TESTING INFECTIVITY OF HCV ISOLATED FROM CELL CULTURE IN CHIMPANZEES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。男性丙型肝炎病毒感染在大约85%的暴露个体中变为慢性感染。目前，美国估计有270万慢性感染者，全球估计有2亿人。目前还没有针对慢性丙型肝炎病毒感染的疫苗，干扰素和利巴韦林的联合治疗也很有限，但治疗成功的患者不到一半。目前存在的这种病毒的细胞培养系统很差，没有证明在实验室之间是可复制的，而且这种病毒的唯一动物模型是黑猩猩。最近，我们成功地在细胞培养中产生了传染性丙型肝炎病毒（称为HCVcc）。这项黑猩猩实验的目的是测试这些颗粒在体内的传染性。对于大多数HCV分离株，适应性突变需要在细胞培养中有效地复制RNA。虽然只有有限数量的这些适应性变化在体内进行了测试，但它们似乎对黑猩猩的复制有害，并且很少在自然HCV分离序列中检测到。在黑猩猩身上测试这种分离物或细胞培养感染性嵌合体对HCV领域具有潜在的重要意义。在此之前，体内（黑猩猩或人类）产生的HCV在细胞培养中是非传染性或传染性较差的。相反，适应在细胞培养中复制的HCV rna在体内复制的能力受到损害。拥有一种可以在细胞培养和体内复制的分离物将是表征HCV复制、进入和发病机制的一个非常有力的工具。这将使我们能够比较肝脏中产生的真正HCV颗粒与细胞培养中产生的HCV颗粒的行为。这对病毒进入和中和的研究尤其重要，也减少了对未来黑猩猩实验的需求。10^6细胞培养将两种不同细胞培养适应病毒的感染单位静脉接种于两种不同的动物。（总容积~1-2mL）。接种物将由浓缩的细胞培养上清组成。动物将每周放血并监测丙型肝炎病毒血症。如果8周后没有感染迹象，将用已知的传染性分离物（HCVH77单克隆病毒）挑战动物以证明感染性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hepatitis C virus infections in man become chronic in approximately 85% percent of individuals exposed. At present there are an estimated 2.7 million chronically infected people in the United States and an estimated 200 million worldwide. There is no vaccine and therapy for chronic HCV infections is limited a combination of interferon and ribavirin, but treatment is successful in less than half the patients. There currently exist poor cell culture systems for the virus which have not proved to be reproducible between labs and the only animal model for the virus is the chimpanzee. Recently we succeeded in producing infectious hepatitis C virus in cell culture (termed HCVcc). The goal of this chimpanzee experiment is to test the infectivity of these particles in vivo. For most HCV isolates, adaptive mutations are required for efficient RNA replication in cell culture. Although only a limited number of these adaptive changes have been tested in vivo, they appear to be deleterious for replication in chimpanzees and have only rarely been detected in sequences of natural HCV isolates. Testing this isolate or cell culture infectious chimeras in chimpanzees is potentially of great importance to the HCV field. Until this point, HCV produced in vivo (from chimpanzees or humans) is non-infectious or poorly infectious in cell culture. Conversely, HCV RNAs adapted to replicate in cell culture are compromised in their ability to replicate in vivo. Having an isolate that can replicate both in cell culture and in vivo would be a very powerful tool for characterizing HCV replication, entry and pathogenesis. This will enable us to compare the behavior of true HCV particles produced in the liver to HCVcc produced in cell culture. The will be particularly important for studies of virus entry and neutralization and also reduce the need for future chimpanzee experiments. 10^6 cell culture infectious units of two different cell culture adapted viruses will be inoculated i.v. into two different animals. (Total volume ~1-2mL). The inoculum will consist of concentrated cell culture supernatant. Animals will be bled weekly and monitored for HCV viremia. If no infection is indicated after 8 weeks the animals will be challenged with a known infectious isolate (HCVH77 monoclonal virus) to demonstrate infectivity.