Detection Of Noroviruses In Shellfish Research Articles

Utility of Droplet Digital PCR Assay for Quantitative Detection of Norovirus in Shellfish, from Production to Consumption in Guangxi, China

ObjectiveShellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. MethodsOyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR). ResultsA total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. ConclusionThis study provides a systematic analysis of norovirus contamination ‘from the farm to the fork’ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.

Detection of Noroviruses in Shellfish and Semiprocessed Fishery Products from a Belgian Seafood Company

Shellfish have been implicated in norovirus (NoV) infection outbreaks worldwide. This study presents data obtained from various batches of shellfish and fishery products from a Belgian seafood company over a 6-month period. For the intact shellfish (oysters, mussels, and clams), 21 of 65 samples from 12 of 34 batches were positive for NoVs; 9 samples contained quantitative NoV levels at 3,300 to 14,300 genomic copies per g. For the semiprocessed fishery products (scallops and common sole rolls with scallop fragments), 29 of 36 samples from all eight batches were positive for NoVs; 17 samples contained quantitative NoV levels at 200 to 1,800 copies per g. This convenience study demonstrated the performance and robustness of the reverse transcription quantitative PCR detection and interpretation method and the added value of NoV testing in the framework of periodic control of seafood products bought internationally and distributed by a Belgian seafood processing company to Belgian food markets.

Human norovirus RNA persists in seawater under simulated winter conditions but does not bioaccumulate efficiently in Pacific Oysters (Crassostrea gigas).

Norovirus (NoV) is the principal agent of bivalve molluscan shellfish-associated gastroenteric illness worldwide. Currently, noncultivable human NoVs can be detected in bivalve molluscan shellfish by using molecular methods such as real-time reverse transcription PCR assays (qRT-PCR). In addition to infectious viruses, this methodology may also detect noninfectious NoV, including fragments of the NoV genome. This study addresses, in part, the implications of qRT-PCR results for the detection of NoV in shellfish in the absence of an infectivity assay. To evaluate environmental persistence, the stability of a short fragment of the NoV genome, spanning the qRT-PCR target in the open reading frame 1/2 junction, was assessed in seawater under artificial environmental conditions simulating winter in the United Kingdom (1 mW/cm² UV irradiation, 8°C) during a 4-week period. Detectable RNA levels decreased exponentially (T₉₀ of approximately 141 h); however, sequences were still detectable for up to 2 weeks. The ability of Pacific oysters (Crassostrea gigas) to bioaccumulate NoV particles (from human feces) and RNA fragments was also compared using qRT-PCR. Oysters exposed to NoV particles subsequently were positive for NoV by qRT-PCR at levels several orders of magnitude in excess of the theoretical limit of detection, whereas oysters exposed to similar quantities of NoV RNA were either negative or positive at significantly lower levels. Therefore, although noninfectious fragments of NoV RNA may persist in the environment under winter conditions, this type of material will not be efficiently bioaccumulated by Pacific oysters and should not significantly contribute to positive qRT-PCR results.

Norovirus Detection in Shellfish Using a Rapid, Sensitive Virus Recovery and Real-Time RT-PCR Detection Protocol

The development and in-house validation of a method for the detection of noroviruses in shellfish are described. The method comprises a simple virus recovery step using proteinase K digestion and extraction of viral ribonucleic acid (RNA) followed by norovirus detection in separate two-step genogroup I (GI) and II (GII) real-time reverse transcription polymerase chain reaction assays. The assay can be completed within 8 h. An internal armored RNA control, which detects the presence of reverse transcription PCR inhibitors, is an integral part of the assay. The method was found to be reliable, fast, robust, reproducible, and sensitive. Sixty New Zealand and imported shellfish samples were tested using the new method, of which 29 (48.3%) samples were associated with recent New Zealand gastroenteritis outbreaks. The nonoutbreak-related samples were submitted for environmental surveillance related to possible sewage contamination events. Norovirus was detected in 30 of 60 (50%) samples, of which 20 were implicated in gastroenteritis outbreaks, and ten were noncommercial shellfish samples from environmental sites. Of the 30 positive samples, 18 (30%) were positive for both GI and GII norovirus, and a further 12 samples (20%) were positive for GII norovirus only. No samples were positive for GI norovirus only. The method is now validated and accredited under ISO 17025. It can be used to monitor norovirus contamination of shellfish in different situations, including outbreak investigations, relaying, environmental monitoring, and following environmental contamination events.

Detection of noroviruses in shellfish in the Netherlands

Shellfish from oyster farms in the Netherlands and imported from other European countries were examined for viral contamination. A method that allows sequence matching between noroviruses from human cases and shellfish was used. The samples of shellfish ( n = 42) were analyzed using a semi-nested RT-PCR that had been optimized for detection of norovirus in shellfish (SR primer sets). In addition, a different genome region was targeted using a second primer set which is routinely used for diagnosis of norovirus infection in humans (JV12Y/JV13I). To improve the detection limit for this RT-PCR a semi-nested test format was developed (NV primer sets). One of 21 oyster samples (4.8%) from Dutch farms was norovirus positive, whereas norovirus was detected in 1 out of 8 oyster samples (12.5%) and 5 out of 13 mussel samples (38.5%) collected directly after importation in the Netherlands. RNA from samples associated with an outbreak of gastro-enteritis in the Netherlands in 2001 was re-analyzed using the NV primer sets. At least one identical sequence (142/142 nt) was found in three fecal and in two oyster samples related to this outbreak. Further surveillance of norovirus by detection and typing of viruses from patients with gastroenteritis and shellfish is warranted to clarify the causes of future outbreaks.