Immortalization of primary sheep embryo kidney cells.

Sheep primary epithelial cells are short-lived in cell culture systems. For long-term in vitro studies, primary cells need to be immortalized. This study aims to establish and characterize T immortalized sheep embryo kidney cells (TISEKC). In this study, we used fetal lamb kidneys to derive primary cultures of epithelial cells. We subsequently immortalized these cells using the large T SV40 antigen to generate crude TISEKC and isolate TISEKC clones. Among numerous clones of immortalized cells, the selected TISEKC-5 maintained active division and cell growth over 20 passages but lacked expression of the oncogenic large T SV40 antigen. Morphologically, TISEKC-5 maintained their epithelial aspect similar to the parental primary epithelial cells. However, their growth properties showed quite different patterns. Crude TISEKC, as well as the clones of TISEKC proliferated highly in culture compared to the parental primary cells. In the early passages, immortalized cells showed heterogeneous polyploidy but in the late passages the karyotype of immortalized cells became progressively stable, identical to that of the primary cells, because the TISEKC-5 cell line has lost the large SV40 T antigen expression, this cell line is a valuable tool for veterinary sciences and biotechnological productions.

Epithelial cells from goat oviduct are highly permissive for productive infection with caprine arthritis-encephalitis virus (CAEV).

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 10(6) TCID(50)/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.

Immortalized goat milk epithelial cell lines replicate CAEV at high level.

Primary milk epithelial cells were isolated from CAEV-uninfected goats and three cell lines designated TIGMEC-1, TIGMEC-2 and TIGMEC-3 were established. The three cell lines retained their morphological characteristics of epithelial cells and expressed specific epithelial cytokeratin marker as well as the immortalizing SV40 large T antigen. The kinetics of growth of TIGMEC1, TIGMEC2 and TIGMEC3 cell lines showed a doubling time of 24-48 hours while the parental cell lines became senescent after the passage 6 in cell culture. Like the parental primary cells, the three cell lines were found to be highly sensitive to CAEV-pBSCA, an infectious molecular clone of CAEV-CO strain, and to a French isolate CAEV-3112. TIGMEC cell lines infected with CAEV-pBSCA became chronically infected producing high virus titers in absence of cytopathic effects. These cell lines may be useful for study of the possible physiological alterations in mammary epithelial cells infected with small ruminant lentiviruses and their consequences on milk quality. On an other hand, these cell lines can be used to study their role in virus transmission and pathogenesis.

Identification and cloning of an 85-kDa protein homologous to RING3 that is upregulated in proliferating endothelial cells.

A central event in angiogenesis is proliferation of blood vessels, which plays a major role in the progression of a number of inflammatory and neoplastic diseases. It is responsible for the switch of endothelial cells from an antiangiogenic to an angiogenic phenotype. To identify novel activated/proliferating-related proteins in human endothelial cells, a subtractive immunization approach was used to elicit a host antibody response against human dermal microvascular endothelial cells (HDMECs) stimulated with a potent angiogenic cytokine such as VPF/VEGF165. In this study, a new mAb, LY9, which is highly specific to VPF/VEGF165-activated HDMECs, was isolated. Stimulation of HDMECs by VPF/VEGF165 or basic fibroblast growth factor (bFGF) resulted in a dose-dependent and time-dependent increase in the binding of LY9. On Western-blot analysis, LY9 identified an 85-kDa protein (p85) in the lysates of several endothelial cells derived from microvascular or large vessel sources, the expression of which is dramatically increased by VPF/VEGF165. The mAb also identified p85 in primary cell cultures of human foreskin keratinocytes but failed to recognize human fibroblasts (MRC5) and a number of different human tumor cell lines, including MG63 osteosarcoma and MCF7 breast carcinoma cells. Immunological screening of a human keratinocyte lambdagt11 cDNA expression library with LY9 identified a partial cDNA clone of 750 bp. DNA sequencing of this clone and predicted amino acids showed more than 93% homology to RING3 kinase, a member of a newly described family of bromodomain-containing proteins that transactivates in the nucleus the promoters of a number of the E2F family of transcription factors. This molecule may represent a new signaling target activated by VPF/VEGF165 and bFGF that allows endothelial cells to enter the proliferative phase of the angiogenic process.

Immunization with plasmid DNA expressing the caprine arthritis-encephalitis virus envelope gene: quantitative and qualitative aspects of antibody response to viral surface glycoprotein.

Saanen goats were vaccinated intradermally with plasmid DNA expressing caprine arthritis-encephalitis virus (CAEV) rev-env (pENV) or tat-rev-env (pTAT-ENV) or vaccinia virus expressing CAEV env (rWR-63). Sera from all vaccinated goats immunoprecipitated CAEV surface (SU) and transmembrane (TM) glycoproteins with a dominant response to SU. Antibody response to CAEV SU induced by plasmid DNA was relatively biased toward IgG2, whereas vaccinia rWR-63 induced predominantly IgG1 antibodies to SU. Differential IgG isotype bias established by immunization with plasmid or vaccinia vectors was maintained following subcutaneous boost with purified CAEV SU in Freund's incomplete adjuvant (FIA). Goats injected with pUC18 control plasmid followed by immunization with SU-FIA also had IgG2 biased responses, whereas SU-FIA immunization of a goat primed with vaccinia rWR-SC11 without the CAEV env gene induced a predominant IgG1 response. We conclude that pUC based plasmids expressing the CAEV env gene promote stable type 1 biased immune responses to plasmid encoded SU. IgG2 biased response may be due to innate type 1 priming capacity of immunostimulatory CpG motifs in the pUC ampicillin resistance gene.

Lack of functional receptors is the only barrier that prevents caprine arthritis-encephalitis virus from infecting human cells.

Barriers to replication of viruses in potential host cells may occur at several levels. Lack of suitable and functional receptors on the host cell surface, thereby precluding entry of the virus, is a frequent reason for noninfectivity, as long as no alternative way of entry (e.g., pinocytosis, antibody-dependent adsorption) can be exploited by the virus. Other barriers can intervene at later stages of the virus life cycle, with restrictions on transcription of the viral genome, incorrect translation and posttranslational processing of viral proteins, inefficient viral assembly, and release or efficient early induction of apoptosis in the infected cell. The data we present here demonstrate that replication of caprine arthritis-encephalitis virus (CAEV) is restricted in a variety of human cell lines and primary tissue cultures. This barrier was efficiently overcome by transfection of a novel infectious complete-proviral CAEV construct into the same cells. The successful infection of human cells with a vesicular stomatitis virus (VSV) G-pseudotyped Env-defective CAEV confirmed that viral entry is the major obstacle to CAEV infection of human cells. The fully efficient productive infection obtained with the VSV-G-protein-pseudotyped infectious CAEV strengthened the evidence that lack of viral entry is the only practical barrier to CAEV replication in human cells. The virus thus produced retained its original host cell specificity and acquired no propensity to propagate further in human cultures.

Experimental infection of Mouflon-domestic sheep hybrids with caprine arthritis-encephalitis virus.

OBJECTIVE: To determine whether monocyte-derived macrophages from Mouflon-domestic sheep hybrids (Ovis musimon X Ovis spp) were susceptible to productive infection with caprine arthritis-encephalitis virus (CAEV) in vitro and whether experimental inoculation of Mouflon-domestic sheep hybrids with a molecularly cloned CAEV would result in persistent infection. ANIMALS: 5 Mouflon hybrids. PROCEDURE: Macrophage monolayers were inoculated with virus in vitro. Three animals were inoculated with virus intratracheally. RESULTS: Productive replication of CAEV was demonstrated in monocyte-derived macrophages following in vitro and in vivo inoculation. Titer of infectious cytopathic CAEV produced by macrophages from the Mouflon hybrids was similar to titers produced by macrophages from an infected goat or by synovial membrane cells. Isolation of virus from monocyte-derived macrophages and use of a semiquantitative polymerase chain reaction assay to amplify a portion of the viral genome demonstrated persistent virus replication in all 3 inoculated animals. Two weeks after inoculation of sheep, approximately 1 of 5,000 monocytes was harboring the virus. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that Mouflon-domestic sheep hybrids are susceptible to infection with isolates of CAEV that cause infection in domestic small ruminants.

Ovine lentivirus-infected macrophages mediate productive infection in cell types that are not susceptible to infection with cell-free virus.

Ovine lentiviruses and caprine arthritis-encephalitis virus (CAEV) are prototypic lentiviruses that replicate predominantly in macrophages of infected animals. In situ hybridization of pathologically affected tissues from diseased animals has shown that viral RNA exists in permissive macrophages as well as in non-macrophage cell types that do not support productive virus replication. These findings raise questions about the cellular tropism of these viruses in vivo and how this may relate to their pathogenesis and the establishment of persistent infections. In this study, the susceptibility of macrophages and fibro-epithelial cells derived from goat synovial membrane (GSM) to infection by 14 North American ovine lentivirus strains was examined. All 14 strains were macrophage-tropic, as indicated by expression of viral proteins and by fusion and development of syncytial cytopathic effects following co-culture of infected macrophages with GSM cells. In contrast, neither viral DNA nor viral proteins was detected in GSM cells inoculated with cell-free virus from nine of the 14 strains. Specific virus proteins were immunoprecipitated from restrictive GSM cells following culture with infected macrophages and serial passage of GSM cells to remove the macrophages. The lack of infection of GSM cells by cell-free virus from some ovine lentivirus field strains was circumvented by cell-associated virus infection from infected macrophages to GSM cells following cell-to-cell contact. This strategy could be one of the mechanisms involved in the escape from immune surveillance and establishment of persistent infection in infected animals.

Goat milk epithelial cells are highly permissive to CAEV infection in vitro.

The main route of small ruminant lentivirus dissemination is the ingestion of infected cells present in colostrum and milk from infected animals. However, whether only macrophages or other cell subtypes are involved in this transmission is unknown. We derived epithelial cell cultures, 100% cytokeratin positive, from milk of naturally infected and noninfected goats. One such culture, derived from a naturally infected goat, constitutively produced a high titer of virus in the absence of any cytopathic effect. The other cultures, negative for natural lentivirus infection, were tested for their susceptibility to infection with the CAEV-CO strain and a French field isolate CAEV-3112. We showed that milk epithelial cells are easily infected by either virus and produce viruses at titers as high as those obtained in permissive goat synovial membrane cells. The CAEV-CO strain replicated in milk epithelial cells in absence of any cytopathic effect, whereas the CAEV-3112 field isolate induced both cell fusion and cell lysis. Our results suggest that CAEV-infected milk epithelial cells of small ruminants may play an important role in virus transmission and pathogenesis.

Neuroinvasion by ovine lentivirus in infected sheep mediated by inflammatory cells associated with experimental allergic encephalomyelitis.

Maedi Visna Virus (MVV) is a prototypic lentivirus that causes infection only in cells of macrophage lineage, unlike the primate lentiviruses which infect both CD4+ T lymphocytes and macrophages. In primates, the earliest viral invasion is associated with the ability of the virus to infect and activate T cells which convey virus to the brain. Infected monocytes in blood rarely cause CNS infection in absence of activation of CD4+ T cells. In the face of lack of infection or activation of T cells by MVV in sheep, the question arises, how does MVV gain access to the brain to cause the classical lesions of visna? In previous studies on experimental induction of visna, sheep were inoculated with virus directly in the brain. In this study, we asked whether neuroinvasion by MVV would occur if sheep were inoculated with virus in a non-neural site. Nine sheep were inoculated intratracheally and all developed systemic infection when examined 3 weeks later. At this time, five were injected intramuscularly with brain white matter homogenized in Freund's complete adjuvant to induce EAE. None of the four animals inoculated with virus alone developed CNS infection despite typical lentiviral infection in lungs, lymphoid tissues and blood-borne mononuclear cells. In contrast, all five of the sheep injected with brain homogenate developed infection in the brain. Virus was produced by macrophages associated with the EAE lesions. This study illustrated that both activated T cells specific for antigen in the CNS and infected macrophages are essential for lentivirus neuropathogenesis.

Immortalization of caprine fibroblasts permissive for replication of small ruminant lentiviruses.

OBJECTIVE: To establish immortalized caprine fibroblastic cell lines permissive for replication of small ruminant lentiviruses. ANIMALS: Carpal synovial membrane explants collected aseptically from a surgically delivered fetus of a lentivirus-seronegative goat. PROCEDURE: Immortalization of goat embryonic fibroblasts was performed by DNA transfection with plasmids coding for simian virus 40 large T antigen. The generated cell lines were phenotypically characterized. Cytogenetics, growth pattern, and sensitivity to viral infection were studied. RESULTS: 3 cell lines, designated TIGEF, mMTSV-54, and mMTSV-93, were generated. They had a more rapid doubling time than did fibroblasts from which they were derived, and retained morphologic and phenotypic fibroblastic characteristics. They were immortalized but not transformed because tumor formation was not promoted after their s.c. injection into athymic nude mice. The 3 cell lines were susceptible to caprine arthritis-encephalitis virus and visna-maedi virus infections, and supported a complete virus replication cycle. CONCLUSIONS: Cultured caprine fibroblastic cells were immortalized, using simian virus 40 large T antigen. The TIGEF, mMTSV-54, and mMTSV-93 immortalized cell lines were permissive to in vitro small ruminant lentivirus replication. CLINICAL RELEVANCE: Because lentivirus detection, as well as detailed studies of host-lentivirus interactions, are hampered by differences in viral susceptibility of each primary culture, permanent cell lines are essential tools for such analysis.

Variations in lentiviral gene expression in monocyte-derived macrophages from naturally infected sheep.

Seventy-nine 1-year-old lambs from three individual farms and a feedlot were examined for natural lentivirus infection. We used three different methods to detect infection and to identify the stage of the ovine lentivirus life cycle in blood-derived macrophages. Cytopathic infectious virus was obtained from 14/14 Border Leicester animals obtained from a naturally infected flock. Neither virus particles, virus proteins, virus specific antibodies nor viral DNA were detected in samples from 34 lambs from two South Kansas City farms. However, among 31 feedlot lambs, we identified 11 infected animals. Specific viral proteins were immunoprecipitated from macrophages of one animal, but no infectious cytopathic virus was isolated from these cells. Cells from ten of the other feedlot animals harboured viral DNA but neither viral particles nor proteins could be detected by our techniques. Thus, in these naturally infected animals, the virus life cycle either proceeded to completion, subject to differentiation of infected precursor cells in blood, or remained arrested at the DNA stage despite maturation of monocytes to macrophages. Sequence analysis of the env gene of viral genomes from two of the ten feedlot sheep showed sequences distinct from those of known ovine and caprine lentiviruses. Surprisingly, these sequences have a higher identity (of nucleotide and derived amino acid sequences) to caprine arthritis-encephalitis virus than to the ovine prototype, maedi-visna virus. These data suggest that the ovine and caprine lentiviruses found in North American sheep may have a common ancestral genotype that is closely related to the caprine virus.

Restrictive type of replication of ovine/caprine lentiviruses in ovine fibroblast cell cultures.

Caprine arthritis-encephalitis virus (CAEV) is a natural lentivirus pathogen of goats. CAEV, like all members of the ovine/ caprine lentivirus family, has an in vivo tropism for cells of the monocyte/macrophage cell lineage and activation of viral gene expression is observed only following differentiation of monocytes to macrophages. In addition to cells of the monocyte/ macrophage lineage, CAEV and the closely related maedi visna virus of sheep (MVV) can also replicate productively in fibro-epithelial cells derived from synovial membrane of goats (GSM). However, these viruses varied greatly in their ability to replicate in fibroblasts. We studied the biological and biochemical properties of CAEV and maedi-visna virus (MVV) of sheep following inoculation into the three ovine/caprine cell types. Our data showed no substantial differences in virus titers, viral protein biosynthesis, or processing of the viral proteins between CAEV and MVV following inoculation into primary macrophages and GSM cells. However, unlike MVV, CAEV failed to replicate productively in ovine fibroblasts (sheep choroid plexus cells). This correlated with a specific but abnormal proteolytic cleavage of the envelope glycoprotein of the virus. This abnormal proteolytic cleavage represents a novel type of host cell restriction of lentivirus replication.

Influence of the nature of the reporter gene and selection pressure on stability of avian retrovirus vectors.

We have compared the long-term stability of 2 avian non-replicative retroviral vectors in an infected permanent cell line from quail fibroblasts (QT6). Vectors NL53 and NPL, expressing both the neo and LacZ genes under control of cis-acting elements originated from avian erythroblastosis virus (AEV), are similar to each other except for the presence of the phleomycin-resistance SHble gene fused upstream the reporter LacZ gene, in NPL vector. The use of such vectors, with an uniform backbone, to infect QT6 cells, allowed us to demonstrate that stability of the beta-galactosidase activity encoded by the SHble-LacZ fusion gene remains higher than that encoded by the native LacZ gene, as determined in the same conditions of culture. Moreover, stability of the provirus was dependent on the selection pressure. Here we show that stability of beta-galactosidase activity in infected QT6 cells was obtained with high dose selection for the selectable SHble-LacZ fusion gene.

Influence of expression and cis-acting sequences from avian leukosis viruses (ALVs) on stability of (ALV)-based retrovirus vectors.

Defective avian leukosis virus (ALV)-based vectors expressing the neo and LacZ genes were constructed under the control of cis-acting elements originated from 4 avian retroviruses: avian erythroblastosis virus (AEV), Rous associated viruses 1 (RAV-1) and 2 (RAV-2), and the Schmidt Ruppin strain of Rous sarcoma virus subgroup D (SR-RSV-D). We used these vectors to study the long-term stability of beta-galactosidase expression (encoded by the LacZ gene) in a permanent cell line from quail fibroblasts (QT6). Infection of the immortalized QT6 cell line with these vectors resulted in unstable beta-galactosidase expression. We determined whether this instability of provirus expression was correlated with: (1) presence of G418 selection; (2) deletion in the proviral genome; (3) hypermethylation of the proviral genome; (4) position of the neo and LacZ genes in the proviral genome; and (5) the transcriptional activity of the long terminal repeat (LTR) elements of proviral vectors. We observed that G418 selection pressure applied to infected QT6 cells lead to a more stable LacZ gene expression. Moreover, our results suggest a correlation between the stability of proviral gene expression and the level of gene expression driven by the LTR elements and depending on the strain origin of these.

Defective retroviral endogenous RNA is efficiently transmitted by infectious particles produced on an avian retroviral vector packaging cell line.

This report describes the contamination of "helper-free" stocks of defective retroviral vector with particles bearing retroviral endogenous RNA. An avian leukosis virus-based packaging cell line was developed from LMH cells that bear the ev1, ev3, and ev6 retroviral endogenous loci. The results show that an endogenous retroviral transcript (ev3) was packaged into virions produced by this packaging cell line and was efficiently transferred along with the vector to target cells. The titer of the ev contaminant particles was estimated at 50-100 CA-p27gag-expressing units/ml of supernatant.

Structure and expression of endogenous retroviral sequences in the permanent LMH chicken cell line.

From DNA mapping data, four endogenous proviral loci have been observed in the chicken permanent cell line LMH. The locus corresponding to endogenous virus (ev) ev1 is present in duplicate whereas the locus corresponding to ev3 is present in one copy. The other loci are probably ev6 and a solitary long terminal repeat. A RNA Northern blot analysis revealed both ev3 and ev6 transcripts but no ev1 transcript was detected. Using avian leukosis virus (ALV)-based vectors, transcomplementing assays were performed. They demonstrate the correct expression and maturation of endogenous env proteins and the absence of production of functional gag and pol components, indicating that these cells are not competent for viral production.

Use of retroviral vectors to introduce and express the beta-galactosidase marker gene in cultured chicken primordial germ cells.

Three methods of isolating primordial germ cells (PGCs) from gonads of 5-day-old chick embryos were compared. PGCs were then cultured in vitro in DMEM/F12 medium containing 10% fetal calf serum. BrdU incorporation showed that at least 10% of the PGC population were dividing, under our culture conditions, during the 2nd day of in vitro culture. During this culture period, PGCs were exposed to avian leukosis sarcoma virus-based retroviral vector pseudotyped with subgroup A envelope, carrying the LacZ reporter gene. X-Gal staining showed that PGCs were permissive to infection, with more than 50% of PGCs expressing the beta-Gal protein. These data represent the first demonstration that PGCs, isolated from gonads of 5-day-old chick embryos, are able to divide in vitro and that it is possible to introduce and express exogenous DNA in chick PGCs maintained in vitro.

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

Newcastle disease virus (NDV) vaccine based on immunization with avian cells expressing the NDV hemagglutinin-neuraminidase glycoprotein.

Newcastle disease virus (NDV) is a paramyxovirus that bears two envelope glycoproteins at the virion surface. These proteins, fusion and hemagglutinin-neuraminidase (HN), are involved in the immune response against NDV infection. Recombinant cells constitutively expressing at their surface the HN protein from the velogenic Texas strain were generated by introducing the HN gene with a helper-free AEV-based vector. These recombinant cells were used to immunize chickens by various protocols, and birds were subsequently challenged with a lethal NDV injection. Both NDV protection and serologic response were observed.