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Foodborne outbreaks caused by noroviruses (NoVs) and hepatitis A virus (HAV) are often linked to consumption of contaminated shellfish. The objective of this study was to identify an appropriate virus recovery method for real-time reverse transcriptase (RT)-PCR detection and subsequently to evaluate this method on shellfish bioaccumulated with virus in a collaborative study. Five methods were compared for recovery of NoV GII.7 and feline calicivirus from spiked digestive tissue of oysters and mussels. A method based on proteinase K digestion followed by NucliSENS miniMAG extraction was found to be the most efficient with a 50% limit of detection (LOD(50)) of 62 and 12 RT-PCR U/1.5 g digestive tissue for NoV GII.7 in oysters and mussels, respectively. Evaluation of the method in four laboratories found the percentage of sensitivity, based on low/high levels of virus bioaccumulated in oysters, to be 33/80 for NoV GI.3b, 13/92 for NoV GII.4 and 50/42 for HAV. A specificity of 100% was found for all three viruses in non-bioaccumulated oysters. As process control Mengovirus (vMC(0)) showed an average recovery of 1.8% from oysters and 1.2% from mussels. The study demonstrates that this recovery method can be useful for harmonized data generation and routine viral analyses of shellfish.

Viral contamination of drinking water is frequently reported as the primary source of gastroenteritis or hepatitis outbreaks. The presence of viruses at low concentration levels in most environmental water poses major analytical problems when determining their concentration. To evaluate the efficiency of different recovery methods of viral RNA from bottled water, a comparison was made of 2 positively and 2 negatively charged membranes that were used for absorbing and releasing HAV virus particles during the filtration of viral spiked bottled water. All the 4 membranes, regardless of charge and pore size, had low level viral recovery. The results show that a considerable number of the virus particles passed through the pores of the membranes instead of being trapped by the electrostatic charges. Two different procedures were then compared using 1.5L polyethylene bottles spiked with 10-fold serial dilutions of HAV and FCV. The first procedure included an ultrafiltration-based method followed by MiniMag RNA extraction, and the second an ultracentrifugation-based method followed by RNA extraction using QIAamp viral RNA mini kit. The ultracentrifugation-based method resulted in a better recovery of HAV and FCV when compared to the ultrafiltration-based method.

Human enteric viruses are detected frequently in various types of environmental water samples, such as irrigation water, wastewater, recreational water, ground or subsurface water and even drinking water, constituting a primary source of gastroenteritis or hepatitis outbreaks. Only a few, but still infective number of viral particles are normally present in water samples, therefore an efficient virus concentration procedure is essential prior to molecular detection of the viral nucleic acid. In this study, a novel chromatographic technology, Convective Interaction Media (CIM) monolithic supports, were optimized and applied to the concentration of hepatitis A virus (HAV) and feline calicivirus (FCV), a surrogate of norovirus (NoV), from water samples. Two-step real-time RT-qPCR was used for quantitation of the virus concentration in the chromatographic fractions. Positively charged CIM QA (quaternary amine) monolithic columns were used for binding of HAV and FCV present in previously inoculated 1.5 l bottled water samples. Column bound viruses were eluted from the monolith using 1M NaCl to a final volume of 15 ml. Elution volume was concentrated further by ultracentrifugation. When the CIM/ultracentrifugation method was compared with another concentration method employing positively charged membranes and ultrafiltration, the recovery of HAV was improved by approximately 20%.