Hepatitis E is caused by hepatitis E virus (HEV), one of the causes of acute viral hepatitis. Domestic pigs are considered as the main reservoir of HEV-3. The recently reported high prevalence of HEV in liver- and meat products on the Dutch market warranted a cross-sectional prevalence study on HEV infection among 5-6 months old pigs slaughtered in the Netherlands (n = 250). For this, liver, caecum content and blood samples were analyzed for the presence of genomic HEV RNA by RT-PCR. In addition, a serological test was performed to detect HEV IgG. Background information was retrieved on the corresponding farms to evaluate potential risk factors for HEV at pig slaughter age. HEV IgG was detected in sera from 167 pigs (67.6 %). HEV RNA was detected in 64 (25.6 %) caecum content samples, in 40 (16.1 %) serum samples and in 25 (11.0 %) liver samples. The average level of viral contamination in positive samples was log₁₀ 4.6 genome copies (gc)/g (range 3.0-8.2) in caecum content, log₁₀ 3.3 gc/ml (range 2.4-5.9) in serum and log₁₀ 3.2 gc/0.1 g (range 1.7-6.2) in liver samples. Sequence analyses revealed HEV-3c only. Ten times an identical strain was detected in two or three samples obtained from the same pig. Each animal in this study however appeared to be infected with a unique strain. The presence of sows and gilts and welfare rating at the farm of origin had a significant effect (p < 0.05) on the distribution over the four groups representing different stages of HEV infection based on IgG or RNA in caecum and/or serum. The observed proportion of tested pigs with viremia (16 %) was higher than in other reported studies and was interestingly often observed in combination with a high number of HEV genome copies in liver and caecum content as detected by RT-qPCR. Data provided will be useful for risk assessment for food safety of pork products, will provide baseline data for future monitoring of HEV infections in pigs and new thoughts for mitigation strategies.

Contamination of bivalve molluscs with viruses is well recognized as a food safety risk. A microbiological criterion for norovirus (NoV) and hepatitis A virus (HAV) in shellfish, however, does not exist in the European Union currently. The aim of this study was to evaluate the contamination levels of these viruses for fluctuation over a long period (2013-2017) in oyster (n = 266) and mussel samples (n = 490) using a method based on ISO/TS 15216-1: 2013. Samples were taken at different points in the food chain, either directly post-harvest, at Dutch dispatch centers or in retail stores, from September until March of each year. Altogether, 53.1% of the mussel and 31.6% of the oyster samples tested positive for NoV RNA. Simultaneous presence of NoV GI and GII RNA was observed in 31.6% of mussel and 10.2% of oyster samples. Contamination levels in NoV positive mussel samples collected post-harvest from B-areas were significantly higher than in those collected post-harvest from A-areas, or at dispatch centers or retail stores. Levels in oysters from dispatch were significantly lower than those collected in retail stores. Ready for sale mussels and oysters contained 2.04 and 1.76 mean log10 transformed NoV genome copies/gram (gc/g), respectively. GII levels were at a constant level in ready for sale mussels throughout all sampling periods in the study. This seemed to be true for oysters as well. HAV RNA was detected in only one of the tested mussel samples (n = 392) (typed HAV 1A) and in none of the tested oyster samples (n = 228). Critical evaluation of NoV and HAV levels in shellfish can be of help for risk assessment and risk management actions.

This report describes an outbreak investigation starting with two closely related suspected food-borne clusters of Dutch hepatitis A cases, nine primary cases in total, with an unknown source in the Netherlands. The hepatitis A virus (HAV) genotype IA sequences of both clusters were highly similar (459/460 nt) and were not reported earlier. Food questionnaires and a case–control study revealed an association with consumption of mussels. Analysis of mussel supply chains identified the most likely production area. International enquiries led to identification of a cluster of patients near this production area with identical HAV sequences with onsets predating the first Dutch cluster of cases. The most likely source for this cluster was a case who returned from an endemic area in Central America, and a subsequent household cluster from which treated domestic sewage was discharged into the suspected mussel production area. Notably, mussels from this area were also consumed by a separate case in the United Kingdom sharing an identical strain with the second Dutch cluster. In conclusion, a small number of patients in a non-endemic area led to geographically dispersed hepatitis A outbreaks with food as vehicle. This link would have gone unnoticed without sequence analyses and international collaboration.

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

Hepatitis E virus genotype 3 (HEV-3) is a food-borne pathogen causative of hepatitis E infections in humans. In Europe, HEV-3 is mainly transmitted through the consumption of raw or undercooked pork. In order to determine the effectiveness of control measures that can be taken in the industry or by the consumer, it is pivotal to determine the infectivity of HEV present in pork products after thermal food-processing steps. First, we implemented a method for the detection of infectious HEV-3c and HEV-3e in a cell culture medium and in extracts from inoculated pork products. Next, we investigated the effect of the thermal inactivation of HEV by mimicking food-processing steps specific for dried sausage and liver homogenate matrices. After four weeks, HEV-inoculated dried sausage subjected to 21 °C or lower temperatures was still infectious. For the liver homogenate, the highest HEV-3c/e inactivation of the conditions tested was observed at 71 °C for five min or longer. Finally, our method was able to successfully detect and estimate viral loads of infectious HEV in naturally infected pig livers. Our data provide a basis for the future use of the quantitative microbial risk assessment of infectious HEV in pork products that are subjected to thermal food processing steps.