Progress 04/27/15 to 03/30/20
Outputs
Target Audience:academia, federal agencies, scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Undergraduate and graduate students had the opportunity to perform research activities in the laboratory How have the results been disseminated to communities of interest?Posters were presented at the IAFP Annual meetings and Center for Food Safety annual meetings What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The Cannon group performed experiemnts to compare PGM-, broadly reactive MAb NV23- and aptamer- binding assays on ELISA plates. The purpose of these preliminary experiments wwere to (1) determine the optiaml concentration of biotinilated aptamer AP25 that will adhere to high binding microtiter plates (2) determine if RNase A addiiton would decrease nonspecific/non-boud RNA, (3) determine if NeutrAvidin coated cells are requires for aptamer binding to microtiter paltes (4) compare the detection limit of GII.4 Sydney in AP21/AP25 coated wells vs PGM coated wells; (5) and compare two GII.2 Sydney stool suspension preparations for PGM binding. A concentration range of 10 to 0.5uM of nucleic acid aptamers resulted in similar quantification cycles (Cq) values on wells with and without NeutrAvidin application. Addiiton of RNase A did not reduce non-specific or non-bound RNA in the assay. AP21, AP25, and PGM have similar binding affinity to GII.4 Nov. As the level of non-specific/non-bound RNA recovered in wells that contained PBS or carbonate bicarbonate buffer (CBS) was greater than expected, a comparison of a GII.4 with a new GII.4 Sydney preparation, SS15. The results indicate that the (12/18/13) GII.4 SYdney stool suspension showed greater affinity for PGM and less nonspecific/ non bound RNA than the newer SS15 preparation. Using medium-binding plates (rather than high-binding plates) and preparation of the virus stock by chloroform extraction were evaluated and a means to reduce non-specific binding of the virus stocks. Both methods used individually resulted in less target virus binding (higher detection limit). Experiments evaluating the porcine gastric mucins (PGM)-binding assays with RNase A treatment and RT-qPCR as a surrogate for detecting infectious was also completed by the Cannon team and was published. Treated and control virus samples were applied to 96-well plates coated with 1ug/ml PGM followed by RNase A (5ng/ul). Average log genome copies per ml (gc/ml) reductions and realtive differences in quantification cycle values after thermal treatment were 1.77/5.62 and 1.71/7.25 (RNase A) annd 1.73/5.50 and 1.56/6.58 (no Rnase A) for GI.1 and GII.4 respectively. Treatment of NoVs with 70% ethanol resulted in 0.05/0.16 (GI.1) and 3.54/10.19 (GII.4) log reductions in gc/ml and the average RD in Cq value, respectively. LV (0.5%) combined with 0.1% SDS provided a greater decrease of GI.1 and GII.4 NoVs with 8.97 and 8.13 average RD in Cq values obtained, respectively than 0.5% LV/0.01SDS. Virus recovery after PGM binding was variable with GII.4>G1.1. PGM biding is a promising surrogate for identifying infectious and non-infectious NoVs after capsid destruction; however, results vary depending on virus strain and inactivation method. The study evaluating MNV-1, HAV and GII.4 human norovirus survival on semi-dried produce and trail mix has been completed. As part of this study, the suitability of the ISO method for virus recovery and detection for semi-dried foods was evalated and he use of MNV-1 and CFV as process control viruses was also evaluated. The Ortega group worked with the surrogate Feline Calicivurus, FCV-9 obtained from ATCC. CRFK cells were propagated as recomended by ATCC and using EMEM medium with Earle's BSS, 2mM glutamine and 10% FBS, 1.5g/L sodium bicarboate, 0.1mM non-essential aminoacids, 1.0mM sodium purivate. Cells were incubated at 35C in 5% CO2 with humidity. Propagation fo the FCV was done using 2% FBS in EMEM without antibiotics. Three types of vinegars were evaluated: Four Monks Cleaning vinegar (Mizkan America, Inc) that was produced from corn (diluted w/water to 6% acidity), pH 2.0. Nakano Rice Vinegar (MIzkan America, Inc) that was made from rice and diluted with water to 4.2% acidity (42 grain) pH 3.02, and Star Italian White Wine Vinegar diluted with water and contains potasium metabisulfite as a preservative, 5% acidity, pH 3.0. The virus suspension (100ul) was incubated with 200 ul of each three types of vinegars. After 5, 10, and 15 min exposuere, the reation was stopped with F-12 meda containing 5% FBS. Serial dilutions of each treatment were then placed on CRFK cell monolayers in 24 well pates containing 800 ul RPMI 5% FBS, 1% antibiotic. The plates were incubated for 2 hrs. Supernatants were aspirated and added 800 ul of 0.75% agarose in RPMI with 5% FBS, incubated at 37C for 3 days and examined for the presence of viral CFU/ml. Non of these three-type vinegars inactivated the virus. The cleaning vinegar although not statistiaslly significant, was the only able to kill 0.7 log CFU when incubated for 15 min. The rice and wine vinegars did not inactivate the FVC even after 15 min incubation.
Publications
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?UNdregraduate students had to opportunity to learn how to work with feline calicivirus in the tissue culture laboratory. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A student was trained to propagate and maintain the feline calicivirus andCrFK cell lines. The antiviral effect of garlic (2.3g/ml)and ginger (2.7g/ml) wasevaluated. Virus and extracts were exposed for 5, 10, and 15 min. Virus suspensions were diluted andadded onto monolayers of CrFK cells and tested for the formation of plaques after 48 hr incubation at 37C 5% CO2. None of these two extracts were effective at inactivating the feline calicivirus under the tested conditions.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Academia, industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Evaluate antimicrobials used by the food industry.
Impacts
What was accomplished under these goals?
Explored three natural vegetable products and their effect on the inactivation of feline calicivirus.
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Academia, food safety scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Evaluate other natural food extracts and disinfectantson the viability of foodborne viruses.
Impacts
What was accomplished under these goals?
Feline Calicivirus , FCV-9 was obtained from ATCC (American Type Culture Collection, Manassas, VA). Cellswere propagated asrecommended by ATCC. CRFK cells werepropagated in Eagle's Minimal Essential medium with Earle's BSS, 2mM l-glutamine and 10% fbs, 1.5g/l sodium bicarbonate, 0.1mM non-essential aminoacids, 1.0 mM sodium pyruvate. Cells were incubated at 35oC in 5% CO2 with humidity. Propagation of the feline calicivirus was done using 2% FBS in EMEM without antibiotics. Vinegars:Four Monks Cleaning Vinegar, Mizkan America, Inc. This distilled vinegar was produced from corn (diluted w/ water to 6% acidity),pH 2.0. Nakano Rice Vinegar, Mizkan America, Inc. This vinegar was made from rice, and diluted with water to 4.2% acidity (42 grain), pH 3.02 andStar Italian White Wine Vinegar diluted with water and contains potassium metabisulfite as a preservative, 5% acidity, pH 3.0.The virus suspension (100 ul) was incubated with 200 ul of each of three-typevinegars. After 5, 10 and 15 min exposure, the reaction was stopped with F-12 media containing 5% FBS. Serial dilutions of each treatment was then placed on CRFK cells monolayers in 24 well plates containing 800 ul RPMI 5% FBS, 1% antibiotic.The plates were incubated for 2 hrs. After that, the supernatant was aspirated and added 800 ul of 0.75% agarose in RPMI with 5% FBS, incubated at 37C for 3 days and examined for the presence of viral CFU/ml. None of the three-type vinegarsinactivatedthe virus. The cleaning vinegar although not statistically significant, was only able to kill 0.7 log CFU when incubated for 15 mins. The rice and wine vinegars did not inactivate the FCV even after15 min incubation.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Academia, food safety scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Determine the recovery efficiencyof foodborne viruses usingholowfiber filters.
Impacts
What was accomplished under these goals?
Experiments evaluating the porcine gastric mucins (PGM)-binding assay with RNase A treatment and RT-qPCR as a surrogate for detecting infectious was published: Afolayan O.T., C.C. Webb and J.L. Cannon. 2015. Evaluation of a Porcine Gastric Mucin and RNase A Assay for the Discrimination of Infectious and Non-infectious GI.1 and GII.4 Norovirus Following Thermal, Ethanol, or Levulinic Acid Plus Sodium Dodecyl Sulfate Treatments. Food and Environmental Virology. Oct. 2015. The study evaluating MNV-1, HAV and GII.4 human norovirus survival on semi-dried produce and trail mix has been completed. A publication is currently in preparation. As part of this study, the suitability of the ISO method for virus recovery and detection from semi-dried foods was evaluated and the use of MNV-1 and FCV as process control viruses was also evaluated.
Publications
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Progress 04/27/15 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Evaluate water testing methods for recovery of MNV from surface irrigationirrigation water. The method will then be used in irrigation water samples collected water sources withhigh and lowcontamination levels.
Impacts
What was accomplished under these goals?
The Cannon group is beginning experiments to compare PGM-, broadly reactive MAb NV23- and aptamer- binding assays on ELISA plates. The purpose of these preliminary experiments were to (1) determine the optimal concentration of biotinylated aptamer AP25 that will adhere to high binding microtiter plates, (2) determine if RNase A addition would decrease non-specific/non-bound RNA, (3) determine if NeutrAvidin coated wells are required for aptamer binding to microtiter plates (4) compare the limit of detection of GII.4 Sydney in AP21/AP25 coated wells versus PGM coated wells; (5) and compare two GII.4 Sydney stool suspension preparations for PGM binding. A concentration range of 10 to 0.5µM of nucleic acid aptamers resulted in similar quantification cycles (Cq) values on wells with and without NeutrAvidin application (objectives 1-4). Addition of RNase A did not reduce non-specific or non-bound RNA in the assay (objective 2). AP21, AP25, and PGM have similar binding affinity to GII.4 NoV (objective 3/4). As the level of non-specific/non-bound RNA recovered in wells that contained PBS or carbonate bicarbonate buffer (CBS) was greater than expected, a comparison of a GII.4 Sydney stool suspension preparation previously used in PGM assays (12/18/13) was compared with a new GII.4 Sydney preparation, SS15. The results indicate that the (12/18/13) GII.4 Sydney stool suspension showed greater affinity for PGM and less non-specific /non-bound RNA than the newer SS15 preparation. Using medium-binding plates (rather than high-binding plates) and preparation of the virus stock by chloroform extraction were evaluated as a means to reduce non-specific binding of the virus stocks. Both methods used individually resulted in less target virus binding (higher detection limit).
Publications
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