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<p>Overall goal is to improve food safety by developing novel detection, prevention and control strategies for human noroviruses and hepatitis a viruses.Objectives:Develop new methods for recovering and detecting human norovirus and Hepatitis A virus from food, water and environmental surfaces and to share this information with the scientific community.Explore the use of electro-separation using a small pore size filter to separate and concentrate foodborne viruses from food matrices.Investigate magnetic spheres coated with histo-blood group antigens as a means of concentrating and purifying infectious human noroviruses from environmental matrices.Expand upon a communication platform for foodborne virus researchers and practitioners.Investigate the survival of human norovirus and hepatitis A virus on hands and environmental surfaces in healthcare facilities, on dried produce, and water.Evaluate the environmental persistence of human noroviruses on hands and environmental surfaces in healthcare facilities during active outbreaks.Explore differences in norovirus and hepatitis A virus survival on dried produce over time.Determine the efficacy of disinfectants used in healthcare facilities for controlling outbreaks human norovirus.Explore naturally-derived liquid antimicrobials effective against human norovirus and hepatitis A virus when used alone or in combination with physical inactivation treatments such as hydrostatic pressure processing (HPP) or pulsed-light or radio frequency (RF) heating.Evaluate the efficacy of chemical and physical virucidal treatments that can be used by healthcare and food industries for inactivating human norovirus and hepatitis A virus.</p>
<p>Viruses, cells and plaque assays: Human norovirus obtained from a NoV virus positive stool specimens (obtained from Jan Vinjé at National Calicivirus Laboratory at the CDC, Atlanta, GA) suspended in a 10% PBS will be used for experiments using human NoV. Viral RNA will be extracted by a Qiagen viral RNA mini kit will be stored at -80°C until use. The cell-culture adapted strain of HAV (HAV HM175; ATCC#VR-1541) will be cultured in FRhK-4 (fetal rhesus kidney) (ATCC#CRL-1688) cells and quantified by plaque assay as previously described (22) with some modifications. MNV (a gift of Dr. Skip Virgin, Washington University School of Medicine, St. Louis, MO) will be cultured in RAW 264.7 (ATCC# TIB-71) cells and quantified by plaque assay as described previously (3). Tulane virus (a gift of Jason Jiang, Cincinnati Children's hospital, Cincinnati, OH) will be cultured in LLC-MK cells as described previously. Briefly, HAV, MNV and TuV will be cultured in ~80-90% confluent monolayers of cells at a multiplicity of infectionof 0.1-0.01. After allowing virus propagation for 1 week for HAV or 48 hrs for MNV and TuV at 37°C in a 5% CO2 environment, cells will be subjected to 3 freeze-thaw cycles and virus stocks will be obtained from cell culture supernatants. To quantify HAV, MNV and TuV eluted from surfaces or food samples, or the cell-culture derived virus stocks, cells grown to ~80-90% confluence in 60 mm petri dishes will be inoculated with 200 µl of sample eluant, or a dilution of the virus stock. After an incubation for 1 hr at 37°C with gentle rocking every 15 min, 5 ml of 1% agarose overlay medium (containing Eagles MEM and 1% agarose LE) will be added to each petri dish and incubated for 7 days for HAV or 48 hrs for MNV at 37°C in a 5% CO2 environment. Then a second layer of 1% agarose overlay media containing 0.75-1% neutral red will be added for plaque visualization. Plates containing 5-100 plaques will be used to determine the virus titer in plaque forming units (PFU).Quantitative RT-PCR: The number of RNA copies will be calculated based on real-time RT-PCR detection of human NoV and MNV RNA compared to a standard curve, generated by in vitro transcription of NoV and MNV partial or full length clones (generous gifts from Jan Vinjé, CDC, Atlanta and Ralph Baric, UNC-Chapel Hill, NC) The following sets of primers and probes will be used: For NoV virus, COG 1F CGY TGG ATG CGI TTY CAT GA; COG 1R CTT AGA CGC CAT CAT CAT TYA C; RING 1A FAM-AGA TYG CGA TCY CCT GTC CA-BHQ (14); for MNV G54763F TGA TCG TGC CAG CAT CGA, G54863R GTT GGG AGG GTC TCT GAG CAT, G54808 FAM-CTA CCC ACC AGA ACC CCT TTG AGA CTC-BHQ (CDC, unpublished) and (Forward primer: 5'-TTGCAGGAGGGTTTCAAGATG-3') (Reverse primer: 5'- 200 CACGGTTTCATTGTCCCCATA-3') (Probe: 5'-FAM-TGATGCACACATGTGGGA- 201 NFQ-3') for Tulane virus (unpublished). A Quantitect Probe kit (Qiagen) will be used with the ABI Step One real-time platform for both viruses.PGM binding assay: Amine-coated magnetic spheres (Dynabeads, Life Technology) will be washed 3 times in PBS/tween (0.05% Tween-20) before adding 100 µl of Porcine Gastric Mucin (Sigma) (1% solution prepared 1 day in advance) in 5% Blotto/tween (0.05% Tween-10) for 1.5 hr at room temperature. After 3 washes with PBS/tween, NoV or TuV (100 µl/well) will be added to replicate wells and incubated for 2 hr at 37°C. The virus bound samples will then be washed 5 times with PBS/tween followed with PBS before resuspension of the virus bound beads in 50 µl PBS. Viral RNA will be extracted by, heat release at 99°C for 5 min and recovered from sample supernatants. Quantitative detection will be achieved by real-time RT-PCR and comparison to a standard curve.</p>