Development of a high pressure processing inactivation model for hepatitis A virus and its application in shellfish processing

Hepatitis A virus (HAV) has been responsible for many large outbreaks of illness throughout the world, often resulting from consuming raw or minimally cooked filter-feeding shellfish contaminated with human faecal effluent. High pressure processing (HPP) is an alternative food preservation technique to heat, preserving the flavour, appearance and nutritional value of high quality foods, including oysters, often with extended shelf life. In this study, the effectiveness of HPP in inactivating HAV was assessed. HAV, suspended in buffered tissue culture media containing either 15 parts per thousand (ppt) or 30 ppt salt (NaCl), was treated with 300, 400 and 500 MPa for between 60 and 600 s. A log-linear function was developed in Microsoft\\(^¬¨vÜ\\) Excel to model the kinetic inactivation data with the effects of NaCl, pressure and treatment time. The model can be used to predict HAV inactivation by interpolation at processing parameters not actually tested for in the laboratory. For the model to be validated in oysters contaminated with HAV, methods for HAV extraction and purification from spiked oyster homogenate were first evaluated. Methods evaluated included the crude extraction method, modified from Kingsley and Richards (2003), and the PEG precipitation method, modified from the glycine-polyethylene glycol (PEG)-Tri reagent-poly(dT) extraction (GPTT) method described by Kingsley and Richards (2001). The PEG precipitation method achieved a mean recovery of 12.6%. With modification, including the use of antibiotic/antimycotic treatment prior to assay, the recovery was improved by up to 27.3%. In comparison, the crude extraction technique, which did not include a virion concentration step, recovered on average more than 40% of the initial spiked titre, and was chosen as a reliable method to extract HAV from oysters for cell culture quantitation. Commercially grown and harvested Pacific oysters (Crassostrea gigas) were contaminated with HAV by natural accumulation, when immersed for up to 24 h in seawater contaminated with 1.1x10\\(^7\\) TCID\\(^{50}\\)/ml HAV. Infectious HAV was detected in only two of the six oysters tested, and less than 1% of the initial contaminating HAV titre was recovered in positive oysters, possibly due to the association of viruses and microalgae with oyster shells and aquarium surfaces throughout the trials. A quantitative real-time reverse transcription PCR (qRT-PCR) method was developed as an alternative method for HAV detection in contaminated oysters to the infectivity assay. HAV was detected by qRT-PCR in all contaminated oysters, including those negative by infectivity assay. An immuno-magnetic bead separation technique was also developed, which additionally purified and concentrated virions, improving the sensitivity of detection by qRT-PCR. The log-linear inactivation model was validated in homogenised oyster meat artificially inoculated with known titres of HAV. Salinity and temperature of samples were adjusted to that of buffered samples, while intermediate times and pressures were chosen for processing. The model tended to underpredict inactivation in homogenised oyster samples; i.e. it was fail-safe. Inactivation tended to be greater in spiked oyster homogenate compared to pure culture in treatments at higher pressures (400 ‚ÄövÑvÆ 500 MPa). The validated model may be a useful reference for Australian oyster processors wishing to implement HPP into their post-harvest processing regime.