The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns

Abstract

f Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ϳ99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by nextgeneration sequencing.

Using populations of human and microbial genomes for organism detection in metagenomes

“…There is a growing recognition of the challenges in using metagenomic sequencing for detecting low abundance pathogens (Naccache et al 2013;Salter et al 2014). The abundance of microbial content found in the HGP data further demonstrates the ongoing challenge to differentiate clinically relevant low abundance microbial content from other sources of biological contamination.…”

Expansion of the Metazoan Virosphere: Progress, Pitfalls, and Prospects

“…Second, the host can be misassigned if samples contain multiple hosts, either naturally or through contamination. Technical nucleic acid contamination can be minimised by good laboratory practice (though see Naccache et al 2013), but highthroughput sequencing technologies are prone to crosscontamination at the point of sequencing. For example, in the absence of dual indexing, 'barcode-switching' in some Illumina platforms can misattribute reads among libraries at a rate of up to 0.3-1% (Kircher et al 2012).…”

Parvovirus-Derived Endogenous Viral Elements in Two South American Rodent Genomes

“…To exclude the possibility that it was somehow derived from contaminating viral DNA (7,8), one intact element (O. degus-4) was independently amplified from genomic DNA by PCR. Tissue was obtained from a fresh male headless O. degus cadaver (kindly donated by Adrian Palacios from Universidad de Valparaiso, Chile).…”