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.The poxviruses (family Poxviridae) cause illness characterized by generalized or localized cutaneous lesions, and most member viruses have broad host ranges. The overall broad host range of this family is demonstrated by the two subfamilies of the Poxviridae. The subfamily Entomopoxvirinae infects insects, and the subfamily Chordopoxvirinae infects vertebrates; the latter consists of eight genera, and other "unclassified chordopoxviruses." The classified genera are Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, and Yatapoxvirus. Poxvirus infections of humans, cattle, sheep, goats, companion animals, birds, and zoo animals have been reported worldwide (2) but in general represent an underappreciated cause of health care utilization. Within the United States, until recently the international classification of diseases (ICD) codes were not available for many of these virus infections. Poxvirus infections can be clinically confused with other cutaneous disease, and other poxviruses are emerging or reemerging infections in various parts of the world. The majority of human poxvirus infections are zoonotic. Poxvirus infections are also a significant burden to agricultural communities. Capripoxvirus infections of ruminants cause significant morbidity and mortality. Parapoxvirus infections of ruminants and their handlers are endemic in the United States and worldwide; infected humans often turn to veterinarians for diagnostic assistance (14). Human monkeypox (an orthopoxvirus) is an emerging infection with smallpox-like characteristics that was introduced into the United States in 2003 via global commerce in the exotic pet animal trade. The evolution of monkeypox virus and its two major clades in Africa represents an incompletely understood emerging infectious risk. Smallpox, caused by variola virus, an eradicated disease, remains a significant biothreat agent of internatio...

Serologic Evidence for Circulating Orthopoxviruses in Peridomestic Rodents From Rural Uganda

“…universal PCR assay (Li et al, 2010). These assays should detect all known viruses within the subfamily Chordopoxviridae, except members of the genus Avipoxvirus.…”

One-step duplex polymerase chain reaction for the detection of swinepox and vaccinia viruses in skin lesions of swine with poxvirus-related disease

“…All other 9 species tested representing 6 genera of the Poxviridae were negative. The presence of viable DNA in all samples was confirmed by a parallel assay using pan-poxvirus universal primers to amplify a 220-bp DNA fragment of poxviruses with low G-C content or a 630-bp DNA fragment of PCPV, the only poxvirus with high G-C content genome tested in this work (Li et al, 2010) (Fig. 1B).…”

“…DNA from 10 scab samples was isolated using the Promega Wizard kit as described (Medaglia et al, 2009). As loading controls for single assays, a panpoxvirus universal PCR was used to detect poxviruses of low-GC and high G-C content genomes (Li et al, 2010). In duplex assays to investigate the presence of VACV in clinical samples, the PCR mixture also included a primer set to detect the hemagglutinin (HA) gene, which is a marker of the genus Orthopoxvirus (Damaso et al, 2000(Damaso et al, , 2007.…”

Orthopoxvirus infection among wildlife in Zambia

“…We validated the sensitivity of detection of the OPXV genome using the rpo18 primers together with primers designed to amplify the 14 kDa protein gene A27L of VACV (Scaramozzino et al, 2007), the DNA polymerase gene E9L (Reynolds et al, 2010), the haemagglutinin gene J7R (Kulesh et al, 2004) and the insulin metalloproteinaselike protein gene G1L (Li et al, 2010). In our assay using a StepOnePlus real-time PCR system, the rpo18 primers were the most sensitive for amplification of the targeted gene from monkeypox (Zr599) virus compared with the other primers (Fig.…”