Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression
This study assesses the ability of quantitative real-time PCR to measure the effects of virus DNA polymerase inhibitors on EBV DNA and late mRNAs syntheses in EBV-producing cell lines. In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs ( BLLF1 gene/gp350/2...
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| Veröffentlicht in: | Journal of virological methods 2007-07, Vol.143 (1), p.38-44 |
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| creator | Ballout, Mirvat Germi, Raphaële Fafi-Kremer, Samira Guimet, Josette Barguès, Gerard Seigneurin, Jean-Marie Morand, Patrice |
| description | This study assesses the ability of quantitative real-time PCR to measure the effects of virus DNA polymerase inhibitors on EBV DNA and late mRNAs syntheses in EBV-producing cell lines.
In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs (
BLLF1 gene/gp350/220,
BVRF2 gene/protease) in P3HR-1 and B95-8 cells induced for EBV production by PMA and exposed to ganciclovir, cidofovir and foscarnet.
The calculated 50% effective concentrations (EC
50) for viral DNA replication inhibition in P3HR-1 cells after 7 days of drug exposure were 0.28
±
0.06, 0.29
±
0.01 and 13.6
±
0.17
μg/mL for ganciclovir, cidofovir and foscarnet, respectively. The EC
50 for B95-8 cells were 0.44
±
0.02, 0.70
±
0.06 and 46.8
±
0.5
μg/mL, respectively. The quantitation of the late viral mRNAs showed a decrease of 79–89% in the mRNA amount after 4 days of antiviral treatment. Nevertheless, a substantial amount of mRNA still remained detectable after drug exposure.
The real-time PCR is an improvement in the attempt to simplify EBV DNA-quantitation for antiviral assays. The quantitation of late mRNA does not appear as more informative than DNA quantitation for the assessment of the DNA polymerase inhibitor activity, but it may be useful to assess the antiviral activity of drugs acting by another mechanism. |
| doi_str_mv | 10.1016/j.jviromet.2007.02.005 |
| format | Article |
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In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs (
BLLF1 gene/gp350/220,
BVRF2 gene/protease) in P3HR-1 and B95-8 cells induced for EBV production by PMA and exposed to ganciclovir, cidofovir and foscarnet.
The calculated 50% effective concentrations (EC
50) for viral DNA replication inhibition in P3HR-1 cells after 7 days of drug exposure were 0.28
±
0.06, 0.29
±
0.01 and 13.6
±
0.17
μg/mL for ganciclovir, cidofovir and foscarnet, respectively. The EC
50 for B95-8 cells were 0.44
±
0.02, 0.70
±
0.06 and 46.8
±
0.5
μg/mL, respectively. The quantitation of the late viral mRNAs showed a decrease of 79–89% in the mRNA amount after 4 days of antiviral treatment. Nevertheless, a substantial amount of mRNA still remained detectable after drug exposure.
The real-time PCR is an improvement in the attempt to simplify EBV DNA-quantitation for antiviral assays. The quantitation of late mRNA does not appear as more informative than DNA quantitation for the assessment of the DNA polymerase inhibitor activity, but it may be useful to assess the antiviral activity of drugs acting by another mechanism.</description><identifier>ISSN: 0166-0934</identifier><identifier>EISSN: 1879-0984</identifier><identifier>DOI: 10.1016/j.jviromet.2007.02.005</identifier><identifier>PMID: 17368820</identifier><identifier>CODEN: JVMEDH</identifier><language>eng</language><publisher>London: Elsevier B.V</publisher><subject>Animals ; Antiviral Agents - pharmacology ; Biological and medical sciences ; Burkitt Lymphoma ; Cell Line ; Cell Survival - drug effects ; Cidofovir ; DNA - biosynthesis ; DNA, Viral - biosynthesis ; EBV ; Epstein-Barr virus ; Epstein-Barr Virus Infections - drug therapy ; Foscarnet ; Fundamental and applied biological sciences. Psychology ; Ganciclovir ; Herpesvirus 4, Human - drug effects ; Herpesvirus 4, Human - physiology ; Humans ; Lymphoid B cell lines ; Microbiology ; Polymerase Chain Reaction ; Real-time PCR ; RNA, Messenger - biosynthesis ; Techniques used in virology ; Viral Load ; Virology ; Virus Replication - drug effects</subject><ispartof>Journal of virological methods, 2007-07, Vol.143 (1), p.38-44</ispartof><rights>2007 Elsevier B.V.</rights><rights>2007 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c427t-f92c2f7dda0937bc3f4ecff2eb8cea0328f164adc52cac7d11d73f7dcf5e9483</citedby><cites>FETCH-LOGICAL-c427t-f92c2f7dda0937bc3f4ecff2eb8cea0328f164adc52cac7d11d73f7dcf5e9483</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jviromet.2007.02.005$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=18808413$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17368820$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ballout, Mirvat</creatorcontrib><creatorcontrib>Germi, Raphaële</creatorcontrib><creatorcontrib>Fafi-Kremer, Samira</creatorcontrib><creatorcontrib>Guimet, Josette</creatorcontrib><creatorcontrib>Barguès, Gerard</creatorcontrib><creatorcontrib>Seigneurin, Jean-Marie</creatorcontrib><creatorcontrib>Morand, Patrice</creatorcontrib><title>Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression</title><title>Journal of virological methods</title><addtitle>J Virol Methods</addtitle><description>This study assesses the ability of quantitative real-time PCR to measure the effects of virus DNA polymerase inhibitors on EBV DNA and late mRNAs syntheses in EBV-producing cell lines.
In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs (
BLLF1 gene/gp350/220,
BVRF2 gene/protease) in P3HR-1 and B95-8 cells induced for EBV production by PMA and exposed to ganciclovir, cidofovir and foscarnet.
The calculated 50% effective concentrations (EC
50) for viral DNA replication inhibition in P3HR-1 cells after 7 days of drug exposure were 0.28
±
0.06, 0.29
±
0.01 and 13.6
±
0.17
μg/mL for ganciclovir, cidofovir and foscarnet, respectively. The EC
50 for B95-8 cells were 0.44
±
0.02, 0.70
±
0.06 and 46.8
±
0.5
μg/mL, respectively. The quantitation of the late viral mRNAs showed a decrease of 79–89% in the mRNA amount after 4 days of antiviral treatment. Nevertheless, a substantial amount of mRNA still remained detectable after drug exposure.
The real-time PCR is an improvement in the attempt to simplify EBV DNA-quantitation for antiviral assays. The quantitation of late mRNA does not appear as more informative than DNA quantitation for the assessment of the DNA polymerase inhibitor activity, but it may be useful to assess the antiviral activity of drugs acting by another mechanism.</description><subject>Animals</subject><subject>Antiviral Agents - pharmacology</subject><subject>Biological and medical sciences</subject><subject>Burkitt Lymphoma</subject><subject>Cell Line</subject><subject>Cell Survival - drug effects</subject><subject>Cidofovir</subject><subject>DNA - biosynthesis</subject><subject>DNA, Viral - biosynthesis</subject><subject>EBV</subject><subject>Epstein-Barr virus</subject><subject>Epstein-Barr Virus Infections - drug therapy</subject><subject>Foscarnet</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Ganciclovir</subject><subject>Herpesvirus 4, Human - drug effects</subject><subject>Herpesvirus 4, Human - physiology</subject><subject>Humans</subject><subject>Lymphoid B cell lines</subject><subject>Microbiology</subject><subject>Polymerase Chain Reaction</subject><subject>Real-time PCR</subject><subject>RNA, Messenger - biosynthesis</subject><subject>Techniques used in virology</subject><subject>Viral Load</subject><subject>Virology</subject><subject>Virus Replication - drug effects</subject><issn>0166-0934</issn><issn>1879-0984</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0c1uEzEQB3ALgWhaeIXKF7jt4o_N2nujjUJBqgBFFVfLsceVo_1IPd4InoDXxlGCeuzJlvybGdt_Qq45qznj7addvTvENA2Qa8GYqpmoGVu-IguuVVexTjevyaLAtuxlc0EuEXesCCXlW3LBlWy1FmxB_m7A9lWOA9Cn2Y45ZpvjAejP1YaGKVGLCIgDjJlOgR5BGWt76tP8SCEEcBmpfbRxxEzXe8wQx-rWpkSLm5Em2PfRlZ7TWKo9Xd_-or3NQIfN9xsKv_eptC-H78ibYHuE9-f1ijx8WT-svlb3P-6-rW7uK9cIlavQCSeC8t6WZ6mtk6EBF4KArXZgmRQ68Lax3i2Fs055zr2SxbuwhK7R8op8PLXdp-lpBsxmiOig7-0I04xGsWWjWs1ehLzTHdfyCNsTdGlCTBDMPsXBpj-GM3OMyuzM_6jMMSrDhClBlMLr84R5O4B_LjtnU8CHM7DobB-SHV3EZ6c10w2XxX0-OSj_doiQDLoIowMfU4nH-Cm-dJd_vBO5IA</recordid><startdate>20070701</startdate><enddate>20070701</enddate><creator>Ballout, Mirvat</creator><creator>Germi, Raphaële</creator><creator>Fafi-Kremer, Samira</creator><creator>Guimet, Josette</creator><creator>Barguès, Gerard</creator><creator>Seigneurin, Jean-Marie</creator><creator>Morand, Patrice</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T7</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20070701</creationdate><title>Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression</title><author>Ballout, Mirvat ; Germi, Raphaële ; Fafi-Kremer, Samira ; Guimet, Josette ; Barguès, Gerard ; Seigneurin, Jean-Marie ; Morand, Patrice</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c427t-f92c2f7dda0937bc3f4ecff2eb8cea0328f164adc52cac7d11d73f7dcf5e9483</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Animals</topic><topic>Antiviral Agents - pharmacology</topic><topic>Biological and medical sciences</topic><topic>Burkitt Lymphoma</topic><topic>Cell Line</topic><topic>Cell Survival - drug effects</topic><topic>Cidofovir</topic><topic>DNA - biosynthesis</topic><topic>DNA, Viral - biosynthesis</topic><topic>EBV</topic><topic>Epstein-Barr virus</topic><topic>Epstein-Barr Virus Infections - drug therapy</topic><topic>Foscarnet</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Ganciclovir</topic><topic>Herpesvirus 4, Human - drug effects</topic><topic>Herpesvirus 4, Human - physiology</topic><topic>Humans</topic><topic>Lymphoid B cell lines</topic><topic>Microbiology</topic><topic>Polymerase Chain Reaction</topic><topic>Real-time PCR</topic><topic>RNA, Messenger - biosynthesis</topic><topic>Techniques used in virology</topic><topic>Viral Load</topic><topic>Virology</topic><topic>Virus Replication - drug effects</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ballout, Mirvat</creatorcontrib><creatorcontrib>Germi, Raphaële</creatorcontrib><creatorcontrib>Fafi-Kremer, Samira</creatorcontrib><creatorcontrib>Guimet, Josette</creatorcontrib><creatorcontrib>Barguès, Gerard</creatorcontrib><creatorcontrib>Seigneurin, Jean-Marie</creatorcontrib><creatorcontrib>Morand, Patrice</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of virological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ballout, Mirvat</au><au>Germi, Raphaële</au><au>Fafi-Kremer, Samira</au><au>Guimet, Josette</au><au>Barguès, Gerard</au><au>Seigneurin, Jean-Marie</au><au>Morand, Patrice</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression</atitle><jtitle>Journal of virological methods</jtitle><addtitle>J Virol Methods</addtitle><date>2007-07-01</date><risdate>2007</risdate><volume>143</volume><issue>1</issue><spage>38</spage><epage>44</epage><pages>38-44</pages><issn>0166-0934</issn><eissn>1879-0984</eissn><coden>JVMEDH</coden><abstract>This study assesses the ability of quantitative real-time PCR to measure the effects of virus DNA polymerase inhibitors on EBV DNA and late mRNAs syntheses in EBV-producing cell lines.
In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs (
BLLF1 gene/gp350/220,
BVRF2 gene/protease) in P3HR-1 and B95-8 cells induced for EBV production by PMA and exposed to ganciclovir, cidofovir and foscarnet.
The calculated 50% effective concentrations (EC
50) for viral DNA replication inhibition in P3HR-1 cells after 7 days of drug exposure were 0.28
±
0.06, 0.29
±
0.01 and 13.6
±
0.17
μg/mL for ganciclovir, cidofovir and foscarnet, respectively. The EC
50 for B95-8 cells were 0.44
±
0.02, 0.70
±
0.06 and 46.8
±
0.5
μg/mL, respectively. The quantitation of the late viral mRNAs showed a decrease of 79–89% in the mRNA amount after 4 days of antiviral treatment. Nevertheless, a substantial amount of mRNA still remained detectable after drug exposure.
The real-time PCR is an improvement in the attempt to simplify EBV DNA-quantitation for antiviral assays. The quantitation of late mRNA does not appear as more informative than DNA quantitation for the assessment of the DNA polymerase inhibitor activity, but it may be useful to assess the antiviral activity of drugs acting by another mechanism.</abstract><cop>London</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>17368820</pmid><doi>10.1016/j.jviromet.2007.02.005</doi><tpages>7</tpages></addata></record> |
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| ispartof | Journal of virological methods, 2007-07, Vol.143 (1), p.38-44 |
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| language | eng |
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| source | MEDLINE; Access via ScienceDirect (Elsevier) |
| subjects | Animals Antiviral Agents - pharmacology Biological and medical sciences Burkitt Lymphoma Cell Line Cell Survival - drug effects Cidofovir DNA - biosynthesis DNA, Viral - biosynthesis EBV Epstein-Barr virus Epstein-Barr Virus Infections - drug therapy Foscarnet Fundamental and applied biological sciences. Psychology Ganciclovir Herpesvirus 4, Human - drug effects Herpesvirus 4, Human - physiology Humans Lymphoid B cell lines Microbiology Polymerase Chain Reaction Real-time PCR RNA, Messenger - biosynthesis Techniques used in virology Viral Load Virology Virus Replication - drug effects |
| title | Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression |
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