Genomic sequence and analysis of a vaccinia virus isolate from a patient with a smallpox vaccine-related complication

Abstract

Background: Vaccinia virus (VACV)-DUKE was isolated from a lesion on a 54 year old female who presented to a doctor at the Duke University Medical Center. She was diagnosed with progressive vaccinia and treated with vaccinia immune globulin. The availability of the VACV-DUKE genome sequence permits a first time genomic comparison of a VACV isolate associated with a smallpox vaccine complication with the sequence of culture-derived clonal isolates of the Dryvax vaccine.

Results: This study showed that VACV-DUKE is most similar to VACV-ACAM2000 and CLONE3, two VACV clones isolated from the Dryvax vaccine stock confirming VACV-DUKE as an isolate from Dryvax. However, VACV-DUKE is unique because it is, to date, the only Dryvax clone isolated from a patient experiencing a vaccine-associated complication. The 199,960 bp VACV-DUKE genome encodes 225 open reading frames, including 178 intact genes and 47 gene fragments. Between VACV-DUKE and the other Dryvax isolates, the major genomic differences are in fragmentation of the ankyrin-like, and kelch-like genes, presence of a full-length Interferon-alpha/beta receptor gene, and the absence of a duplication of 12 ORFs in the inverted terminal repeat. Excluding this region, the DNA sequence of VACV-DUKE differs from the other two Dryvax isolates by less than 0.4%. DNA sequencing also indicated that there was little heterogeneity in the sample, supporting the hypothesis that virus from an individual lesion is clonal in origin despite the fact that the vaccine is a mixed population.

Conclusion: Virus in lesions that result from progressive vaccinia following vaccination with Dryvax are likely clonal in origin. The genomic sequence of VACV-DUKE is overall very similar to that of Dryvax cell culture-derived clonal isolates. Furthermore, with the sequences of multiple clones from Dryvax we can begin to appreciate the diversity of the viral population in the smallpox vaccine.

Figure 1

Genome map of VACV-DUKE. Predicted genes are numbered and represented by colored arrows (green: structure and assembly; light blue: RNA metabolism; dark blue: DNA metabolism; dark purple: host range; pink: immunomodulators or virulence factors; black: unknown function; red staggered: fragmented orthologs of ORFs present in other OPVs; red straight arrow: truncated orthologs of ORFs present in other OPVs). Open arrowheads indicate an ORF is split over 2 lines of the diagram. Scale is shown in kb. Thick black lines represent the ITRs.

Figure 2

Phylogenetic tree of the orthopoxviruses. The tree was generated using an 82 kb DNA sequence conserved among all fully sequenced OPVs. Sampling number for bootstrap is 1000; only bootstrap values above 500 are shown.

Figure 3

Map of genomic differences among DUKE, CLONE3 and ACAM2000. A) Map of the region of major genomic differences among the three Dryvax^® clones Solid black line: viral genome; dashed black line: deletion. ORFs are shown as colored arrows. Purple: IFN-α/β receptor homolog which is intact in DUKE and CLONE3 but truncated 90 amino acids from the C terminus in ACAM2000; Red: Ankyrin-like gene, which is intact in CLONE3, fragmented in DUKE and absent from ACAM2000; Light blue: fragments of Kelch-like gene; Dark blue: genes present in the right ITR of ACAM2000 due to a duplication; Green: Serpin (SPI) gene. Gene lengths are not drawn to scale. B) Overview of the positions of substitutions (blue bars), insertions (green bars) and deletions (red bars) among the genomes of three Dryvax^® clones, and VACV-Cop, a VACV strain not derived from Dryvax^®. Substitutions, insertions and deletions are relative to the consensus sequence. Genomes were aligned using Clustal W and edited in Base-By-Base. Numbers indicate the nucleotide position.