Development of a primary tamarin hepatocyte culture system for GB virus-B: a surrogate model for hepatitis C virus
Development of a primary tamarin hepatocyte culture system for GB virus-B: a surrogate model for hepatitis C virus
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DOI：
10.1128/jvi.74.24.11764-11772.2000
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发表时间：
2000-12-01
影响因子：
5.4
通讯作者：
Lanford, RE
中科院分区：
文献类型：
--
作者：
Beames, B;Chavez, D;Lanford, RE
GB病毒-B（GBV-B）引起绢毛猴的急性肝炎，其特征是丙氨酸转氨酶水平升高，随着病毒被清除，丙氨酸转氨酶水平迅速恢复正常。在系统发生学上，GBV-B与丙型肝炎病毒（HCV）关系最近，因此，绢毛猴的GBV-B感染是研究HCV的一个强有力的替代模型系统。在这项研究中，使用实时5 '核酸外切酶（TaqMan）逆转录-PCR测定来确定血清中GBV-B的水平，在绢毛猴中感染GBV-B的过程。峰值病毒血症水平超过10（9）基因组当量/ml，随后在14至16周内清除病毒。已清除感染的动物的再激发导致病毒血症，其限于1周，表明强烈的保护性免疫应答。用原代培养的绢毛猴肝细胞建立了一个稳定的GBV-B组织培养系统。从GBV-B感染的动物获得的肝细胞在培养42天内保持高水平的细胞相关病毒RNA和病毒体分泌。正常肝细胞的体外感染导致细胞相关病毒RNA的快速扩增，并分泌高达10（7）基因组当量/ml培养物上清。此外，可以通过免疫荧光染色GBV-B非结构NS 3蛋白来监测感染。该模型系统克服了目前HCV研究的许多障碍，包括病毒复制水平低、缺乏小型灵长类动物模型以及缺乏可重复的组织培养系统。
GB virus-B (GBV-B) causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Phylogenetically, GBV-B is the closest relative to hepatitis C virus (HCV), and thus GBV-B infection of tamarins represents a powerful surrogate model system for the study of HCV. In this study, the course of infection of GBV-B in tamarins was followed using a real-time 5 ' exonuclease (TaqMan) reverse transcription-PCR assay to determine the level of GBV-B in the serum. Peak viremia levels exceeded 10(9) genome equivalents/ml, followed by viral clearance within 14 to 16 weeks. Rechallenge of animals that had cleared infection resulted in viremia that was limited to 1 week, suggestive of a strong protective immune response. A robust tissue culture system for GBV-B was developed using primary cultures of tamarin hepatocytes. Hepatocytes obtained from a GBV-B-infected animal maintained high levels of cell-associated viral RNA and virion secretion for 42 days of culture. In vitro infection of normal hepatocytes resulted in rapid amplification of cell-associated viral RNA and secretion of up to 10(7) genome equivalents/ml of culture supernatant. In addition, infection could be monitored by immunofluorescence staining for GBV-B nonstructural NS3 protein. This model system overcomes many of the current obstacles to HCV research, including low levels of viral replication, lack of a small primate animal model, and lack of a reproducible tissue culture system.