Detection of highly pathogenic orthopoxviruses (Poxviridae: Chordopoxvirinae: Orthopoxvirus) by dot immunoassay in a high-containment biological safety laboratory

Introduction. Highly pathogenic for humans orthopoxviruses (OPV) – Variola virus (VARV) and Monkeypox virus (MPXV) can cause systemic diseases characterized by high contagiousness and often leading to death. In previous publications (https://doi.org/10.3390/v14112580), we reported on the development of a sensitive, rapid and easy-to-use immunochemical test potentially suitable for use in the infection foci and in laboratory conditions with a high level of biosecurity. The prototype of the diagnostic kit was tested on non-pathogenic and low-pathogenic for humans OPV and specifically detected them with a sensitivity within the range of 10³ to 10⁴ PFU/mL.

The aim of this work was to evaluate the sensitivity of this assay in detecting highly pathogenic for humans MPXV and VARV, as well as the applicability of the assay in a laboratory with a high level of biosafety (BSL-4).

Materials and methods. The efficiency of virus detection in cryolysates of CV-1 cell culture samples infected with VARV and MPXV was assessed using a one-step dot immunoassay method in a laboratory with biosafety level BSL-4.

Results. It was shown that the one-step dot assay allows detection of MPXV at a concentration of 2.5 × 10³ PFU/mL, and VARV at a concentration of 1.0 × 10⁴ PFU/mL. It was noted that vapors from disinfectants (hydrogen peroxide/formaldehyde) applied in biosafety cabinet decontamination may interfere with assay performance and affect result interpretation.

Conclusion. The one-step dot immunoassay can be performed in a BSL-4 laboratory. When limiting the contact of the detecting system with formaldehyde vapors, the sensitivity of the test for detection of VARV and MPXV falls within the previously declared range of 10³–10⁴ PFU/mL.