Optimized Buffer for Preservation of Hepatitis E Virus During Freeze-Thaw Cycles.

Hepatitis E virus (HEV) is a zoonotic pathogen responsible for approximately 20 million infections annually worldwide. The lack of robust cell culture systems and the absence of approved antiviral therapies have hindered HEV research and drug development. A major technical challenge is the rapid loss of viral infectivity during freeze-thaw cycles following virus purification. Here, we describe a simple and reproducible method to preserve HEV infectivity during storage. We systematically evaluated the effects of salt, serum, and sucrose on viral stability under freezing conditions. We identified an optimized buffer containing 2% fetal bovine serum (FBS), 150 mM NaCl, and 7% sucrose, which significantly maintained the infectivity of non-enveloped HEV (nHEV) and quasi-enveloped HEV (eHEV) following freeze-thaw cycles based on immunofluorescence. The buffer also demonstrated good stability across three independent repeat infection experiments. This protocol provides a practical and scalable approach for maintaining HEV infectivity and will facilitate HEV-related virological studies. Key features • Optimized buffer formulation for preserving HEV infectivity during freeze-thaw cycles. • Compatibility with nHEV and eHEV. • Simple and low-cost formulation using FBS, NaCl, and sucrose. • Downstream validation using an infectivity assay.