Objectives:
Objective 1: Develop and quantify the sensitivity of two first-level detection methods for infectious HAV on frozen strawberries and irrigation water by specific detection of viral capsid-protected RNA.
Objective 2: Replicate these two methods in separate laboratories, and validate these two methods comparing their performance against infectious HAV, inactivated HAV and free HAV RNA.
Abstract: In response to four US outbreaks associated with frozen berries contaminated with norovirus and hepatitis A virus (HAV), the US FDA began a surveillance assignment on the detection of norovirus and HAV RNA on frozen berries, including frozen strawberries. Industry was concerned with the surveillance methods because standard viral amplification methods (RT-qPCR) detect viral RNA but not infectious virus. For example, extracted viral RNA may be environmentally present and detectable by RTqPCR for weeks (our work) without an intact capsid for infection. Thus, a positive RT-qPCR signal from standard viral detection methods (ISO 15216-2:2019, FDA BAM chapter 26) does not represent an intact, functional virus, may not lead to illness, and should not be the sole basis for recalls and market withdrawals. In response, several groups proposed combining RNA amplification methods with a proxy of viral infectivity, such as a capsid integrity assay, such as RNase pretreatment. For example, RNA amplification preceded by RNase degrades free, whole, or fragmented viral RNA from partially destroyed capsids. This method may discriminate non-infectious viruses. We propose two validated methods to detect infectious HAV and discriminate non-infectious HAV, including free RNA, on frozen strawberries and irrigation water. Both methods will integrate RNase pretreatment, to detect capsid integrity, into its amplification step. Method 1 will be the standard method for HAV detection on soft fruit, ISO 15216-2:2019. Method 2 will be the same, except it will replace the RT-qPCR step with a novel, more sensitive, lower cost, and more rapid amplification step – a CRISPR-Cas HAV RNA amplification. Method 2 can be easily performed in a laboratory that already conducts HAV detection methods (RT-qPCR). Both Method 1 and 2 will be replicated in separate laboratories, validated with inactivated and infectious HAV, and statistically analyzed for their sensitivity. Throughout this one-year project, we will work with the American Frozen Food Institute and our Offices of Technology Transfer to identify rapid commercialization strategies at project conclusion. Methods 1 and 2 will impact the frozen berry industry because they will address their concerns for false associations of infectious HAV with frozen strawberries and irrigation water. Specifically, this project will support the frozen industry’s efforts to protect their consumers from HAV illness, instill consumer confidence in the frozen produce supply chain, focus recall and destruction activities on product with infectious HAV, and reduce waste from unnecessarily recalled produce. The research team has expertise in viral detection methods for foodborne viruses, including HAV, CRISPR-Cas assay development, and HAV validation studies.