title: Optimization of virus detection in cells using massively parallel sequencing.
abstract: Massively parallel sequencing (MPS)-based virus detection has potential regulatory applications. We studied the ability of one of these approaches, based on degenerate oligonucleotide primer (DOP)-polymerase chain reaction (PCR), to detect viral sequences in cell lines known to express viral genes or particles. DOP-PCR was highly sensitive for the detection of small quantities of isolated viral sequences. Detected viral sequences included nodavirus, bracovirus, and endogenous retroviruses in High Five cells, porcine circovirus type 1 and porcine endogenous retrovirus in PK15 cells, human T-cell leukemia virus 1 in MJ cells, human papillomavirus 18 in HeLa cells, human herpesvirus 8 in BCBL-1 cells, and Epstein-Barr Virus in Raji cells. Illumina sequencing (for which primers were most efficiently added using PCR) provided greater sensitivity for virus detection than Roche 454 sequencing. Analyzing nucleic acids extracted both directly from samples and from capsid-enriched preparations provided useful information. Although there are limitations of these methods, these results indicate significant promise for the combination of nonspecific PCR and MPS in identifying contaminants in clinical and biological samples, including cell lines and reagents used to produce vaccines and therapeutic products.
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
authors.forename: Shasta D
authors.initials: SD
authors.lastname: McClenahan
authors.affiliation: Highly Pathogenic Viruses ZBS1, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Uhlenhaut
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA. Electronic address: philip.krause@fda.hhs.gov.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Krause
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D016133:Polymerase Chain Reaction; D014780:Viruses / Q000145:classification / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers
keywords: Massively parallel sequencing (MPS); Non-specific PCR; Virus detection