Specific detection of monkeypox virus by polymerase chain reaction

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Monkeypox virus (MPV) is an orthopoxvirus with potential public health implications.
• The A-type inclusion body protein (ATI) is a key viral protein in orthopoxviruses.
• Understanding genetic variations within MPV is crucial for diagnostics and epidemiology.

Purpose of the Study:

• To sequence and analyze the open reading frame (ORF) of the ATI gene in MPV strains.
• To identify genetic markers for differentiating MPV from other orthopoxviruses.
• To develop a molecular diagnostic tool for MPV identification.

Main Methods:

• Sequencing of the ATI ORF from two MPV strains.
• Nucleotide sequence comparison with other orthopoxvirus ATI sequences.
• Design of a primer pair targeting an 8-bp deletion in the MPV ATI ORF.
• PCR amplification of a 601-bp fragment specific to MPV.
• Restriction fragment length polymorphism (RFLP) analysis using BglII.

Main Results:

• MPV ATI ORFs showed 72-95.3% homology with other orthopoxviruses.
• A consistent 8-bp deletion causing a frameshift and premature stop codon was identified in MPV ATI ORFs.
• A primer pair successfully amplified a 601-bp fragment specific to 19 MPV strains.
• RFLP analysis of amplicons confirmed MPV specificity.

Conclusions:

• The identified 8-bp deletion in the MPV ATI gene serves as a reliable genetic marker.
• A PCR-based assay utilizing this marker can accurately identify and differentiate MPV strains.
• This molecular tool aids in the surveillance and diagnosis of MPV infections.