Teimourpour Roghayeh, Meshkat Zahra, Gholoubi Aida, Nomani Hosein, Rostami Sina
Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, IR Iran.
Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, IR Iran.
Jundishapur J Microbiol. 2015 May 31;8(5):e19279. doi: 10.5812/jjm.8(5)2015.19279. eCollection 2015 May.
Previous studies using cell culture systems for the replication of hepatitis C virus have opened new research dimensions, and paved the ways for further and detailed studies of the virus in vitro.
The purpose of the present study was to cultivate hepatitis C virus in a cell culture system and evaluate viral amplification.
In order to propagate hepatitis C virus, cloned whole genome of virus, JFH-1, was used. JFH-1 cDNA was introduced into strain JM109 of Escherichia coli and plasmid, containing the viral genome was purified from transformed bacteria. After XbaI digestion, RNA synthesis was induced using T7 RNA polymerase enzyme. Next, eukaryotic cell line Huh 7.5 was transfected by the purified RNA. Finally, Huh-7.5 cell line was infected with replicated virus and viral load was determined using real-time PCR (Polymerase Chain Reaction).
The amount of viral load, which was measured using real-time PCR was 17592 IU/mL.
In the present study, using cell culture, a high titer (in acceptable range) of infectious hepatitis C virus was produced. This method could be used in future studies.
以往利用细胞培养系统进行丙型肝炎病毒复制的研究开辟了新的研究维度，为该病毒在体外的进一步深入研究铺平了道路。
本研究旨在利用细胞培养系统培养丙型肝炎病毒并评估病毒扩增情况。
为了繁殖丙型肝炎病毒，使用了病毒的克隆全基因组JFH-1。将JFH-1 cDNA导入大肠杆菌JM109菌株，并从转化细菌中纯化出含有病毒基因组的质粒。经XbaI酶切后，使用T7 RNA聚合酶诱导RNA合成。接下来，用纯化的RNA转染真核细胞系Huh 7.5。最后，用复制的病毒感染Huh-7.5细胞系，并使用实时聚合酶链反应（PCR）测定病毒载量。
使用实时PCR测定的病毒载量为17592 IU/mL。
在本研究中，通过细胞培养产生了高滴度（在可接受范围内）的传染性丙型肝炎病毒。该方法可用于未来的研究。
