + 33-24068-7714; fax: + 33-24068-7748 E-mail address: [email protected] (A. Lamara). 0168-1702/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 2 ) 0 0 0 8 2 - 5
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1. Introduction Artificial reproductive techniques are increasingly used for the expansion of stocks of highvalue goats from genetically selected parents. In view of the widespread prevalence of the caprine arthritis–encephalitis virus (CAEV) in goats in most regions of the world (Adams et al., 1984; Robinson and Ellis, 1986; Hanel, 1991), it is desirable to avoid any associated propagation of this economically damaging virus. CAEV is a lentivirus which chiefly targets cells of the monocyte/macrophage lineage in the infected animal (Narayan et al., 1982, 1983), and which is transmitted to the offspring through colostrum and milk (Kennedy-Stoskopf et al., 1985; McGuire, 1987). Infection is lifelong, and disease can affect multiple organs resulting in disability or even death (Narayan and Clements, 1989). CAEV, like other lentiviruses, has been shown to be capable of infecting epithelial cells from several organs, and viral transcripts can be found in kidney or small intestine (Zink et al., 1990; Brodie et al., 1995) or mammary gland (Mselli-Lakhal et al., 1999) epithelia from infected animals. Infection of epithelial cells by HIV-1 may contribute to milk transmission (Toniolo et al., 1995) or to primary infection of the reproductive tract (Howell et al., 1997). Infection of epithelial cells has also been observed with feline immunodeficiency virus (Park et al., 1995), bovine immunodeficiency virus (Zhang et al., 1997 and equine infectious anaemia virus (Carpenter and Chesebro, 1989). While CAEV screening of the parental donors and the surrogate dams for artificial goat reproduction is possible and desirable, it may not be sufficient to exclude the risk of virus transmission. Several potentially infectable epithelial cell tissues are employed in the artificial preparation of embryos for implantation. We have previously shown that granulosa cells, which are necessary for the efficient pre-fertilisation maturation of the oocytes, and which are often derived from untested slaughterhouse animals, can sustain infection by CAEV (Lamara et al., 2001). For the post-fertilisation development of the embryo, a feeder culture (Prichard et al., 1992; Crozet et
al., 1995; Martino et al., 1995) of caprine oviduct epithelial cells (COEC) is used to provide essential support for its early development (Galli and Moor, 1991; Crozet, 1991). In the present paper, we investigate the susceptibility of COEC to infection by CAEV. Control of cultures used as feeders in artificial reproduction of goats has not been specifically addressed, although the International Embryo Transfer Society (IETS, 1990) and the Office Internationale des Epizooties (Singh, 1985) have produced recommendations for the certification of donors, stringent washing procedures and rejection of embryos with a damaged zona pellucida (ZP). Some control may be desirable, because bovine viral diarrhoea virus (Booth et al., 1995) has been shown to be transmitted from such cells to the embryo during culture (Stringfellow et al., 2000).
2. Materials and methods
2.1. Animals and cells Oviducts from healthy 1–3-year-old Alpine or Saanen goats from certified CAEV-free herds in the Deux Se`vres region of France were removed surgically, rinsed and trimmed free of fat and connective tissue in modified phosphate buffered saline (PBS, Eurobio, France). Each oviduct was closed with a clip at one end and filled with a solution of trypsin (0.25%) in calcium and magnesium-free PBS supplemented with 0.02% EDTA (Gibco-BRL, France). The distal end was also sealed and the filled oviduct was incubated at 37 °C for 15 min. The liberated mucosal tissue was dissociated in 10 ml of minimal essential medium (MEM, Gibco-BRL) in a conical tube by repeated gentle syringing through an 18 g needle. Cells were harvested by centrifugation at 660 g for 5 min, washed twice with MEM and re-suspended in 10 ml of R10 medium, consisting of RPMI (Gibco-BRL) supplemented with 10% heat inactivated (56 °C for 30 min) foetal bovine serum (FBS, Sigma, France). The COEC were seeded at 106 cells per 25 ml culture flask
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(Nunc, Polylabo, France) and incubated at 37 °C in 5% CO2 in a moist atmosphere for 24 h, when non-adherent cells were removed and fresh R10 medium was added. Thereafter, medium was replaced at 3-day intervals until a near-confluent cell monolayer developed. GSM cells, originally derived from the explanted carpal synovial membrane of a colostrumdeprived newborn goat (Narayan and Clements, 1989), were cultured in MEM supplemented with 10% FBS for the production of virus.
2.2. Viruses CAEV-pBSCA was produced as described previously (Mselli-Lakhal et al., 1999), by transfecting GSM cells with the pBSCA plasmid, harvesting the medium and titrating the released virus on fresh GSM cells. CAEV-3112 is a field isolate obtained by culture of inflammatory cells aspirated from the synovium of a swollen carpus of a naturally-infected arthritic goat from the Lyon region of France (Blondin et al., 1989). CAEV-3112 produced in GSM cells regularly titres at 106 TCID50/ml on either GSM cells or caprine macrophages.
2.3. COEC infection and 6irus titration COEC were seeded at 105 cells per well in 2 ml R10 + 10% FBS in six-well plates (Polylabo, France), and incubated at 37 °C in 5% CO2 until they reached sub-confluence, when they were infected with either CAEV-pBSCA or CAEV-3112 at a multiplicity of infection (MOI) of 1. After 6 days further incubation, they were fixed in 10% formalin, stained with May-Gru¨ nwald-Giemsa stain and microscopically examined for the presence of multinucleated giant cells, the typical cytopathic effect (CPE) indicating CAEV infection. Virus was titrated in serial tenfold dilutions of supernatants from COEC cultures after filtration through 0.45 mm membranes (Polylabo, France) on GSM cells in 24-well plates (Polylabo, France). After 6 days, CPE was detected as above, and the titre calculated as TCID50/ml following Reed and Muench (1938).
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2.4. Immunocytochemistry Epithelial cell markers and CAEV antigens were detected by incubating infected or control COEC, cultured to sub-confluence in eight-chamber slides (Lab-Tek, France) and fixed in cold acetone, with specific antisera for 1 h. Cytokeratin was detected using a 1:100 dilution of K813 mouse monoclonal antibody (Sigma) and CAEVp25 using the 30-5A1 mouse monoclonal antibody (VMRD, France) at a dilution of 1:500 in PBS containing 1% bovine serum albumin (BSA). After washing, antibody uptake was revealed by incubation with a 0.5% solution of biotinylated goat anti mouse IgG, then with streptavidin peroxidase (Kit K0377, Dako, France) in PBS with 1% BSA at room temperature for 30 min. After washing, the slides were stained with diaminobenzidine for 8 min, counterstained with hematoxylin and mounted (Mselli-Lakhal et al., 1999). Antibody negative controls were unrelated mouse antisera of the same IgG subclass, and appropriate epithelial and infected cells served for positive controls.
2.5. Immunoprecipitation of 6iral proteins Cells were seeded into six-well plates at a density of 5× 105/well, and then were infected at a MOI of 1 with either CAEV-pBSCA or CAEV 3112 24 h later. After culture for 6 days, the medium was replaced by MEM lacking methionine and cysteine and 100 mCi of [35S]methionine/ cysteine (Amersham, France). After overnight culture, the supernatants were recovered and the cells were lysed in RIPA buffer. Viral proteins were detected by overnight incubation with 10 ml of undiluted polyclonal serum from a goat that had received multiple injections of a mixture of three different CAEV isolates and K1514 MaediVisna virus (G9615), and recovered using protein A-sepharose (Sigma). Bands separated by SDSPAGE were visualised by autoradiography.
2.6. PCR amplification of pro6iral DNA Freshly prepared or infected COEC (105 cells) were lysed as previously described (Chebloune et
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al., 1996) and CAEV proviral DNA was investigated by nested PCR (Guiguen et al., 2000). Sequences from the gag region were amplified using primers GEX5 (5%-GAAGTGTTGCTGCGAGAGGTGTTG-3% and GEX3 (5%-TGGCTGATCCATGTTAGCTTGTGC-3%), corresponding to bases 393–416 and the complement of bases 1268–1291 of CAEV-CO (Saltarelli et al., 1990). An initial denaturation at 94 °C for 3 min was followed by 35 rounds of amplification (denaturation: 92 °C for 1 min, annealing: 56 °C for 90 s and extension at 70 °C for 3 min). Five microliters of the product of this reaction was used as template for a second round of amplifications using the internal primers GIN5 (5%GATAGAGACATGGCGAGGCAAGT-3%) and GIN3 (5%-GAGGCCATGCTGCATTGCTACTGT-3%), situated at positions 524– 546 and 1013– 1036 in CAEV-CO. Sample DNA integrity was controlled by amplifying the b-actin gene using primers based on the human sequence (Joag et al., 1994). Amplified bands were visualised by ethidium bromide staining after electrophoresis through 1.5% agarose gel. This technique has been shown to be capable of detecting less than ten infected cell samples containing 106 or 107 cells (Chebloune et al., 1996).
3. Results
3.1. Morphology and properties of cultured COEC When seeded at a density of 106 cells in a 25 cm2 culture flask, the majority of oviduct epithelial cells attach to the plastic during the initial 24 h of culture. They form discrete disseminated foci of fibro-epithelial or typically epithelial (polygonal) cells then grow out to confluence around the 7th day. The initial monolayers are typically strewn with sparse scattered macrophages, which only disappear after the second or third passage. Cultures of COEC remained vigorous and healthy even after numerous passages. Cultured COEC retain their epithelial charac-
teristics as exemplified by cytokeratin, which is constitutively expressed by 100% of the cells in the cultures, irrespective of morphology (Fig. 1a, b). In contrast, cells from CAEV-seronegative donor animals were never recognised by a monoclonal antiserum specific for CAEV-p25 antigen. In addition, PCR amplification of DNA recovered from any of these cell cultures never produced a band corresponding to the CAEV gag sequences targeted, although actin gene amplification confirmed that the DNA samples were of adequate quality (see Section 3.2). Taken together, these results suggest that COEC taken from seronegative nannies from a certified CAEV-free flock are indeed negative for the presence of the virus.
3.2. Susceptibility of COEC to infection by CAEV Typical development of multinucleated giant cells occurs some 6 days after inoculation of COEC cultures with either CAEV-pBSCA or CAEV-3112, but the extent of CPE is consistently greater after infection with CAEV-3112 (Fig. 2). The cells show specific staining by the mouse monoclonal antibody directed towards CAEV p25 antigen (Fig. 1c). PCR analysis detects proviral sequences in the DNA from infected cells only (Fig. 3, lane 3 vs. lane 1), whereas actin-specific sequences are easily visible in both samples. No bands are visible in the DNA-free control (Fig. 3, lane 2). The normal set of viral proteins appear to be produced in cells infected by either of the two virus strains tested, as demonstrated by the presence of all the expected precursor and mature protein bands on radioimmunoprecipitation with polyvalent goat serum (Fig. 4). The mature components were also released into the supernatant medium (Fig. 4). These results suggest that COEC are competent for CAEV replication and this was confirmed by productive infection of GSM cells with the supernatants from infected COEC cultures. Titres of 105 and 106 TCID50/ml were obtained for CAEV-pBSCA and CAEV-3112 infections, respectively.
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Fig. 1. Immunocytochemical detection of cellular and viral proteins. Expression of cytokeratin as detected by the K813 monoclonal antibody on cultured COEC: (a) PBS negative control ( × 100), (b) K813 antibody ( × 200). Expression of CAEV antigens on cultured COEC: (c) Presence of positive cells after reaction with anti p25 Gag monoclonal antibody ( ×100).
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Fig. 2. Multinucleated giant cells induced by CAEV infection of COEC. Uninfected controls. CAEV-infected CEOC.
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4. Discussion Artificial reproduction of goats involves a series of cellular manipulations to achieve a viable embryo for implantation into a surrogate dam. First, the recovered oocytes require a feeder layer of granulosa cells to allow their proper maturation before in vitro fertilisation. We have already shown that these granulosa cells, which are often obtained from uncontrolled slaughterhouse nannies, can be productively infected by CAEV (Lamara et al., 2001). After fertilisation, the zygote again needs a feeder layer, this time of
Fig. 4. Viral proteins produced in COEC after infection by two CAEV strains. Autoradiographic visualisation of proteins immunoprecipitated by G9615 serum from the supernatant (SN) or cellular lysate (C) of cultured COEC, either mock infected (1), or infected with CAEV-pBSCA (2) or CAEV-3112 (3). Arrows mark the major viral Gag (p25) and Env (gp175; gp135) proteins; the molecular weight ladder is on the left.
Fig. 3. Presence of CAEV proviral DNA in infected COEC. DNA fragments amplified by nested PCR from (1) uninfected COEC from healthy does, (2) control lacking template DNA, and (3) cultured COEC 6 days after infection with CAEV. Bands were separated by electrophoresis through 1.5% agarose and stained with ethidium bromide.
oviduct epithelial cells, to supply the factors required for early embryonic development. These cells are, again, often obtained from slaughterhouse animals and represent a further potential source of infection for the high-value embryos produced by in vitro methods. CAEV occurs in a range of natural variants, which may have different biological properties, so we tested two different isolates for their capacity to infect COEC. Both the infectious molecular clone CAEV-pBSCA and a laboratory-maintained field isolate CAEV-3112 proved easily able to infect COEC, with later release of high titres of infectious virus.
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A crucial question is whether these accessory cells are, in fact, infected in CAEV-positive animals. At present it is impossible to provide a formal demonstration because, as we show here, the primary cultures always contain cells of the monocyte/macrophage lineage, and mature macrophages are regularly seen on the cultures at early passages. Macrophage lineage cells are the main centre of infection in the virus-positive animal (Narayan et al., 1983; Gorell et al., 1992; Singh et al., 1999), and so the presence of virus in the early cultures would not determine the cellular source. Although the macrophages disappear on continued passage, CAEV is produced efficiently by maturing macrophages, and we have shown that in vitro cultures of COEC are highly sensitive to infection. Presence of virus in cultures of this nature from CAEV-infected animals is almost inevitable and would not determine the in vivo source of infection. Nevertheless, many lentiviruses have some capacity to infect epithelial cells in vivo (Carpenter and Chesebro, 1989; Park et al., 1995; Zhang et al., 1997; Mselli-Lakhal et al., 1999), and the present demonstration that the epithelial cells of the proximal goat reproductive tract are easily infected raises concerns about their potential activity as a viral reservoir. Any infection present would undoubtedly be amplified greatly under the culture conditions of artificial reproduction. Such infections with bovine viral diarrhoea virus have been observed (Booth et al., 1995) and may cause infection of the embryo (Bielanski et al., 1998; Stringfellow et al., 2000). In goat artificial reproduction, the developing embryo could be directly infected, or adherent virus could infect the surrogate dam, and perhaps later the late stage foetus or the newborn kid. It is not known whether the early goat embryo can be infected with CAEV, although cells from 8– 16 cell embryos were shown to be able to transmit the CAEV in vitro, whereas intact embryos with an unruptured ZP failed to transmit the virus (data not shown). The ZP frequently acts as a protection against infectious agents (Philipott, 1993), although some enveloped viruses may penetrate it, or adhere strongly enough to resist washing (Shisong and Warthall, 1989). Rupture of the ZP does not stop embryo
development in vitro, and indeed may be deliberately performed to facilitate hatching, thereby opening a breach for potential infection. While the present study cannot demonstrate conclusively whether COEC are actively infected in the CAEV-positive goat, their susceptibility to infection in vitro and their capacity for viral amplification suggests caution in choosing their origins. Goats produced using in vitro fertilisation methods will mainly be high-value animals for improved agronomic use, or genetically modified animals, perhaps destined to produce pharmaceutical products. Secretion of biologically active molecules in the milk of a modified animal is an attractive way to harvest such products, and so any risk of contamination by a lentivirus naturally transmitted through milk should be avoided. In the absence of specific recommendations, and in view of the efficient CAEV multiplication in COEC that we show here, it would be prudent to use only COEC from CAEV-certified donors for the in vitro reproduction of goats.
References Adams, D.S., Oliver, R.E., Ameghino, E., De Martini, J.C., Verwoerd, D.W., Houwers, D.J., Waghela, S., Gorham, J.R., Hyllseth, B., Dawson, M., Trigo, F.J., McGuire, T.C., 1984. Global survey of serological evidence of caprine arthritis-encephalitis virus infection. Veterinary Res. 115, 493 – 495. Bielanski, A., Sapp, T., Lutze-Wallace, C., 1998. Association of bovine embryos produced by in vitro fertilization with a noncytopathic strain bovine viral diarrhea virus type II. Therio 49, 1231 – 1238. Blondin, I., Grillet, C., Thiogane, Y., 1989. Syncitia formation in cultures and analysis of the protein composition of various strains of caprine arthritis encephalitis virus (CAEV). Ann. Rech. Ve´ te´ rinaires 20, 153 – 158. Booth, P.J., Stevens, D.A., Collins, M.E., Brownlie, J., 1995. Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle. J. Reprod. Fertility 105, 17 –24. Brodie, S.J., Pearson, L.D., Zink, M.C., Bickle, H.M., Anderson, B.C., Marcom, K.A., DeMartini, J.C., 1995. Ovine lentivirus expression and disease virus replication, but not entry, is restricted to macrophages of specific tissues. Am. J. Pathol. 146, 250 – 263. Carpenter, S., Chesebro, B., 1989. Change in host cell tropisme associated with in vitro replication of equine infectious anemia virus. J. Virol. 63, 2492 – 2496.
A. Lamara et al. / Virus Research 87 (2002) 69–77 Chebloune, Y., Sheffer, D., Karr, B.M., Stephens, E., Narayan, O., 1996. Restrictive type of replication of ovine/ caprine lentiviruses in ovine fibroblast cell cultures. Virology 222, 21 – 30. Crozet, N., 1991. Manipulation of oocytes and in vitro fertilization. J. Reprod. Fertility Suppl. 43, 235 –243. Crozet, N., Ahmed-Ali, M., Dubos, M.P., 1995. Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J. Reprod. Fertility 103, 293 –298. Galli, C., Moor, R.M., 1991. Developmental of immature bovine oocytes into viable embryos in vitro. Bull. Assoc. Anatomistes 75, 67 – 71. Gorell, M.D., Brandon, M.R., Sheffer, D., Adams, R.J., Narayan, O., 1992. Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes. J. Virol. 66, 2679 – 2688. Guiguen, F., Mselli-Lakhal, L., Durand, J., Du, J., Favier, C., Fornazero, C., Grezel, D., Balleydier, S., Hausmann, E., Chebloune, Y., 2000. Experimental infection of Mouflondomestic sheep hybrids with caprine arthritis –encephalitis virus. Am. J. Vet. Res. 61 (4), 456 –461. Hanel, A., 1991. The serological diagnosis of caprine arthritis encephalitis in goats and visna-maedi in sheep. Tierarztliche Umschau. 46, 665 –673. Howell, A.L., Edkins, R.D., Rier, S.E., Yeaman, G.R., Stern, J.E., Franger, M.W., Wira, C.R., 1997. Human immunodeficiency virus type 1 infection of cells and tissues from the upper and lower human female reproductive tract. J. Virol. 71, 3498 –3506. IETS, Stringfellow, D.A., Saidel, S.M. (Eds.), 1990. Manual of the International Embryo Transfer Society, second ed. IETS, Champaign, IL. Joag, S.V., Stephens, E.B., Adams, R.J., Foresman, L., Narayan, O., 1994. Pathogenesis of SIV mac infection in Chinese and Indian rhesus macaques: effects of splenectomy on virus burden. Virology 200, 436 –446. Kennedy-Stoskopf, S., Narayan, O., Strandberg, J.D., 1985. The mammary gland as a target organ for infection with caprine arthritis encephalitis virus. J. Comp. Pathol. 95, 609 – 617. Lamara, A., Fieni, F., Mselli-Lakhal, L., Tainturier, D., Chebloune, Y., 2001. Efficient replication of caprine arthritis– encephalitis virus in goat granulosa cells. Virus Res. 79, 165 – 172. Martino, A., Mogas, T., Palomo, M.J., Paramio, M.T., 1995. In vitro maturation and fertilization of prepubertal goat oocytes. Therio 43, 473 –485. McGuire, T.C., 1987. The immune response to viral antigens as determinant of arthritis in caprine arthritis – encephalitis virus infection. Vet. Immunol. Immunopathol. 17, 465 – 470. Mselli-Lakhal, L., Guiguen, F., Fornazero, C., Jian, D., Favier, C., Durand, J., Grezel, D., Balleydier, S., Mornex, J.F., Chebloune, Y., 1999. Goat milk epithelial cells are highly permissive to CAEV infection in vitro. Virology 259, 67 – 73.
77
Narayan, O., Clements, J.E., 1989. Biology and pathogenesis of lentiviruses. J. Gen. Virol. 70, 1617 – 1639. Narayan, O., Wolinsky, J.S., Clements, J.E., Strandberg, J.D., Griffin, D.E., Cork, L.C., 1982. Slow virus replication: the role of macrophages in the persistence and expression of Visna viruses of sheep and goats. J. Gen. Virol. 59, 345 – 356. Narayan, O., Kennedy-Stoskopf, S., Sheffer, D., Griffin, D.E., Clements, J.E., 1983. Activation of caprine arthritis – encephalitis virus expression during maturation of monocytes to macrophages. Infect. Immunol. 41, 67 – 73. Park, H.S., Kyaw-Tanner, M., Thomas, J., Robinson, W.F., 1995. Feline immunodeficiency virus replicates in salivary gland ductual epithelium during the initial phase of infection. Vet. Microbiol. 46, 257 – 267. Philipott, M., 1993. The danger of disease transmission by artificial insemination and embryo transfer. Br. Vet. J. 149, 339 – 369. Prichard, J.F., Thibodeaux, J.K., Pool, S.H., Blakewood, E.G., Me´ ne´ zo, Y., Godke, R.A., 1992. In vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells. Human Reproduction 7, 553 – 557. Reed, L., Muench, H., 1938. A simple method for estimating fifty per cent points. Am. J. Hygien. 27, 413 – 497. Robinson, W.F., Ellis, T.M., 1986. Caprine arthritis – encephalitis virus infection: from recognition to eradication. Austr.Vet. J. 5, 399 –406. Saltarelli, M., Querat, G., Konings, D.A., Vigne, R., Clements, J.E., 1990. Nucleotide sequence and transcriptional analysis of molecular clones of CAEV which generate infectious virus. Virology 179, 347 – 364. Shisong, C., Warthall, A.E., 1989. The importance of the zona pellucida for diseases control in livestock by embryo transfer. Br. Vet. J. 145, 129 – 140. Singh, E.L., 1985. Disease control: procedures for handling embryos. Rev. Sci. Technol. l’Office Int. Epizooties 4, 867 – 872. Singh, D., Chebloune, Y., Mselli-Lkhal, L., Karr, B., Narayan, O., 1999. Ovine lentivirus-infected macrophages mediate productive infection in cell types that are not susceptible to infection with cell-free virus. J. Gen. Virol. 80, 1437 – 1444. Stringfellow, D.A., Ridell, K.P., Galik, P.K., Damiani, P., Bishop, M.D., Wright, J.C., 2000. Quality controls for bovine viral diarrhea virus-free IVF embryos. Therio 53 (3), 827 – 839. Toniolo, A., Serra, C., Conaldi, P.G., Basolo, F.V., Dolei, A., 1995. Productive HIV-1 infection of normal human mammary epithelial cells. AIDS 9, 859 – 866. Zhang, S., Troyer, D.L., Kapil, S., Zheng, L., Kennedy, G., Weiss, M., Xue, X., Wood, C., Minocha, H.C., 1997. Detection of proviral DNA of bovine immunodeficiency virus in bovine tissues by polymerase chain reaction (PCR) and PCR in situ hybridization. Virology 236 (2), 249 – 257. Zink, M.C., Yager, J.A., Myers, J.D., 1990. Pathogenesis of caprine arthritis – encephalitis virus. Cellular localization of viral transcripts in tissues of infected goats. Am. J. Pathol. 136, 843 – 854.