A novel and sensitive real-time PCR system for universal detection of poxviruses

Abstract

Success in smallpox eradication was enabled by the absence of non-human reservoir for smallpox virus. However, other poxviruses with a wider host spectrum can infect humans and represent a potential health threat to humans, highlighted by a progressively increasing number of infections by (re)emerging poxviruses, requiring new improved diagnostic and epidemiological tools. We describe here a real-time PCR assay targeting a highly conserved region of the poxvirus genome, thus allowing a pan-Poxvirus detection (Chordopoxvirinae and Entomopoxvirinae). This system is specific (99.8% for vertebrate samples and 99.7% for arthropods samples), sensitive (100% for vertebrate samples and 86.3% for arthropods samples) and presents low limit of detection (< 1000 DNA copies/reaction). In addition, this system could be also valuable for virus discovery and epidemiological projects.

Monkeypox: another test for PCR

“…Unlike severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in early 2020, monkeypox virus is not a new virus and several published assays are already available [5][6][7][8][9][10][11], some developed by diagnostics manufacturers. When choosing a PCR assay to detect monkeypox, in silico tools can be used to predict their performance and aid selection (Table ); Supplementary Table S1 provides a detailed list of in silico-analysed targets collated by the authors; the underlying method and choice of targets are described in the Supplement.…”

“…Monkeypox virus shares genetic similarities with other viruses in the Poxviridae family, however, as these other family members are generally rare their undesired detection is not likely to impact on test specificity. Members of the Poxviridae family have very large genomes with many potential PCR targets of varying specificities: assays can range from pan- Orthopoxvirus detection [ 8 ] to specific detection of monkeypox virus [ 9 ] ( Table ); see Supplementary Table S1 for a more detailed analysis of targets. The cause of chickenpox, varicella zoster virus, which may result in clinically suspected cases of monkeypox, is an unrelated herpesvirus that should not be detectable by an Orthopoxvirus -specific diagnostic PCR.…”

Current and Perspective Sensing Methods for Monkeypox Virus

“…Multiplex detection can significantly reduce the misidentification of coexisting pathogens [ 63 , 64 ]. A multicolor, multiplex approach for MPXV detection was reported where MPXV was specifically detected in the presence of the variola virus (VARV) and the varicella-zoster virus (VZV) [ 65 ].…”

“…The detection approach is based on conventional PCR, and PCR amplicons are evaluated by Taq I RFLP patterns. In a similar line of work, a real-time PCR assay for the universal detection of orthopoxviruses was reported [ 64 ]. The system was reported to be able to detect poxviruses excluded in a previous study [ 66 ] as well as those from the subfamily Entomopoxvirinae .…”

Molecular detection of monkeypox and related viruses – Challenges and opportunities

“…Compared to smallpox, monkeypox has a much lower infectivity or human to human transmission with R0 < 1 (Learned et al, 2005;Sklenovská and Van Ranst, 2018), perhaps ≈0.576 (McMullen, 2015), and an attack rate of ≈50% (Luciani et al, 2021). While antiviral drug ST-246, a potent Orthopoxvirus egress inhibitor, can protect non-human primates from VARV or MPXV (Huggins et al, 2009), smallpox vaccine (for example, Dryvax) seems 85% effective in humans against monkeypox (Edghill-Smith et al, 2005).…”