Short communicationInactivation of orthopoxvirus for diagnostic PCR analysis
Introduction
Accurate and timely diagnostics of viral infections include a variety of PCR and genotyping methods. Many diagnostic laboratories now utilize automated sample extraction systems such as MagNA Pure (Roche) or other. A number of human virus pathogens require high-biological containment for targeted research and virus cultivation, whereas equally high containment is generally not required for untargeted laboratory work, such as common diagnostic procedures. However, inactivation of possible hazardous virus in patient samples is preferred before performing diagnostic PCR; for example if an infection is suspected to include a potential bioterror agent. The majority of such virus includes enveloped RNA virus (e.g. members of the Flavivirus, Filovirus, Bunyavirus and Alphavirus genera) but also a few enveloped DNA virus (e.g. Orthopoxvirus) (Mahy, 2003, Whitley, 2003). Among the Orthopoxvirus genus the major human pathogens include Variola virus, Vaccinia virus, Monkeypox virus and Cowpox virus (Esposito and Fenner, 1996). In cases where inactivation is desirable this should be completed at earliest possible time, preferably at the time of sample collection (bed-side).
Although it is assumed that nucleic acid extraction inactivates the viral pathogens, surprisingly little documentation is available. Only one study has been published demonstrating inactivation of pathogenic RNA virus using two commercially available lysis buffers; AVL (Qiagen) and TRIzol® (Invitrogen) (Blow et al., 2004). Here, it is shown that two species of Orthopoxvirus (Vaccinia virus and Cowpox virus) are efficiently inactivated by a commercially available Roche MagNA Pure lysis/binding buffer. This buffer contains both strong detergent (20% Triton X-100) and chaotropic salt (>25% guanidine isothiocyanate).
Inactivation reagents containing detergent and chaotropic salt are toxic and thereby problematic in cell culture experiments. Their cytotoxic effect on target cells often appears visually indistinguishable from virus-mediated cytopathic effect (CPE) in dilutions less than 102 to 104. As a result the presence of residual infectious activity cannot be excluded in high-tittered samples. Using the relatively large orthopoxvirus as model for variola, a method was developed in which the cytotoxic effect of the lysis buffer is eliminated before applying onto the CPE assay target cells.
The results support the finding of Blow et al. (2004) that demonstrated inactivation of enveloped RNA virus, and provides documentation for inactivation of orthopoxvirus. The use of inactivation buffers for safe bed-side inactivation of patient samples should be considered in extraordinary cases where high containment would otherwise be required.
Section snippets
The virus isolates
The following human pathogens was included belonging to the Orthopoxvirus genus. Vaccinia virus was selected as a model for variola. The Western Reserve strain was expanded to titers greater than 1 × 109 PFU/ml. The cowpox virus (CoPV/Den98) was isolated from a Danish patient (Christensen et al., 2005) and was cultivated to titers of >1 × 105 PFU/ml.
Assay for cytopathic effect (CPE)
Vaccinia and cowpox assays were performed in Vero cells cultivated in MEM with Hanks’ salts, 10% FCS, 100 U/ml pen. strep. and 10 mM Hepes. In some
Inactivation of vaccinia virus and cowpox virus by commercially available lysis/binding buffer
The results of the orthopoxvirus inactivation experiments are shown in Table 1. Initial experiments showed an apparent inactivation of vaccinia virus between 4 × 108 and 2.1 × 109 pfu/ml. However, cytotoxic effect of the inactivation reagent was observed in dilutions ≤104 (Fig. 1A) that is difficult to distinguish from virus-mediated cytopathic effects. To eliminate the cytotoxic effect a 1-h high-speed (20,000 × g) centrifugation step was included into the protocol to pellet any infectious material
Discussion
In the absence of centrifugation the cell cultures were affected by the cytotoxicity of the inactivation reagent in dilutions 101 to 104, as measured by MTT assay (Fig. 1A). The data show that the cytotoxic effect of the inactivation agent is eliminated by applying the centrifugation protocol (Fig. 1B). Before centrifugation we added excess volumes of media ensuring that any differences in liquid density between inactivation buffer and media are minimal. Upon centrifugation small uniform
Acknowledgements
We thank Laurids Siig Christensen for providing the Cowpox isolate (CoPV/Den98) and Ms. Anne Lyhning Jensen and Maria Nørtoft for technical assistance.
References (7)
- et al.
Virus inactivation by nucleic acid extraction reagents
J. Virol. Methods
(2004) An overview on the use of a viral pathogen as a bioterrorism agent: why smallpox?
Antiviral Res.
(2003)Smallpox: a potential agent of bioterrorism
Antiviral Res.
(2003)
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