Application of an Optimized Direct Lysis Method for Viral RNA Extraction Linking Contaminated Dates to Infection With Hepatitis A Virus.

Consumption of dates has not been considered a common risk of hepatitis A virus (HAV) infection. In January 2018, an outbreak of hepatitis was identified with cases resident in all regions of Denmark. All the detected strains belonged to HAV genotype 3A. Epidemiological investigations through patients' interviews, case-control and trace-back studies pointed toward different batches of dates from a single producer as the vehicle of infection. Boxes of dates from suspected batches were collected from homes of patients and healthy families and analyzed using a recently reported optimized direct lysis method, consisting of simultaneous viral RNA elution and extraction from dates followed by purification of the nucleic acids. Extracts were analyzed for HAV and norovirus (NoV) RNA using RT-qPCR, while detected HAV were genotyped by Sanger sequencing. Among 20 nucleic acid extracts representing eight batches of dates, RNA of HAV (9.3 × 102 genome copies/g) and NoV genogroup (G)II (trace amounts) were detected in one batch, while NoV GII RNA (trace amounts) was detected in another. Average extraction efficiency of spiked process control murine norovirus was 20 ± 13% and the inhibitions of RT-qPCR detection of NoV GI, NoV GII, and HAV were 31 ± 34, 9 ± 9, and 3 ± 7%, respectively. The HAV genome detected in the dates matched by sequence 100% to the HAV genotype 3A detected in stool samples from cases implicated in the outbreak. This confirmed, to our knowledge, for the first time a sequence link between HAV infection and consumption of contaminated dates, suggesting dates to be an important vehicle of HAV transmission.

An Optimised Direct Lysis Method for Viral RNA Extraction and Detection of Foodborne Viruses on Fruits and Vegetables.

Detection of norovirus (NoV) and hepatitis A virus (HAV) on fruits and vegetables using current standard methodologies can be inefficient. Method optimisation focussing on ease, rapidity and increased viral RNA recovery is needed for efficient reverse transcription (RT)-qPCR detection of viruses. A simple and quick direct lysis method for RNA extraction was optimised (method A) to achieve increased viral RNA recovery and minimised RT-qPCR inhibition by increasing the volume of lysis buffer and inclusion of pectinase, Plant RNA Isolation Aid and OneStep PCR Inhibitor Removal Kit. Method A and an internal method structurally comparable to the ISO 15216 standard (method B) were compared for their efficiencies to recover viral RNA from the process controls, mengovirus (MC0) and murine norovirus (MNV), spiked in 13 types of fruits, vegetables, compound foods or seeds/nuts. All extracts (> 61) were also analysed for RT-qPCR inhibition and for natural contamination of NoV and HAV. The overall mean extraction efficiencies of MC0 and MNV were 36 ± 31 and 44 ± 38%, respectively, for method A and 9 ± 16 and 5 ± 11%, respectively, for method B. Inhibition of RT-qPCR amplification of RNA from NoV genogroup (G)I, NoV GII, and HAV ranged from 5 ± 10 to 13 ± 14% for method A and 34 ± 36 to 48 ± 40% for method B. NoV GII was detected in samples of strawberries and seaweed processed by both methods. In conclusion, the new direct lysis method showed an overall better performance compared to the modified ISO 15216 standard and should be validated for implementation in analysis of viruses in foods of plant origin.

Inactivation of hepatitis A virus and murine norovirus on surfaces of plastic, steel and raspberries using steam-ultrasound treatment.

The leading causes of foodborne viral disease outbreaks are human norovirus and hepatitis A virus (HAV). Their environmental persistence enables contamination of kitchen surfaces and crops often consumed raw, such as berries. Many decontamination procedures are inefficient and unsuitable for surfaces of industrial kitchen environments and soft fruits. In this study, we investigated the efficiency of a novel surface decontamination technology, combining steam and ultrasound (steam-ultrasound). Plastic, steel or raspberry surfaces were spiked with the norovirus surrogate, murine norovirus (MNV), and HAV, and steam-ultrasound treated at 85, 90 and 95 °C for 0-5 s. Post treatment viruses were titrated for survival by plaque assay and for genome stability by real-time quantitative PCR (RT-qPCR) of nucleic acid extracts. Survival of viruses were estimated in a log-linear model and the treatment time requirements for each decimal reduction (D value) in viral survival were calculated. The estimated D values of MNV or HAV were 0.4-0.2 or 1.1-0.8 s on plastic, 0.9-0.7 or 1.4-0.8 s on steel and 1.6-1.7 or 3.2-4.7 s on raspberries. No clear trend of genome reduction was observed with tested treatment parameters. Raspberries treated up to 4 s retained its natural texture and visual appeal similar to untreated controls whilst monitored for 7 days. In conclusion, steam-ultrasound treatment can within seconds reduce the titre of foodborne viruses on surfaces of plastic, steel and raspberries. This may particularly benefit industrial scale production of soft fruits for raw consumption and for swift non-hazardous decontamination of industrial kitchen surfaces.