Plaque Reduction Neutralization Test — PRNT

Both express the serum dilution that reduces plaque counts relative to a virus-only control, but at different stringency levels. PRNT50 is the titer at which plaques are reduced by 50%; PRNT90 requires a 90% reduction and is therefore a more demanding, more specific endpoint. PRNT90 is preferred for flavivirus serology because it better discriminates type-specific from cross-reactive antibodies, though it also yields lower (less sensitive) reported titers.

ELISA is faster and higher throughput, making it suitable for initial screening. However, ELISA detects binding antibodies, not neutralizing antibodies, and is more prone to cross-reactivity among related viruses. PRNT is typically used to confirm positive or equivocal ELISA results, to resolve cross-reactive patterns, or whenever a biologically meaningful neutralizing titer is required — for example, in vaccine efficacy studies or regulatory submissions.

Vero cells (African green monkey kidney) are the most widely used cell line for PRNT because they support replication of a broad range of arboviruses, form uniform monolayers, and produce well-defined plaques. BHK-21 (baby hamster kidney) cells are commonly used for foot-and-mouth disease virus and some bunyaviruses. Primary or secondary cell cultures may be required for viruses that do not replicate efficiently in continuous cell lines.

The timeline depends primarily on virus growth kinetics. For fast-replicating viruses such as some alphaviruses, plaques may be readable in 2–4 days. For dengue and West Nile virus, 6–10 days are typical. Foot-and-mouth disease virus results can be read in 48–72 hours. Adding serum preparation, overlay removal, fixation, and counting, the practical turnaround for most veterinary PRNT applications is 5–14 days from sample receipt to final reported titer.