Cultured early goat embryos and cells are susceptible to infection with caprine encephalitis virus

Department of Research into the Health Risk and Biotechnology of Reproduction ENVN/DGER, National Veterinary School, BP 40706, 44307 Nantes Cedex 03, France.
Virology (Impact Factor: 3.32). 10/2006; 353(2):307-15. DOI: 10.1016/j.virol.2006.06.007
Source: PubMed


Zona-pellucida-free embryos at 8-16 cell stage were co-cultured for 6 days in an insert over a mixed cell monolayer infected with CAEV-pBSCA. Embryos were washed and transferred to an insert on CAEV indicator goat synovial membrane cells for 6 h, then they were washed and cultivated in B2 Ménézo for 24 h, finally, embryo cells were dissociated and cultivated on a feeder monolayer for 8 days. After 5 weeks, multinucleated giant cells typical of CAEV infection were observed in indicator GSM cell monolayers. In the acellular medium, the early embryonic cells produced at least 10(3.25) TCID50/ml over 24 h. The monolayer of cultivated embryonic cells developed cytopathic lesions within 8 days, and CAEV RNA, CAEV proviral DNA and protein p28 of the capsid were detected. All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with CAEV and that infection with this virus is productive.

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    • "-PCRn em ovócitos (Tabela 1 e Fig. 1), o que indica que o CAEV está presente neste material na forma livre, possivelmente aderido à zona pelúcida. A PCRn identificou apenas uma amostra de ovócito positivo dentre as 11 avaliadas (9,1%), indicando a presença do DNA pró-viral nesta amostra (Tabela 1). Estes resultados corroboram os de Ali Al Ahmad et al. (2006) que, utilizando técnicas moleculares como a PCRn e a RT-PCRn, verificaram que células embrionárias são suscetíveis e permissivas ao CAEV. Diferentemente, Ali Al Ahmad et al. (2005) não verificaram a presença do DNA pró-viral do CAEV em ovócitos de folículos antrais, mas sim em células da granulosa de cabras infectadas naturalmente. A té"
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    ABSTRACT: Caprine arthritis-encephalitis (CAE) is an infectious disease caused by the caprine arthritis-encephalitis virus (CAEV), belonging to the lentivirus genus. The presence of the virus has been observed in the nervous system, respiratory tract and mammary gland, and also in the male and female genital tract. The objective of this study is to identify the virus in oocyte and uterine fluid of infected goats by molecular diagnostic techniques, in order to assess the possibility of CAEV transmission with reproduction. Thirteen infected goats were selected and submitted to euthanasia for the collection of the reproductive system, aspiration of the uterine fluid and dissection of ovaries for oocyte collection. In order to identify the CAEV in the collected material, in the protovirus and free forms, it was submitted to the nRT-PCR and nPCR techniques, respectively. As a result, it was observed that 53.8% of oocytes were positive to nRT-PCR, while only 9.1% were positive to nPCR. The nRT-PCR also identified the virus in the uterine fluid of 46.1% of the tested females. Even though the 13 goats had CAEV, 30.8% presented negative results in nPCR and nRT-PCR in all of the analyzed samples (oocyte and uterine fluid). This work concludes that nRT-PCR and nPCR can be used in the diagnosis of CAE for the analysis with oocytes and uterine fluid, and that the presence of CAEV in these materials points out to the risk of CAEV transmission through reproductive technologies used in females.
    Full-text · Article · Dec 2012
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    • "In this study, we used co-culture systems either in direct contact or with membrane separation between Vero cell monolayers and the embryos [23]. This insert co-culture system helped to exclude the contamination of cultured embryos or embryo cell cultures with feeder Vero cells from the monolayer. "
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    ABSTRACT: The three objectives of this study were to investigate whether cells of early goat embryos isolated from in vivo fertilized goats interact with bluetongue virus (BTV) in vitro, whether the embryonic zona pellucida (ZP) protects early embryo cells from BTV infection, and whether the 10 wash cycles recommended by the International Embryo Transfer Society (IETS) for bovine embryos effectively decontaminates caprine embryos exposed to Bluetongue Virus (BTV) in vitro. Donor goats and bucks were individually screened and tested negative for the virus by RT-PCR detection of BTV RNA in circulating erythrocytes. ZP-free and ZP-intact 8-16 cell embryos were co-cultured for 36 h in an insert over a Vero cell monolayer infected with BTV. Embryos were washed 10 times in accordance with IETS recommendations for ruminant and porcine embryos, before being transferred to an insert on BTV indicator Vero cells for 6 h, to detect any cytopathic effects (CPE). They were then washed and cultured in B2 Ménézo for 24 h. Non-inoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls. The Vero cell monolayer used as feeder cells for BTV inoculated ZP-free and ZP-intact embryos showed cytopathic effects (CPE). BTV was found by RT-qPCR in the ten washes of exposed ZP-free and ZP-intact embryos. In the acellular medium, the early embryonic cells produced at least 10(2.5) TCID(50)/ml. BTV RNA was detected in ZP-free and ZP-intact embryos using RT-qPCR. All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with BTV and that infection with this virus is productive. The washing procedure failed to remove BTV, which indicates that BTV could bind to the zona pellucida.
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    • "The risk of viral infection of the goat embryo during embryo transfer has been demonstrated in vivo through the demonstration of CAEV proviral DNA in the embryo harvesting fluid [7], as well as in the cells of the cumulus oophorus surrounding the oocytes in the ovarian follicle [8]. In vitro, the epithelial cells of the oviduct [9], the cells of the granulosa [10], and the embryonic blastocytes [11] were shown to be sensitive to infection with CAEV and capable of producing virus. However, washing in vitroinfected embryos with an intact zona pellucida (intact- ZP) 10 times over [12], or washing oocytes with intact- ZP, in which the in vivo CAEV-infected cumulus oophorus cells have been physically and enzymatically removed, 10 times over [8], resulted in the production of virus-free female gametes or embryos that can be used for in vitro fertilisation or embryo transfer. "
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    ABSTRACT: The aim of this study was to demonstrate that embryo transfer can be used to produce CAEV-free kids from CAEV-infected biological mothers when appropriate procedure is implemented. Twenty-eight goats that had tested positive for CAEV using PCR on vaginal secretions were used as embryo donors. Embryos with intact-ZP were selected and washed 10 times; they were then frozen and used for transfer into CAEV-free recipient goats. Nineteen of the 49 recipient goats gave birth, producing a total of 23 kids. Three blood samples were taken from each recipient goat, 10 days before, during, and 10 days after parturition; these were tested for CAEV antibodies using ELISA and for CAEV proviral DNA using PCR. The mothers were then euthanized. Tissue samples were taken from the lungs, udder, and retromammary and prescapular lymph nodes. The kids were separated from their mothers at birth. Seven of them died. At 4 months of age, 16 kids were subjected to drug-induced immunosuppression. Blood samples were taken every month from birth to 4 months of age; samples were then taken on days 15, 21, and 28 after the start of the immunosuppressive treatment. The kids were then euthanized and tissue samples taken from the carpal synovial membrane, lung tissue, prescapular lymph nodes, inguinal and retromammary lymph nodes, and uterus. All samples from the 19 recipient goats and 23 kids were found to be negative for CAEV antibodies and/or CAEV proviral DNA. Under acute conditions for infection this study clearly demonstrates that embryo transfer can be safely used to produce CAEV-free neonates from infected CAEV donors.
    Full-text · Article · Apr 2008 · Theriogenology
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