{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T03:52:50Z","timestamp":1774929170521,"version":"3.50.1"},"reference-count":47,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2025,7,23]],"date-time":"2025-07-23T00:00:00Z","timestamp":1753228800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Porcine endogenous retroviruses (PERVs) are integrated into the genome of all pigs. As they can be released as infectious virus particles capable of infecting human cells in vitro, they pose a potential risk for xenotransplantation involving pig cells or organs. To assess whether pigs produce infectious human-tropic viruses, infection assays with human cells are required. There are three main types of assays. First is the incubation of human target cells with gamma-irradiated pig cells. This method ensures that viral transmission is assessed in the absence of replicating pig cells. However, gamma irradiation may alter gene expression in pig cells, potentially affecting the results. Second is the co-culture in a double-chamber system in which pig and human cells are separated by a porous membrane, preventing direct cell-to-cell contact. While this method allows for the detection of infection by free virus particles, it does not account for infection via cell-to-cell transmission, which is a common mode of retroviral infection. And third is the co-culture of pig cells with human cells expressing a resistance gene. The resistance gene allows selective elimination of pig cells upon the addition of a selection medium. This assay enables both free virus and cell-to-cell transmission as well as complete removal of pig cells, which may not be fully achieved in the first type of assay. The third assay best simulates the conditions of in vivo xenotransplantation. However, in all cases the selection of donor and recipient cells is crucial to the experimental outcome. Results only indicate whether a specific pig cell type releases PERVs and whether a specific human cell type is susceptible to infection. A negative infection result does not necessarily reflect the in vivo situation, in which a transplanted organ consists of multiple pig cell types interacting with a diverse range of human cells within a living organism. Knowledge of these limitations is important for authorities regulating clinical applications for xenotransplantation.<\/jats:p>","DOI":"10.3390\/ijms26157111","type":"journal-article","created":{"date-parts":[[2025,7,23]],"date-time":"2025-07-23T14:22:44Z","timestamp":1753280564000},"page":"7111","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Co-Cultivation Assays for Detecting Infectious Human-Tropic Porcine Endogenous Retroviruses (PERVs)"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3244-6085","authenticated-orcid":false,"given":"Joachim","family":"Denner","sequence":"first","affiliation":[{"name":"Institute of Virology, Free University Berlin, 14163 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1128\/CMR.05011-11","article-title":"Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses","volume":"25","author":"Denner","year":"2012","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1038\/nm0397-282","article-title":"Infection of human cells by an endogenous retrovirus of pigs","volume":"3","author":"Patience","year":"1997","journal-title":"Nat. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1006\/viro.2001.0934","article-title":"Productive infection of human primary cells and cell lines with porcine endogenous retroviruses","volume":"285","author":"Specke","year":"2001","journal-title":"Virology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1111\/xen.12150","article-title":"Porcine endogenous retrovirus infection of human peripheral blood mononuclear cells","volume":"22","author":"Denner","year":"2014","journal-title":"Xenotransplantation"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1034\/j.1399-3089.2000.00052.x","article-title":"Productive infection of primary human endothelial cells by pig endogenous retrovirus (PERV)","volume":"7","author":"Martin","year":"2000","journal-title":"Xenotransplantation"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"692","DOI":"10.1016\/S0140-6736(98)07144-X","article-title":"Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells","volume":"352","author":"Martin","year":"1998","journal-title":"Lancet"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Halecker, S., Krabben, L., Kristiansen, Y., Kr\u00fcger, L., M\u00f6ller, L., Becher, D., Laue, M., Kaufer, B., Reimer, C., and Denner, J. (2022). Rare isolation of human-tropic recombinant porcine endogenous retroviruses PERV-A\/C from Gottingen minipigs. Virol. J., 19.","DOI":"10.1186\/s12985-022-01742-0"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3082","DOI":"10.1128\/JVI.72.4.3082-3087.1998","article-title":"Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells","volume":"72","author":"Wilson","year":"1998","journal-title":"Virol. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1128\/JVI.74.1.49-56.2000","article-title":"Extended analysis of the in vitro tropism of porcine endogenous retrovirus","volume":"74","author":"Wilson","year":"2000","journal-title":"Virol. J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"13871","DOI":"10.1128\/JVI.78.24.13871-13879.2004","article-title":"Determinants of high titer in recombinant porcine endogenous retroviruses","volume":"78","author":"Harrison","year":"2004","journal-title":"Virol. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1016\/S0042-6822(03)00428-8","article-title":"Genetic alterations of the long terminal repeat of an ecotropic porcine endogenous retrovirus during passage in human cells","volume":"314","author":"Denner","year":"2003","journal-title":"Virology"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Denner, J. (2020). Sensitive detection systems for infectious agents in xenotransplantation. Xenotransplantation.","DOI":"10.1111\/xen.12594"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1111\/xen.12154","article-title":"Review on porcine endogenous retrovirus detection assays--impact on quality and safety of xenotransplants","volume":"22","author":"Godehardt","year":"2015","journal-title":"Xenotransplantation"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/j.repbio.2014.01.006","article-title":"Detection of porcine endogenous retrovirus in xenotransplantation","volume":"14","author":"Gola","year":"2014","journal-title":"Reprod. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Kono, K., Kataoka, K., Yuan, Y., Yusa, K., Uchida, K., and Sato, Y. (2020). A highly sensitive method for the detection of recombinant PERV-A\/C env RNA using next generation sequencing technologies. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-78890-2"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Denner, J. (2022). What does the PERV copy number tell us?. Xenotransplantation, 29.","DOI":"10.1111\/xen.12732"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Godehardt, A.W., Fischer, N., Rauch, P., Gulich, B., Boller, K., Church, G.M., and T\u00f6njes, R.R. (2020). Characterization of porcine endogenous retrovirus particles released by the CRISPR\/Cas9 inactivated cell line PK15 clone 15. Xenotransplantation, 27.","DOI":"10.1111\/xen.12563"},{"key":"ref_18","first-page":"42","article-title":"Effects of different doses of X-ray irradiation on cell apoptosis, cell cycle, DNA damage repair and glycolysis in HeLa cells","volume":"17","author":"Zhao","year":"2019","journal-title":"Oncol. Lett."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"578","DOI":"10.1016\/j.clon.2013.06.007","article-title":"Biological consequences of radiation-induced DNA damage: Relevance to radiotherapy","volume":"25","author":"Lomax","year":"2013","journal-title":"Clin. Oncol. (R Coll. Radiol.)"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Delso-Vallejo, M., Kollet, J., Koehl, U., and Huppert, V. (2017). Influence of Irradiated Peripheral Blood Mononuclear Cells on Both Ex Vivo Proliferation of Human Natural Killer Cells and Change in Cellular Property. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.00854"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Lee, H., Kang, S.H., Jeong, G.H., Lee, S.S., Chung, B.Y., Kim, G.J., and Bai, H.W. (2024). Gamma irradiation-engineered macrophage-derived exosomes as potential immunomodulatory therapeutic agents. PLoS ONE, 19.","DOI":"10.1371\/journal.pone.0303434"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1016\/j.transproceed.2008.01.051","article-title":"Porcine endogenous retrovirus transmission characteristics from a designated pathogen-free herd","volume":"40","author":"Garkavenko","year":"2008","journal-title":"Transplant. Proc."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1111\/xen.12032","article-title":"Screening pigs for xenotransplantation: Prevalence and expression of porcine endogenous retroviruses in Gottingen minipigs","volume":"20","author":"Semaan","year":"2013","journal-title":"Xenotransplantation"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1111\/xen.12081","article-title":"Comparison of porcine endogenous retroviruses infectious potential in supernatants of producer cells and in cocultures","volume":"21","author":"Fischer","year":"2014","journal-title":"Xenotransplantation"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1159\/000081745","article-title":"Porcine endogenous retroviruses PERV-A and PERV-B infect neither mouse cells in vitro nor SCID mice in vivo","volume":"48","author":"Irgang","year":"2005","journal-title":"Intervirology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.virol.2010.12.031","article-title":"Cell-to-cell transmission of retroviruses: Innate immunity and interferon-induced restriction factors","volume":"411","author":"Jolly","year":"2011","journal-title":"Virology"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Calado, M., Pires, D., Concei\u00e7\u00e3o, C., Ferreira, R., Santos-Costa, Q., Anes, E., and Azevedo-Pereira, J.M. (2023). Cell-to-Cell Transmission of HIV-1 and HIV-2 from Infected Macrophages and Dendritic Cells to CD4+ T Lymphocytes. Viruses, 15.","DOI":"10.3390\/v15051030"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Ivanusic, D., Madela, K., Bannert, N., and Denner, J. (2021). The large extracellular loop of CD63 interacts with gp41 of HIV-1 and is essential for establishing the virological synapse. Sci. Rep., 11.","DOI":"10.1038\/s41598-021-89523-7"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1038\/ncb1544","article-title":"Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission","volume":"9","author":"Sherer","year":"2007","journal-title":"Nat. Cell Biol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Jin, J., Sherer, N.M., Heidecker, G., Derse, D., and Mothes, W. (2009). Assembly of the murine leukemia virus is directed towards sites of cell-cell contact. PLoS Biol., 7.","DOI":"10.1371\/journal.pbio.1000163"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3248","DOI":"10.1128\/JVI.02155-09","article-title":"Directional spread of surface-associated retroviruses regulated by differential virus-cell interactions","volume":"84","author":"Sherer","year":"2010","journal-title":"Virol. J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.coviro.2012.11.004","article-title":"Cell-to-cell transmission of viruses","volume":"3","author":"Zhong","year":"2013","journal-title":"Curr. Opin. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Sewald, X., Gonzalez, D.G., Haberman, A.M., and Mothes, W. (2012). In vivo imaging of virological synapses. Nat. Commun., 3.","DOI":"10.1038\/ncomms2338"},{"key":"ref_34","first-page":"49","article-title":"Selectable markers for eukaryotic cells","volume":"8","author":"Vile","year":"1992","journal-title":"Methods Mol. Biol."},{"key":"ref_35","first-page":"59","article-title":"No evidence for PERV release by islet cells from German landrace pigs","volume":"13","author":"Irgang","year":"2008","journal-title":"Ann. Transplant."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Kono, K., Kataoka, K., Yuan, Y., Yusa, K., Uchida, K., and Sato, Y. (2021). Infectivity assessment of porcine endogenous retrovirus using high-throughput sequencing technologies. Biologicals, 71.","DOI":"10.1016\/j.biologicals.2021.05.001"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Denner, J. (2016). How Active Are Porcine Endogenous Retroviruses (PERVs)?. Viruses, 8.","DOI":"10.3390\/v8080215"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Kr\u00fcger, L., Kristiansen, Y., Reuber, E., M\u00f6ller, L., Laue, M., Reimer, C., and Denner, J. (2019). A Comprehensive Strategy for Screening for Xenotransplantation-Relevant Viruses in a Second Isolated Population of G\u00f6ttingen Minipigs. Viruses, 12.","DOI":"10.3390\/v12010038"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1159\/000068120","article-title":"Differences in release and determination of subtype of porcine endogenous retroviruses produced by stimulated normal pig blood cells","volume":"46","author":"Tacke","year":"2003","journal-title":"Intervirology"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1111\/j.1399-3089.2009.00515.x","article-title":"Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation","volume":"16","author":"Dieckhoff","year":"2009","journal-title":"Xenotransplantation"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Denner, J., and Schuurman, H.J. (2021). High Prevalence of Recombinant Porcine Endogenous Retroviruses (PERV-A\/Cs) in Minipigs: A Review on Origin and Presence. Viruses, 13.","DOI":"10.3390\/v13091869"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3045","DOI":"10.1128\/JVI.76.6.3045-3048.2002","article-title":"Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine","volume":"76","author":"Oldmixon","year":"2002","journal-title":"Virol. J."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"154","DOI":"10.2152\/jmi.54.154","article-title":"Generation and characterization of APOBEC3G-positive 293T cells for HIV-1 Vif study","volume":"54","author":"Piroozmand","year":"2007","journal-title":"J. Med. Invest."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Fuchs, N.V., Loewer, S., Daley, G.Q., Izsv\u00e1k, Z., L\u00f6wer, J., and L\u00f6wer, R. (2013). Human endogenous retrovirus K (HML-2) RNA and protein expression is a marker for human embryonic and induced pluripotent stem cells. Retrovirology, 10.","DOI":"10.1186\/1742-4690-10-115"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Mattiuzzo, G., Matouskova, M., and Takeuchi, Y. (2007). Differential resistance to cell entry by porcine endogenous retrovirus subgroup A in rodent species. Retrovirology, 4.","DOI":"10.1186\/1742-4690-4-93"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Mattiuzzo, G., and Takeuchi, Y. (2010). Suboptimal porcine endogenous retrovirus infection in non-human primate cells: Implication for preclinical xenotransplantation. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0013203"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Denner, J. (2018). Why was PERV not transmitted during preclinical and clinical xenotransplantation trials and after inoculation of animals. Retrovirology, 15.","DOI":"10.1186\/s12977-018-0411-8"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/15\/7111\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:14:50Z","timestamp":1760033690000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/15\/7111"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,23]]},"references-count":47,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["ijms26157111"],"URL":"https:\/\/doi.org\/10.3390\/ijms26157111","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,23]]}}}