Search For New and Emerging Etiologic Agents

寻找新的和正在出现的病原体

• 批准号: 8555744
• 负责人: Robert H. Purcell
• 金额: $ 30.43万
• 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/8555744
• 关键词: Accounting; Acute; Animals; Antibodies; Biochemical; Biological; Blood donor; Cattle; Child; Chronic; Chronic Hepatitis; Cirrhosis; Clinical; Collaborations; Communities; Contracts; Developed Countries; Developing Countries; Development; Diagnosis; Diagnostic; Disease; Disease Outbreaks; Domestic Animals; Egypt; Epidemiology; Etiology; Evaluation; Family; Family suidae; Feces; Food Chain; Gastroenteritis; Gastrointestinal Diseases; Genotype; 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; Norovirus; Pakistan; Pan Genus; Patients; Phase; Plasma; Predisposition; Prevention; Primates; Procedures; Public Health; RNA Viruses; Rat Strains; Rat virus; Rattus; Rattus norvegicus; Sampling; Saudi Arabia; Serial Passage; Seroepidemiologic Studies; Serological; Serum; Sheep; Small RNA; Source; Techniques; Technology; Testing; Time; Tissues; Transfusion; United States; Virus; Virus Diseases; Zoonotic Infection; base; clinical material; epidemiology study; experience; follower of religion Jewish; man; next generation; nonhuman primate; research study; 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. In 2011 we have continued to refine extraction and amplification techniques for the identification of new hepatitis viruses from primate-derived clinical materials, in anticipation of additional "next-generation" sequencing experiments. Known viruses of known titer have been used as controls to optimize procedures. In other collaborative studies, the epidemiology of emerging norovirus infections among children were studied in stored clinical samples obtained between 1975 and 1991. Evolutionary analysis revealed that norovirus genotype GII.3 viruses evolved at a rate comparable to that of other small RNA viruses and that these viruses evolve at a relatively steady state. Understanding the evolutionary dynamics of prevalent noroviruses is relevant to the development of effective prevention and control strategies. In 2012 we studied rat HEV strains originally discovered in Germany and found that they naturally infected rats in Los Angeles. Attempts to infect rats with human HEV strains and to infect nonhuman primates with rat strains indicated that rat HEV is probably not a threat to humans.

正在研究急性或慢性非A，B，C，D，E肝炎，用于传播药物的生物学，血清学或分子证据。暴发性非A到肝炎仍然是一种诊断性的谜，可能代表一种或多种以前未识别的疾病。我们试图发现这种疾病的病因。我们在印度，埃及和沙特阿拉伯的血清ePIDEMIologic研究中存在额外的水传播肝炎病毒的证据。肝炎病毒可能比以前想象的更大。 我们正在研究其在全球发展和工业化国家的流行病学。 获得了肝炎病毒（HEV）感染猪的血清学证据。从受感染的猪中回收了一种新的独特的HEV菌株，并表征了。 它被证明具有全球分布。对猪处理者和匹配的献血者的血清ePIDEMIologology研究表明，在猪处理程序中对HEV的抗体过多，这表明该病毒可能会在人畜共形上传播。此外，在整个2009年和2010年中，我们一直在评估抗体对HEV（抗HEV）的重要性，这是人类食物链的一部分，尤其是牛，绵羊和山羊。 在所有物种中都发现了抗HEV，尽管没有在猪中发现它。 猪很少是肝炎E的重要人畜共患病来源，尤其是在工业化国家中，但这并不能说明伊斯兰和犹太文化中抗HEV的高流行。 通过与XJ Meng合作，我们确定了山羊与公认的HEV菌株感染的敏感性，并试图从年轻的山羊中恢复类似HEV的药物，这些山羊被深入监测，以了解血清学和分子感染的证据。 还获得了类似的血清学证据，以鉴定出野生大鼠用HEV感染野生大鼠，并且已经鉴定出感染剂。迄今为止，我们已经成功地从被困在洛杉矶的大鼠传播到了同一物种的实验室大鼠（Rattus Norvegicus）。但是，很难传播，表明该病毒在低滴度下复制。在2010年，使用用于检测德国新大鼠HEV的PCR引物，我们能够从以前传播给其他大鼠的洛杉矶大鼠HEV菌株中恢复序列。 该序列与德国恢复的大鼠HEV的序列非常相似。 我们已经确认将大鼠HEV传播到其他大鼠，并且已经确定了来自感染大鼠的几个临床样本（包括粪便，血清和肝组织）的感染性滴度和大鼠HEV的PCR滴度。 我们已经证明，这些临床材料的感染性滴度和病毒的PCR滴度相对较低，从而为难以一致的传播提供了解释。 我们还通过PCR证实，HEV的人菌株不可传导到实验室大鼠，而大鼠HEV不能被人类的代孕鼠尾草传播。 在大鼠中检测到的低病毒滴度，将病毒传播到大鼠的困难以及无法将大鼠病毒传播到非人类的灵长类动物中，该物种容易受到人类和猪HEV菌株感染的感染，这会导致我们得出结论，大鼠病毒可能不会对人动物感染对人类的威胁。 近年来，现代的分子生物学技术已用于发现新病毒。这些现在已应用于血清的血清中，来自输血相关或社区获得性肝炎的患者，以寻找新的肝炎病毒，这些病毒可能会在美国引起多达2％的此类肝炎，而在发展中国家则最多可导致三分之一的肝炎。此外，大量慢性肝炎，肝硬化和肝癌病例仍未诊断。 为了提高病毒发现的敏感性，我们将微阵列技术应用于将新药物传输到黑猩猩的尝试，这是人类以外唯一受到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，大约一半的非细菌胃炎病例没有公认的病因。 与流行病学部分LID合作，我们正在采用相同的方法来尝试鉴定新的肠胃炎剂。 在2009财年，在评估了被实验感染了丙型肝炎病毒（HEV）的黑猩猩的先天和适应性免疫反应（HEV）时，我们发现了两种在巴基斯坦爆发肝炎中因HEV感染HEV的黑猩猩的异常先天免疫反应。 基于黑猩猩对所有五种公认的人类肝炎病毒的先天和适应性免疫反应的丰富经验，我们假设这些动物的反应是对肝脏复制的第二种药物的反应。 通过尝试从第二个先天免疫反应发作到新黑猩猩的传播，我们证明了与原始感染中与乙型肝炎无关的感染的证据。 它仅与肝炎的次要生化证据有关，但对于黑猩猩的HEV感染也是如此。 我们计划确定这种推定的药物是否会对连续传递产生更严重的感染，以及它是否与肝炎或其他肝病（例如肝癌）有关。 肝癌是五个公认的人肝炎病毒中的三个感染的常见后遗症。 在2010年，我们试图从一位黑猩猩的临床材料中鉴定出具有新肝炎剂感染的临床材料中的病因学剂。 根据与SAIC的NCI合同，对当时使用的“病毒芯片”测试了临床样品。 尽管根据检测到的序列，发现几种病毒正在感染黑猩猩，但没有发现急性相样样本是独有的，但不是感染前样品。 随后，对黑猩猩的感染前和急性相位血浆样品均通过454技术进行了“下一代”深度测序。 检测到已知病毒家族的两个序列（在某些情况下与病毒芯片检测到的序列相同）和独特的序列。 这些目前正在进行深入分析，以确定它们是否包括一种或多种先前未识别的病毒。 在2011年，我们继续完善提取和放大技术，从灵长类动物衍生的临床材料中鉴定出新的肝炎病毒，以期预期其他“下一代”测序实验。 已知滴度的已知病毒已被用作对照来优化程序。 在其他合作研究中，在1975年至1991年之间获得的储存临床样本中研究了新兴诺如病毒感染的流行病学。进化分析表明，诺如病毒基因型GII.3病毒以与其他小RNA病毒的速率相当，这些病毒与其他小RNA病毒的速度相当，并且这些病毒在相关稳定的状态中进化。 了解普遍的北病毒的进化动态与有效的预防和控制策略的发展有关。 2012年，我们研究了最初在德国发现的老鼠静脉菌株，发现它们在洛杉矶自然感染了大鼠。 试图用人类肝素菌株感染大鼠并用大鼠菌株感染非人类灵长类动物，这表明大鼠HEV可能不是对人类的威胁。