Influence of NaCl on the Inactivation of Murine Norovirus-1 and Hepatitis A Virus in Korean Traditional Salted Oyster Called “Eoriguljeot” during Storage

Introduction: Enteric noroviruses (NoV) and hepatitis A virus (HAV) are the leading causes of non-bacterial shellfish-borne gastroenteritis. Raw fresh oysters (Crassostrea gigas) in Korea are processed into a salted and seasoned traditional food called Eoriguljeot. However, it is currently not known whether the addition of NaCl and storage times are sufficient to inactivate NoV and HAV in Eoriguljeot, and there is still lack of information and scientific data on the non-thermal inactivation or reduction of specific pathogenic viruses in diverse seafood products (National Advisory Committee on Microbiological Criteria for Foods, 2008).

Purpose: In the present study we examined the effects of various NaCl concentrations on the survival of murine norovirus-1 (MNV-1) as a surrogate of NoV and HAV inoculated in raw oysters to simulate storage conditions for home-made Eoriguljeot.

Methods: Fresh oyster of 10 g were infected with 100 μl of the appropriate dilution of each viral stock to obtain the final infection levels of 5.83 log TCID₅₀ for MNV-1 and 5.30 log TCID₅₀ for HAV.  NaCl at 3, 5, 7, or 10% was added to MNV-1 or HAV-contaminated raw oyster samples which were then stored at 10°C for 72h. The 50% tissue culture infectious dose (TCID₅₀) method was used to estimate virus titers during the storage. Decimal reduction times at a given concentration of NaCl were also calculated using D = - 1/slope.

Results: Both MNV-1 and HAV titers were significantly (P < 0.05) decreased by stepwise increase in NaCl and storage time; however, MNV-1 was more resistant to NaCl than HAV.  The overall reductions in MNV-1 and HAV titers in oysters incubated with 5%, 3%, and 0% NaCl were 0.61 and 1.20, 0.50 and 0.89, 0.35 and 0.64, 0.25 and 0.42, and 0.07 and 0.25 log TCID₅₀ (50% tissue culture infectious dose)/ml, respectively.  The 1.23 log (94.11%) reduction of MNV-1 and 2.45 log (99.65%) reduction of HAV survival also occurred in 10% NaCl-containing oysters by 72h of storage.  Using the first-order reaction model we determined that in 10% NaCl the MNV-1 decimal reduction time (D-value) was 64h, whereas for HAV D-values of 63h, 47h, and 31h were achieved in 5%, 7%, and 10% NaCl, respectively.

Significance: This study suggests that NaCl in concentrations over 10% could be potentially used in conventional Eoriguljeot as an antiviral agent because HAV was almost completely inactivated (99%) by 10% NaCl.