Optimizing methods for the detection and quantification of infectious human norovirus from fresh berries using human intestinal enteroids

Consuming berries is part of a healthy diet; however, berries are occasionally implicated in viral foodborne outbreaks. Berries can become contaminated with viral pathogens that originate in human feces. Sources of berry contamination are numerous and include unhygienic food handlers, the use of unclean water, and/or unclean food-contact surfaces. Human norovirus and hepatitis A virus are the most implicated culprits in berry-associated outbreaks. Both of these viruses can cause acute gastrointestinal diseases, with chronic complications and potentially death in vulnerable populations. Detection of viral pathogens in food, such as berries, is complicated by the fact that viruses are usually present in low numbers and the recovery efficiency of current methods is low. In addition, detection of these viruses by standard methods relies on detecting pieces of viral RNA, which does not provide information on the infectivity of the virus. Recent advances in human norovirus cell culture allow for the detection of infectious noroviruses. This project will harness the latest technology in norovirus cell culture to fill critical knowledge gaps regarding detection and persistence of infectious viruses in berries. The early detection of these viruses in berries will provide better protection for consumers, avoid product recalls, and boost confidence in consuming safe berries.

Foodborne viruses, such as human norovirus (HuNoV) and hepatitis A virus (HAV), are responsible for the majority of foodborne outbreaks associated with berries. Identification of these outbreaks relies on epidemiological investigations; however, these viruses are often not isolated from implicated berries. The current FDA standard method for HuNoV and HAV detection in berries relies on detection of small pieces of viral RNA by molecular assays (realtime RT-qPCR). Historically, HuNoV lacked a permissive cell line to estimate its infectivity; in contrast HAV can be adapted to replicate in cell culture such as the FRhK-4 cells (rhesus monkey kidney epithelial cells). A recent breakthrough in HuNoV cell culture allowed the detection of infectious norovirus. This cell culture system is derived from human stem cells that are grown as 3D human intestinal enteroids (HIE), forming mini-guts with similar morphology and functions to the human gut. Therefore, in this project, we propose to optimize the various steps of the FDA standard method for recovery of infectious HuNoV and HAV from berries using HIE and FRhK-4 cells, respectively. The optimized method’s detection limit, recovery efficiency, as well as the relationship between viral RNA or Ct values and infectivity, will be determined for both viruses using various berries (blueberry, blackberry, raspberry and strawberry). The optimized method will be used to investigate the persistence of infectious HuNoV and HAV on various berries under postharvest conditions. Using the novel cell culture system for HuNoV, this project is uniquely designed to optimize the FDA standard method for detection of infectious HuNoV and HAV from various berries and to generate the necessary log-reduction data for future QMRA studies.

1. Optimize the FDA-based method for the detection of infectious HuNoV from various berries using HIE, establish the method’s detection limit for infectious HuNoV from various berries, and determine the relationship between infectious HuNoV and viral RNA copies recovered from various berries. Tulane virus (TV; a norovirus surrogate) and HAV will be initially used to optimize the method. 

2. Determine the persistence of infectious HuNoV and HAV on various fresh berries under postharvest conditions.

Human norovirus (HuNoV) and hepatitis A virus (HAV) are responsible for a number of foodborne illness outbreaks associated with berries. The FDA/BAM chapter 26 and ISO 15216 standardized methods consist of three main steps that basically allow the elution, separation and concentration of viruses from berries. The final detection of viruses is achieved by RT-qPCR which quantifies viral RNA through cycle threshold (Ct) values. These Ct values are inversely proportional to the amount of viral RNA; however, their relationship to virus infectivity is not well delineated. A recent breakthrough allowed the detection of infectious HuNoV by using 3D human intestinal enteroids (HIE).  In this project, it was found that the first step of the FDA/BAM method, i.e. virus elution, is the most critical step that affected recovery of infectious virus from strawberries. By lowering the elution buffer beef percentage and pH, higher recovery for infectious Tulane virus, a surrogate for human norovirus, was achieved (~75%). The RTqPCR Ct limit of detection of the FDA/BAM method was found to be similar to the ISO 15216 method for both hepatitis A virus and Tulane virus, ranging from 35-36 and 37-38, respectively. Whether using the BAM or ISO methods, infectious HAV and Tulane virus are less likely to be directly recovered from strawberries when the RT-qPCR Ct >36. Furthermore, Ct >40-44 is less likely to predict the presence of infectious viruses per 50 g of strawberries. On day 7 of strawberries stored at 4°C, infectious HAV was significantly reduced by ~0.6 log, while HuNoV was not detected by HIE. Overall, results from this study provide better insights into the interpretation of virus Ct values obtained from strawberries whether using the BAM or ISO methods and enhance our knowledge into infectious virus persistence on strawberries stored under refrigeration.