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.

项目摘要/摘要血液产物输血的安全依赖于排除艾滋病毒，HBV，HCV和 WNV。 HIV，WNV，DENV和CHIKV等艾滋病毒的最近出现以及其他高度致病性病毒表明仍然未表征的病毒仍然对输血接受者的健康，血液仍然存在持续的威胁和血浆捐献者和普通人群。我们已经开发并成功地使用了宏基因组学方法发现新病毒，并用它来识别和表征许多人类和动物DNA和RNA病毒。首先是无偏的病毒颗粒内核酸的纯化 RNA和DNA扩增，然后通过大量平行的pyro测序。为了识别已知和新颖以及高度发散的病毒家族，属或物种，由组装的重叠群和单线序列编码的蛋白质与完整的病毒蛋白数据库相比，读取的读数是在近距离或遥远的序列相似性中进行的。这宏基因组方法将用于遗传表征血浆的病毒含量，从广泛的抽样中不同的人群并确定新的新兴病毒。这项宏基因组学研究将包括1200个大型等离子体池用于净化血液产品以输血，每种由数千个单独的等离子体标本组成补偿血浆供体。为了丰富暴露于Arbovirus感染的样品的队列，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收集的血浆标本，200 来自美国的WNV RNA+献血者。为了将测序集中在有症状的个体上，我们将分析来自 1300名美国献血者在捐赠后五天内报告发烧。进一步的深度测序将是在800名病毒式暴露量高的人的血浆上进行，包括：发热MSM和IDU 未感染艾滋病毒；有和没有HIV和HCV感染的IDU；和高级艾滋病患者的样本。我们将通过使用Pyrosequence读取所有新鉴定的病毒的完整基因组使用PCR填充序列间隙。然后，实时PCR将用于测量血浆样品中的病毒患病率从900名美国献血者那里收集。新鉴定的病毒物种内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确定。这种方法将提供公正的采样种群血浆中存在的所有病毒核酸的表征，第一个近似人血浆病毒蛋白。美国献血者的患病率测量将确定新近的传播程度确定的病毒。因此，这些宏基因组学研究将确定人血浆中的新病毒，并确定其美国献血者的遗传多样性和患病率。这些研究还将以病毒核酸和基因组序列，需要进行进一步研究以测量血清偏差和疾病关联并确定将需要采取哪些公共卫生反应（如果有）将这些病毒排除在血液之外供应。