Viability RT-qPCR to detect potentially infectious enteric viruses on heat processed berries

Berries have frequently been cited as causing gastroenteritis and acute hepatitis outbreaks due to enteric virus contamination, including human norovirus and hepatitis A virus (HAV). Model experiments were performed to evaluate the potential use of viability RT-qPCR to assess the thermal inactivation of norovirus genotype I (GI), GII, and HAV on raspberries, blueberries and strawberries. Initially, two viability markers, platinum chloride and propidium monoazide (PMAxx (TM)), were compared using thermally inactivated norovirus GI and GII suspensions. The results showed better performance of PMAxx (TM) pretreatment in discriminating native and inactivated viruses. Thus, the pretreatment was optimized in raspberry concentrates that were obtained following the ISO15216-1:2017 procedure and were artificially contaminated with norovirus GI, GII and HAV and finally heated at 60 degrees C, 72 degrees C and 95 degrees C for 15 min. Optimized viability pretreatment was further assayed in artificially inoculated blueberry, strawberry and frozen raspberry concentrates. Finally, the improved viability pretreatment was tested in inoculated raspberry and strawberry samples that were exposed to increasing thermal treatments. A complete inhibition of amplification signals was observed for norovirus GII and HAV in raspberries and for norovirus GII in strawberries, all heated at 95 degrees C for 15 min. Notably, significant differences (p < 0.05) were observed for norovirus GI and GII comparing PMAxx (TM)-RT-qPCR and RT-qPCR for all thermal treatments tested. In conclusion, although this viability procedure may still overestimate viral infectivity, the PMAxx (TM) pretreatment represents progress for improving interpretation of the quantification data obtained according to ISO15216:2017-1 in berries.