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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR & BIOCHEMICAL DETECTION & INTERVENTION METHODS FOR BACTERIAL AND VIRAL PATHOGENS IN AQUACULTURE PRODUCTS Title: Rapid detection of hepatitis A virus and murine norovirus in hemocytes of contaminated oysters

Authors
item Dancho, Brooke
item Kingsley, David

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 17, 2010
Publication Date: July 21, 2010
Citation: Dancho,B.,Kingsley,D. 2010. Rapid detection of hepatitis A virus and murine norovirus in hemocytes of contaminated oysters [abstract].American Society of Virology.Bozeman, Montana. p.1.

Technical Abstract: The human enteric pathogens, hepatitis A virus and human norovirus, have been shown to contaminate molluscan shellfish and cause foodborne disease in consumers. Rapid viral extraction methods are needed to replace current time consuming methods, which use whole oysters or dissected tissues. In our laboratory, we showed that Eastern oyster (Crassostrea virginica) hemocytes are a site of viral persistence. Therefore, hemocytes might be utilized in screening for contaminated shellfish instead of processing oyster tissues. In this study, oysters were placed in natural seawater containing 1x106 PFU of hepatitis A virus and murine norovirus, a surrogate for human norovirus, per oyster and allowed to bioaccumulate the viruses for 16 hours at room temperature. RNA extracts were prepared from oyster hemocytes using TRI Reagent (Sigma), Dynabeads Oligo(dT)25 (Invitrogen), Illustra RNAspin Mini Kit (GE Healthcare), and RNeasy Mini Kit (Qiagen), and were compared to RNA extracts prepared from whole oysters using the GPTT protocol (Kingsley, D.H., and G.P. Richards. 2001. Appl. Environ. Microbiol. 67:4152-7). Duplex real time RT-PCR was used to detect hepatitis A virus and murine norovirus RNA in the hemocyte and whole oyster RNA extracts. We were able to detect virus-contamination in oysters with all the hemocyte extraction methods evaluated in a fraction of the time necessary to perform the GPTT protocol on whole oysters. These results suggest that the analysis of oyster hemocytes may be a rapid and useful tool to monitor shellfish safety.

Last Modified: 12/18/2014