Abstract
There is a need for a broad and efficient testing strategy for the detection of both known and novel viral adventitious agents in vaccines and biologicals. High-throughput sequencing (HTS) is an approach for such testing; however, an optimized testing method is one with a sample-processing pipeline that can help detect any viral adventitious agent that may be present. In this study, 11 commercial methods were assessed for efficient extraction of nucleic acids from a panel of viruses. An extraction strategy with two parallel arms, consisting of both the Invitrogen PureLink™ Virus RNA/DNA kit for total nucleic acid extraction and the Wako DNA Extractor® kit with an RNase A digestion for enrichment of double-stranded nucleic acid, was selected as the strategy for the extraction of all viral nucleic acid types (ssRNA, dsRNA, and dsDNA). Downstream processes, such as double-strand DNA synthesis and whole-genome amplification (WGA), were also assessed for the retrieval of viral sequences. Double-stranded DNA synthesis yielded larger numbers of viral reads, whereas WGA exhibited a strong bias toward amplification of double-stranded DNA, including host cellular DNA. The final sample-processing strategy consisted of the dual extraction approach followed by double-stranded DNA synthesis, which yielded a viral population with increased detection of some viruses by 8600-fold. Here we describe an efficient extraction procedure to support viral adventitious agent detection in cell substrates used for biological products using HTS.
Highlights
Vaccines are among the most cost-effective public heath medical products available to date
porcine circovirus (PCV) is not known to be infectious to humans and in the study by Victoria et al high-throughput sequencing (HTS) was useful in the discovery of contaminants, despite the lack of pathogenicity in humans
Eleven commercially available extraction kits were tested for their efficient extraction of nucleic acid from HeLa cells spiked with a panel of four viruses that represent diverse biochemical and biophysical properties across different viral families: enveloped versus non-enveloped, double-stranded DNA (Epstein-Barr virus; EBV); double-stranded RNA (Reovirus 3; Reo3); single-stranded RNA (Feline leukemia virus; FeLV, and respiratory syncytial virus; RSV); and segmented genetic material (Reo[3]; Supplementary Information—Table 1)
Summary
Vaccines are among the most cost-effective public heath medical products available to date. Guidelines for testing for adventitious agents in vaccines are provided by regulatory agencies.[2] Viral adventitious agent testing includes in vivo assays and cell culture-based in vitro assays These current testing methods are limited and are unable to detect a number of viral families where no suitable animal model or appropriate culturing method exists.[3] To address these gaps in testing, targetspecific nucleic acid testing (NAT) methods, such as quantitative PCR (qPCR), are used to detect the presence of viruses of interest.[4] The use of PCR-based methods relies on a prior knowledge of the nucleic acid sequence of the viral adventitious agent for purposes of primer design, which may not always be available especially for poorly characterized or novel viruses. PCV is not known to be infectious to humans and in the study by Victoria et al HTS was useful in the discovery of contaminants, despite the lack of pathogenicity in humans
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