Metagenomic detection of emerging viruses in the blood supply

描述（由申请人提供）：血液制品输血的安全性依赖于排除受HIV、HBV、HCV和WNV污染的献血。最近出现的HIV、虫媒病毒如WNV、DENV和Chikv以及其他高致病性病毒表明，尚未表征的病毒仍然对输血接受者、血液和血浆供体以及一般人群的健康构成持续威胁。我们已经开发并成功地使用了宏基因组学方法来发现新的病毒，并使用它来识别和表征许多人类和动物DNA和RNA病毒的全基因组。病毒颗粒内核酸的纯化之后，首先是它们的无偏RNA和DNA扩增，然后是大规模平行焦磷酸测序。为了鉴定已知的以及新的和高度不同的病毒科、属或种，将由组装的重叠群和单重序列读段编码的蛋白质与完整的病毒蛋白质数据库进行计算比较，以获得近或远的序列相似性。这种宏基因组学方法将用于从不同人群的广泛采样中遗传表征血浆中的病毒含量，并识别新出现的病毒。这项宏基因组学研究将包括1200个大型血浆池，用于净化输血用血液制品，每个血浆池由数千份来自有偿血浆捐献者的个体血浆标本组成。为了丰富暴露于虫媒病毒感染的样本的队列，我们将分析在登革热病毒爆发期间从洪都拉斯和巴西的献血者以及来自美国的200名WNV RNA+献血者中收集的2000份血浆标本。为了将测序重点放在有症状的个体上，我们将分析来自1300名美国献血者的血浆，这些献血者在献血后5天内报告发烧。将对来自800名高水平病毒暴露者的血浆进行进一步的深度测序，包括：未感染HIV的发热MSM和IDU;有和无HIV和HCV感染的IDU;以及来自晚期艾滋病患者的样本。我们将通过使用焦磷酸序列读数作为使用PCR填补序列缺口的遗传立足点来表征所有新鉴定的病毒的全基因组。实时PCR将用于测量从900名美国献血者收集的血浆样本中的病毒流行率。新发现的病毒种属内的遗传多样性范围将通过对最不同毒株的基因组进行测序来确定。该方法将提供采样人群血浆中存在的所有病毒核酸的无偏倚表征，这是人血浆病毒组的第一近似值。美国献血者的流行率测量将确定新发现病毒的传播程度。因此，这些宏基因组学研究将识别人类血浆中的新型病毒，并确定它们在美国献血者中的遗传多样性和流行率。这些研究还将以病毒核酸和基因组序列的形式提供必要的起始材料，以启动进一步的研究，衡量血清流行率和疾病关联，并确定需要采取何种公共卫生应对措施，以将这些病毒从血液供应中排除。 公共卫生相关性：新出现的病毒对血液供应和一般人群的安全构成持续威胁。识别和遗传特性以前未知的病毒是衡量其流行率，致病性和输血传播性所需的第一步。本文提出的人血浆中无偏倚病毒监测计划将使用宏基因组方法为新出现的病毒感染提供早期预警系统，并有助于防止最近输血传播病毒病原体的复发。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Emerging viruses present a constant threat to the safety of the blood supply and the general population. Identifying and genetically characterizing previously unknown viruses is a required first step for measuring their prevalence, pathogenicity, and transfusion transmissibility. The unbiased virus surveillance program in human plasma proposed here will use a metagenomic approach to provide an early warning system for emerging viral infections, and help prevent a recurrence of the recent transfusion-transmissions of viral pathogens.