Summary
Hepatitis A virus (HAV) outbreaks occur worldwide from consumption of contaminated fresh and frozen berries. Unlike bacterial pathogens like Salmonella, there are no methods for detecting “live,” infectious HAV, rather scientists rely on molecular approaches that detect the virus’ genetic material (RNA). Unfortunately, viral RNA is stable and can be detected long after the live virus has been “killed” or degraded, meaning a positive result can overestimate disease risk because it is detecting virus that is no longer infectious. Yet, these molecular methods are highly sensitive and best suited for detecting low concentrations of virus anticipated in naturally contaminated berries. We hypothesize that molecular methods can be modified to eliminate RNA from inactive virus using relatively simple enzymatic and/or chemical pre-treatments that ‘chew up or bind’ naked RNA to the exclusion of that associated with fully encapsidated infectious virus. In this study, we will optimize and compare these methods for their ability to exclude non-infectious virus from detection. The best approach(es) will then be tested against fully and partially inactivated HAV added to berries. This will provide a simple, inexpensive method to exclusively detect infectious virus that is also compatible with the existing international methods for HAV detection in berries.
Technical Abstract
Outbreaks of hepatitis A virus (HAV) have occurred worldwide from consumption of contaminated fresh and frozen berries. Competent authorities have instituted sampling and surveillance, and some large retailers are requiring testing of frozen berries for direct consumer market. While internationally validated methods are available for detecting HAV in these products, the wildtype strains present in berries cannot be laboratory-grown. Hence, these methods are reliant on the use of molecular amplification (RT-qPCR) to detect virus genome fragments. Such fragments can be stable long after the virus has been inactivated by physical or chemical means. Therefore, RT-qPCR does not have a direct correlation to virus infectivity and may overestimate virus concentration and disease risk associated with a positive test result. The central hypothesis is that the standard RT-qPCR-based methods for detecting HAV in berries can be modified to minimize detection of RNA associated with damaged viral protein capsids that are presumably non-infectious. This will allow for the continued use of highly sensitive and validated (standardized) molecular amplification, without reliance on detecting intact HAV capsid proteins directly, to effectively detect infectious HAV to the exclusion of non-infectious virus. Previous studies have evaluated the efficacy of various infectivity ‘proxies’ for the detection of non- cultivable enteric viruses, HAV and human norovirus (hNoV). Of note are enzyme-based (RNase/proteinase) and nucleic acid intercalation [e.g., propidium monoazide (PMA/PMAxx) and platinum chloride (PtCl4)] methods, both being pre-treatments to prevent RT-qPCR amplification of viral RNA arising from damaged or destroyed virus particles. Most of this work has been done on hNoV, not HAV, and has primarily focused on heat inactivated viruses, neglecting inactivation by chemical disinfectants. The purpose of this project is to develop a prototype screening (rapid) assay that can be used to detect infectious HAV to the exclusion of non-infectious virus. Three specific objectives have been identified. The first focuses on optimization of the performance of enzyme and nucleic acid intercalation-based methods (RNase/proteinase, PMA, PMAxx, and PtCl4) specifically for infectivity discrimination of HAV using RT-qPCR, compared directly to cell culture infectivity assay using the cultivatable HAV strain HM-175 18f. After correlation analyses to determine which of the optimized methods performs best, one or two of them (alone or combined) will be selected for Objective 2, focusing on evaluation of method performance using HAV that has been gradually inactivated by heat or chlorine yielding differential proportions of infectious:non-infectious virus. Thereafter, Objective 3 will evaluate the efficacy of the top-performing method(s) in a frozen berry matrix seeded with HAV (fully infectious, partially inactivated, and fully inactivated) and processed for detection using the ISO 15216 protocol for soft fruits. We anticipate delivery of an RT-qPCR-based detection method that is consistent with the ISO 15216 standard by the simple addition of one or two enzymatic or chemical pre-treatments. This method will provide greater assurance that test results are indicative of virus infectivity; be field- deployable by commercial laboratories; and have utility in providing vital data on inactivation of infectious virus, and relationship to public health risk, than what is currently available.
Research Objectives
Objective 1: Methods Optimization: Improve the performance of candidate molecular-based infectivity discrimination methods (RNase/proteinase; and PMAxx, or PtCl4 pre-treatments, in conjunction with RT-qPCR) for detection of HAV and compare their performance to infectivity assay.
Objective 2: Screening: Perform direct comparison between optimized methods and infectivity assay as applied to fully infectious HAV, and that partially and fully inactivated by exposure to heat and sodium hypochlorite.
Objective 3: Matrix Performance: Evaluate the efficacy of the top-performing method(s) in a frozen berry matrix seeded with HAV (fully infectious, partially inactivated, and fully inactivated) processed for detection using the ISO 15216 protocol.
Findings & Recommendations
This project is ongoing. A final report will be provided when the project is finished.