| The consumption of foods contaminated with the Norwalk-like viruses (NLVs) was recently estimated to be the leading cause of foodborne illness in the U.S. In order to address previous detection and transmission issues, the objectives of this project were to (i) develop an enzyme immunoassay (EIA) to quantitatively detect Norwalk virus (NV)-specific IgG and IgA in saliva; (ii) improve the sensitivity and speed of virus detection from foods by reverse transcription-polymerase chain reaction (RT-PCR); and (iii) investigate the stability and transfer of NV on various environmental surfaces and in foods.; Initially, the level of NV-specific antibodies in oral fluid samples was measured by EIA for 38 volunteers challenged with NV. Fifteen of 19 (79%) infected subjects showed a ≥4-fold increase in NV-specific salivary IgG and IgA antibody titers, when comparing pre-challenge saliva to saliva collected post-challenge. There were strong correlations between IgG seroconversion and ≥4-fold increases in NV-specific salivary IgA and IgG antibody titers.; Secondly, alternative RNA extraction methods and primer sets for NV were evaluated to improve the sensitivity of virus detection from non-shellfish foods. The use of TRIzol with the QIAshredder™ Homogenizer (TRIzol/Shred) yielded the best RT-PCR detection limits and the NVp110/NVp36 primer set was the most efficient for detecting NV from seeded food samples. A one-step RT-PCR protocol using the TRIzol/Shred extraction method and the NVp110/NVp36 primer set demonstrated improved assay detection limits over the routinely used two-step RT-PCR method.; Environmental persistence studies showed that NV RNA could be detected by RT-PCR on NV-inoculated stainless steel, ceramic, and formica surfaces for up to 7 days. However, RT-PCR detection of purified NV RNA was possible on these same surfaces for only 24 hr. NV inoculum could be readily transferred from stainless steel to lettuce at levels approaching 20%, although transfer efficiency was affected by variables such as constant pressure, inoculum drying time, and the presence of water.; This research will improve our ability to diagnose, detect, and investigate viral foodborne disease. Taken together, these studies will aid in the design and implementation of practical control strategies to reduce the public health risks associated with viral contamination of foods. |
