Researchers at the University of Barcelona presented this poster presentation at the Center for Produce Safety (CPS) Research Symposium 2024.
Summary
Viruses commonly associated with illness from consuming leafy greens and fresh or frozen berries include human noroviruses (HuNoV) and hepatitis A virus (HAV), which can lead to acute gastroenteritis and hepatitis, respectively.
Detecting viral contamination remains difficult due to the absence of straightforward automated methods. Moreover, new approaches are needed to confirm the infectivity of identified viral genomes. The overall goal of the project is to optimize current methods to improve efficiency, reduce turnaround time, and assess viral infectivity. This will involve refining capsid-integrity molecular assays and investigating the suitability of salivary-gland continuous cell lines to detect infectious HuNoV. Optimized procedures will be employed to gain a deeper understanding of how infectious viruses persist in leafy greens and berries throughout post-harvest storage and disinfection processes.
Objectives
1. Optimize the currently standardized methods for the recovery of infectious enteric viruses from leafy greens and berries.
2. Optimize capsid-integrity molecular assays to better estimate infectious HuNoV
and HAV.
3. Investigate persistence of infectious viruses, capsid-protected genomes, and
total genomes in lettuce and berries under different post-harvest conditions.
Methods
During the first trimester of the project, the team developed two alternative virus concentration protocols widely used in the field: aluminum hydroxide adsorption-precipitation (AlCl3), and Concentrating Pipette Select (CP, InnovaPrep). These methods’ effectiveness was assessed in comparison to the current FDA and ISO reference protocols. Leafy greens and strawberries obtained from retail were spiked at 105 genome equivalents of HAV (pHM175 43c HAV) and HuNoV GI.6 and GII.4 per 25 g of produce. Mengovirus was used as a process control virus.
The increased performance of the PMAxx viability RTqPCR assay to minimize detection of inactivated HAV (subjected to 5 min at 99ºC) was evaluated by
increasing the length of the RTqPCR amplicon approximately fourfold. This was
then compared against the FDA and ISO RTqPCR protocols.
Results to Date
Method characterization for viral extraction/concentration procedures and novel
capsid integrity PMAxx assays are presented.
Viral recoveries from four extraction/concentration methods were assessed on lettuce and strawberries for the target viruses. Results showed significantly
higher recoveries for FDA and Concentrating Pipette from leafy greens, with average recoveries for all 3 viruses of 62.5%±18.9 and 77.6%±22.3, respectively. On strawberries, FDA, AlCl3 and Concentrating Pipette methods performed better than ISO, with average recoveries of 38.8%±44.2, 37.1%±26.8, and 35.6%±18.1, respectively.
Overall, the Concentrating Pipette method shows a lower turnaround time and satisfactory recoveries for both matrices.
Combination of PMAxx with a novel RTqPCR protocol based on a long amplicon
completely reduced detection of nonviable heat-inactivated HAV, as compared to
ISO and FDA protocols.
Benefits to the Industry
The current project aims to provide the following benefits to the fresh produce processing industry:
• Optimized user-friendly Standard Operating Procedures for detection and quantification of HuNoV and HAV from leafy greens and berries, with a reduced turnaround time and independent of ultracentrifugation equipment.
• Optimized set of methods to better estimate HuNoV infectivity on contaminated samples.
• Knowledge of die-off rates of infectious HuNoV and HAV in leafy greens and berries in post-harvest scenarios to support design of effective practices.
View the Poster - Improving Methods for the Assessment of Infectious Human Enteric Virus Survival in Produce, Guix, et al. PhD University of Barcelona Enteric Virus Laboratory, 2024
