[The bovine immunodeficiency virus: 1990-1992 update]. / Le virus de l'immunodéficience bovine: mise au point 1990-1992.

Substantial progress has been shown on the bovine immunodeficiency-like virus (BIV) in 1990-1991 and up to mid-1992. The genomic sequences of BIV were analysed in detail and several subgenomic RNAs were identified. Nucleic acid molecular probes, PCR (polymerase chain reaction) amplification and a novel Western blotting procedure have been of great assistance for the experimental diagnosis of BIV. Antibody response after BIV infection has shown that antibodies to p26 antigen were always present in naturally and experimentally infected animals. The experimental infection of sheep, goats and rabbits was confirmed. BIV causes an infection with no pathognomonic clinical signs in cattle and sheep for at least 3 and 4 years, respectively. Finally, there is not yet any evidence of BIV immune response disturbances similar to that of human AIDS.

[Bovine immunodeficiency virus: short review]. / Le virus de l'immunodéficience bovine: brève revue.

A bovine visna-like virus was isolated by Van Der Maaten et al (1972) but it did not draw attention since, at that time, most efforts were directed towards research on bovine leukemia virus. However, new interest was shown on the bovine visna-like virus after the isolation of the human immunodeficiency virus (HIV), because of the urgent need for developing animal models for the acquired immunodeficiency syndrome (AIDS). The purpose of this paper is to describe the different stages of the identification of the bovine virus and to up-date knowledge about it. The bovine visna-like virus has recently been named the bovine immuno-deficiency-like virus (BIV) and is the sole bovine lentivirus known to-date. BIV shares morphologic, antigenic and genomic characteristics with other lentiviruses. It grows and induces large syncytia in vitro and generates virus-productive and latent infections in cell culture. It causes persistent infection and slow progressive disease in cattle and probably in sheep. As target cells of the virus are leukocytes, the type of which is unknown, perturbations of the immune system are expected. Consequently, BIV may potentiate the occurrence of secondary infections and play a role in retroviral, multiple infections. It is not oncogenic. Transmission appears to occur in cattle by contact, but evidence of transmission in human beings has not been shown. Finally, BIV may be a potential model in vitro and in vivo for HIV and AIDS.

Replication of the bovine immunodeficiency-like virus in diploid and aneuploid cells: permanent, latent and virus-productive infections in vitro.

Bovine immunodeficiency-like virus (BIV) is an infectious and leukotropic retrovirus, the sole lentivirus candidate which has been isolated from cattle. Although BIV has recently been shown to be related to the human immunodeficiency virus, there is very limited information on the replication and the pathogenesis of BIV. It is reported here that BIV can permanently infect diploid and aneuploid cells from four different species: bovine, canine, ferret and ovine. With the exceptions of a bovine diploid and a canine aneuploid cell line, all lines were virus non-productive. However, BIV was rescued by co-cultivation of virus non-productive cells with homologous BIV-free or canine cells (Cf2Th). A permanent and BIV-productive infection was established for 90-serial subcultures in a canine cell line. A BIV titre of 1 x 10(6)/0.1 ml was observed in stationary culture and 1 x 10(10)/0.1 ml in suspension culture. The canine cell line above was used for production of BIV antigens, whereas BIV-free canine cells were routinely used to isolate BIV from BIV non-productive cells infected in vitro and from blood from experimentally BIV-infected cattle. The different steps of virus maturation were similar by electron microscopy to those of lentiviruses. BIV results are compared to those of lentiviruses.

A comparison of the bovine leukemia and bovine syncytial virus status in utero-tubal cells recovered from fluids used to flush the uterus and oviducts of BLV-infected, superovulated cattle.

Twenty-four cell lines were established from uterine-oviductal flush fluid (UOFF) cells from 20 bovine leukosis virus (BLV)-infected embryo-donor cows and 4 BLV-free control cows harvested by the Ficoll-gradient technique. Similar epithelial-like and fibroblast-like cells were observed in the primary cultures of UOFF from both groups. BLV-antigens were not detected with direct immunofluorescence test in any of the cell-lines from the 20 positive BLV-cows but a positive reaction was observed with the competitive radioimmunoassay in one cell line only. Bovine syncytial virus (BSV) was detected (multinucleated cells) in five of the 20 cows, bovine virus diarrhea (BVD) in six and infectious bovine rhino-tracheitis (IBR) in one (by D-IF). Some of the cell lines had antigens to one (3/20) two (2/20) or three (1/20) different viruses as demonstrated by D-IF. There were no antigens to BLV, BSV or IBRV demonstrated in the BLV-free cows by both immunofluorescence test and radioimmunoassay. Permissiveness to the growth of BLV in the cell lines of bovine utero-tubal (BUT) origin was demonstrated by inoculating three of the 20 cell lines from BLV-infected cows and three cell lines from the 4 BLV-free cow by BLV suspensions. All six cell lines permitted the growth of BLV as shown by syncytia, and positive reactions with the immunofluorescence test but only three out of six lines were BLV-positive by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Analogy between lymphotropic human retroviruses and large animal retroviruses.

The family Retroviridae comprises some fifty viruses in three subfamilies: Oncoviridae, Lentiviridae and Spumaviridae. A better understanding of retroviral pathobiology has resulted from the rapid developments in knowledge of the molecular biology of normal and cancerous cells as well as retroviruses. Genomic relatedness was found between two human T cell leukemia viruses and bovine leukemia virus, similarly, some relatedness appears possible between human AIDS (acquired immunodeficiency syndrome) virus and lentiviruses of large animals. Because of their genomic relatedness, retroviruses from man and animals could theoretically form recombinants during in vitro manipulation. Therefore persons who work with retroviral materials should follow established laboratory practices to control infectious agents.

The persistent infection of a canine thymus cell line by equine infectious anaemia virus and preliminary data on the production of viral antigens.

Equine infectious anaemia virus (EIAV) was adapted to the Cf2Th cell line, a heterologous malignant line from canine thymus. A persistent infection was monitored for 100 serial passages by demonstrating the presence of virus and viral antigens at each 10th passage by electron-microscopy, immunodiffusion and immunofluorescence. Chromosome analysis of EIAV-infected cells indicated they had a karyotype resembling the control cells of similar passage history. Virus-infected cells, grown in roller cultures for 65 days without subculturing, continuously produced viral antigens into supernatant fluids which were harvested every 3-4 days. Antigen peaks were observed at approximately 12-day intervals. Immunoprecipitin lines of identity were demonstrated between ether-extracted antigens from virus-infected canine cell line and known positive EIAV antigen extracted from infected equine spleen and a commercial source. Replication of a non-oncogenic retrovirus (EIAV) resulted in the continuous release of viral antigens from a persistently infected and infinite cell line.

Analogie entre les rétrovirus humains lymphotropes et les rétrovirus des grands animaux.

The family Retroviridae comprises some fifty viruses in three subfamilies: Oncoviridae, Lentiviridae and Spumaviridae. A better understanding of retroviral pathobiology has resulted from the rapid developments in knowledge of the molecular biology of normal and cancerous cells as well as retroviruses. Genomic relatedness was found between two human T cell leukemia viruses and bovine leukemia virus; similarly, some relatedness appears possible between human AIDS (acquired immunodeficiency syndrome) virus and lentiviruses of large animals. Because of their genomic relatedness, retroviruses from man and animals could theoretically form recombinants during in vitro manipulation. Therefore persons who work with retroviral materials should follow established laboratory practices to control infectious agents.

Embryo transfer in relation to bovine leukemia virus control and eradication.

Two hundred and seven, zona pellucida-intact bovine embryos were collected from bovine leukemia virus-infected donors, washed, and transferred to uninfected recipients: 111 of these embryos were sired by bovine leukemia virus-infected bulls. Fifty live calves were obtained from the 57 pregnancies resulting from the transfers. None of the recipients or calves developed antibodies to bovine leukemia virus. Nine zona-intact ova, 12 zona-intact morulae and 15 hatched blastocysts, exposed "in vitro" to bovine leukemia virus, washed and then tested for bovine leukemia virus were negative. Twenty-seven, zona-intact embryos and 14 hatched embryos were similarly exposed and washed prior to being transferred in groups to two uninfected recipients: no pregnancies resulted, nor did the recipients develop antibodies to bovine leukemia virus up to 120 days posttransfer. The conclusion from these and other bovine leukemia virus studies is that zona-intact embryos can be transferred from bovine leukemia virus-infected donors, including those bred by bovine leukemia virus-infected bulls, without risk of transmitting bovine leukemia virus, providing that they are properly washed prior to transfer.

[Chromosomes and carcinogenesis. Study on the evolution of an epithelial cell line of porcine origin]. / Chromosomes et cancérogenèse. Etude de l'évolution d'une lignée cellulaire épithéliale d'origine porcine (PFT).

The PFT cell line was established in 1969 from diploid cells of the inner lining of a uterine tube of a 2 year-old sow and has been continuously subcultured more than 500 times over a decade. Three chromosomal rearrangements have occurred during this time. The first translocation was shown at the 100th passage with the concomitant and spontaneous release of an endogeneous type C virus. The second translocation was observed at the 290th passage along with the appearance of gap junctions and the induction of malignant tumors in athymic nude mice following the inoculation of PFT cells. The third translocation was found towards the 470th passage with the simultaneous appearance of annulate lamellae. Since the translocations were accompanied by the spontaneous release of a retrovirus and then by malignancy of PFT cells when inoculated in athymic nude mice, it is likely that the chromosomal abnormalities are associated with the viral carcinogenesis of the cell line. The third translocation appears to confirm the perenniality of the multistep evolution hypothesis of malignancy.

Isolation of bovine syncytial virus from lymphocytes recovered from fluids used to flush uterus and oviducts of superovulated cattle.

A Ficoll-gradient method was applied to isolate lymphocytes from fluids used to flush the uterus and oviducts of superovulated cows. Bovine syncytial virus antigens were demonstrated in 15 of 19 cows by cocultivation of lymphocytes with fetal lamb spleen cells and examining them with direct immunofluorescence. Viral serum antibodies were found in the same 15 of 19 cows as above by the modified direct complement-fixation test. The virus was also detected in one of four uterotubal cell cultures.

Ultrastructural comparison of Oncovirinae (type C), Spumavirinae, and Lentivirinae: three subfamilies of Retroviridae found in farm animals.

The successive steps of maturation of seven retroviruses from five species of farm animals and one retrovirus from a mouse were compared in cell cultures. The viruses included three type C oncoviruses, one spumavirus, and three lentiviruses. Although members of the 3 subfamilies shared some gross morphologic features such as budding on plasma membranes, core, and surface projections, differences were noted in the ultrastructural detail of these features. Type C oncoviruses did not show any structural differentiation in identifiable form in the cytoplasm as opposed to characteristic features observed in the spumavirus and lentivirus subfamilies, respectively. Budding viruses were distinct among the 3 subfamilies. The type C bovine leukemia virus budding on vacuole membranes differed from the two other type C viruses by lacking an electron-lucent intermediate layer as did the lentiviruses. Differentiation between type C oncoviruses and lentiviruses could be confusing because of the similarity of the fully mature virions appearing in the intercellular space. However, each subfamily of retroviruses can be readily differentiated from one another when each morphologic stage of virus replication is examined by electron microscopy.

Enzyme linked immunosorbent assay (ELISA) for the detection of antibodies to porcine cytomegalovirus.

The ELISA and indirect immunofluorescence test were compared on 56 porcine sera which were tested for antibodies to porcine cytomegalovirus. Viral antigens were prepared in cells of a pig fallopian tube line. The ELISA was found to be a sensitive reproducible and practical test to measure specific antibodies to this infection.

Attempts to isolate bovine leukemia and bovine syncytial viruses from blood, uterine flush fluid, unfertilized ova and embryos from infected donor cattle.

Blood leucocytes, sediments of uterine flush fluid (UFF), eggs and embryos from 25 BLV-positive donor cows were tested for bovine leukemia (BLV) and bovine syncytial (BSV) viruses by cocultivation with fetal lamb spleen cells and by applying syncytium induction and immunofluorescence tests. BLV was diagnosed in 11/15 (73.3%) leucocyte and 4/25 (16.0%) UFF-sediment specimens as compared to BSV in 14/15 (93.3%) and 21/25 (84.0%) of the similar specimens and neither BLV or BSV were found in 26 eggs and 60 embryos collected from 20 of the 25 cows. Detection of BLV antigens by immunofluorescence was hampered by the competitive replication of both BLV and BSV and competitive growth in indicator cells and uterine cells. As BLV has not been observed in cells of UFF sediments, it was probably isolated from leucocytes present in the lumen of uterus or from blood seeping out from inapparent vessel damage during flushing. Isolation of BLV in UFF sediments gives additional evidence to the concept of a transplacental transmission by a not yet elucidated mechanism. The high rate of BSV recovery from cells of UFF sediments indicated that this virus is more wide-spread than previously shown and that it may play a role in causing disorders of the reproductive tract.

Nontumoral, benign ad malignant stages of transformation of a diploid pig cell line. A review.

The PFT cell line originated from diploid endometrial cells and was subcultured more than 500 times. Nontumoral, benign and malignant transformation appeared at different stages. Four populations (F1, F2, F3 and F4) of cells were demonstrated sequentially by chromosomes analysis. These consisted of eight phenotypes corresponding to the sequential appearance of markers which included three chromosomal aberrations. The PFT malignant transformation in vitro is a progressive process through qualitatively different stages and may reflect neoplastic development in vivo. It is suggested that the benign and malignant transformations were under separate genetic control since they occurred after two chromosomal translocations on two different chromosomes. The spontaneous expression of a pig endogenous type-C virus which occurred concomitantly with the first translocation appeared to be related to oncogenesis.

Concanavalin A agglutination assays in the multistage transformation of a pig cell line.

Cells of a pig Fallopian tube line were agglutinated by concanavalin A (Con A). Similar ConA agglutination was shown in diploid and nontumoral transformed cells with one (T1) translocation until the 112th subculture. Then a progressive increase of agglutination was shown until the 149th subculture and the rate of agglutination remained constant thereafter. The progression of ConA agglutination paralleled the progression of anchorage independence. The highest ConA agglutination occurred when anchorage independence was well established and persisted until the 193rd T1 subculture when benign tumorigenicity was demonstrated. After the appearance of the second translocation (T2), the self-agglutination of the cells was directly related to malignancy and to high tumor incidence in mice. Anchorage independence and the highest ConA agglutination were directly related to benign transformation and indirectly related to malignant transformation of the pig Fallopian tube cell line.

Multisequential transformation of a pig cell line (PFT): correlations between tumorigenicity and chromosome and ultrastructural markers.

Cells from representative subcultures of a continuous cell line of pig uterine tube (PFT) and carrying none, one, or two chromosome markers were inoculated in athymic nude mice (RCN) to determine the occurrence of the malignant transformation in this line. No tumors developed in mice after implantation of cells from either the 16th and 65th subcultures (no chromosome markers) or the 106th subculture (one chromosome marker, anchorage-dependent). Three adenomas (11.1%) were found in 27 mice that had been inoculated with anchorage-independent cells having one chromosome marker. Of 27 mice inoculated with cells showing two chromosome markers and gap junctions, 23 mice (85.1%) developed undifferentiated carcinomas. Ultrastructural traits and karyotypes of tumors were generally similar to those of the PFT cell inocula. However, annulate lamellae were found in 8 of 13 tumors examined by electron microscopy but were not seen in PFT cell inocula. The occurrence of a multisequential transformation was indicated by serial examination of the first 400 subcultures of the line and by comparison of the markers observed in three successive populations of cells differing in their genetic constitution.

Production of tumors in hamsters by cells of a pig uterine tube line.

Tumorigenicity was investigated in a pig established cell line continuously subcultured more than 400 times. Tumors were not found in hamsters' cheek pouches inoculated with cells from the first 200 subcultures. Fibroma developed in five out of 13 animals (9/26 pouches) inoculated with cells of the 313th and 314th subcultures. Two chromosome markers were found in the 290th subculture, in the 313th subculture inoculum and in tumor cells.

Cell ultrastructure during the development and the transformation of a pig--uterine tube cell line.

Observations by electron microscopy disclosed ultrastructural features which were not apparent by light microscopy during the three stages of the development of a pig uterine tube cell line. Ciliated cells without dynein arms were found naturally occurring in the first 100 subcultures (pseudo-diploid stage). They were not observed in the routinely growing cultures after the concomitant detection of the first chromosome marker and expression of a type C virus, unless the cells were treated with estradiol (transformed stage). Gap junctions and increased numbers of lamellar and lysosomic bodies and vacuole-like structures were observed in clusters of tumoral cells in the high subcultures if a new chromosome marker occurred (malignant stage). Type C viruses were found as early as the 106th subculture (transformed stage) in populations of actively dividing cells 3 days after seeding, but not in populations of nondividing cells a week after seeding. Both viruses and viral reverse transcriptase were continuously released in the 300 subsequent subcultures.

Evidence of a mixed population from uterine tube epithelium in a continuous line of pig cells.

The pig uterine tube (PFT) cell line is composed of a mixed population which is undifferentiated. However, specific markers indicating the original tissue of the uterine tube were shown if cells differentiated into epithelial cells forming spheroids in the 254th subculture. Ciliated and secretory cells, and cells with a basal lamina and interstitial collagen were observed in the spheroids. Microlumina found in the spheroids appeared morphologically similar to the lumen of the uterine tube. These observations indicate that undifferentiated cells can multiply in vitro and keep their potentiality of differentiation for future expression. It is proposed that the PFT cell line was partly derived from epithelial cells originally harvested from the PFT.