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 international concern and the subject of World Health Organization-approved research activities. A recent study on health care utilization for the other nonzoonotic human poxvirus infections (21
) began to define the burden (22/10,000 health care visits) of molluscum contagiosum infections in the community. Over the past 5 years, an increasing number of potential poxvirus therapies have been studied. Better diagnostics targeting poxviruses as the cause of cutaneous infections will decrease the inappropriate treatment of these infections. For instance, parapoxvirus infections are often misdiagnosed as cutaneous anthrax, which unnecessarily contributes to overuse of antibacterial agents. By using effective therapies and prevention measures, after appropriate diagnosis, the interhuman transmissibility of poxvirus infections will be reduced, and days lost from work will be reduced.
Poxviruses represent one of largest viruses known and replicate in the cytoplasm of the infected cell and encode most enzymes for their life cycle. The genomes of poxviruses are a linear double-stranded DNA genome in the range of 134 kb (Parapoxvirus) to 330 kb (Avipoxvirus) and encode more than 130 genes. The central region, comprised of nearly 100 genes which encode viral gene expression, DNA replication, and virion formation, has a structural arrangement that is conserved in most chordopoxviruses. Between genera of chordopoxviruses, host specificity and genome sequence have diverged. Interestingly, the chordopoxviruses have two distinct types of genome based on GC content: parapoxviruses, molluscipoxviruses, and crocodilepox virus (an unclassified poxvirus most similar to molluscipoxvirus) have high GC contents (>60%); the genomes of the other six genera of chordopoxvirus have a low GC (30 to 40%) content. The evolutionary factors for this divergence are unknown.
The preponderance of data suggests that although the poxviruses can readily recombine under tissue culture conditions (23
), the genomes are quite stable in evolutionary time (15
). To develop a PCR assay, or assays, which could easily be used to screen for the presence of a poxvirus in a clinical sample, we developed two PCR signatures, one that would be expected to amplify nucleic acid from poxviruses with high GC content (high-GC PCR) while the other would be expected to amplify nucleic acid from most poxviruses with low GC content (low-GC PCR).
In this study, we describe the design and validation of the two new pan-chordopoxvirus standard PCR assays using over 150 chordopoxvirus isolates. We also describe the use of these new PCR assays to diagnose and discover previously unknown poxviruses as the cause of infections based on the sequence information from the resultant PCR amplicons.