Challenges to Develop a Detection Method for Hepatitis A Virus of Culture or Clinical Origin from Frosting Containing Berries

Introduction: The number of reported outbreaks caused by foodborne viruses and more specifically human noroviruses and hepatitis a virus (HAV) has been on the rise. However, even though the means of transmission can be identified as foodborne, the virus cannot always be isolated from the suspected food vehicle.

Purpose: Berries have frequently been associated with HAV outbreaks worldwide. A study was undertaken to detect HAV from artificially contaminated strawberries in a cake frosting mix.

Methods:  HAV was used as inoculum from either purified cell culture lysate or clinical samples to seed a 30 g of strawberries-frosting mix sample. HAV was eluted with a 0.1 M Tris-HCl, 0.05 M glycine, 1% beef extract, pH 9.2 (TGBE) containing 2% Polyvinyl Pyrrolidone (PVP). After a brief spin, the eluate was combined with chloroform to separate the fats from the frosting, and after a second re-extraction was precipitated with 10% polyethylene glycol overnight. The next day the pellet was washed with chloroform-butanol, the virus was eluted with TGBE buffer and concentrated with a second PEG precipitation. Finally, virus RNA was isolated from the pellet with a commercial kit and detected with a real-time RT-PCR.

Results: HAV could be detected at a level of seeding of at least 10⁴ PFU of purified cell culture lysate per 30 g of sample. When the frosting-strawberries mix was inoculated with a clinical isolate of HAV, recovery was achieved at a low contamination level of less than 200 RNA copies per 30 g sample.

Significance: We developed a rigorous methodology to isolate HAV in a strawberries-frosting mix, that also provides insight into useful steps for reducing the inhibitory effect of polyphenolic and fat substances often present in produce and produce-related food items, with the aim of providing a tool for critical response during disease outbreaks.