Improving methods for the assessment of infectious human enteric virus survival in produce

Produce may occasionally become contaminated by human viruses and cause sporadic cases of disease as well as outbreaks in the community. Viruses most commonly associated with disease from consumption of produce are human noroviruses and hepatitis A virus, which may cause acute gastroenteritis and hepatitis, respectively. Detection of viruses in produce items, such as leafy greens and berries, is difficult because contamination usually occur at low numbers and because available methods show limited recovery efficiencies and provide information on contamination by viral genomes, which do not always indicate a real risk of infection. This project aims at optimizing current norovirus and hepatitis A concentration and detection methods in leafy greens and berries, to increase their efficiency, reduce their turnaround time and provide estimates of viral infectivity and risk of infection, by combining them with a recently published cell-culture system for noroviruses based on salivary-gland continuous cell lines. The optimized methods will be consistently compared to the two reference methods currently available for virus analysis in foods. Optimized methods will also be implemented at laboratory scale experiments to fill critical knowledge gaps regarding detection and persistence of infectious viruses in leafy greens and berries during post-harvest storage and disinfection scenarios.

Despite the occurrence of human enteric viruses like norovirus (HuNoV) and hepatitis A virus (HAV) in produce is documented, data are limited mainly due to the lack of rapid economically feasible methods for the analysis of viral contamination along the food chain. Understanding of the risk associated to produce contamination by viruses and the best industry practices to prevent it is hampered by the difficulty of measuring HuNoV and HAV infectivity. Detection of viral pathogens in produce is challenging due to the usual low concentrations of contaminating viruses, the low process recoveries of current extraction methods, the time required for analysis, and the reliability solely on standardized molecular detection of viral genome equivalents, which do not correspond to infectious viruses. Despite Human Intestinal Organoids (HIOs) have represented a breakthrough in supporting HuNoV replication, there are not available for a routine bases due to the high cost and dependence on proficiency skilled personnel for manipulation. Early this year, salivary gland (SG)-derived continuous cell lines have been reported to support HuNoV replication, suggesting their potential for generating a scalable and manageable system of production. Facing difficulties to detect/quantify infectious enteric viruses from foods, recent studies have also proposed the use of viability PCR-based methods for assessing capsid-integrity and estimating infectivity of enteric viruses in several types of food and water samples, but complete elimination of signals from unviable viruses has remained a challenge. In this proposal, our main aim is to optimize current HuNoV and HAV concentration and detection methods in leafy greens and berries, to increase their efficiency, reduce their turnaround time and provide estimates of viral infectivity. Two alternative virus concentration protocols based on fast procedures widely used for the concentration of viruses (aluminum hydroxide adsorption-precipitation method and InnovaPrep Concentrating Pipette) will be tested and compared to the FDA and ISO reference protocols. We also want to validate the use of SG-derived cell culture system to detect infectious HuNoV, adapt it as a novel method to determine HuNoV infectivity in produce, and answer critical knowledge gaps regarding survival of infectious virus under selected post-harvest scenarios, as compared to HAV. A correlation analysis between the SG-derived cell culture system, standardized direct RTqPCR genome quantification, and optimized capsid-integrity alternative assays will also be performed to provide information on die-off rates and infectious:GE ratios over time. An improved understanding of the efficacy of current industry practices and other alternative potential decontamination methods will help to develop interventions to minimize contamination and mitigate the risk of viral infections through the consumption of produce.

This research project aims at improving available methods for the quantification of infectious enteric viruses and identifying fundamental knowledge gaps regarding their persistence on produce. To achieve these central research objectives, we propose the following specific objectives: 

1. Optimize the currently standardized methods for the recovery of infectious enteric viruses from leafy greens and berries 

1.1. Optimize HuNoV and HAV concentration and extraction procedures, and analytical comparison with current standardized FDA and ISO protocols 

1.2. Develop a method using a salivary gland (SG)-derived continuous cell lines to detect and quantify infectious HuNoV 

1.3. Occurrence of enteric viruses in fresh produce 

2. Optimize capsid-integrity molecular assays to better estimate infectious HuNoV and HAV 

2.1. Optimize performance of PMAxx viability RTqPCR assays to improve correlation with infectivity 2.2. Develop a specific RT-Loop-mediated isothermal amplification (RT-LAMP) viability assay 

3. Investigate persistence of infectious, capsid-protected GE, and total GE HuNoV and HAV on harvested lettuce and berries under different post-harvest conditions 

3.1. Enteric virus die-off rate on berries and leafy greens in post-harvest storing conditions 

3.2. Efficacy of chlorine, chlorine and peracetic acid washes for viral disinfection of lettuce 2023 Request for Proposal 7 The performance of these objectives will be possible through the collaboration between two research groups: the Enteric Laboratory at the University of Barcelona (UB, http://www.ub.edu/virusenterics/ ) and the Environmental Virology and Food Safety lab (VISAFELab) at the Institute of Agrochemistry

This project is ongoing. A final report will be provided when the project is finished.