Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation
Abstract Background Porcine cytomegalovirus (PCMV) is a porcine roseolovirus (PCMV/PRV) which is widely distributed in pigs. Transmission of PCMV/PRV in preclinical xenotransplantations was shown to significantly reduce the survival time of the pig transplants in non-human primates. PCMV/PRV was als...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-01-01
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| Series: | Virology Journal |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12985-023-01975-7 |
| _version_ | 1828051937083260928 |
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| author | Sabrina Hansen Konrad Fischer Ludwig Krabben Alexander Rinke Carrapeiro Bernhard Klinger Angelika Schnieke Benedikt Kaufer Joachim Denner |
| author_facet | Sabrina Hansen Konrad Fischer Ludwig Krabben Alexander Rinke Carrapeiro Bernhard Klinger Angelika Schnieke Benedikt Kaufer Joachim Denner |
| author_sort | Sabrina Hansen |
| collection | DOAJ |
| description | Abstract Background Porcine cytomegalovirus (PCMV) is a porcine roseolovirus (PCMV/PRV) which is widely distributed in pigs. Transmission of PCMV/PRV in preclinical xenotransplantations was shown to significantly reduce the survival time of the pig transplants in non-human primates. PCMV/PRV was also transmitted in the first transplantation of a pig heart into a human patient. To analyze how PCMV/PRV could be introduced into pig breeds, especially considering cloned transgenic pigs, and subsequently spread in breeding facilities, we screened ovaries and derived materials which are used to perform somatic cell nuclear transfer (SCNT). Methods DNA was isolated from ovarian tissues, follicular fluids, oocytes with cumulus cells, denuded oocytes and parthenotes. A real-time PCR with PCMV/PRV-specific primers and a probe was performed to detect PCMV/PRV. Furthermore, a Western blot assay using a recombinant fragment of the gB protein of PCMV/PRV was performed to screen for virus-specific antibodies in the follicular fluids. Results PCMV/PRV was found by real-time PCR in ovarian tissues, in the follicular fluid and in oocytes. In parthenotes the virus could not be detected, most-likely due to the low amount of DNA used. By Western blot assay specific antibodies against PCMV/PRV were found in 19 of 20 analyzed follicular fluids. Conclusion PCMV/PRV was found in ovarian tissues, in the follicular fluids and also in denuded oocytes, indicating that the virus is present in the animals of which the oocytes were taken from. Despite several washing steps of the denuded oocytes, which are subsequently used for microinjection or SCNT, the virus could still be detected. Therefore, the virus could infect oocytes during genetic modifications or stay attached to the surface of the oocytes, potentially infecting SCNT recipient animals. |
| first_indexed | 2024-04-10T19:45:50Z |
| format | Article |
| id | doaj.art-f63f122561c448e19f3f25e6befd92b1 |
| institution | Directory Open Access Journal |
| issn | 1743-422X |
| language | English |
| last_indexed | 2024-04-10T19:45:50Z |
| publishDate | 2023-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Virology Journal |
| spelling | doaj.art-f63f122561c448e19f3f25e6befd92b12023-01-29T12:04:17ZengBMCVirology Journal1743-422X2023-01-012011810.1186/s12985-023-01975-7Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantationSabrina Hansen0Konrad Fischer1Ludwig Krabben2Alexander Rinke Carrapeiro3Bernhard Klinger4Angelika Schnieke5Benedikt Kaufer6Joachim Denner7Institute of Virology, Free University BerlinChair of Animal Biotechnology, TUM School of Life Sciences Weihenstephan, Technical University MunichInstitute of Virology, Free University BerlinChair of Animal Biotechnology, TUM School of Life Sciences Weihenstephan, Technical University MunichChair of Animal Biotechnology, TUM School of Life Sciences Weihenstephan, Technical University MunichChair of Animal Biotechnology, TUM School of Life Sciences Weihenstephan, Technical University MunichInstitute of Virology, Free University BerlinInstitute of Virology, Free University BerlinAbstract Background Porcine cytomegalovirus (PCMV) is a porcine roseolovirus (PCMV/PRV) which is widely distributed in pigs. Transmission of PCMV/PRV in preclinical xenotransplantations was shown to significantly reduce the survival time of the pig transplants in non-human primates. PCMV/PRV was also transmitted in the first transplantation of a pig heart into a human patient. To analyze how PCMV/PRV could be introduced into pig breeds, especially considering cloned transgenic pigs, and subsequently spread in breeding facilities, we screened ovaries and derived materials which are used to perform somatic cell nuclear transfer (SCNT). Methods DNA was isolated from ovarian tissues, follicular fluids, oocytes with cumulus cells, denuded oocytes and parthenotes. A real-time PCR with PCMV/PRV-specific primers and a probe was performed to detect PCMV/PRV. Furthermore, a Western blot assay using a recombinant fragment of the gB protein of PCMV/PRV was performed to screen for virus-specific antibodies in the follicular fluids. Results PCMV/PRV was found by real-time PCR in ovarian tissues, in the follicular fluid and in oocytes. In parthenotes the virus could not be detected, most-likely due to the low amount of DNA used. By Western blot assay specific antibodies against PCMV/PRV were found in 19 of 20 analyzed follicular fluids. Conclusion PCMV/PRV was found in ovarian tissues, in the follicular fluids and also in denuded oocytes, indicating that the virus is present in the animals of which the oocytes were taken from. Despite several washing steps of the denuded oocytes, which are subsequently used for microinjection or SCNT, the virus could still be detected. Therefore, the virus could infect oocytes during genetic modifications or stay attached to the surface of the oocytes, potentially infecting SCNT recipient animals.https://doi.org/10.1186/s12985-023-01975-7Porcine cytomegalovirusPorcine roseolovirusXenotransplantationOocytesSomatic cell nuclear transferCloning |
| spellingShingle | Sabrina Hansen Konrad Fischer Ludwig Krabben Alexander Rinke Carrapeiro Bernhard Klinger Angelika Schnieke Benedikt Kaufer Joachim Denner Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation Virology Journal Porcine cytomegalovirus Porcine roseolovirus Xenotransplantation Oocytes Somatic cell nuclear transfer Cloning |
| title | Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation |
| title_full | Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation |
| title_fullStr | Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation |
| title_full_unstemmed | Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation |
| title_short | Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation |
| title_sort | detection of porcine cytomegalovirus a roseolovirus in pig ovaries and follicular fluid implications for somatic cells nuclear transfer cloning and xenotransplantation |
| topic | Porcine cytomegalovirus Porcine roseolovirus Xenotransplantation Oocytes Somatic cell nuclear transfer Cloning |
| url | https://doi.org/10.1186/s12985-023-01975-7 |
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