Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation

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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.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12985-023-01975-7.

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Most cited references 45

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Genetically Modified Porcine-to-Human Cardiac Xenotransplantation

Bartley P Griffith, Corbin Goerlich, Avneesh Singh … (2022)

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Herpesvirus latency

Jeffrey I Cohen (2020)

Herpesviruses infect virtually all humans and establish lifelong latency and reactivate to infect other humans. Latency requires multiple functions: maintaining the herpesvirus genome in the nuclei of cells; partitioning the viral genome to daughter cells in dividing cells; avoiding recognition by the immune system by limiting protein expression; producing noncoding viral RNAs (including microRNAs) to suppress lytic gene expression or regulate cellular protein expression that could otherwise eliminate virus-infected cells; modulating the epigenetic state of the viral genome to regulate viral gene expression; and reactivating to infect other hosts. Licensed antivirals inhibit virus replication, but do not affect latency. Understanding of the mechanisms of latency is leading to novel approaches to destroy latently infected cells or inhibit reactivation from latency.

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Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines.

M Gemeiner, J. Catharina Duvigneau, S J Groiss … (2005)

We have established an easy real-time PCR assay, which allows the precise quantification of changes in the expression level of 6 relevant porcine cytokines, and 3 housekeeping genes. This assay simultaneously detects 9 sequences by measuring 3 x 3 targets in a triplex-format. The mRNA of the lymphokines IL-2, IL-4, IL-10, and IFN-gamma, of the proinflammatory cytokines IL-1alpha and IL-6, and of the housekeeping genes are quantified using TaqMan-probes by means of standard dilution series on the iCycler iQ. The standard consists of equal aliquots of the experimental cDNAs under investigation. Simultaneously the most suitable combination of 3 out of the four housekeeping genes beta-actin, HPRT, GAPDH, and cyclophilin can be selected, and their averaged expression values constitute a normalisation factor. The raw data of all targets of interest is then calculated relative to this normalisation factor, making eventual changes of the relative expression level of the single housekeeping genes controllable and quantifiable. We have applied this assay to quantify changes in the cytokine mRNA levels of porcine stimulated with various concentrations of LPS and ConA, known to induce different cytokine expression patterns. We have shown, that even small differences in the expression level (less than 2-fold) can be precisely quantified, and reveal statistically significant changes, when using the normalisation factor. This assay will be useful for studying changes in the expression of relevant porcine cytokines and will help to further improve the investigation of immune responses in the pig.

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Author and article information

Contributors

Joachim Denner: Joachim.Denner@fu-berlin.de

Journal

Journal ID (nlm-ta): Virol J

Journal ID (iso-abbrev): Virol J

Title: Virology Journal

Publisher: BioMed Central (London )

ISSN (Electronic): 1743-422X

Publication date (Electronic): 27 January 2023

Publication date PMC-release: 27 January 2023

Publication date Collection: 2023

Volume: 20

Electronic Location Identifier: 15

Affiliations

[1 ]GRID grid.14095.39, ISNI 0000 0000 9116 4836, Institute of Virology, , Free University Berlin, ; Berlin, Germany

[2 ]GRID grid.6936.a, ISNI 0000000123222966, Chair of Animal Biotechnology, TUM School of Life Sciences Weihenstephan, , Technical University Munich, ; Freising, Germany

Article

Publisher ID: 1975

DOI: 10.1186/s12985-023-01975-7

PMC ID: 9881377

PubMed ID: 36707837

SO-VID: 4d140fd2-2ad0-49eb-8ce5-8cc5d2af4c9e

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 17 October 2022

Date accepted : 20 January 2023

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;

Award ID: TR127

Funded by: Freie Universität Berlin (1008)

Open Access :

Open Access funding enabled and organized by Projekt DEAL.

Custom metadata

ScienceOpen disciplines: Microbiology & Virology

Keywords: porcine cytomegalovirus,porcine roseolovirus,xenotransplantation,oocytes,somatic cell nuclear transfer,cloning

Data availability:

ScienceOpen disciplines: Microbiology & Virology

Keywords: porcine cytomegalovirus, porcine roseolovirus, xenotransplantation, oocytes, somatic cell nuclear transfer, cloning

Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation

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Abstract

Background

Methods

Results

Conclusion

Supplementary Information

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Role of Microbes in Soil Fertility and Human Health

Most cited references 45

Genetically Modified Porcine-to-Human Cardiac Xenotransplantation

Herpesvirus latency

Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines.

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