Table 3.

Methods involving live virus work have very few alternatives for performance at Biosafety level 2 (BSL-2).

Method or Assay	Alternative
Live virus quantitation: plaque assay, TCID50, focus forming or live cell assay for viral titration of a virus stock or an experimental sample	Any live virus quantification in samples must be done inside the suite. Plaque assays could be counted on photographs rather than inside the suite, but the assay must be performed entirely at BSL-4. Viral genomic burden in a sample can be measured by qRT-PCR.
Neutralization assay for antibody or chemical activity against virus	Plaque-reduction neutralization tests on native viruses must be done inside the suite. Use of BSL-2 pseudotyped or surrogate viruses, if available, could be substituted for testing antibody binding.
ELISA or plate-based assays using any reagent that is either live virus as substrate or capture antigen, or where the reagent (such as immune serum) under test could be potentially infectious	No immune serum can come out of the suite unless irradiated and safety tested. Irradiation may destroy serum proteins of interest before they can be assayed.
Flow cytometry on infected cells	Infected cells can be permeabilized and fixed in formalin and brought out for flow methods, provided that the antibody staining is not inhibited by the fixation.
Protein analysis methods	Some co-immunoprecipitation and native PAGE methods may require BSL-4 containment.
Cell culture based drug screening assays for antiviral activity	No alternative unless a pseudotyped or surrogate virus exists for use in screens.
Animal observations and veterinary assessments	No alternative, animals must be tended in person at BSL-4.
Quality Assurance audits supporting regulated (GLP) studies	No realistic alternative, most work cannot be closely audited from windows or security cameras.