Metagenomic detection of emerging viruses in the blood supply

血液供应中新出现病毒的宏基因组检测

基本信息

批准号：
8402145
负责人：
ERIC L DELWART
金额：
$ 46.89万
依托单位：
VITALANT
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8402145
关键词：
Acquired Immunodeficiency Syndrome Algorithms Amino Acid Sequence Animal Sources Animal Viruses Animals Antibodies Antigens Arbovirus Infections Arboviruses Bioinformatics Blood Blood Donations Blood Tests Blood Transfusion Blood donor Blood-Borne Pathogens Brazil Country DNA Sequence DNA Viruses DNA amplification Data Dengue Dengue Virus Detection Disease Association Disease Outbreaks Distant Evolution Exclusion Family Fever General Population Genetic Genetic Databases Genetic Variation Genome HIV HIV Infections Health Hepatitis B Virus Hepatitis C Hepatitis C virus Honduras Human Human Virus Individual Infection Injecting drug user Laboratories Location Measurement Measures Metagenomics Methods Nucleic Acids Pathogenicity Patients Peptide Sequence Determination Persons Plasma Population Prevalence Primer Extension Protein Databases Proteins Proteome Public Health Qualifying RNA RNA Viruses RNA amplification Reading Recurrence Reporting Research Risk Safety Sampling Sensitivity and Specificity Specimen Surveillance Program Symptoms System Technology Testing Time Transfusion Vascular blood supply Viral Viral Genome Viral Proteins Virus Virus Diseases West Nile virus abstracting arm base blood product cohort deep sequencing design experience genetic variant genome sequencing high risk improved men who have sex with men novel novel strategies novel virus nucleic acid purification particle pathogen prevent programs pyrosequencing response transmission process viral DNA viral RNA virome

项目摘要

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.

项目概要/摘要血液制品输注的安全性依赖于排除受 HIV、HBV、HCV 污染的献血西尼罗河病毒。最近出现的 HIV、虫媒病毒（例如 WNV、DENV 和 ChikV）以及其他高致病性病毒病毒，表明仍然未知的病毒仍然对输血接受者、血液和血液的健康构成持续威胁以及血浆捐献者和普通人群。我们开发并成功使用了宏基因组学方法新病毒的发现，并用它来识别和表征许多人类和人类的完整基因组动物DNA和RNA病毒。病毒颗粒内核酸的纯化之后首先是它们的公正性 RNA 和 DNA 扩增，然后进行大规模并行焦磷酸测序。为了识别已知的和新颖的和高度分歧的病毒科、属或种，由组装的重叠群和单线序列编码的蛋白质通过计算将读数与完整的病毒蛋白质数据库进行比较，以获得接近或遥远的序列相似性。这宏基因组方法将用于从广泛的血浆样本中对血浆病毒含量进行遗传表征。不同人群并识别新出现的病毒。这项宏基因组学研究将包括 1200 个大型血浆池用于纯化输血用血液制品，每个血液制品由数千个单独的血浆样本组成，来自补偿血浆捐献者。为了丰富暴露于虫媒病毒感染的样本队列，我们将分析 2000 登革热病毒爆发期间从洪都拉斯和巴西的献血者身上采集的血浆样本以及 200 来自美国的 WNV RNA+ 献血者。为了将测序重点放在有症状的个体上，我们将分析来自 1300 名美国献血者在献血后五天内报告发烧。进一步深度测序将对 800 名病毒暴露水平较高的个体的血浆进行了检测，其中包括：发热的 MSM 和注射吸毒者没有感染艾滋病毒；感染或未感染 HIV 和 HCV 的注射吸毒者；以及晚期艾滋病患者的样本。我们将通过使用焦磷酸序列读数作为遗传立足点来表征所有新发现的病毒的完整基因组使用 PCR 填补序列空白。然后将使用实时 PCR 来测量血浆样本中的病毒流行率从 900 名美国献血者身上采集。新发现的病毒物种的遗传多样性范围将是通过对最不同菌株的基因组进行测序来确定。这种方法将提供一个公正的样本群体血浆中存在的所有病毒核酸的特征，第一近似值人血浆病毒组。美国献血者的患病率测量将决定新冠病毒的传播程度识别出病毒。因此，这些宏基因组学研究将鉴定人血浆中的新型病毒并确定其美国献血者的遗传多样性和患病率。这些研究还将提供起始材料，其形式为病毒核酸和基因组序列，是启动进一步研究以测量血清流行率和疾病所必需的关联并确定需要采取哪些公共卫生措施（如果有的话）来将这些病毒从血液中排除供应。