Orthopoxvirus detection in environmental specimens during suspected bioterror attacks: inhibitory influences of common household products

Abstract

After terrorists attacked the United States in 2001, the appearance of letters and other objects containing powdery substances with unknown potentials for biological threat focused attention on the speed, sensitivity, and reliability of diagnostic methods. This study summarizes the abilities and limitations of real-time PCR, electron microscopy (EM), and virus isolation when used to detect potential bioweapons. In particular, we investigated the inhibitory influences of different common household products present in environmental specimens on PCR yield, EM detection, and virus isolation. We used vaccinia virus as a model for orthopoxviruses by spiking it into specimens. In the second part of the study, we describe modifications of diagnostic methods to overcome inhibitory effects. A variety of PCR amplification enhancers, DNA extraction protocols, and applications of internal controls were evaluated to improve diagnostic simplicity, speed, and reliability. As a result, we strongly recommend using at least two different frontline techniques in parallel, e.g., EM and PCR. A positive result obtained by any one of these techniques should be followed by a biological method to confirm the putative diagnosis. Confirmatory methods include virus isolation followed by an agent-specific immunofluorescence assay to confirm the presence of replication-competent particles.

FIG. 1.

EM diagnosis of VACV particles. Comparison of virus morphologies after incubation of VACV with different household products. (a) Typical appearance of VACV: brick-shaped with short surface projections. Virion with unstable membrane in combination with washing detergent (b) or with covered surface structures in combination with coffee powder (c); both combinations make it very difficult to differentiate between possible virus particles and background structures. Negative staining with 1% uranyl acetate. Bar = 100 nm.