Molecular Characterization of Novel Astroviruses

描述(申请人提供)：在世界范围内，胃肠道感染和腹泻疾病导致严重的发病率和死亡率，每年约有200万人死亡。然而，目前的诊断技术无法在30%-50%的病例中确定病因。已知星状病毒(ASTV)可引起人类、其他哺乳动物和禽类宿主的急性胃肠炎。已知的感染人类的所有8种ASTV基因类型在系统发育上都有密切联系，代表了星病毒科中的一个ASTV物种。使用高通量测序和简并PCR相结合的方法，我们在来自不同大陆的人类粪便样本中检测了12个高度差异的ASTV序列。对全基因组和部分基因组序列的比较分析表明，这些高度分化的ASTV变异株可分为四个新的ASTV物种，它们在遗传上都与动物ASTV物种更接近，而不是先前已知的人类ASTV物种。为了防止这些新病毒及其疾病在世界范围内在人类中进一步传播，关键是要确定它们感染人类的能力、遗传多样性、流行率和疾病关联。产生的数据将有助于设计敏感的分子试剂，并为未来对这些新型病毒的研究提供基础。我们的假设是，这四个新的ASTV物种包括几个不同的基因类型，这些基因能够感染人类，并与胃肠炎有关。我们提出了两个必要的具体目标，以实现我们的长期目标，即了解和减少由这些新型病毒造成的人类疾病负担。具体目标1是确定病毒遗传多样性的全部特征，开发准确的诊断分析并确定疾病关联。我们已经收集了来自墨西哥、美国、尼泊尔、巴基斯坦、印度、尼日利亚、突尼斯和法国的大量临床和现场标本，这将使我们能够进行这些研究。由于ASTV只能在来自其自然宿主的细胞系中繁殖，因此特定的目标2是通过细胞培养来确定这些新病毒的宿主取向并开发血清学检测方法。来自几个哺乳动物物种的细胞系将被用于感染分析和制造纯病毒株。在血清学方面，病毒中和和荧光素酶免疫沉淀系统(LIPS)分析将得到优化，以检测和测量人类血清样本中的抗病毒抗体，从而确认或驳斥它们是人类病毒。同样的检测也将用于确定病毒血清阳性率，因为后者将表明易受这些病毒及其疾病感染或免疫的人类人口的比例。提出的这些新型病毒的分子和生物学特征对于了解它们的致病潜力和开发防止或阻断它们传播的方法至关重要。 公共卫生相关性：与急性胃肠炎相关的新型病毒的分子特征对于开发敏感和准确的诊断分析至关重要。血清阳性率研究将确定易受这些新型星状病毒引起的疾病影响的人群比例。这项研究的结果将有助于制定阻断这些新病毒及其疾病在人类体内传播的战略。

DESCRIPTION (provided by applicant): Worldwide, gastrointestinal infections and diarrheal diseases result in significant morbidity and mortality with approximately two million deaths each year. However, current diagnostic techniques fail to determine a causal infectious agent in 30-50 percent of cases. Astroviruses (AstV) are known to cause acute gastroenteritis in humans, other mammals and avian hosts. All eight genotypes of AstV known to infect humans are phylogenetically closely related and represent a single AstV species within the Astroviridae family. Using a combination of high throughput sequencing and degenerate PCR, we detected sequences of twelve highly divergent AstV in human stool samples from different continents. Comparative analysis of complete and partial genome sequences suggest that these highly divergent AstV variants can be classified into four novel AstV species, all of which are genetically closer to animal AstV species than to the previously known human AstV species. To prevent further spread of these novel viruses and their disease in humans worldwide, it's crucial to determine their capability of infecting humans, genetic diversity, prevalence and disease association. Data generated will help design sensitive molecular reagents and provide groundwork for future research on these novel viruses. Our hypothesis is that these four novel AstV species include several divergent genotypes which are capable of infecting humans and are associated with gastroenteritis. We propose two specific aims necessary to achieve our long term goal of understanding and reducing human disease burden caused by these novel viruses. Specific aim 1 is to characterize the full range of viral genetic diversity, develop accurate diagnostic assays and determine disease association. We have accumulated a large collection of clinical and field specimens from Mexico, Unites States, Nepal, Pakistan, India, Nigeria, Tunisia and France, that will allow us to perform these studies. Since AstV can only be propagated in cell lines derived from their natural host, specific aim 2 is to define the host tropism of these novel viruses using cell culture and to develop serological assays. Cell lines from several mammalian species will be used for infection assays and to make pure virus strains. For serology, virus neutralization and luciferase immunoprecipitation system (LIPS) assays will be optimized to detect and measure anti-virus antibodies in human serum samples thereby confirming or refuting them as human viruses. The same assays will also be used to determine viral seroprevalence, because the latter will indicate the proportion of the human populations susceptible or immune to infection by these viruses and their diseases. The proposed molecular and biological characterization of these novel viruses is critical to understand their pathogenic potential and to develop methods to prevent or interrupt their transmission. PUBLIC HEALTH RELEVANCE: Molecular characterization of the novel viruses associated with acute gastroenteritis is crucial to develop sensitive and accurate diagnostic assays. Seroprevalence studies will determine the proportion of the human populations susceptible to diseases caused by these novel astroviruses. Results of this study will help to formulate strategies to interrupt transmission of these new viruses and their diseases in humans.