Full annotation of serum virome in Chinese blood donors with elevated alanine aminotransferase levels

Abstract

Background: A serum alanine aminotransferase (ALT) test is currently demanded for blood donation in China. One of the major reasons to include such a test is possible etiology of known or unknown hepatotropic viruses. However, this hypothesis has never been examined convincingly.

Study design and methods: The study recruited 90 Chinese blood donors that were divided into three groups based on their ALT values. Serum virome from these donors was explored using a metagenomics approach with enhanced sensitivity resolved at single sequencing reads.

Results: Anellovirus and pegivirus C (GBV-C) were detected among these donors. None of them were found solely in donors with abnormal liver enzyme. Anellovirus was highly prevalent (93.3%) and the co-infection with multiple genera (alpha, beta, and gammatorquevirus) were more common in the donors with normal ALT values in comparison to those with elevated ALT (single/double/triple Anellovirus genera, 1/3/24 vs. 7/7/14 or 6/7/13, p = 0.009). For unmapped reads that accounted for 15 ± 14.9% of the data, similarity-based (BLASTN, BLASTP, and HMMER3) and similarity-independent (k-mer frequency) analysis identified several circular rep encoding ssDNA (CRESS-DNA) genomes. Direct PCR testing indicated these genomes were likely reagent contaminants.

Conclusion: Viral etiology is not responsible for elevated ALT levels in Chinese blood donors. The ALT test, if not abandoned, should be adjusted for its cutoff in response to donor shortage in China.

Figure 1.. Detection of known viruses by read mapping.

Original virus-mapped read numbers were normalized based on the average reads among 90 donors (1,424,365), expressed as the number of reads per million of total reads, and presented in the form of heatmap (27). Of 59 viruses detected, their corresponding viral families were indicated.

Figure 2.. The bioinformatics pipeline for the analysis of unmapped reads.

The pipeline is featured by the circumvention of computation-limited steps.

Figure 3.. Comparative analysis of intra-donor Anellovirus diversity among three groups.

First, linear regression analysis showed a weak association between the number of Anellovirus genus and viral concentration inferred from read counts (A); Second, there were significantly different compositions with regard to the donors infected with single, double, triple or more Anellovirus genera between group 3 and group 1 or group 2 (B); Finally, the neighbor-joining trees were constructed with 187 and 294 Anellovirus ORF1 sequences respectively from alphatorquevirus (each 882 nt) and gammatorquevirus (each 474 nt). Bootstrap values were indicated on major branches. For both genera, no apparent clustering was found three groups of donors, i.e., group 1 (red), group 2 (blue), and group 3 (black).