Abstract Title
Challenges in the culture of norovirus isolated from oyster tissues
Presenter
Marion Desdouits, Ifremer
Co-Author(s)
Flora Marteau (1), Cécile Le Mennec (1), Marine Cuau (1), Mary K. Estes (2), Soizick Le Guyader (1) and Marion Desdouits (1). (1) : Laboratoire Santé Environnement Microbiologie, MASAE, IFREMER, Nantes, France(2) : Baylor College of Medicine, Houston, Texas, USA
Abstract Category
Food & Environmental Virology-I (Food)
Abstract
Originating from human wastewaters, norovirus particles are bio-accumulated in the digestive tissue of oysters grown in sewage-polluted coastal areas. Current mitigation procedures involve the purification of shellfish in seawater tanks, usually for a few days. The report of shellfish-borne outbreaks following depuration illustrates the long persistence of infectious norovirus in shellfish tissues. While the virus can remain infectious in seawater for weeks, no data are available yet quantifying the decay of norovirus in shellfish. For this, a method to isolate infectious norovirus from shellfish tissues is first needed.
We compared methods for their ability to elute different strains of norovirus from oysters based on quantitative PCR, for the impact of the eluate on enteroids cultures and viral replication, and finally for the isolation of infectious norovirus previously bioaccumulated in oysters. Most simple methods did not allow efficient elution of the virus, when compared to the ISO 15-216 method based on proteinase K. However, this latter method includes a heating step that is both necessary and detrimental to norovirus infectivity. An optimized method using chloroform-butanol elution and PEG-concentration allowed recovery of infectious norovirus GII.3, but was not efficient to elute GII.4. Despite moderate inhibition of the viral replication by the oyster eluate, this method could be applied to measure the infectious titer of a GII.3 strain isolated from oyster tissues.
In conclusion, a method is now available to assess the persistence of norovirus GII.3 in shellfish tissues. Development of a universal protocol for all norovirus strains is ongoing.
We compared methods for their ability to elute different strains of norovirus from oysters based on quantitative PCR, for the impact of the eluate on enteroids cultures and viral replication, and finally for the isolation of infectious norovirus previously bioaccumulated in oysters. Most simple methods did not allow efficient elution of the virus, when compared to the ISO 15-216 method based on proteinase K. However, this latter method includes a heating step that is both necessary and detrimental to norovirus infectivity. An optimized method using chloroform-butanol elution and PEG-concentration allowed recovery of infectious norovirus GII.3, but was not efficient to elute GII.4. Despite moderate inhibition of the viral replication by the oyster eluate, this method could be applied to measure the infectious titer of a GII.3 strain isolated from oyster tissues.
In conclusion, a method is now available to assess the persistence of norovirus GII.3 in shellfish tissues. Development of a universal protocol for all norovirus strains is ongoing.