Purpose: The objectives were to concentrate, extract, detect and characterize norovirus (GI, GII, and GIV), Aichivirus, adenovirus, enterovirus, hepatitis A (HAV) and hepatitis E viruses (HEV) in cooked and uncooked individually quick frozen (IQF) breaded oysters implicated in an outbreak. Male-specific coliphage (MSC), a municipal sewage indicator, was also enumerated.
Methods: IQF breaded oysters were analyzed, pre- and post-cooked, for norovirus genogroups (I, II, IV,) enteroviruses, adenoviruses, Aichivirus, HAV, and HEV. Ultracentrifugation was used for concentration of enteric viruses from implicated shellfish samples with murine norovirus (MNV) as an extraction control. Qiagen protocols and RT-qPCR/PCR assays were used for RNA/DNA extraction and detection. Conventional RT-PCR or RT-qPCR/qPCR and big-dye terminal sequencing was used to generate amplicons for characterization. MSC was enumerated by a double agar overlay technique.
Results: Levels of MSC in the uncooked oysters were 110 PFU/100 g. Norovirus GI, GII, GIV, enteroviruses, adenovirus, Aichivirus, and HEV were detected in uncooked oysters with average levels of 3129, 18296, 1835, 194, 877, 35780, 2160 RT-PCR/PCR units/100 g of digestive diverticula (DD), respectively. HAV was not detected. Levels persisted in post-cooked oysters for GI, GII, GIV, enteroviruses, adenovirus, Aichivirus, and HEV with averages of 824, 1687, <10, 234, 704, 6370, and 1429 RT-qPCR/qPCR units/100 g of DD, respectively. The average extraction efficiency of MNV was 68%. Sequence analysis for norovirus revealed ≥99% homology with GI.2, GI.5A, and GII.6.
Significance: This was the first reported incidence of norovirus associated illnesses due to consumption of fried breaded oysters. This is also the first instance of detection of norovirus genogroup IV in the US from shellfish implicated in an outbreak.