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. 2023 Jul 11;22(2):140–149. doi: 10.2450/BloodTransfus.451

Full annotation of viral metagenomics in different components from Chinese blood donors using next-generation sequencing

Jingjing Zhang 1,2, Yanmin He 1,2, Ji He 1,2, Yanling Ying 1,2,*, Faming Zhu 1,2,*
PMCID: PMC10920063  PMID: 37458723

Abstract

Background

Emerging viruses in the blood of healthy/qualified donors can seriously affect transfusion safety. However, the virus characteristics in different healthy blood donors and blood components are still not fully understood.

Materials and methods

Buffy coat (BC) and plasma specimens were collected from 32 whole blood donors, and platelet (PLT) and BC specimens from 30 apheresis platelet donors to explore the full annotation of viral metagenomics in different blood components from Chinese blood donors using next-generation sequencing technology.

Results

The study detected 56 viruses in the plasma and BC groups of whole blood donors. The plasma group had a significantly higher viral abundance and more types of viruses than the BC group. We detected 20 viruses in the PLT and BC groups of apheresis platelet donors. Viral abundance and types were significantly lower in the BC group than in the PLT group. According to β-diversity analysis, the plasma group had a significantly different community structure and composition than the BC group.

Discussion

Viral nucleic acid is found in the blood of healthy Chinese blood donors, with the highest concentration in plasma, which could explain the distribution of viruses in the blood of healthy individuals.

Keywords: viral metagenomics, next-generation sequencing, Chinese blood donors, plasma, platelet

INTRODUCTION

Blood safety remains a global health priority and is constantly threatened by emerging transfusion-transmitted pathogens1. The pathogens can be prevented and blocked from spreading to other subjects by excluding blood donations from donors infected with some well-known dangerous viruses. However, the number of viruses that can be detected in blood donation is limited2. Other infectious viruses, including undetected and/or unknown viruses, may be transmitted to recipients via the blood components of donors. Although routine nucleic acid tests for virus-specific tests have become more sensitive, these safeguards are only applicable to a small number of known viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV)3.

Blood donors can be classified into whole blood and apheresis platelet donors. Previous data revealed that the residual risk of HIV, HCV, and HBV in blood donors in China was higher than in the United States and some other countries4. The four pathogen (HBV, HCV, HIV, and Treponema pallidum) markers undergo mandatory testing in China3. However, West Nile virus and human T-cell leukemia virus tests in blood donation have been added in the USA, while cytomegalovirus (CMV) and hepatitis E virus (HEV) tests have been carried out in several countries of the European Union5–8. The Standard Operating Procedures for Blood Services (SOPFBS, 2019 Edition) in China also specifically stipulated that blood services should conduct marker tests for local and time-limited transfusion-related infectious diseases, as stipulated by national and provincial health administrations. The pathogen prevalence in blood donors must be determined to allow the development of a detection strategy for blood donation.

Optimal detection methods are needed to identify overall pathogen distribution in blood from donors, but most early approaches focused on amplifying a single known pathogen. For instance, parvovirus B19 DNA in Chinese whole blood donors was detected using polymerase chain reaction (PCR) with a 0.06% positive rate9. However, complete pathogen distribution data in blood donors are rarely available. Next-generation sequencing (NGS) technology allows entire viral patterns in blood donors to be explored, potentially improving blood transfusion safety10. Lau et al.11 reported that the common viruses in Swiss blood donors were Anelloviruses and human pegivirus, and the astrovirus MLB2 was identified and characterized in fresh frozen plasma. Xu et al.12 conducted metagenomic analysis on blood donor plasma specimens from the Luzhou region of Southwest China. The results revealed that CMV had the highest incidence, followed by HEV. All the above studies demonstrated a certain distribution of the pathogens among blood donors, but there were differences according to different populations and blood components.

In this study, we collected buffy coat (BC) and plasma specimens from whole blood donors, and platelet (PLT) and BC specimens from apheresis platelet donors to analyze the distribution of viruses in different blood components of blood donors in China. We then explored the diversity and composition of the viruses in different blood components using NGS, which can help identify potential viruses that may cause transfusion-transmitted infections.

MATERIALS AND METHODS

Population and blood specimen collection

This study was conducted on 62 healthy blood donors (32 whole blood and 30 apheresis platelet donors) in the Blood Center of Zhejiang Province, in Eastern China. The project was approved by the Ethics Committee of the Blood Center of Zhejiang Province. All participants provided written informed consent, completed the health consultation form before donation, and then performed the tests according to the blood donation guidelines in China. After topical skin disinfection, one EDTA whole blood was collected from all healthy donors. All donors were tested for hepatitis B and C, HIV, HTLV, and Treponema pallidum, with negative results.

Blood component preparation

Plasma and BC components were prepared from the whole blood of 32 whole blood donors after centrifugation at 2,500 × g for 15 minutes (min) at 4°C. PLT were extracted directly from apheresis platelet products from another 30 blood donors, and BC was isolated from the whole blood of the same individuals. All blood specimens were processed under a Class II biological safety cabinet and immediately frozen in liquid nitrogen before being stored at −80°C until DNA/RNA extraction.

Specimen preprocessing

The plasma was centrifuged at 1,000xg for 5 min before being filtered with a 0.45 μM filter into a new 1.5 mL EP tube to remove excess cells. Then 20 μL 10xTurBo buffer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and 4 μL TurBo Dnase I were added, and, after mixing, incubated at 37°C for 20–30 min to remove human DNA. DNase inactivation reagent (1/10 volume) was added to each specimen, incubated at room temperature for 5 min, and then vortexed twice during incubation. Finally, the supernatant was transferred to a new EP tube after centrifugation at 10,000 × g for 90 seconds (s).

Initially, the BC components were pretreated with Erythrocyte Lysis Buffer (Qiagen, Hilden, Germany) to remove red blood cells. Subsequently, 600 μL of leukocyte lysate prepared with the ratio of RNeasy Lysis Buffer (Qiagen): β-Mercaptoethanol (1: 100) was added to the BC and mixed well. The specific operations were performed according to the kit operating manual.

Nucleic acid extraction, reverse transcription, and two-strand synthesis

After processing all specimens, DNA/RNA were extracted from PLT, plasma, and BC specimens using QIAamp MinElute Virus Spin Kits (Qiagen) according to the manufacturer’s instructions. The quality and quantity of DNA/RNA were detected using an ultraviolet spectrophotometer (Multiscan Go, Thermo Scientific, Waltham, MA, USA). RNA were reverse transcribed into cDNA using the SuperScript™ III First-Strand Synthesis System for reverse transcription (RT)-PCR Kit. Then the second strand of cDNA was synthesized using the Klenow fragment (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instruction.

Library preparation and sequencing

The library was prepared using the Nextera® XT DNA Kit according to the manufacturer’s instruction (Illumina, San Diego, CA, USA). The primary procedures involved labeling genomic DNA, amplification, purification, normalization, and library pooling. After purification, the final sequencing libraries were obtained and quality checked using the Aligent 4200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA, USA) to ensure that the average fragment length was approximately 250 bp. All specimens were homogenized to the same concentration; BC and plasma groups from whole blood donors were pooled in equal quantities into one tube, while PLT and BC groups from apheresis platelet donors were pooled in equal quantities into another tube. The library was denatured with 0.2 mol/L NaOH, diluted to a final concentration of 10 pmmol/L, and then sequenced using an Illumina MiSeq instrument with a v3 Reagent Kit (150-cycle; Illumina). The original data were formatted as a fastq file using the software in the MiSeq instrument.

Sequencing analysis

Viral classification data were analyzed using the CLC Microbial Genomics Module (version 21, Qiagen). The reads were pruned using the Data Quality Control (QC) and Clean Host DNA workf low to trim reads, create a QC report, and clean the dataset of host DNA, leaving only the reads that did not match the host genome. These data were then compared using NCBI RefSeq viral genome database (2019-04-24) in the CLC software. The identical sequences were then performed for species annotation and classification. The viruses with false sequences or a maximum combined abundance of less than 10 were eliminated.

Bioinformatics analysis

Species compositions in different groups were visualized using stacked bar charts, sunburst view, heat maps, and β-diversity analysis. The OTU abundance table was visualized as a stacked bar. Only the family and species levels were visualized to ensure the visual effect of the figure. Trimmed mean of M values (TMM) normalization was performed for the species abundance using Create Heat Map for Abundance Table tool (Qiagen) and a z-score normalization to make features comparable. The features of the hierarchical clustering groups were clustered using the similarity features of their genomes on a set of specimens. β diversity was analyzed for the similarity of viral community structure among different groups. The distance matrix was calculated using principal co-ordinate analysis (PCoA) in CLC Genomics workbench software based on Jaccard and Bray-Curtis methods.

Statistical analysis

Statistical analyses were conducted using the GraphPad Prism 5 (GraphPad Software, Boston, MA, USA). p<0.05 was considered statistically significant.

RESULTS

Abundance and types of viruses were significantly more enriched in the plasma than in the buffy coat group in whole blood donors

Figure 1 presents viral composition in two different blood components of whole blood donors. We discovered 56 viruses in blood specimens from whole blood donors. Virus abundance was significantly lower (p<0.0001) in the BC (11.19±3.273) than in the plasma (882.7±77.84) (Figure 1A). A sunburst view analysis of virus data detected in BC and plasma groups revealed that Human betaherpesvirus 5 was the most dominant virus (Figure 1B). Separately, Peribunyaviridae (44%) and Microviridae (42%) were highly abundant in the BC group, while Herpesviridae (27%), Inoviridae (18%), Polyomaviridae (14%), and Peribunyaviridae (14%) were highly abundant in the plasma group at the family level (Figure 1C). At a deeper taxonomic level, Escherichia virus phiX174 and Shamonda orthobunyavirus were significantly enriched in the BC group, while Human betaherpesvirus 5, Escherichia virus M13, Macaca mulatta polyomavirus 1, and Shamonda orthobunyavirus were significantly enriched in the plasma group (Figure 1D). β diversity was analyzed using two assessment methods (Bray-Curtis and Jaccard) among the BC and plasma group community composition (Figures 1E, F). The distance between the specimens intuitively reflects the differences in species abundance between them. The results demonstrated significant differences in the overall viral species abundance and community composition between the BC and the plasma groups.

Figure 1.

Figure 1

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The virus species, abundance analysis, and β diversity of buffy coat (BC ) and plasma groups from whole blood donors

A: Statistical analysis of viral abundance among different blood components. B: Sunburst view of the viral community showing all taxa belonging to the kingdom viruses. C and D: Stacked bar of the viral community among BC and plasma at the family and species levels, respectively. E and F: β-diversity results are seen as 2D PCoA, with coloring carried out according to taxonomic abundance values. BC (green dot): 1–32; plasma (red dot): 33–64.

The analysis further visualized the species composition in the combined abundance of each specimen in the form of a heat map (Figure 2). Genes or specimens with similar expression patterns are classified into one category and are closer together in the evolutionary tree. The overall different colored small squares present obvious differences in virus expression between the plasma group and the BC group, with the viruses having higher expression levels in the plasma group than the BC group. The left evolutionary tree reveals that transfusion-transmitted viruses, such as torque teno virus (TTV, Alphatorquevirus), torque teno mini virus (TTMV, Betatorquevirus), and torque teno midi virus (TTMDV, Gammatorquevirus), are also commonly present in the plasma group.

Figure 2.

Figure 2

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Heat map analysis of 56 microbiota in plasma and buffy coat (BC ) groups

Abundance and species of viruses were significantly lower in the buffy coat than in the platelet group in apheresis platelet donors

Figure 3 indicates viral composition in BC and PLT components of the apheresis platelet donors. We discovered 20 viruses in blood specimens from the apheresis platelet donors. The virus abundance was significantly higher (p<0.0001) in the PLT (200.7±23.58) than in the BC (8.267±4.394) group (Figure 3A). A sunburst view analysis of virus data detected in the PLT and BC groups revealed that the Burkholderia phage KS10 was the most dominant (Figure 3B). Separately, Myoviridae (66%) was highly abundant in the PLT group, while Microviridae (73%) was highly abundant in the BC group, followed by Peribunyaviridae (21%) at the family level (Figure 3C). At a deeper taxonomic level, Burkholderia phage KS10 was significantly enriched in the PLT group, while Escherichia virus phiX174 and Shamonda orthobunyavirus were significantly enriched in the BC group (Figure 3D). These results demonstrate that the PLT group had significantly more virus abundance and species than the BC group; they also had different dominant virus species. The heat map (Figure 4) shows that the PLT group also contains certain Gammatorquevirus, and that there is a significant difference in virus species expression between the PLT and BC groups. The PLT group had a higher abundance and more types of virus expression than the BC group.

Figure 3.

Figure 3

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The virus species and abundance analysis of buffy coat (BC ) and platelet (PLT) groups from apheresis platelet donors

A: Statistical analysis of viral abundance among different blood components. B: Sunburst view of the viral community showing all taxa belonging to the kingdom viruses. C and D: Stacked bar of the viral community among BC and PLT at the family and species levels, respectively. ***p<0.001.

Figure 4.

Figure 4

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Heat map analysis of 20 microbiota in buffy coat (BC ) and platelet (PLT) groups

Differences in virus species between the plasma and platelet groups

The study discovered 56 viruses in the plasma group of whole blood donors and 20 in the PLT group of the apheresis platelet donors, with 12 species shared by both groups. PLT also contained the highest levels of Human betaherpesvirus 5, Escherichia virus M13, Macaca mulatta polyomavirus 1, and Shamonda orthobunyavirus in the plasma group. However, Burkholderia phage KS10, which has the highest abundance in platelets, was not detected in the plasma group (Figure 5).

Figure 5.

Figure 5

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The virus species in plasma and platelet (PLT) groups and the percentage of each virus to the total virus abundance in the group

Red box shows viruses shared by both groups.

DISCUSSION

Today, viruses are among the most abundant micro-organisms receiving special attention11. New or re-emerging virulent epidemics often become a global public health concern, while blood is an important vehicle in transmitting many viruses. Pathogens, such as the Torque teno (TT) virus and human hepegivirus-1, are widespread in human blood11,13. Studies have reported that potential pathogens in blood donors originate primarily from the blood, the body surface of the puncture site, or contamination during the blood collection process14,15. However, blood donors do not have the corresponding disease after most infections; therefore, they cannot be identified through routine medical examinations and consultations, resulting in risk of infection with the corresponding disease through transfusion16. Therefore, prevention and control strategies, and detection techniques should be formulated for potential pathogens in blood donors to reduce the risk of transfusion transmission; these require systematic identification of the possible distribution of pathogens in blood donors. However, there is an urgent need to investigate the overall distribution of pathogens in blood donors because there are few data and information in this area.

Metagenomics based on NGS technology has recently been applied to detect blood pathogens. Metagenomics can reflect the genetic composition and community function of all pathogens in the specimens by extracting nucleic acid of all pathogens in the specimen15,17,18. It provides a new method and approaches for comprehensively annotating the virus meta-genome in blood donors.

This study discovered 56 and 20 viruses in whole blood and apheresis PLT donors using NGS technology, respectively. However, the virus activities were not analyzed; therefore, only DNA/RNA fragments from the viruses were confirmed. Whether it is a live virus or in a latent state needs further study. Human betaherpesvirus 5 was the most abundant virus in the plasma group of whole blood donors, and the result of this study is similar to that of the Guo19 report. Guo et al. identified 14 viruses with a >10% viral genome coverage in the unmapped sequencing reads from the ChinaMAP dataset and discovered that Human betaherpesvirus 5 was widely detected in the blood. Burkholderia phage KS10, which lacks potential virulence factors, was the virus with the highest abundance in the PLT group from apheresis platelet donors20. However, Burkholderia phage KS10 was not detected in the whole blood donors, and, thus, requires further study. The types and abundances of viruses in the plasma and PLT groups were greater than in the BC group. In contrast, Escherichia virus phiX174 was identified primarily in BC rather than plasma and PLT groups. Escherichia virus phiX174 belongs to Microviridae and is a common intestinal microbe21. We speculated that Escherichia virus Phix174 might enter the blood via the intestinal mucosal barrier leading to phagocytosis by leukocytes. Whether these viruses cause the disease still has to be verified.

The highest abundance of virus types in blood donors and the distribution of virus species in different blood components differed slightly from those reported in the literature. This difference could be due to different populations and regions, or it could be due to the presence of a large number of human genomes in BC specimens. When removing the interference of the human genome, viral genomes in the specimens were also removed, so the types and abundance of viruses were lower in the BC group than in the plasma and PLT groups, or it may be that since the NGS method detects viral genome fragments, the plasma content is higher than the BC content. Additionally, when we collect PLT specimens from apheresis PLT donors, the donor’s blood has undergone multiple cycles in the body, whereas the collected BC only contains the donor’s local circulating blood, so the types and abundance of viruses may be higher in PLT than in BC.

Notably, transfusion-transmitted viruses (namely α, β, and γTTV) were detected in plasma and PLT, as reported in most of the literature13,19,22. In Japan, the infection rate in the Anelloviridae among healthy/eligible people is almost 100%, compared to 10% in the US and UK23. There are few data regarding these viruses in Chinese blood donors. High-loading Anellovridae has caused some clinical symptoms in humans24,25; however, whether they cause disease is unclear. Conversely, studies point to the potential benefits of Anelloviruses for human health. For example, in a newborn infected with the Anelloviridae, the virus can promote the development and maturation of the child’s immune system26. Therefore, we must continue researching TTV and its related viruses, and explore their pathogenicity and infectivity when conditions allow.

According to the above results, many viruses are also present in healthy/eligible blood donors, which can lead to further contamination if blood collection or preparation procedures are not appropriately managed. Therefore, blood collection and supply should strictly follow the relevant (or even higher) standards when collecting and preparing blood for therapeutic use.

We were unable to comprehensively analyze the prevalence of existing or new viruses in healthy/eligible blood donors in Zhejiang Province, China, due to the limitations of specimen size and population, and time constraints. This metagenomics analysis of viruses in blood from qualified donors using NGS technology only provides a reference for the certain virus prevalence. Neither did we evaluate the clinical impact of these pathogens in blood transfusion. However, a virus genome identified using metagenomics would have a clinical impact on the recipient such as to be considered a transfusion-transmitted virus. Viral metagenomics relies on NGS technology, has the potential to screen large numbers of viruses, and is an ideal technical approach for detecting emerging or potentially dangerous viruses in the population. NGS technology is expected to be used in future routine diagnosis and treatment as science and technology develop.

CONCLUSIONS

This experiment aimed to detect the viral genomes of different blood components from healthy Chinese blood donors using a metagenomics method based on NGS technology. The results of this study indicated that there were viral sequence fragments in the blood of healthy donors. The species diversity and abundance of viruses were the most abundant in plasma and the least in BC. A thorough understanding of the blood microbiome of healthy donors is critical to transfusion safety, which depends on using sensitive and specific methods to detect viruses in the blood. Systematic identification of pathogen distribution will facilitate the application of different control measures for different blood products.

Footnotes

ETHICAL CONSIDERATIONS: The project was approved by the ethics committee of the Blood Center of Zhejiang Province. All participants gave written informed consent, filled the health consultation form before donation, and then performed the tests according to the guidelines for blood donation in China (GB18467-2011).

AUTHORSHIP: JZ, YY and YH performed the experiments. JZ, JH and YY analyzed the data. JZ, YY and FZ wrote the paper.

The Authors declare no conflicts of interest.

FUNDING: This work was sponsored by the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents.

DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article are available at the NCBI Sequence Read Archive (SRA) with the accession number PRJNA931776.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The raw data supporting the conclusions of this article are available at the NCBI Sequence Read Archive (SRA) with the accession number PRJNA931776.

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