Development and use of new methods and technologies to study and control foodborne viruses on fruits, greens, and surfaces of plastic and steel

Sheikh Md Rajiuddin*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis


Human norovirus (NoV) causes 120 million foodborne gastroenteritis cases while hepatitis A virus (HAV) causes 14 million cases of foodborne jaundice worldwide (Bosch et al. 2018). NoV in particular but recently also HAV are frequently reported to cause disease outbreaks among consumers of contaminated fruit and greens (Chatziprodromidou et al. 2018). Next to person-to person transmission, contaminated food or food contact surfaces are the sources of most NoV and HAV outbreak (Fankhauser et al. 2002; Franck et al. 2015). Despite this, genome extraction for the detection of these viruses, using standard methods can be challenging (Bartsch et al. 2016), in addition many currently applied methods for decontamination of surfaces of soft fruit and industrial kitchen environments are inefficient (EFSA 2011) or unsuitable (Hirneisen et al. 2010). Moreover, the primary method used for detection of foodborne viruses, real-time quantitative PCR (RT-qPCR) lack the ability to distinguish between infectious and non-infectious viruses (de Roda Husman et al. 2009) limiting accurate risk estimate and control measures.
The aims of this PhD project were to i) develop and evaluate a sensitive, easy and simple method for viral genome extraction of fruits and leafy greens for subsequent RT-qPCR detection and demonstrate its application on samples suspected to have caused foodborne outbreaks, ii) evaluate the efficiencies of a 3rd generation steam-ultrasound technology to inactivate the NoV surrogate, murine norovirus (MNV), and hepatitis A virus (HAV) on surfaces of plastic, steel and raspberries, and study the mechanism of steam-ultrasound viral inactivation.
Initially, a previously described method for direct lysis of viral genome on soft fruit was optimized (Method A) for the efficiency to extract viral RNA of spiked mengovirus (MC0) and MNV on fruits and leafy greens with limited accumulation of RT-qPCR inhibitors for NoV GI, GII and HAV detection (Paper I). In comparison with the efficiency of a modified ISO 15216:2017 standard (method B), method A showed better potential in recovering foodborne viruses from a broad variety of samples of fruits and leafy greens without compromising co-concentration of RT-qPCR inhibitors. In addition method A is quicker, easier and cheaper to apply, which can facilitate more laboratories to implement viral analysis and reduce response time during investigations of foodborne viral outbreaks and surveillance.
Method A was subsequently applied in the analytical investigation of samples of dates suspected to be the cause of a HAV outbreak (Paper II). Among the 10 samples tested, one was found positive for HAV and two for NoV GII. The detected HAV sequence in the dates matched 100 % to the HAV genotype 3A detected in the stool sample of cases involved in the outbreak. This confirmed, to our knowledge, for the first time a sequence link between a HAV infection and consumption of contaminated dates.
Secondly, we investigated the efficiency of a novel surface decontamination technology, combining steam and ultrasound (steam-ultrasound), for the efficiency to inactivate foodborne viruses on surfaces of plastic, steel and raspberries (Paper III). We found the estimated treatment time requirement for each decimal reduction (D-value) of MNV and HAV at treatment temperature 85, 90 or 95 °C, to be 0.4-0.2 or 1.1-0.8 seconds on plastic, 0.9-0.7 or 1.4-0.8 seconds on steel and 1.6-1.7 or 3.2-4.3 second on raspberries. With these results, we conclude that steam-ultrasound treatment can reduce viral titer on contaminated surfaces of plastic, stainless steel and raspberries in seconds.
The mechanism behind steam-ultrasound viral inactivation was investigated (Paper IV). The capsid integrity was studied using the genome intercalating reagents, PMAxx and PtCl4, and the RNA degrading enzyme, RNase, with the aim to prevent detection of released genomes from damaged viral capsids. In addition, the integrity of capsid proteins was studied using cell binding affinity assays. The use of in particularly PMAxx and to lesser extent PtCl4 and RNase showed a reduction in detected HAV genomes with increased treatment time, following the trend of reduction in the titter of infectious viruses. However, no pronounced reduction in genome titter as observed for the infectious titter of MNV could be shown using any of the three reagents. On the contrary, using cell binding affinity assays, we observed to some degree a loss in cell binding capacity for MNV but not for HAV. Thus we could conclude that decreased titter of infectious viruses following steam ultrasound treatment could in part be explained by decreased capsid integrity for HAV and decreased protein integrity for MNV.
In conclusion, with this PhD thesis, an optimized direct lysis method for foodborne virus detection on a broad variety of fruits and leafy greens was developed, evaluated and demonstrated more efficient, quicker, easier and cheaper compared to the modified ISO standard. The method showed fit for purpose on naturally NoV and HAV contaminated samples, and allowed in a HAV outbreak investigation of dates, for the first time, to establish a 100% match between a HAV strain detected in the suspected dates and outbreak involved cases. The method may facilitate more laboratories to implement viral analysis and reduce response time during investigations of foodborne viral outbreaks and surveillance. Finally, it was demonstrated that few seconds of steam-ultrasound treatment could be used to decontaminate surfaces of plastic, stainless steel and raspberries contaminated with foodborne viruses. It was further shown that the mechanism of inactivation most likely can be explained by reduced viral protein integrity for MNV and reduced capsid integrity for HAV This steam Ultrasound technology can be particularly useful for industrial scale production of foods for raw consumption and for swift non-hazardous decontamination of industrial kitchen surfaces.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages238
Publication statusPublished - 2019


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