Abstract
BackgroundOrthopoxvirus (OPV) and Parapoxvirus (PPV) have been associated with worldwide exanthematic outbreaks. Some species of these genera are able to infect humans and domestic animals, causing serious economic losses and public health impact. Rapid, useful and highly specific methods are required to detect and epidemiologically monitor such poxviruses. In the present paper, we describe the development of a nested-multiplex PCR method for the simultaneous detection of OPV and PPV species directly from exanthematic lesions, with no previous viral isolation or DNA extraction.Methods and ResultsThe OPV/PPV nested-multiplex PCR was developed based on the evaluation and combination of published primer sets, and was applied to the detection of the target pathogens. The method showed high sensitivity, and the specificity was confirmed by amplicon sequencing. Exanthematic lesion samples collected during bovine vaccinia or contagious ecthyma outbreaks were submitted to OPV/PPV nested-multiplex PCR and confirmed its applicability.ConclusionThese results suggest that the presented multiplex PCR provides a highly robust and sensitive method to detect OPV and PPV directly from clinical samples. The method can be used for viral identification and monitoring, especially in areas where OPV and PPV co-circulate.
Highlights
Orthopoxvirus (OPV) and Parapoxvirus (PPV) have been associated with worldwide exanthematic outbreaks
Several species included in these genera are related with worldwide acute exanthematic disease in humans and domestic animals, which cause serious economic losses and impact public health [1,2]
There are three zoonotic OPV species known, Monkeypox virus (MPXV), Cowpox virus (CPXV) and Vaccinia virus (VACV), and their presence is associated with an increased number of outbreaks in Africa, Europe, South America and Asia [3,4,5,6]
Summary
Multiplex PCR setting and sensitivity tests The OPV/PPV multiplex PCR was designed based on computer simulation of different combinations of several published primer pairs, using software available online [19]. The nested-multiplex was able to detect OPV and PPV DNA until reactions in which there was 1 ng of vgf or b2l genes. Nested-multiplex applicability tests: clinical samples from exanthematic outbreaks Vesicle contents and dried scabs from cattle udders and milkers' hands were collected during Brazilian BV outbreaks or from sheep and goats during CE outbreaks. This collection was accomplished using 1-ml insulin syringes, 0.45 mm×13 mm needles, and cotton swabs or a pair of forceps. A total of 64 clinical samples were collected and submitted to OPV/PPV nested-multiplex PCR (Table 2). Among the BV clinical samples, the OPV/PPV nested-multiplex PCR detected OPV DNA in 53 scabs/vesicles (94.4%). No co-infection case was detected in this molecular screening
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
