Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus

Abstract

Metagenomics and a panmicrobial microarray were used to examine eight live-attenuated viral vaccines. Viral nucleic acids in trivalent oral poliovirus (OPV), rubella, measles, yellow fever, varicella-zoster, multivalent measles/mumps/rubella, and two rotavirus live vaccines were partially purified, randomly amplified, and pyrosequenced. Over half a million sequence reads were generated covering from 20 to 99% of the attenuated viral genomes at depths reaching up to 8,000 reads per nucleotides. Mutations and minority variants, relative to vaccine strains, not known to affect attenuation were detected in OPV, mumps virus, and varicella-zoster virus. The anticipated detection of endogenous retroviral sequences from the producer avian and primate cells was confirmed. Avian leukosis virus (ALV), previously shown to be noninfectious for humans, was present as RNA in viral particles, while simian retrovirus (SRV) was present as genetically defective DNA. Rotarix, an orally administered rotavirus vaccine, contained porcine circovirus-1 (PCV1), a highly prevalent nonpathogenic pig virus, which has not been shown to be infectious in humans. Hybridization of vaccine nucleic acids to a panmicrobial microarray confirmed the presence of endogenous retroviral and PCV1 nucleic acids. Deep sequencing and microarrays can therefore detect attenuated virus sequence changes, minority variants, and adventitious viruses and help maintain the current safety record of live-attenuated viral vaccines.

FIG. 1.

Viral genome sequencing coverage. Depth of sequence coverage at each genomic position for OPV, Attenuvax, YF-Vax, Varivax, MMR-II, Rotarix (PCV1), and Rotateq. For vaccines containing multiple genomes (MMRII) or multiple segments (Rotateq) of variable lengths, coverage is normalized to genetic position as percentage of fragment length. Depth of coverage for rotavirus and rubella virus from Rotarix and Meruvax-II, respectively, are not shown due to low coverage.

FIG. 2.

Taxonomic distribution of sequences in 8 live-attenuated viral vaccines (a to h) and water (i). Reads were classified using BLASTx as either viral (V), eukaryotic (E), or bacterial/unclassifiable (E/U) (see Materials and Methods). Vaccine viral input (PFU) is listed based on manufacturers’ reported titers as either the 50% tissue culture infectious dose (TCID₅₀), PFU, or infectious units (IU).