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KMID : 0364820090450030286
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Korean Journal of Microbiology
2009 Volume.45 No. 3 p.286 ~ p.290
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Deterimination of an Optimal Time Point for Analyzing Transcriptional Activity and Analysis of Transcripts of Avian Influenza Virus H9N2 in Cultured Cell
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Na Gi-Youn
Lee Young-Min
Koo Yong-Bum
Byun Sung-June
Jeon Ik-Soo
Kown Jun-Hun
Park Jong-Hyeon
Lee Yun-Jung
Joo Yi-Seok
Cho In-Soo
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Abstract
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The transcription of mRNA of avian influenza virus is regulated temporally during infection. Therefore, the measurement of transcript level in host cells should be performed before viral release from host cells because errors can occur in the analysis of the transcript levels if the viruses released from the infected cells re-infect cells. In this study, the timing of viral release was determined by measuring the level of viral RNA from viruses released from H9N2-infected chicken fibroblast cell line UMNSAH/DF-1 by semi-quantitative RT-PCR. The viral genomic RNA was isolated together with mouse total RNA which was added to the collected medium as carrier to monitor the viral RNA recovery and to use its GAPDH as an internal control for normalizing reverse transcription reaction as well as PCR reaction. It was found that viral release of H9N2 in the chicken fibroblast cell line UMNSAH/DF-1 took between 16 and 20 h after infection. We measured all 8 viral mRNA levels. Of the 8 transcripts, 7 species of viral mRNAs (each encoding HA, NA, PB1, PB2, NP, M, NS, respectively) except
PA mRNA showed robust amplification, indicating these mRNA can be used as targets for amplification to measure transcript levels. These results altogether suggest that the method in this study can be used for screening antiviral materials against viral RNA polymerase as a therapeutic target.
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KEYWORD
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avian influenza virus, H9N2, RT-PCR
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