B-220 Development of Reference Materials for Detection of Monkeypox

Abstract Background Monkeypox virus (MPXV) is a member of Poxviridae family and belongs to the genus of orthopoxvirus. The Monkeypox virus has a double stranded DNA genome of approximately 197 kb with almost 190 nonoverlapping ORFs. Monkeypox virus encodes all the enzymes needed for transcription and replication process of the viral genome. It has been hypothesized that progressive gene loss, primarily at the terminal end of the genome, has been the justification behind evolution of a new adaptive virus capable of attacking humans and causing serious illness. Monkeypox virus infection in human can lead to a smallpox-like illness with almost 11% fatality rate in individuals who are non-vaccinated. Monkeypox virus infects a wide range of mammalian cells without the need for specific host receptors, cell entry molecules and replication machinery. No specific approved treatment exists for Monkeypox. The symptoms could be managed by treating secondary bacterial infections, reducing symptoms, and providing supportive care. ST-246 (tecovirimat), FDA approved drug against Smallpox virus, has shown some efficacy against Monkeypox, and therefore, accurate diagnosis, usually through PCR-based testing is critical. LGC Clinical Diagnostics has developed AccuPlex Monkeypox Reference Material, which is a non-infectious, stable, and reproducible standard to aid nucleic acid testing for Monkeypox virus. Methods AccuPlex™ Monkeypox Reference Material consists of recombinant Adeno virus bearing the complete Monkeypox genes: J2L (TNF receptor gene), F8L (DNA polymerase gene), F3L (Double-stranded RNA-binding protein, inhibitor of IFN signaling), and N3R (orthopoxvirus MHC class I-like protein -OMCP). The design of the recombinant virus is based on the sequence of ON585038.1 USA strain from the recent outbreak. A digital PCR assay was developed using the E9L Non-variola (NVAR) Orthopox Generic Assay design described in Y. Li et al. /Journal of Clinical Virology 36 (2006) 194–203. Digital PCR testing using the BioRad QX-200 droplet digital PCR system was used to quantitate the viral copies/mL and guide formulation. The final product is targeted as a low positive control, approximately 2–3 times the detection limit of commercial PCR testing methods and is formulated in defibrinated human plasma. Results AccuPlex™ Monkeypox Reference Material gave an average value of 5.33E+03 copies/mL ± 6.34E+02 copies/mL by digital PCR. Along with the undiluted sample, 1:2 and 1:4 dilutions were made which were then tested on Quest Diagnostics Qualitative RT-PCR platform to verify performance and compatibility with the assay detection limits. All three samples were positively detected. Conclusion LGC Clinical Diagnostics has developed a stable, non-infectious reference material using recombinant Adenovirus technology for assays that detect Monkeypox DNA. Our study helps ensure that the control will be low positive for Monkeypox virus and could be used for monitoring the sensitivity of the assay. Data presented indicates the compatibility of the reference material with commercially available real time PCR assays. These materials may be used by labs for development, validation, training, and ongoing QC of Monkeypox detection assays and workflows.