GC content-based pan-pox universal PCR assays for poxvirus detection

Abstract

Chordopoxviruses of the subfamily Chordopoxvirinae, family Poxviridae, infect vertebrates and consist of at least eight genera with broad host ranges. For most chordopoxviruses, the number of viral genes and their relative order are highly conserved in the central region. The GC content of chordopoxvirus genomes, however, evolved into two distinct types: those with genome GC content of more than 60% and those with a content of less than 40% GC. Two standard PCR assays were developed to identify chordopoxviruses based on whether the target virus has a low or high GC content. In design of the assays, the genus Avipoxvirus, which encodes major rearrangements of gene clusters, was excluded. These pan-pox assays amplify DNA from more than 150 different isolates and strains, including from primary clinical materials, from all seven targeted genera of chordopoxviruses and four unclassified new poxvirus species. The pan-pox assays represent an important advance for the screening and diagnosis of human and animal poxvirus infections, and the technology used is accessible to many laboratories worldwide.

FIG. 1.

Sequence alignment of the low- and high-GC PCR amplicons. (A) Alignment of amplicon sequences of low-GC PCR assay: 12 predicted amplicon sequences represent 12 species within five chordopoxvirus genera and one unclassified poxvirus. The names for the virus abbreviations used on the figure are given in Table 1. (B) Alignment of amplicon sequences of high-GC PCR assay: five predicted amplicon sequences represent four species of three chordopoxvirus genera and one unclassified poxvirus. The primer positions are indicated with arrow bars.

FIG. 2.

Low- and high-GC PCR and TaqI RFLP assays. (A) Low-GC PCR amplicons and TaqI RFLP patterns. The top panel shows low-GC PCR amplicons from 20 poxvirus DNA samples. The bottom panel shows corresponding TaqI restriction endonuclease RFLP patterns. (B) High-GC PCR amplicons and TaqI RFLP patterns. The left panel shows high-GC PCR amplicons from nine clinical poxvirus DNA samples. The right panel shows the corresponding TaqI restriction endonuclease RFLP patterns.

FIG. 3.

Phylogenetic analysis of a chordopoxvirus clinical specimen. (A) The phylogram of low-GC PCR amplicon sequences from which new poxvirus isolates were inferred (labeled in blue). POX_08040 forms a cluster with the genus Leporipoxvirus, and an unknown poxvirus (POX_01960) forms a distinct clade to all known chordopoxviruses. (B) The phylogram of high-GC PCR amplicon sequences confirms the clinical diagnostics of the PCPV and BPSV specimens, which form distinct clades relative to ORFV. MOCV08031 forms a relatively large divergence with molluscum contagiosum virus type I (MOCV_T1).