Rapid Detection And High-Pressure Inactivation Of Noroviruses In Shellfish

Noroviruses (NoVs), which belong to the family Caliciviridae, are the single most common cause of outbreaks as well as sporadic cases of acute gastroenteritis. Shellfish can bioaccumulate microbial pathogens at high levels from surrounding marine and estuarine waters. The consumption of raw or minimally cooked oysters, which is a common custom practiced worldwide, can be a great transmission route of NoVs. Nowadays, NoVs are a great concern as a threat to the safety of edible shellfish worldwide.Since firstly detected by immune electron microscopy, further study has always hampered by the lack of suitable animal models and the inability to propagate NoVs in cell cultures. Aside from the high cost and low sensitivity of electron microscopy, immunological methods are also with several drawbacks. As the development of molecular biology, the whole genome of many NoVs strains has been determined. Molecular method based on PCR is becoming most commonly used because of its rapidness and high sensitivity.High-pressure processing (HPP) has recently been developed as a potential means for reducing pathogens within raw shellfish. With the use of this technology, the appearance, flavor, texture, and nutritional qualities of unprocessed foods like oysters can be retained. In addition to enhancing the safety and extending the shelf-life of seafood, HPP treatment has the additional advantage of shucking shellfish, thus rendering this technology particularly useful to the shellfish-processing industry and consumers alike.In this study, reverse transcription polymerase chain reaction (RT-PCR), RT-seminested PCR, RT-Real Time PCR and reverse transcription loop mediated isothermal amplification (RT-LAMP) assays were compared to detect norovirus in oysters. In comparison to other approaches, the amplification program of RT-LAMP is simple, and the reaction time is short. Without special apparatus needed, RT-LAMP can be performed under isothermal condition and the results may be visually ascertained by a simple color reaction using SYBR Green I dye. Therefore, RT-LAMP is of great potential to become a valuable method for rapid detection of norovirus in oysters.Murine norovirus-1 (MNV-1) was used as a surrogate to assess the effects of high-pressure processing (HPP) at 200-400 MPa on contaminated oysters by using a flow-through seawater system. Plaque assays demonstrated that at 0℃, the onset of inactivation was observed at 200 MPa, and treatment with 400 MPa for 5min was sufficient to inactivate MNV-1 within the oysters such that the viral load is undetectable.The mechanism of MNV-1 inactivation by HPP was investigated:HPP-treated MNV could not bind to its target receptor and therefore could not initiate infection of mouse RAW cells. The integrity of its genome RNA and the antigenic epitope on capsid was not affect by HPP. Partial motif changes of the viral capsid caused by HPP were accessed by induced sensitivity to proteinase K.Both of LAMP and HPP belong to novel techniques which are still at their initial stages being applied in NoVs research. This study supplys theoretical basis for a rapid detection of NoVs in shellfish and a minimal processing for industry regarding to this issue.