REPLICATION AND CAPSID ANTIGENS OF HEPATITIS A VIRUS

甲型肝炎病毒(HAV)引起的最流行的 人类感染，并且仍然是一个世界性的公共卫生问题。 在培养细胞中传播病毒的能力，以及 新近构建的cDNA克隆及其活性的测定 病毒基因组的核苷酸序列，已经成为可能 许多新的方法来理解生物学和 甲型肝炎病毒感染的发病机制 潜在亚单位疫苗的开发。在此应用程序中，我们 提出旨在识别特定病毒序列的实验方案 导致缓慢而延长的复制周期 这种病毒的特征，这是它区别于所有其他病毒的特征 它的属(肠道病毒)和科(短小航病科)的成员。 将构建由以下成分组成的重组病毒 HAV5‘端调控序列和脊髓灰质炎病毒编码序列， 或甲型肝炎病毒聚合酶编码序列取代同源 小儿麻痹症序列。努力插入这些潜在的 将甲型肝炎病毒序列下调为快速生长、裂解的 脊髓灰质炎病毒将通过检查斑块大小和 形态、温度敏感性、病毒RNA和蛋白质 合成，然后病毒产生。另一种未鉴定的甲型肝炎病毒基因， 将分析2A型蛋白水解酶编码区的功能 通过以下方式处理宿主细胞相互作用和病毒形态发生 表达含2A的基因工程蛋白 体内和体外的序列。最后，我们制作了 两种原核生物中的甲型肝炎病毒衣壳蛋白(VP1)抗原 和真核细胞(杆状病毒感染的SF9)细胞。我们计划 系统评价不同抗原蛋白的能力， 从各种不同的载体中表达并纯化 不同的程序，以诱导中和抗体和/或 启动免疫系统，以产生中和的记忆反应。 我们希望这些研究将确定一种重组蛋白 作为亚单位的候选免疫原将是有效的。 疫苗。

Hepatitis A virus (HAV) causes one of the most prevalent infections of man, and remains a worldwide public health problem. The ability to propagate the virus in cultured cells, as well as the recent construction of cDNA clones and the determination of the nucleotide sequence of the viral genome, have made possible numerous new approaches to understanding the biology and pathogenesis of HAV infection and provided insight towards the development of potential subunit vaccines. In this application, we propose experiments designed to identify specific viral sequences that are responsible for the slow and protracted replication cycle of this virus, a characteristic that distinguishes it from all other members of its genus (enterovirus) and family (Picornaviridae). Recombinant viruses will be constructed that are composed of HAV 5' end regulatory sequences and poliovirus coding sequences, or HAV polymerase coding sequences replacing the homologous polio sequences. The efforts of the insertion of these potentially down-regulating HAV sequences into a rapidly-growing, lytic poliovirus will be evaluated by examining plaque size and morphology, temperature sensitivity, viral RNA and protein synthesis, and virus yields. Another uncharacterized HAV gene, the 2A protease coding region, will be analyzed for functions dealing with host cell interaction and virus morphogenesis by expressing genetically engineered proteins containing 2A sequences, both in vivo and in vitro. Finally, we have produced HAV capsid protein (VP1) antigens in both prokaryotic (E. coli) and eukaryotic (baculovirus-infected SF9) cells. We plan to systematically evaluate the ability of different antigenic proteins, expressed from a variety of different vectors and purified by different procedures, to induce neutralizing antibodies and/or to prime the immune system for a neutralizing anamnestic response. We hope that these studies will identify a recombinant protein that will be effective as a candidate immunogen for a subunit vaccine.