Metagenomics to identify viral indicators in the produce chain

Fresh produce is an important vector for the transmission of human pathogenic viruses like norovirus (NV), hepatitis A virus (HAV), and hepatitis E virus (HEV) to the human host. These viruses are transmitted through the fecal-oral route and cause diarrheic and hepatic diseases in the population. NVs, for instance, are the leading cause of foodborne outbreaks of acute gastroenteritis, with an estimate of 120 million cases each year. Surface irrigation water and stools (that may contaminate agricultural and produce handling facilities by food handlers) represent the main transmission sources of viruses to produce. Therefore, there is a clear need to identify microorganisms that can easily and reliably indicate the presence or absence of human pathogenic viruses along the produce chain. Current fecal indicator bacteria (FIB; i.e. enterococci and Escherichia coli) and emerging microbial source tracking (MST) methods may indicate the presence and even the likely source of bacterial contamination of water and food, but they are less effective at determining the potential risk to health from human pathogenic viruses. Bacteriophages (including F-specific RNA, somatic coliphages and Bacteroides spp. phages) are generally better predictors of human pathogenic virus persistence and environmental behavior than FIB because of structural similarities. However there is no consensus on the best phage to indicate human pathogenic viral contamination. Due to the lack of good reliable indicators, it has been suggested that searching for the target human pathogenic virus is the only way to check fecal contamination by viruses, however this is expensive, time consuming, and even difficult to interpret. Recently next generation sequencing (NGS) tools open a great opportunity to identify reliable viral indicators. The main objective of this proposal is to determine the viriome (presence and relative abundance of sequences from viruses, including phages and human pathogenic viruses) of irrigation waters samples, produce and stools (that may contaminate agricultural and produce handling facilities by food handlers) by using NGS. The proposed project aims at identifying a more significant indicator/s of the presence or absence of human pathogenic viruses in agricultural inputs, within the on-farm agricultural environment, in/on produce commodities and in produce handling facilities (similarly to the use of E. coli numbers associated with the presence of human bacterial pathogens). This insight will facilitate the development of a tool for a rapid detection and quantification of the viral indicator/s. This proposal benefits from having a large panel of stools and reclaimed and surface irrigation water samples positive for NV GI and GII, HAV and hepatitis E virus (HEV).