Metagenomic detection of emerging viruses in the blood supply

项目摘要/摘要 输血产品的安全性依赖于排除受艾滋病毒、乙肝病毒、丙型肝炎病毒和 西尼罗河畔。最近出现的艾滋病毒、虫媒病毒如西尼罗河病毒、DENV和CHIKV以及其他高致病性病毒 病毒，表明仍未确定特征的病毒仍然是对输血者健康的持续威胁，血液 和血浆捐献者，以及普通民众。我们已经开发并成功地使用了元基因组学方法来 新病毒的发现，并用它来识别和表征许多人类和 动物DNA和RNA病毒。提纯病毒颗粒中的核酸之后，首先是它们的无偏 RNA和DNA扩增，然后通过大规模平行焦化测序。为了识别已知的和新奇的 和高度不同的病毒家族、属或物种，由组装的重叠群和单一序列编码的蛋白质 通过计算将读数与完整的病毒蛋白数据库进行比较，以获得近距离或远距离的序列相似性。这 将使用元基因组方法从大范围样本中确定血浆中病毒含量的基因特征 不同的人群，并识别新出现的病毒。这项元基因组学研究将包括1200个大型血浆池 用于提纯供输血的血液产品，每个血液产品由数千个单独的血浆样本组成，来自 有补偿的血浆捐献者。为了丰富暴露于虫媒病毒感染的样本队列，我们将分析2000 在登革热病毒暴发期间从洪都拉斯和巴西的献血者以及200名献血者身上采集的血浆样本 来自美国的西尼罗河病毒RNA+献血者。为了集中对有症状的个人进行测序，我们将分析来自 1300名美国献血者在献血后5天内报告发烧。进一步的深度测序将是 对800名有高病毒暴露水平的人的血浆进行了检测，其中包括：发烧的MSM和IDU，他们是 没有感染艾滋病毒；有或没有感染艾滋病毒和丙型肝炎病毒的注射吸毒者；以及晚期艾滋病患者的样本。我们 将通过使用焦化序列读数作为遗传立足点来表征所有新发现的病毒的全基因组 用聚合酶链式反应填补序列空白。然后，将使用实时聚合酶链式反应来测量血浆样本中的病毒流行率 从900名美国献血者那里收集的。新发现的病毒物种内的遗传多样性范围将是 通过对最具差异性的菌株的基因组进行测序来确定。这种方法将提供一个不偏不倚的 样本人群血浆中存在的所有病毒核酸的特征，第一近似值 人类血浆病毒。美国献血者的流行率测量将确定新的病毒传播程度 识别出的病毒。因此，这些元基因组学研究将识别人类血浆中的新病毒，并确定它们的 美国献血者的遗传多样性和流行率。这些研究还将以下列形式提供起始材料： 病毒核酸和基因组序列，是启动进一步研究以衡量血清流行率和疾病所必需的 协会并确定需要什么公共卫生对策(如果有的话)才能将这些病毒从血液中排除 供给。

Project Summary/Abstract The safety of blood product transfusions relies on the exclusion of donations contaminated with HIV, HBV, HCV, and WNV. The recent emergence of HIV, arboviruses such as WNV, DENV, and ChikV, as well as other highly pathogenic viruses, indicates that still uncharacterized viruses remain an ongoing threat to the health of transfusion recipients, blood and plasma donors, and the general population. We have developed and successfully used a metagenomics approach for the discovery of new viruses and have used it to identify and characterize the full genomes of numerous human and animal DNA and RNA viruses. Purification of the nucleic acids within viral particles is followed first by their unbiased RNA and DNA amplification and then by massively parallel pyro-sequencing. In order to identify known as well as novel and highly divergent viral families, genera, or species, the proteins encoded by the assembled contig and singlet sequence reads are computationally compared to the complete viral protein database for close or distant sequence similarities. This metagenomic approach will be used to genetically characterize the viral content of plasma from a wide sampling of different populations and identify novel emerging viruses. This metagenomics study will include 1200 large plasma pools used to purify blood products for transfusion, each made up of thousands of individual plasma specimens, from compensated plasma donors. To enrich the cohort for samples exposed to arbovirus infections, we will analyze 2000 plasma specimens collected from blood donors in Honduras and Brazil during outbreaks of dengue virus and from 200 WNV RNA+ blood donors from the US. To focus sequencing on symptomatic individuals, we will analyze plasma from 1300 US blood donors who reported fever within five days after their donations. Further deep sequencing will be performed on plasma from 800 individuals with high levels of viral exposures including: febrile MSM and IDU who are not infected with HIV; IDU with and without HIV and HCV infections; and samples from advanced AIDS patients. We will characterize the full genome of all newly identified viruses by using the pyrosequence reads as genetic footholds for filling sequence gaps using PCR. Real-time PCR will then be used to measure viral prevalence in plasma samples collected from 900 US blood donors. The range of genetic diversity within newly identified viral species will be determined by sequencing the genomes of the most divergent strains. This approach will provide an unbiased characterization of all the viral nucleic acids present in the plasma of the sampled populations, the first approximation of the human plasma virome. Prevalence measurements in US blood donors will determine the extent of spread of the newly identified viruses. These metagenomics studies will therefore identify novel viruses in human plasma and determine their genetic diversity and prevalence in US blood donors. These studies will also provide the starting material, in the form of the viral nucleic acids and genome sequences, necessary to initiate further studies to measure sero-prevalence and disease association and determine what public health responses, if any, will be needed to exclude these viruses from the blood supply.