Viral Metagenomics of Blood Donors and Blood-Derived Products Using Next-Generation Sequencing

Abstract

Transfusion-transmitted infections remain a permanent threat in medicine. It keeps the burden of the past, marked by serious infections transmitted by transfusion, and is constantly threatened by emerging viruses. The global rise of immunosuppression among patients undergoing frequent transfusions exacerbates this problem. Over the past decade, criteria for donor selection have become increasingly more stringent. Although routine nucleic acid testing (NAT) for virus-specific detection has become more sensitive, these safety measures are only valuable for a limited number of select viruses. The scientific approach to this is however changing, with the goal of trying to identify infectious agents in donor units as early as possible to mitigate the risk of a clinically relevant infection. To this end, and in addition to an epidemiological surveillance of the general population, researchers are adopting new methods to discover emerging infectious agents, while simultaneously screening for an extended number of viruses in donors. Next-generation sequencing (NGS) offers the opportunity to explore the entire viral landscape in blood donors, the so-called metagenomics, to investigate severe transfusion reactions of unknown etiology. In the not too distant future, one could imagine this platform being used for routine testing of donated blood products.

Keywords: Blood products; Metagenomics; Next-generation sequencing; Transfusion-associated infections; Virus safety.

Fig. 1

Nature and localization of nucleic acids in blood products. This scheme represents the various types of nucleic acids found in the different compartments of blood-derived products. The main source is from nucleated cells present within the product. It is mainly from residual leukocytes present in PC, RBCU and FFP. It is noteworthy that during the preparation of FFP, the freezing process results in a release of free or altered nuclei/cells, which in turn alters the residual leukocytes. Within these residual nucleated cells, the entire human genome is present as a double-stranded DNA (dsDNA), with the possibility of viral dsDNA integration. dsDNA or single-stranded DNA (ssDNA) of viral origin, which is not integrated in the host genome can also be found. In addition to genomic DNA, nucleated cells contain various types of RNAs, including mRNA, tRNA, siRNA, rRNA and small RNAs. DNA viruses at their replication phase will also produce RNA. rRNA, ribosomal RNA; tRNA, transfer RNA; vRNA, viral RNA; sRNA, small RNA; gDNA, genomic DNA; vDNA, viral DNA.

Fig. 2

Interpretation of depth and genome coverage values. A The depth of sequencing indicates how many times a read, i.e. a fragment sequence (nucleic acids are generally fragmented prior to sequencing) attributed to a viral sequence, is detected. Sequence reads with a high depth reflect a high abundance of a virus in the sample. B The second parameter, namely the percentage of genome coverage, refers to the percentage of the viral genome covered by detected reads. C A high percentage of genome coverage is highly suggestive of good specificity and the correct attribution to one defined virus. D In contrast, a low percentage of genome coverage, focused on one fragment of the genome, is more indicative of a misattribution, generally due to homologous sequences of the human genome not being correctly filtered out at the initial stages of data processing.