Evaluation of an automated ultrafiltration system as a concentration method for recovery of infectious norovirus from berries

Efstathia (Efi) Papafragkou, US Food and Drug Administration

Efstathia Papafragkou, Cameron Boerner, and Samantha Q. Wales

Food & Environmental Virology-I (Food)

Detection of enteric virus contamination in foods is mostly done with molecular based methods. The InnovaPrep® Concentrating Pipette Select (CP) technology was evaluated for the first time to concentrate infectious human norovirus recovered from fresh strawberries coupled with infection using the Human Intestinal Enteroid (HIE) system. Fifty grams of strawberries were seeded with approximately 10^6 RNA copies from a 10% stool suspension of a GII.4 norovirus strain. After 24-hour storage at refrigeration temperature, the virus was eluted from the fruits, the supernatant was filtered through a 0.22 μm pore-size filter, followed by concentration through the CP. Several parameters were evaluated in total of 47 samples including different hollow fiber polysulfone CP tips, elution buffers, and instrument settings. Viral particles collected on the filter tips were eluted with phosphate-buffered saline buffer containing Tween 20 and they were either processed to infect the HIE (J2 line) or subjected to RNA extraction, followed by RT-qPCR. Recovery efficiency, evaluated in RNA copy number, varied with an average close to 3%. The hollow instrument setting and 0.05 CP tip seemed to have an effect, while overall, exclusion of beef extract in the tris-glycine elution buffer did not improve recovery. When recovery of 25 of those samples was evaluated with virus growth in the HIE model, 7 replicated successfully. We demonstrated that the conditions tested do not interfere with norovirus infectivity in the HIE system and with additional optimization, this technology could be integrated into protocols to capture infectious norovirus from representative food matrixes.