Orthopoxvirus pan-genomic DNA assay

Area of Science:

• Virology
• Molecular Biology
• Genomics

Background:

• Orthopoxviruses (OPVs) cause significant human diseases, including Monkeypox and Variola virus.
• Accurate and rapid laboratory confirmation of OPV infections is crucial for public health.
• Existing diagnostic methods may have limitations in speed or scope.

Purpose of the Study:

• To develop and describe a novel genome-spanning assay for the laboratory confirmation of OPV infections.
• To provide a method for differentiating between various OPV isolates, including Vaccinia, Monkeypox, and Variola viruses.
• To establish a diagnostic tool applicable to natural, recombinant, and bioengineered OPVs.

Main Methods:

• Utilized a series of overlapping polymerase chain reaction (PCR) amplicons to cover the entire OPV genome.
• Employed restriction fragment length polymorphism (RFLP) gel electrophoresis to compare PCR amplicons from different viral isolates.
• Demonstrated that viral growth is not always necessary for DNA extraction for RFLP analysis.

Main Results:

• The described assay successfully spans the approximately 200 kilobase pair linear DNA genome of OPVs.
• RFLP patterns of corresponding amplicons allow for the comparison and differentiation of viral isolates.
• The PCR step can generate sufficient DNA for RFLP analysis without requiring extensive viral cultivation.

Conclusions:

• The developed genome-spanning assay provides a robust method for laboratory confirmation of OPV infections.
• This assay serves as a valuable prelude to whole or partial genome sequencing of OPVs.
• The technique is applicable to a wide range of OPVs, including potentially hazardous bioengineered variants.