Molecular Characterization of Novel Astroviruses

描述（由申请人提供）：在世界范围内，胃肠道感染和消化道疾病导致显著的发病率和死亡率，每年约有200万人死亡。然而，目前的诊断技术无法确定30- 50%的病例中的致病性感染原。已知星状病毒（AstV）在人类、其他哺乳动物和禽类宿主中引起急性胃肠炎。已知感染人类的所有八种AstV基因型在系统发育上都密切相关，代表了星状病毒科中的单一AstV物种。使用高通量测序和简并PCR的组合，我们检测了来自不同大陆的人类粪便样本中的12个高度不同的AstV序列。完整和部分基因组序列的比较分析表明，这些高度不同的AstV变种可以分为四个新的AstV物种，所有这些都是遗传上更接近动物AstV物种比以前已知的人类AstV物种。为了防止这些新型病毒及其疾病在全球人类中的进一步传播，确定它们感染人类的能力、遗传多样性、流行率和疾病关联至关重要。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奠定基础。我们的假设是，这四个新的AstV物种包括几个不同的基因型，能够感染人类，并与胃肠炎。我们提出了两个必要的具体目标，以实现我们的长期目标，了解和减少这些新病毒引起的人类疾病负担。具体目标1是表征病毒遗传多样性的全部范围，开发准确的诊断测定法并确定疾病关联。我们已经从墨西哥、美国、尼泊尔、巴基斯坦、印度、尼日利亚、突尼斯和法国收集了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只能在源自其天然宿主的细胞系中繁殖，因此具体目标2是使用细胞培养物确定这些新型病毒的宿主向性并开发血清学测定。来自几种哺乳动物物种的细胞系将用于感染测定和制备纯病毒株。对于血清学，将优化病毒中和和荧光素酶免疫沉淀系统（LIPS）试验，以检测和测量人血清样本中的抗病毒抗体，从而确认或排除其为人病毒。同样的试验也将用于确定病毒血清阳性率，因为后者将表明对这些病毒及其疾病的感染易感或免疫的人群比例。这些新病毒的分子和生物学特征对于了解其致病潜力和开发预防或中断其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灵敏和准确的诊断方法至关重要。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战略，以阻断这些新病毒及其疾病在人类中的传播。

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.