Purpose: The objectives were to concentrate, extract, detect and characterize norovirus viruses in oysters implicated in an outbreak.
Methods: Five bags of oysters were received and analyzed for NoV genogroups I and II, male specific coliphage (MSC) and coliforms. An ultracentrifugation protocol, with the inclusion of murine norovirus (MNV) as an extraction control, was used for concentration of enteric viruses from the implicated shellfish samples. For extraction and detection, Qiagen’s ® silica-based protocol and a real-time RT-qPCR assay was used, respectively. Conventional RT-PCR and big-dye terminal sequencing was used to generate amplicons for characterization of norovirus. MSC were enumerated by a double agar overlay technique while coliforms were enumerated using the APHA MPN protocol.
Results: Levels of MSC, fecal coliforms, and E. coli in the oysters were <11 PFU/100g, 9.3-14 MPN/ 100g and 2.0- 4.5 MPN/ 100g, respectively. Only NoV genogroup I was detected with an average level of 2972 RT-PCR units/100g of digestive diverticula. Extraction efficiency of MNV from oysters was 85%. Sequence analysis and genotyping of the 329 bp fragment for norovirus revealed sequence 100 % homology to NoV and was characterized as NoV genogroup I.4. Norovirus genogroup I.4 was also identified in the clinical samples associated with this outbreak.
Significance: In the absence of cell culture techniques for the propagation of norovirus, the ability to detect and characterize norovirus in foodborne associated outbreaks was an integral part of this outbreak investigation. Detecting and characterizing GI.4 in the shellfish and clinical samples created the integral link between the consumer and the implicated shellfish.