Search For New and Emerging Etiologic Agents

寻找新的和正在出现的病原体

• 批准号: 8156822
• 负责人: Robert H. Purcell
• 金额: $ 60.78万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8156822
• 关键词: Accounting; Acute; Animals; Antibodies; Biochemical; Biological; Blood donor; Cattle; Chronic; Chronic Hepatitis; Cirrhosis; Clinical; Collaborations; Communities; Contracts; Developed Countries; Developing Countries; Diagnosis; Diagnostic; Disease; Disease Outbreaks; Domestic Animals; Egypt; Epidemiology; Etiology; Evaluation; Family; Family suidae; Feces; Food Chain; Gastroenteritis; Gastrointestinal Diseases; Germany; Goals; Goat; Hepatic; Hepatitis; Hepatitis A; Hepatitis E; Hepatitis E virus; Hepatitis Viruses; High Prevalence; Human; Immune response; India; Infection; Laboratory Rat; Liver; Liver diseases; Los Angeles; Macaca mulatta; Malignant neoplasm of liver; Medical; Microarray Analysis; Minor; Molecular; Molecular Biology Techniques; Monitor; New Agents; Pakistan; Pan Genus; Patients; Phase; Plasma; Predisposition; Public Health; Rat Strains; Rat virus; Rattus; Rattus norvegicus; Sampling; Saudi Arabia; Serial Passage; Seroepidemiologic Studies; Serological; Serum; Sheep; Source; Technology; Testing; Time; Tissues; Transfusion; United States; Virus; Virus Diseases; Zoonotic Infection; base; clinical material; experience; follower of religion Jewish; man; next generation; nonhuman primate; response; transmission process

Acute or chronic non-A, B, C, D, E hepatitis is being studied for biological, serological or molecular evidence of transmissible agents. Fulminant non-A to E hepatitis remains a diagnostic enigma and may represent one or more previously unrecognized diseases. We are attempting to discover the etiology of this disease. Evidence for the existence of an additional water-borne hepatitis virus has come from our seroepidemiologic studies in India, Egypt and Saudi Arabia. Hepatitis E virus may be emerging as a greater public health problem than previously thought. We are studying its epidemiology in developing and industrialized countries worldwide. Serologic evidence of infection of swine with hepatitis E virus (HEV) was obtained. A new and unique HEV strain was recovered from infected swine and characterized. It was shown to have a worldwide distribution. Seroepidemiological studies of swine handlers and matched blood donors have shown an excess of antibody to HEV in swine handlers, suggesting that the virus may be zoonotically spread. In addition, throughout FY 2009 and 2010, we have been evaluating the significance of antibody to HEV (anti-HEV) in domestic animals that are part of the human food chain, especially cattle, sheep and goats. Anti-HEV has been found in all of the species, although not to the extent that it is found in swine. Rarely, swine are an important zoonotic source of hepatitis E, especially in industrialized countries, but these don't account for the high prevalence of anti-HEV in Islamic and Jewish cultures. In collaboration with XJ Meng, we are determining the susceptibility of goats to infection with the recognized HEV strains and attempting to recover HEV-like agents from young goats that are being intensively monitored for serologic and molecular evidence of infection. Similar serologic evidence for infection of wild rats with HEV has also been obtained and the infecting agent has been identified. To date we have successfully transmitted the agent from rats trapped in Los Angeles to laboratory rats of the same species (Rattus norvegicus). However, transmission was difficult, suggesting that the virus replicates at low titer. In 2010, using PCR primers developed for detecting a new rat HEV in Germany, we were able to recover sequence from the Los Angeles strain of rat HEV that we had transmitted to other rats previously. The sequence was very similar to the sequence of rat HEV recovered in Germany. We have confirmed transmission of rat HEV to other rats and we have determined both the infectivity titer and the PCR titer of rat HEV in several clinical samples from infected rats, including feces, serum and liver tissue. We have documented that both the infectivity titer and the PCR titer of the virus is relatively low in these clinical materials, thus providing an explanation for difficulty in consistent transmission. We have also confirmed by PCR that human strains of HEV were not transmissible to laboratory rats and that the rat HEV was not transmissible to rhesus monkeys, a surrogate of man. The low virus titers detected in rats, the difficulty in transmitting the virus to rats and the inability to transmit the rat virus to a nonhuman primate species that is susceptible to infection by human and swine HEV strains leads us to conclude that the rat virus probably does not pose a threat of zoonotic infection to humans. Modern techniques of molecular biology have been used to discover new viruses in recent years. These are now being applied to sera from patients with transfusion-associated or community-acquired hepatitis in a search for new hepatitis viruses that may cause up to 2% of such hepatitis in the US and up to one-third of hepatitis in developing countries. In addition, a significant number of cases of chronic hepatitis, cirrhosis and liver cancer remain undiagnosed. In an attempt to increase the sensitivity of virus discovery, we are applying microarray technology to attempts to transmit new agents to chimpanzees, the only species other than man that is susceptible to all five recognized human hepatitis viruses. Preliminary results are promising. Similarly, approximately one half of nonbacterial gastroenteritis cases have no recognized etiology. In collaboration with the Epidemiology Section, LID, we are applying the same approaches to attempts to identify new gastroenteritis agents. In FY 2009, while evaluating the innate and adaptive immune responses of chimpanzees that had been experimentally infected with hepatitis E virus (HEV), we discovered an aberrant innate immune response in two chimpanzees that had been infected with HEV from an outbreak of hepatitis E in Pakistan. Based on extensive experience with innate and adaptive immune responses to all five recognized human hepatitis viruses in chimpanzees, we postulated that the response in these animals was to a second agent replicating in the liver. By attempting transmission from the second innate immune response episode to new chimpanzees, we demonstrated evidence for an infection not related to hepatitis E in the original infection. It has been associated with only minor biochemical evidence of hepatitis, but that is also true for HEV infection of chimpanzees. We plan to determine if this putative agent produces more severe infection on serial passage and whether it can be associated with hepatitis or other liver disease, such as liver cancer. Liver cancer is a common sequela of infection with three of the five recognized human hepatitis viruses. In 2010, we attempted to identify an etiologic agent in clinical materials from one of the chimpanzees with evidence of infection with a possible new hepatitis agent. Clinical samples were tested on the "virus Chip" being utilized at the time under the NCI contract with SAIC. Although, based on sequences detected, several viruses were found to be infecting the chimpanzee, none was found to be unique to the acute phase sample but not the pre-infection sample. Subsequently, both pre-infection and acute phase plasma samples from the chimpanzee were subjected to "next-generation" deep sequencing with 454 technology. Both sequences of known virus families (in some cases the same as those detected by the virus Chip) and unique sequences were detected. These are currently being subjected to in-depth analysis to determine if they include one or more previously unrecognized viruses.

正在研究急性或慢性非甲、乙、丙、丁、戊型肝炎，以寻找传染源的生物学、血清学或分子证据。暴发性非甲型至戊型肝炎仍然是一个诊断之谜，可能代表一种或多种以前未被识别的疾病。我们正在尝试找出这种疾病的病因。我们在印度、埃及和沙特阿拉伯进行的血清流行病学研究提供了另一种水源性肝炎病毒存在的证据。戊型肝炎病毒可能正在成为比以前想象的更严重的公共卫生问题。 我们正在研究其在全世界发展中国家和工业化国家的流行病学。 获得了猪感染戊型肝炎病毒（HEV）的血清学证据。从受感染的猪身上发现了一种新的、独特的 HEV 毒株并进行了鉴定。 它被证明在全球范围内都有分布。对养猪员和匹配献血者的血清流行病学研究表明，养猪员体内存在过量的 HEV 抗体，表明该病毒可能会在人畜共患病中传播。此外，在2009财年和2010财年，我们一直在评估作为人类食物链一部分的家畜，特别是牛、绵羊和山羊，HEV抗体（抗HEV）的重要性。 在所有物种中都发现了抗HEV，但程度不如在猪中发现的程度。 猪很少成为戊型肝炎的重要人畜共患来源，尤其是在工业化国家，但这并不能解释伊斯兰和犹太文化中抗HEV的高流行率。 我们与孟晓杰合作，正在确定山羊对已识别的 HEV 毒株感染的敏感性，并试图从正在密切监测感染的血清学和分子证据的幼山羊中回收 HEV 样病原体。 野生大鼠感染 HEV 的类似血清学证据也已获得，感染因子也已确定。迄今为止，我们已成功地将病毒从被困在洛杉矶的老鼠身上传播到实验室的同种老鼠（褐家鼠）身上。然而，传播很困难，这表明该病毒的复制滴度较低。 2010年，使用为检测德国新型大鼠HEV而开发的PCR引物，我们能够从之前传播给其他大鼠的洛杉矶大鼠HEV株中恢复序列。 该序列与在德国回收的大鼠 HEV 序列非常相似。 我们已证实大鼠 HEV 会传播给其他大鼠，并在受感染大鼠的一些临床样本（包括粪便、血清和肝组织）中测定了大鼠 HEV 的感染滴度和 PCR 滴度。 我们已经证明，这些临床材料中病毒的感染滴度和PCR滴度都较低，这为一致传播的困难提供了解释。 我们还通过 PCR 证实，人类 HEV 株不会传染给实验室大鼠，并且大鼠 HEV 不会传染给人类替代品——恒河猴。 在大鼠中检测到的病毒滴度低、将病毒传播给大鼠的困难以及无法将大鼠病毒传播给易受人类和猪HEV毒株感染的非人灵长类动物物种的结论使我们得出结论，大鼠病毒可能不会对人类构成人畜共患感染的威胁。 近年来，分子生物学的现代技术已被用来发现新病毒。这些现在被应用于输血相关性或社区获得性肝炎患者的血清，以寻找新的肝炎病毒，这种病毒可能导致美国高达 2% 的此类肝炎，以及发展中国家高达三分之一的肝炎。此外，大量慢性肝炎、肝硬化和肝癌病例仍未确诊。 为了提高病毒发现的敏感性，我们正在应用微阵列技术来尝试将新的病毒传播给黑猩猩，黑猩猩是除人类之外唯一对所有五种公认的人类肝炎病毒敏感的物种。初步结果是有希望的。同样，大约一半的非细菌性胃肠炎病例没有公认的病因。 我们与 LID 流行病学部门合作，采用相同的方法来尝试识别新的胃肠炎病原体。 2009 财年，在评估实验性感染戊型肝炎病毒 (HEV) 的黑猩猩的先天性和适应性免疫反应时，我们发现两只因巴基斯坦爆发戊型肝炎而感染了 HEV 的黑猩猩出现了异常的先天免疫反应。 根据黑猩猩对所有五种公认的人类肝炎病毒的先天性和适应性免疫反应的丰富经验，我们推测这些动物的反应是对在肝脏中复制的第二种药物的反应。 通过尝试从第二次先天免疫反应事件传播给新的黑猩猩，我们证明了与原始感染中的戊型肝炎无关的感染证据。 它仅与肝炎的少量生化证据相关，但黑猩猩的 HEV 感染也是如此。 我们计划确定这种假定的病原体在连续传代时是否会产生更严重的感染，以及它是否与肝炎或其他肝脏疾病（例如肝癌）有关。 肝癌是五种公认的人类肝炎病毒中的三种感染的常见后遗症。 2010年，我们试图从一只黑猩猩的临床材料中鉴定出一种病原体，该黑猩猩有证据表明可能感染了一种新的肝炎病原体。 根据 NCI 与 SAIC 的合同，当时使用的“病毒芯片”对临床样本进行了测试。 尽管根据检测到的序列，发现有几种病毒感染黑猩猩，但没有发现任何病毒是急性期样本所独有的，而不是感染前样本所独有的。 随后，利用 454 技术对黑猩猩的感染前和急性期血浆样本进行“下一代”深度测序。 检测到已知病毒家族的序列（在某些情况下与病毒芯片检测到的序列相同）和独特序列。 目前正在对这些病毒进行深入分析，以确定它们是否包含一种或多种以前未识别的病毒。