A Multicenter Study To Evaluate the Performance of High-Throughput Sequencing for Virus Detection

Abstract

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses makes it a powerful tool for broad microbial investigations, such as evaluation of novel cell substrates that may be used for the development of new biological products. However, like any new assay, regulatory applications of HTS need method standardization. Therefore, our three laboratories initiated a study to evaluate performance of HTS for potential detection of viral adventitious agents by spiking model viruses in different cellular matrices to mimic putative materials for manufacturing of biologics. Four model viruses were selected based upon different physical and biochemical properties and commercial availability: human respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), feline leukemia virus (FeLV), and human reovirus (REO). Additionally, porcine circovirus (PCV) was tested by one laboratory. Independent samples were prepared for HTS by spiking intact viruses or extracted viral nucleic acids, singly or mixed, into different HeLa cell matrices (resuspended whole cells, cell lysate, or total cellular RNA). Data were obtained using different sequencing platforms (Roche 454, Illumina HiSeq1500 or HiSeq2500). Bioinformatic analyses were performed independently by each laboratory using available tools, pipelines, and databases. The results showed that comparable virus detection was obtained in the three laboratories regardless of sample processing, library preparation, sequencing platform, and bioinformatic analysis: between 0.1 and 3 viral genome copies per cell were detected for all of the model viruses used. This study highlights the potential for using HTS for sensitive detection of adventitious viruses in complex biological samples containing cellular background. Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms.

Viral contamination in biologic manufacture and implications for emerging therapies

“…While it is challenging to test for adventitious viruses in the presence of recombinant viral stocks, approaches to develop successful virus tests have been developed and used 56 . Additionally, new detection technologies, such as high-throughput sequencing (HTS), have detected adventitious viral contaminants in the presence of virus product 57 and are being explored for use in the industry 58,59 .…”

“…HTS provides a greater breadth of detection than PCR and is seeing widespread interest from the vaccine and recombinant protein industry 59 . However, current HTS sample preparation approaches and bioinformatic pipelines are not as rapid as PCR and can take 7-10 days 58 . Additionally, determining if the contaminant identified in a nucleic acid-based assay is biologically active may require a different method, though we would note that HTS of viral RNA has been used to demonstrate a virus is biologically active 64 .…”

Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

“…The plenitude and breadth of nucleic acid recovery, a very important first step to sample preparation, will influence the overall sensitivity of virus detection by an assay. The selection of the extraction methods can best be evaluated in the context of the entire pipeline using a panel of different virus types to determine its sensitivity before the implementation of any specific pipeline [ 20 , 21 ].…”

Current Trends in Diagnostics of Viral Infections of Unknown Etiology

“…Considering that metagenomics focuses on relative quantities of nucleic acids rather than absolute counts, establishing appropriate thresholds for positive and negative results becomes challenging. In cases of cell cultures and tissues, calculating copies per cell (CPC) has been suggested [89]; however, this parameter would not work for other types of samples, e.g., swabs and washes. High content of a host's nucleic acids might artificially lower CPC, falsely attributing negative results to samples.…”