Single Lab Validation of Dead-end Ultrafiltration for Isolation of Hepatitis A Virus and Human Norovirus in Agricultural Water and RT-qPCR Detection

Christine Yu, U.S. Food and Drug Administration

Qianru Yang, Michael Kulka Division of Food and Environmental Safety, Virology and Parasitology Branch, Office of Applied Microbiology and Technology, Office of Laboratory Operations and Applied Science, Human Foods Program, U.S. Food and Drug Administration, Laurel, MD 20708

Food & Environmental Virology-I (Food)

Fresh produce is a leading vehicle for foodborne outbreaks. Agricultural water, such as irrigation water, is considered a potential route for enteric virus contamination. Currently, there are no regulatory methods for the sampling and detection of viruses in agricultural water. This study aimed to conduct a single-lab validation of Dead-end Ultrafiltration (DEUF) method for the isolation of hepatitis A virus (HAV) and human norovirus from surface water, utilizing RT-qPCR detection. Fecal suspension from two norovirus strains (genogroup I and II) and a cell culture lysate of a HAV strain (HM175/18f) were spiked into 10L of surface water. A murine norovirus strain (MNV-1) was spiked as a process control. For each target, three levels of spiking were tested representing the high, fractional, and zero detection levels. ElisioTM-25H filters were used for ultrafiltration, which was followed by virus concentration, RNA extraction, and RT-qPCR detection. To reach 100% detection rate, high levels of viruses were spiked at 5.68E+06 genome copies (gc) for HAV, 1.38E+06 gc for norovirus GI, and 1.91E+06 gc for GII. The fractional level was set at 4.88E+05 gc for HAV, 1.26E+05 gc for norovirus GI, and 1.20E+05 gc for GII, which generated a detection rate of 51.4%, 59.1%, and 64.0%, respectively, in 20 replicates. The limit of detection (LOD50) for HAV, norovirus GI, and GII was 4.56E+05, 9.77E+04, and 8.14E+04 gc/10L, respectively. This study provided an extension of the DEUF method for recovery of enteric viruses in surface water, supporting the FDA’s efforts to ensure the safety of fresh produce.