Fish viruses: buffers and methods for plaquing eight agents under normal atmosphere

Area of Science:

• Aquatic Virology
• Fish Cell Culture
• Plaque Assay Development

Background:

• Accurate diagnosis of fish viral diseases is crucial for aquaculture.
• Existing plaque assay methods for fish viruses often require specific conditions, such as CO(2) incubators.
• A need exists for a standardized, efficient, and accessible plaque assay for a broad range of fish viruses.

Purpose of the Study:

• To develop a universal plaque assay procedure for eight significant fish viruses.
• To compare different cell culture media and buffering systems for optimal plaque assay performance.
• To identify a method that enhances assay efficiency, sensitivity, and simplifies diagnostic procedures.

Main Methods:

• Tested plaque assay procedures for eight fish viruses using five fish cell lines.
• Compared Eagle minimal essential medium with sodium bicarbonate-CO(2) buffer (Earle's salt solution) against organic buffers (Tris and HEPES).
• Evaluated two-phase (gel-liquid) media at temperatures ranging from 15 to 33°C under normal atmosphere and CO(2) enriched conditions.

Main Results:

• Five fish cell lines supported the growth of all tested viruses.
• Organic buffers, particularly tris(hydroxymethyl)aminomethane, provided greater efficiency and sensitivity compared to bicarbonate-CO(2) buffering.
• The optimized assay allowed plaquing in a normal atmosphere, eliminating the need for CO(2) incubators.

Conclusions:

• A universal plaque assay procedure using organic buffers is feasible for diagnosing multiple fish viruses.
• Tris(hydroxymethyl)aminomethane buffer offers superior performance for fish virus plaque assays.
• This method simplifies fish virus diagnostics, improving accessibility and efficiency in epizootiological studies.