Inactivation of Hepatitis A Virus and Human Norovirus in Clams Subjected to Heat Treatment

The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.

The epidemiological data clearly demonstrates that filter feeding bivalve shellfish can, and do, act as efficient vehicles for the transmission of enteric viruses transmitted by the faecal-oral route. This identified hazard has been documented as a cause for concern by various international agencies and has a long history. Disease outbreaks can occur on an epidemic scale as graphically illustrated by an outbreak of Hepatitis A in Shanghai, China in 1988 involving about 300,000 cases. Improvement of harvesting area water quality offers the most sustainable route to improvement in the virological quality of bivalve shellfish sold live. However there is growing awareness, and concern, that current regulatory standards based on faecal coliform monitoring do not fully protect the shellfish consumer from viral infection. New viral test methods based on PCR, and the development of alternative more reliable faecal pollution indicators, offer new approaches for the further development of public health controls. However, further work is required to build a scientific consensus and to understand the implications of their introduction into legislation.

In the present work, we aimed at determining the relationship between the hepatitis A virus (HAV) numbers in imported frozen coquina clams involved in two hepatitis outbreaks, as well as the risk for human health. Due to HAV unculturability, a standardized TaqMan real-time reverse transcription-PCR controlling the virus/nucleic acid extraction and enzyme efficiencies was employed to figure the exposure dose for clams responsible for hepatitis cases. HAV numbers were then employed to figure the risk of infection based on a dose-response model for echovirus 12. The estimated risk of infection after consumption of lightly cooked clams matched actual attack rates. Our data show that prospective monitoring of bivalve samples may fail to prevent the occurrence of outbreaks, since HAV was detected in 44% of samples directly associated with cases but was undetectable in samples that were randomly collected from the importers and belonged to the same batches. A correlation was nevertheless observed between the prevalence of hepatitis A cases in the harvesting areas and positive HAV isolation in clams, which points to the need to identify and prevent hazards rather than relying on random sampling of finished products to ensure safety. However, when evidence shows that a critical limit of viral contamination has been exceeded in the potential sources of contamination discharging into the shellfish-growing beds, quantitative virological analysis addressing quality assurance and quality control requirements should be performed with the bivalves. This work provides the first evidence of accurate HAV levels in shellfish involved in outbreaks that could be of use for risk assessment purposes.