A rapid Orthopoxvirus purification protocol suitable for high-containment laboratories

Abstract

Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron^®. Genetron^® is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new Orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield.

Keywords: High-containment; Monkeypox; Orthopoxvirus; Purification; Variola.

Fig. 1

Electron Microscopy images of (A–C) Genetron^® purified samples and (D–F) Benzonase^® purified samples. Figures shown representative images of (A and D) the purity of the preparations, (B and E) image of aggregates, and (C and F) an individual Variola virus particle.

Fig. 2

Western blot of cellular membrane protein contaminants: Though both methods of purification appear to produce similar levels of cellular contaminants, the nature of the contaminants are markedly different. The Benzonase^® purification method consists primarily of nuclear membrane proteins with a very slight amount of mitochondrial contamination, while the Genetron^® purification method contaminants are comprised primarily of plasma membrane proteins with some cystolic contaminants and varying mitochondrial contaminants.