Estimating the infectivity of norovirus during the depuration of bivalve shellfish

Abstract

Human norovirus (HuNoV) represents the leading cause of acute gastroenteritis globally. Consumption of shellfish presents a notable risk for HuNoV-related gastroenteritis as they are filter feeders and thus bio-accumulators. Shellfish can be purified of microbial contaminants (such as bacteria and viruses) using a process called “depuration”, which allows the bivalve shellfish to purge contaminants in a sanitised environment. This is achieved by placing shellfish in a tank of recirculating water which is sterilised by ultraviolet light or ozone. Studies debate both the efficacy and duration required for depurating HuNoV as post-process testing often reveals the presence of residual HuNoV RNA. The ISO 15216-1:2017 method (the standard for the quantification of HuNoV and Hepatitis A viral RNA from environmental samples) is unable to distinguish between intact viral capsids (likely infectious) and free (non-infectious) RNA. Testing should reflect viral infectivity rather than RNA presence alone. Unfortunately, the current culture-based gold standard for determining infectivity is unsuited for use with HuNoV from environmental samples, leading for a need to establish other methods. The ISO accredited method (ISO 15216-1:2017) potentially overstates the risk of viral infectivity, and methods estimating the infectivity of viruses known as capsid integrity assays are being investigated. A capsid integrity assay, using protein-coated magnetic beads, to estimate HuNoV viability, has yet to be applied to shellfish depuration. Practical experience of shellfish farmers suggests 10 days of depuration reduces the risk of HuNoV infection to consumers. Validating this timeframe would improve public health protection and consumer confidence, negating the need for any costly extension of depuration time.

This study primarily aimed to evaluate the effectiveness of capsid integrity assays for estimating the infectivity of HuNoV, during the depuration of shellfish. The study specifically focused on HuNoV genogroups GI and GII. These genogroups are commonly associated with people suffering from gastroenteritis after the consumption of raw or lightly cooked shellfish. Oysters and mussels were artificially contaminated and independently depurated for a period of 10 days in a recirculating system at ambient water temperature (11°C). Shellfish samples were taken every 24 hours, in triplicate. An intact viral recovery method from the shellfish matrix, permitted subsequent culture-based infectivity assays. These assays covered indicator and surrogate viruses; HuNoV, murine norovirus, human adenovirus and MS2 bacteriophage. A capsid integrity assay (protein coated magnetic beads) was used on norovirus samples to assess infectivity. The capsid integrity assay was run in parallel with the established ISO 15216-1:2017 method, permitting method comparison. Since HuNoV cannot be successfully cultured invitro from environmental samples, surrogate and indicator viruses were monitored during depuration. Measuring the infectivity of surrogates enabled result comparison with the HuNoV capsid integrity assay.

The findings revealed that during oyster and mussel depuration, RNA detected by the standard ISO 15216-1:2017 method persisted longer than measures of virus infectivity determined by culture-based approaches. While HuNoV RNA did not clear within the 10-day timeframe, the protein coated magnetic bead capsid integrity assay demonstrated that the virus seemed to maintain infectivity in shellfish for only 6 days. This agrees with earlier suggestions that the ISO 15216-1:2017 method overstates the risk of infectious viruses to consumers. This study agrees with previous literature showing HuNoV RNA is very stable in shellfish digestive tissue during depuration. The use of protein coated magnetic beads gave novel indications that HuNoV viability in shellfish may be shorter than previously thought, even under cooler unheated conditions, however extensive further research is required due to the intact viral extraction method used being deemed inefficient. Given that a safe shellfish depuration timeframe of 10 days, is currently suggested by some shellfish farmers, it is plausible that HuNoV infectivity can potentially decline after 6 days as shown in this study, and further research is warranted.

Date of Award	2024
Original language	English
Supervisor	Shelagh Malham (Supervisor) & Kata Farkas (Supervisor)