RESUMO
Recent reports demonstrated the susceptibility of epithelial cells from different organs to caprine arthritis-encephalitis virus (CAEV) both in vitro and in vivo. Since granulosa cells (GC) are of epithelial origin and currently used for in vitro oocyte maturation, we addressed the question whether these cells are susceptible or resistant to CAEV infection. GC were isolated from goats from certified CAEV-free herds. PCR analysis on GC DNA using CAEV specific primers confirmed the absence of CAEV infection and immunocytochemistry using specific K813 anti-cytokeratin monoclonal antibodies confirmed the epithelial nature of GC. These cells were then inoculated with CAEV using two strains: the CAEV-pBSCA molecular clone and the CAEV-3112 French field isolate. 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 an hyperimmune serum. Supernatant of infected cells were shown to contain high titers (ranging 10(5) tissue culture infectious doses 50 per ml: TCID(50) per ml) of infectious cytopathic viruses when assayed onto the indicator goat synovial membrane (GSM) cells. Our findings demonstrate the large cell tropism of CAEV and suggest that GC could serve as a reservoir for the virus during the sub-clinical phase of infection. Furthermore, given the high seroprevalence of CAEV in the all industrialised countries and the large number of ovaries derived from unknown serological status animals used for in vitro goat embryo production, one can conclude that these feeder cell cultures might be a potential source of early transmission of CAEV to goat embryos.
Assuntos
Vírus da Artrite-Encefalite Caprina/fisiologia , Células da Granulosa/virologia , Animais , Vírus da Artrite-Encefalite Caprina/genética , Vírus da Artrite-Encefalite Caprina/metabolismo , Células Cultivadas , Feminino , Cabras , Células da Granulosa/citologia , Replicação ViralRESUMO
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.
Assuntos
Vírus da Artrite-Encefalite Caprina/crescimento & desenvolvimento , Tubas Uterinas/citologia , Animais , Células Cultivadas , Células Epiteliais/virologia , Feminino , CabrasRESUMO
The aim of this study was to investigate whether cells of early goat embryos isolated from in vivo-fertilized goats interact with the caprine arthritis-encephalitis virus (CAEV) in vitro and whether the embryonic zona pellucida (ZP) protects early embryo cells from CAEV infection. ZP-free and ZP-intact 8-16 cell embryos were inoculated for 2 h with CAEVat the 10(4) tissue culture infectious dose 50 (TCID50)/ml. Infected embryos were incubated for 72 h over feeder monolayer containing caprine oviduct epithelial cells (COECs) and CAEV indicator goat synovial membrane (GSM) cells. Noninoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls. Six days postinoculation, infectious virus assay of the wash fluids of inoculated early goat embryos showed typical CAEV-induced cytopathic effects (CPE) on indicator GSM monolayers, with fluids of the first two washes only. The mixed cell monolayer (COEC + GSM) used as feeder cells for CAEV inoculated ZP-free embryos showed CPE. In contrast, none of the feeder monolayers, used for culture of CAEV inoculated ZP-intact embryos or the noninoculated controls, developed any CPE. CAEV exposure apparently did not interfere with development of ZP-free embryos in vitro during the 72 h study period when compared with untreated controls (34.6 and 36% blastocysts, respectively, P > 0.05). From these results one can conclude that the transmission of infectious molecularly cloned CAEV-pBSCA (plasmid binding site CAEV) by embryonic cells from in vivo-produced embryos at the 8-16 cell stages is possible with ZP-free embryos. The absence of interactions between ZP-intact embryos and CAEV in vitro suggests that the ZP is an efficient protective embryo barrier.
Assuntos
Vírus da Artrite-Encefalite Caprina/fisiologia , Embrião de Mamíferos/virologia , Cabras/embriologia , Cabras/virologia , Infecções por Lentivirus/transmissão , Animais , Vírus da Artrite-Encefalite Caprina/genética , Clonagem Molecular , Técnicas de Cultura , Efeito Citopatogênico Viral , Sincronização do Estro , Superovulação , Zona Pelúcida/fisiologiaRESUMO
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.
Assuntos
Vírus da Artrite-Encefalite Caprina , Infecções por Lentivirus/veterinária , Doenças dos Ovinos/virologia , Animais , Vírus da Artrite-Encefalite Caprina/genética , Vírus da Artrite-Encefalite Caprina/fisiologia , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Infecções por Lentivirus/virologia , Macrófagos/virologia , Ovinos , Replicação ViralRESUMO
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.
Assuntos
Lentivirus Ovinos-Caprinos/fisiologia , Lentivirus Ovinos-Caprinos/patogenicidade , Macrófagos/virologia , Membrana Sinovial/virologia , Replicação Viral , Animais , Células Cultivadas , Técnicas de Cocultura , Efeito Citopatogênico Viral , DNA Viral/genética , Cabras , Testes de Precipitina , Ovinos , Membrana Sinovial/citologia , Proteínas Virais/isolamento & purificaçãoRESUMO
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.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Artrite-Encefalite Caprina/imunologia , Produtos do Gene env/imunologia , Glicoproteínas , Cabras/imunologia , Proteínas de Membrana , Plasmídeos/genética , Vacinas de DNA/imunologia , Proteínas Virais , Animais , Vírus da Artrite-Encefalite Caprina/genética , Células COS , Produtos do Gene env/química , Produtos do Gene env/genética , Produtos do Gene tat/genética , Vetores Genéticos/genética , Imunização Secundária , Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas/imunologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinação , Vacinas de DNA/genética , Vaccinia virus/genética , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
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.
Assuntos
Vírus da Artrite-Encefalite Caprina/patogenicidade , Glicoproteínas de Membrana , Receptores Virais/metabolismo , Replicação Viral , Animais , Vírus da Artrite-Encefalite Caprina/fisiologia , Células Cultivadas , Cabras , Humanos , Testes de Precipitina , Receptores Virais/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Transfecção , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
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.
Assuntos
Vírus da Artrite-Encefalite Caprina/fisiologia , Células Epiteliais/virologia , Glândulas Mamárias Animais/citologia , Leite/citologia , Animais , Divisão Celular , Linhagem Celular , Feminino , Doenças das Cabras/virologia , Cabras , Imuno-Histoquímica/veterinária , Cinética , Infecções por Lentivirus/veterinária , Infecções por Lentivirus/virologia , Glândulas Mamárias Animais/virologia , Mastite/veterinária , Mastite/virologia , Leite/virologia , Transfecção/veterinária , Replicação Viral/fisiologiaRESUMO
Replication defective retroviral vectors are regularly used for transfer and expression of exogenous genes into dividing cells and in animals. Since lentiviruses are able to infect terminally differentiated and non-dividing cells, their use to produce replication defective vectors may overcome this limitation. We developed two replication-defective lentiviral vectors based on the genome of Caprine Arthritis Encephalitis Virus (CAEV). The first vector (pBNL2) carries the neo and lacZ marker genes. Neo gene is expressed from a genomic RNA and lacZ gene from a subgenomic RNA. The second vector (pCSHL) carries a single fusion gene encoding both phleomycin resistance and beta-galactosidase activity. Replication-competent CAEV was used as helper virus to provide the viral proteins for transcomplementation of these vectors. Our data demonstrated that the genomes of both vectors were packaged into CAEV virions and transduced into goat synovial membrane cells following infection. However, the vector titers remained 3 to 4 logs lower than those of CAEV. Further analysis showed a lack of accumulation of unspliced pBNL2 RNA into the cytoplasm of producer cells resulting in the packaging of pBNL2 sub-genomic RNA only. In contrast, RNA produced from pCSHL vector was correctly transported to the cytoplasm and more efficiently packaged than the pBNL2 sub-genomic RNA as revealed by slot-blot and quantitative RT/PCR analyses. However this higher packaging efficiency of pCSHL genome did not result in a higher transduction efficiency of lacZ gene.
Assuntos
Vírus da Artrite-Encefalite Caprina/genética , Vetores Genéticos , RNA Viral/metabolismo , Montagem de Vírus , Animais , Linhagem Celular , Citoplasma/virologia , Vírus Defeituosos/genética , Genoma Viral , Cabras , Óperon Lac , Transfecção , Vírion/genéticaRESUMO
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.