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.
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, pathogencicity, 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.
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