Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment

Foodborne illnesses associated with contaminated fresh produce are a common public health problem and there is an upward trend of outbreaks caused by enteric viruses, especially human noroviruses (HNoVs) and hepatitis A virus (HAV). This study aimed to assess the use of DNase and RNase coupled to qPCR and RT-qPCR, respectively, to detect intact particles of human adenoviruses (HAdVs), HNoV GI and GII and HAV in fresh produce. Different concentrations of DNase and RNase were tested to optimize the degradation of free DNA and RNA from inactivated HAdV and murine norovirus (MNV), respectively. Results indicated that 10 µg/ml of RNase was able to degrade more than 4 log₁₀ (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84–2.5 log₁₀ of free DNA depending on the fresh produce analysed. The treatment with nucleases coupled to (RT)-qPCR was applied to detect potential infectious virus in organic lettuce, green onions and strawberries collected in different seasons. As a result, no intact particles of HNoV GI and GII were detected in the 36 samples analysed, HAdV was found in one sample and HAV was present in 33.3% of the samples, without any reasonable distribution pattern among seasons. In conclusion, RT-qPCR preceded by RNase treatment of eluted samples from fresh produce is a good alternative to detect undamaged RNA viruses and therefore, potential infectious viruses. Moreover, this study provides data about the prevalence of enteric viruses in organic fresh produce from Brazil.     

Optimization of Methods for Detecting Hepatitis A Virus in Food

Hepatitis A virus (HAV) is currently recognized as an important human food borne pathogen, and it is one of the most resistant enteric RNA viruses, is highly infectious, and may lead to widespread outbreaks. The aim of this study was to optimize the methods to detect HAV from artificially contaminated food. To this end, strawberry and lettuce were experimentally contaminated with HAV suspension containing 6 × 10⁶ copies/ml. After contamination, HAV persistence and washing procedure were evaluated at 0, 1, 3, 7, and 9 days of storage. Five elution buffers (PBS (pH 7.4)/0.1% Tween80; 50 mM glycine/3% (wt/vol) beef extract (pH 9.5); PBS (pH 7, 4); 25 mM glycine/0.1 Tween80; and 1 M sodium bicarbonate) were used to elute the virus, and qualitative and quantitative PCR were used for HAV detection. HAV was detected by qualitative and quantitative PCR using any of the five elution buffers, but PBS was the most effective. Even after washing, HAV was detected up to 9 days after contamination by quantitative PCR. Quantitative PCR was more sensitive than qualitative PCR since samples containing viral load lower than 1.4 × 10³ copies/ml could not be detected by qualitative PCR. Quantitative PCR can be used for rapid detection of food borne viruses and will help in the monitoring and control of food borne disease.     

Comparative Uptake of Enteric Viruses into Spinach and Green Onions

Root uptake of enteric pathogens and subsequent internalization has been a produce safety concern and is being investigated as a potential route of pre-harvest contamination. The objective of this study was to determine the ability of hepatitis A virus (HAV) and the human norovirus surrogate, murine norovirus (MNV), to internalize in spinach and green onions through root uptake in both soil and hydroponic systems. HAV or MNV was inoculated into soil matrices or into two hydroponic systems, floating and nutrient film technique systems. Viruses present within spinach and green onions were detected by RT-qPCR or infectivity assays after inactivating externally present viruses with Virkon^®. HAV and MNV were not detected in green onion plants grown up to 20 days and HAV was detected in only 1 of 64 spinach plants grown in contaminated soil substrate systems up to 20 days. Compared to soil systems, a drastic difference in virus internalization was observed in hydroponic systems; HAV or pressure-treated HAV and MNV were internalized up to 4 log RT-qPCR units and internalized MNV was shown to remain infectious. Understanding the interactions of human enteric viruses on produce can aid in the elucidation of the mechanisms of attachment and internalization, and aid in understanding risks associated with contamination events.     

Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log₁₀ between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

     

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

     

Occurrence of Norovirus and Hepatitis A Virus in U.S. Oysters

Noroviruses (NoV) and hepatitis A virus (HAV) are the leading causes of non-bacterial gastroenteritis in shellfish consumers worldwide. This study determined the seasonal and geographical distribution of NoV (genogroups I and II) and HAV in live U.S. market oysters. Samples were analyzed to determine the occurrence and levels of NoV and HAV using RT-qPCR and conventional RT-PCR. NoV and HAV were detected in 3.9 and 4.4%, respectively. NoV genogroups I and II were detected, with genogroup II predominating. Sequencing identified genotypes II.4, II.3, and II.7. The GII.4 strain showed ≥98% similarity with 2006–2007 circulating strains, Minerva and Laurens. HAV sequences from the 5′ non-coding region (NCR) of the genome were from genotypes I, II, or III. The incidence of NoV in oysters harvested from Atlantic Coast states was higher than that in oysters from other regions and its occurrence was greatest during the cooler months (December to February). HAV was detected at a higher frequency in shellfish harvested from the Gulf Coast and also predominated during cooler months. The seasonal occurrence of viruses in this study corresponded to the reported incidence of shellfish-associated viral illnesses. This investigation provides an overview of the occurrence and distribution of NoV and HAV in U.S. market shellfish.     

Temperature Dependent Depuration of Norovirus GII and Tulane Virus from Oysters (Crassostrea gigas)

Raw oysters are considered a culinary delicacy but are frequently the culprit in food-borne norovirus (NoV) infections. As commercial depuration procedures are currently unable to efficiently eliminate NoV from oysters, an optimisation of the process should be considered. This study addresses the ability of elevated water temperatures to enhance the elimination of NoV and Tulane virus (TuV) from Pacific oysters (Crassostrea gigas). Both viruses were experimentally bioaccumulated in oysters, which were thereafter depurated at 12 °C and 17 °C for 4 weeks. Infectious TuV and viral RNA were monitored weekly for 28 days by TCID₅₀ and (PMAxx-) RT-qPCR, respectively. TuV RNA was more persistent than NoV and decreased by?<?0.5 log₁₀ after 14 days, while NoV reductions were already?>?1.0 log₁₀ at this time. For RT-qPCR there was no detectable benefit of elevated water temperatures or PMAxx for either virus (p?>?0.05). TuV TCID₅₀ decreased steadily, and reductions were significantly different between the two temperatures (p?<?0.001). This was most evident on days 14 and 21 when reductions at 17 °C were 1.3–1.7 log₁₀ higher than at 12 °C. After 3 weeks, reductions?>?3.0 log₁₀ were observed at 17 °C, while at 12 °C reductions did not exceed 1.9 log₁₀. The length of depuration also had an influence on virus numbers. TuV reductions increased from?<?1.0 log₁₀ after seven days to?>?4.0 log₁₀ after 4 weeks. This implies that an extension of the depuration period to more than seven days, possibly in combination with elevated water temperatures, may be beneficial for the inactivation and removal of viral pathogens.

     

Virus Genome Quantification Does not Predict Norovirus Infectivity After Application of Food Inactivation Processing Technologies

When determining the effect of food processing on the infectivity of any contaminating virus, it is necessary to distinguish unambiguously between infectious and non-infectious viruses present. However, this can be difficult in the particular case of noroviruses (NoVs) because no reliable cell culture model is available. The aim of this study was to assess the use of molecular methods—RT real-time PCR (RT-qPCR) and enzymatic treatment (ET) coupled to RT-qPCR—to quantify the infectivity of NoV after application of various inactivating food-processing technologies. RT-qPCR and ET-RT-qPCR gave significantly different (P < 0.01) results concerning the reduction in viral genome counts by all inactivation procedures and conditions used, except for HHP treatment at 600 MPa for 5 min. These findings indicate that the ET prior to RT-qPCR has an effect on the estimation of the reduction of virus genome counts, and may eliminate genomes of affected virus particles. However, no correlation was found between the results obtained by ET-RT-qPCR and those obtained by cell culture. Therefore, the effect is presumably only partial, and not adequate to allow accurate estimation of virus inactivation. Consequently, our results indicate that the quantification of virus genomes by PCR, regardless of prior ET, is not adequate for establishing virus inactivation and/or infectivity. In addition, our results also illustrate that the general effect of virus inactivation is not directly correlated to effects on the integrity of virus genome and protein capsid. Presumably, inactivation by food processing is the consequence of effects on proteins involved in adhesion and invasion stages.     

Effect of the Shellfish Proteinase K Digestion Method on Norovirus Capsid Integrity

Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log₁₀ was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log₁₀ loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log₁₀, with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.     

Estimation of Human Norovirus Infectivity from Environmental Water Samples by In Situ Capture RT-qPCR Method

Human noroviruses (HuNoVs) are highly infectious viruses for which water is an important medium of transmission. In this study, we explored a new in situ capture RT-qPCR (ISC-RT-qPCR) methodology to estimate the infectivity of HuNoV in environmental water samples. This assay was based on capturing encapsidated HuNoV by viral receptors, followed by in situ amplification of the captured viral genomes by RT-qPCR. We demonstrated that the ISC-RT-qPCR did not capture and enable signal amplification of heat-denatured Tulane Virus (TV) and HuNoVs. We further demonstrated that the sensitivity of ISC-RT-qPCR was equal or better than that of conventional RT-qPCR procedures for the detection of HuNoV GI and GII. We then utilized the ISC-RT-qPCR to detect HuNoV in environmental water samples for comparison against that from a conventional RT-qPCR procedure. TV was used as a process control virus. While complete inhibition of TV genomic signal was observed in 27% of samples tested by RT-qPCR, no inhibition of TV genomic signal was observed by ISC-RT-qPCR. From 72 samples tested positive for HuNoV GI signal by RT-qPCR, only 20 (27.8%) of these samples tested positive by ISC-RT-qPCR, suggesting that 72.2% of RT-qPCR-positive samples were unlikely to be infectious. From 16 samples tested positive for HuNoV GII signal by RT-qPCR, only one of these samples tested positive by ISC-RT-qPCR. Five samples that had initially tested negative for HuNoV GII signal by RT-qPCR, was tested as positive by ISC-RT-qPCR. Overall, ISC-RT-qPCR method provided an alternative assay to estimate infectivity of HuNoV in environmental samples.     

Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR (PMA-qPCR)

The detection of viable bacteria in wastewater treatment plants(WWTPs) is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide(PMA) combined with the quantitative polymerase chain reaction(PMA-qPCR) to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 μmol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.     

Survival of Coronaviruses in Water and Wastewater

The advent of severe acute respiratory syndrome and its potential environmental transmission indicates the need for more information on the survival of coronavirus in water and wastewater. The survival of representative coronaviruses, feline infectious peritonitis virus, and human coronavirus 229E was determined in filtered and unfiltered tap water (4 and 23°C) and wastewater (23°C). This was compared to poliovirus 1 under the same test conditions. Inactivation of coronaviruses in the test water was highly dependent on temperature, level of organic matter, and presence of antagonistic bacteria. The time required for the virus titer to decrease 99.9% (T_99.9) shows that in tap water, coronaviruses are inactivated faster in water at 23°C (10 days) than in water at 4°C (>100 days). Coronaviruses die off rapidly in wastewater, with T_99.9 values of between 2 and 4 days. Poliovirus survived longer than coronaviruses in all test waters, except the 4°C tap water.     

High Pressure Inactivation of HAV Within Mussels

The potential of high pressure processing to inactivate hepatitis A virus (HAV) within Mediterranean mussels (Mytilus galloprovincialis) and blue mussels (Mytilus edulis) was evaluated. HAV was bioaccumulated within mussels to approximately 6-log₁₀ PFU by exposure of mussels to HAV-contaminated seawater. After shucking, 5 min pressure treatments of 300, 325, 350, 375, and 400 MegaPascals (MPa) were performed at room temperature (18–22°C). For blue mussels, log₁₀ PFU reductions of HAV averaged 2.1 and 3.6 for treatments of 350 and 400 MPa, while for Mediterranean mussels reductions of 1.7 and 2.9 log₁₀ PFU MPa were observed for equivalent treatments. These results demonstrate that high pressure processing is capable of inactivating HAV within mussels.     

Application of Pesticide Sprays to Fresh Produce: A Risk Assessment for Hepatitis A and <Emphasis Type="Italic">Salmonella</Emphasis>

The purpose of this study was to quantify the transfer of viral and bacterial pathogens in water used to dilute pesticides sprayed onto the surfaces of cantaloupe, iceberg lettuce, and bell peppers. The average percent transfer of bacteria was estimated to range from 0.00021 to 9.4%, while average viral transfer ranged from 0.055 to 4.2%, depending on the type of produce. Based on these values the concentrations of hepatitis A virus (HAV) and Salmonella in water necessary to achieve a 1:10,000 annual risk of infection were calculated. Under worst case scenario assumptions, in which a pesticide is applied on the same day that the produce is harvested and when maximum transfer values are used, concentrations of 1.5 × 10⁻³ CFU Salmonella or 2.7 × 10⁻⁷ MPN HAV per 100 ml of the water used for application would result in 1:10,000 annual infection risk to anyone who consumes the fresh produce. If harvesting does not occur until at least 14 days after the application, to produce the same risk of infection, the numbers of Salmonella in 100 ml of water used to dilute the pesticides will be greater by up to five orders of magnitude, while the HAV numbers will have increased by up to two orders of magnitude. Based on the reported concentrations of enteric viruses in surface and ground waters in the United States, a 1:10,000 annual risk of infection could easily be exceeded with some groundwater sources used in the United States. To reduce the risks associated with the consumption of fresh produce, water used to prepare pesticides in spray applications should be evaluated for its microbiological quality.