Optimizing human coronavirus OC43 growth and titration.

Area of Science:

• Virology
• Infectious Diseases
• Molecular Biology

Background:

• Coronaviruses, including SARS-CoV-2, necessitate specialized biosafety containment, limiting research accessibility.
• Less pathogenic human coronaviruses (HCoV), such as HCoV-OC43, offer safer alternatives for study in standard laboratories.
• Standardized protocols for propagating and titering HCoV-OC43 are lacking, hindering research progress.

Purpose of the Study:

• To determine optimal conditions for propagating HCoV-OC43, a surrogate for SARS-CoV-2.
• To evaluate different cell lines and titration methods for efficient HCoV-OC43 stock generation.
• To establish standardized, high-yield protocols for HCoV-OC43 research.

Main Methods:

• Tested five cell lines (MRC-5, Huh7.5, Vero, HCT-8, HRT-18) for HCoV-OC43 propagation.
• Assessed the impact of temperature (33°C vs. 37°C), serum concentration, and sonication on viral yields.
• Compared four titration methods (plaque assay, TCID50-CPE, TCID50-IFA, TCID50-IPA) using VeroE6 and HRT-18 cells.

Main Results:

• Physiologically relevant MRC-5 cells and rapidly growing HRT-18 cells yielded high viral titers.
• Culturing at 33°C (upper respiratory tract temperature) significantly improved viral yields compared to 37°C.
• The TCID50-IPA method using HRT-18 cells proved most sensitive, rapid, and cost-effective for titration, achieving titers of 10^8 TCID50/ml.

Conclusions:

• Optimized protocols using specific cell lines, temperature, and titration methods enable high-titer HCoV-OC43 stock production.
• These findings provide standardized tools for researchers studying HCoV-OC43 as a surrogate for more pathogenic coronaviruses.
• The developed methods facilitate broader research into human betacoronaviruses in standard laboratory settings.