Sensitive genotyping of foodborne-associated human noroviruses and hepatitis A virus using an array-based platform

Authors: Quinones, Beatriz; Lee, Bertram; MARTINSKY, TODD - Arrayit Corporation; Yambao, Jaszemyn; HAJE, PAUL - Arrayit Corporation; SCHENA, MARK - Arrayit Corporation

Submitted to: Sensors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/2017
Publication Date: 9/20/2017
Citation: Quinones, B., Lee, B.G., Martinsky, T.J., Yambao, J.C., Haje, P.K., Schena, M. 2017. Sensitive genotyping of foodborne-associated human noroviruses and hepatitis A virus using an array-based platform. Sensors. 17(9):2157. https://doi.org/10.3390/s17092157.
DOI: https://doi.org/10.3390/s17092157

Interpretive Summary: Human noroviruses (NoV) are a leading cause of foodborne illness and are responsible for over half of the foodborne outbreaks in the United States. NoV are a genetically diverse group of single-stranded RNA, non-enveloped viruses in the Caliciviridae family and can be transmitted through person-to-person contact, contact with aerosolized vomitus from an infected person, fomites, or fecal contamination of water or food. Foodborne transmission vehicles include vegetable row crops, fruits, mollusks and ready-to-eat foods. The molecular characterization of the NoV complete capsid sequences led to the six genogroup classification, and in particular, genogroups I and II are responsible for most human illness. Another important foodborne enteric virus is hepatitis A virus (HAV), which are a small non-enveloped spherical virus in the family Piconoroviridae with a single positive strand of RNA. There are six genotypes, of which genotypes I and III are the most prevalent among human isolates, and subtype IA is the most common worldwide. HAV can be transmitted by person-to-person contact and through consumption of contaminated water or foods. Foodborne HAV outbreaks have been associated with ready-to-eat foods, shellfish, and produce such as lettuce, green onions and berries. Although the incidence of HAV is low in developed countries, there has been a recent emergence of HAV outbreaks associated with the increasing trade of food products. Although the incidence of laboratory-confirmed illnesses by leading foodborne pathogens has remained stable over the past decade, there is still a critical need for the development of advanced technologies to accurately identify and determine the causative agent. Specifically, improved methods that can rapidly and accurately detect and characterize virulent strains would be highly relevant and valuable for use in surveillance and attribution. Molecular-based technologies, such as DNA microarrays, offer a viable alternative to screen multiple markers simultaneously. The use of these multiplex methods will enable the identification of virus genotypic profiles, and this information will assist in the implementation of measures for prevention and recognition of transmission routes.

Technical Abstract: The viral pathogens, human norovirus (NoV) and hepatitis A virus (HAV), are significant contributors of foodborne associated outbreaks. To develop a typing tool for foodborne viruses, a focused, low-density DNA microarray was developed in conjunction with a rapid and high-throughput fluorescent method. Genotype specific probes (25-mer) were designed to target the NoV variable genomic region C at the 5’-end of ORF2, encoding the major capsid in both NoV genogroup I (GI) and genogroup II (GII). Additional capture probes were designed to target the VP1/P2A junction region at the end of the capsid region and beginning of the non-structural proteins in HAV genotypes IA and IB. Validation experiments indicated that signals specific for the tested genotypes, belonging to NoV GI and GII, were on average 200-times higher than those detected for non-targeted genotypes. The specific fluorescent signals detected for probes targeting HAV genotypes IA and IB were 38 times higher than those detected for other probes included on the array. To assess the sensitivity of the detection method, the hybridization efficiency of a probe to its intended target was assessed by hybridizing various known amounts of in vitro cRNA transcripts on the array, and the results indicated that the use of microarrays has a potential detection limit of <10 transcript copies for NoV GII strains. Our findings led us to conclude that the use of low density array-based method enabled the accurate, rapid, and sensitive detection of NoV and HAV genotypes, associated with foodborne illness. This foodborne virus typing assay has proven to be a practical method that can be implemented for routine pathogen surveillance and for the identification of risk factors in pre- and post-harvest environments.