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However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral\n            <jats:italic>E1A 13S<\/jats:italic>\n            and\n            <jats:italic>12S<\/jats:italic>\n            mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.\n          <\/jats:p>","DOI":"10.1128\/jvi.00166-17","type":"journal-article","created":{"date-parts":[[2017,3,16]],"date-time":"2017-03-16T00:40:40Z","timestamp":1489624840000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":16,"title":["Simultaneous Single-Cell\n            <i>In Situ<\/i>\n            Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection"],"prefix":"10.1128","volume":"91","author":[{"given":"Tomasz","family":"Krzywkowski","sequence":"first","affiliation":[{"name":"Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sibel","family":"Ciftci","sequence":"additional","affiliation":[{"name":"Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden"},{"name":"Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Farzaneh","family":"Assadian","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mats","family":"Nilsson","sequence":"additional","affiliation":[{"name":"Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0561-367X","authenticated-orcid":false,"given":"Tanel","family":"Punga","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00116-13"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1006\/viro.1996.0295"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0154814"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.76.21.10608-10616.2002"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02392-08"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.32.3.727-733.1979"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1987.tb02468.x"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(79)90036-6"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrm827"},{"key":"e_1_3_2_11_2","first-page":"177","article-title":"Regulation of adenovirus E2 transcription unit","volume":"199","author":"Swaminathan S","year":"1995","unstructured":"Swaminathan S Thimmapaya B . 1995. 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