Shellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious viruses. The aim of the present study was to optimize a pretreatment using PMAxx to better discriminate between intact and heat-treated HuNoVs in shellfish and sewage. To this end, the optimal conditions (30min incubation with 100μM of PMAxx and 0.5% of Triton, and double photoactivation) were applied to mussels, oysters and cockles artificially inoculated with thermally-inactivated (99°C for 5min) HuNoV GI and GII. This pretreatment reduced the signal of thermally-inactivated HuNoV GI in cockles and HuNoV GII in mussels by >3 log. Additionally, this pretreatment reduced the signal of thermally-inactivated HuNoV GI and GII between 1 and 1.5 log in oysters. Thermal inactivation of HuNoV GI and GII in PBS, sewage and bioaccumulated oysters was also evaluated by the PMAxx-Triton pretreatment. Results showed significant differences between reductions observed in the control and PMAxx-treated samples in PBS following treatment at 72 and 95°C for 15min. In sewage, the RT-qPCR signal of HuNoV GI was completely removed by the PMAxx pretreatment after heating at 72 and 95°C, while the RT-qPCR signal for HuNoV GII was completely eliminated only at 95°C. Finally, the PMAxx-Triton pretreatment was applied to naturally contaminated sewage and oysters, resulting in most of the HuNoV genomes quantified in sewage and oyster samples (12 out of 17) corresponding to undamaged capsids. Although this procedure may still overestimate infectivity, the PMAxx-Triton pretreatment represents a step forward to better interpret the quantification of intact HuNoVs in complex matrices, such as sewage and shellfish, and it could certainly be included in the procedures based on RT-qPCR.
Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness, with common vehicles including bivalve molluscan shellfish, soft fruit and various vegetables. Outbreaks of viral illness due to contamination of the surfaces of foods, or food preparation surfaces by for example infected food handlers are also common. Virus analysis of food matrices can contribute towards risk management for these hazards and a two-part technical specification for determination of Hepatitis A virus and norovirus in food matrices (ISO/TS 15216:2013) was published jointly by the European Committee for Standardisation and the International Organization for Standardization in 2013. As part of the European Mandate No. M381 to validate 15 standards in the field of food microbiology, an international validation study involving 18 laboratories from 11 countries in Europe was conducted between 2012 and 2014. This study aimed to generate method characteristics including limit of detection, limit of quantification, repeatability and reproducibility for ISO 15216 - Part 1, the method for quantification, in seven food matrices. The organization and results of this study, including observations that led to improvements in the standard method are presented here. After its conclusion, the method characteristics generated were added to the revised international standard, ISO 15216-1:2017, published in March 2017.
dHepatitis E virus (HEV), an enteric pathogen of both humans and animals, is excreted by infected individuals and is therefore present in wastewaters and coastal waters. As bivalve molluscan shellfish are known to concentrate viral particles during the process of filter feeding, they may accumulate this virus. The bioaccumulation efficiencies of oysters (Crassostrea gigas), flat oysters (Ostrea edulis), mussels (Mytilus edulis), and clams (Ruditapes philippinarum) were compared at different time points during the year. Tissue distribution analysis showed that most of the viruses were concentrated in the digestive tissues of the four species. Mussels and clams were found to be more sensitive to sporadic contamination events, as demonstrated by rapid bioaccumulation in less than 1 h compared to species of oysters. For oysters, concentrations increased during the 24-h bioaccumulation period. Additionally, to evaluate environmental occurrence of HEV in shellfish, an environmental investigation was undertaken at sites potentially impacted by pigs, wild boars, and human waste. Of the 286 samples collected, none were contaminated with hepatitis E virus, despite evidence that this virus is circulating in some French areas. It is possible that the number of hepatitis E viral particles discharged into the environment is too low to detect or that the virus may have a very short period of persistence in pig manure and human waste.
The aim of this study was to evaluate the presence of human enteric viruses in shellfish collected along the Mediterranean Sea and Atlantic Coast of Morocco. A total of 77 samples were collected from areas potentially contaminated by human sewage. Noroviruses were detected in 30 % of samples, with an equal representation of GI and GII strains, but were much more frequently found in cockles or clams than in oysters. The method used, including extraction efficiency controls, allowed the quantification of virus concentration. As in previous reports, results showed levels of contamination between 100 and 1,000 copies/g of digestive tissues. Sapoviruses were detected in 13 % of samples mainly in oyster and clam samples. Hepatitis A virus was detected in two samples, with concentrations around 100 RNA copies/g of digestive tissues. Only two samples were contaminated with enterovirus and none with norovirus GIV or Aichi virus. This study highlights the interest of studying shellfish samples from different countries and different production areas. A better knowledge of shellfish contamination helps us to understand virus levels in shellfish and to improve shellfish safety, thus protecting consumers.
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