Inactivation of Viruses and Bacteriophages as Models for Swine Hepatitis E Virus in Food Matrices

Hepatitis E virus has been recognised as a food-borne virus hazard in pork products, due to its zoonotic properties. This risk can be reduced by adequate treatment of the food to inactivate food-borne viruses. We used a spectrum of viruses and bacteriophages to evaluate the effect of three food treatments: high pressure processing (HPP), lactic acid (LA) and intense light pulse (ILP) treatments. On swine liver at 400 MPa for 10 min, HPP gave log₁₀ reductions of ≥4.2, ≥5.0 and 3.4 for feline calicivirus (FCV) 2280, FCV wildtype (wt) and murine norovirus 1 (MNV 1), respectively. Escherichia coli coliphage ?X174 displayed a lower reduction of 1.1, while Escherichia coli coliphage MS2 was unaffected. For ham at 600 MPa, the corresponding reductions were 4.1, 4.4, 2.9, 1.7 and 1.3 log₁₀. LA treatment at 2.2 M gave log₁₀ reductions in the viral spectrum of 0.29–2.1 for swine liver and 0.87–3.1 for ham, with ?X174 and MNV 1, respectively, as the most stable microorganisms. The ILP treatment gave log₁₀ reductions of 1.6–2.8 for swine liver, 0.97–2.2 for ham and 1.3–2.3 for sausage, at 15–60 J cm^?2, with MS2 as the most stable microorganism. The HPP treatment gave significantly (p < 0.05) greater virus reduction on swine liver than ham for the viruses at equivalent pressure/time combinations. For ILP treatment, reductions on swine liver were significantly (p < 0.05) greater than on ham for all microorganisms. The results presented here could be used in assessments of different strategies to protect consumers against virus contamination and in advice to food producers. Conservative model indicators for the pathogenic viruses could be suggested.     

江苏省某市部分农村河道水体和污水处理厂主要工艺环节病毒污染状况调查

为研究江苏省某市农村河道水体和污水处理厂主要工艺环节病毒的污染状况,分析常见理化因素与病毒污染之间的相关性,对江苏省某市部分农村河道水体和污水处理厂主要工艺环节7个水样、1个污泥和2个沉积物样点进行为期9个月的病毒(包括诺如病毒G Ⅰ、诺如病毒G Ⅱ、轮状病毒、札如病毒、腺病毒、星状病毒、肠道病毒)污染状况监测采样(共90份样本),通过阴离子膜吸附-洗脱法进行富集,荧光定量RT-PCR检测法对病毒进行检测,分析病毒污染状况;同时,监测水温、pH、COD(化学耗氧量)、TP(总磷)、TN(总氮)、EC(电导率)和DO(溶解氧)等7个理化指标的变化情况,探讨这些常见理化因素与病毒污染之间的相关性. 结果表明:90份样本中,诺如病毒G Ⅱ检出率为36.67%,肠道病毒检出率为30%,其他病毒检出率均低于诺如病毒G Ⅱ和肠道病毒. 污水处理后出水中有诺如病毒G Ⅱ、肠道病毒和轮状病毒检出,相关性分析显示,水温与诺如病毒G Ⅱ检出率呈显著负相关(P < 0.05). 研究显示,江苏省某市农村河道水体和污水处理厂主要工艺环节存在病毒污染,诺如病毒G Ⅱ为优势毒株;目前的污水消毒处理工艺并不能完全去除病毒,人群在接触外环境水体时,有潜在病毒暴露风险.      

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.     

The Effect of Heat and Free Chlorine Treatments on the Surface Properties of Murine Norovirus

Heat and free chlorine are among the most efficient and commonly used treatments to inactivate enteric viruses, but their global inactivation mechanisms have not been elucidated yet. These treatments have been shown to affect at least the capsid proteins of viruses and thus may affect the surface properties (i.e. electrostatic charge and hydrophobicity) of such particles. Our aim was to study the effects of heat and free chlorine on surface properties for a murine norovirus chosen as surrogate for human norovirus. No changes in the surface properties were observed with our methods for murine norovirus exposed to free chlorine. Only the heat treatment led to major changes in the surface properties of the virus with the expression of hydrophobic domains at the surface of the particles after exposure to a temperature of 55 °C. No modification of the expression of hydrophobic domains occurred after exposure to 60 °C, and the low hydrophobic state exhibited by infectious and inactivated particles after exposure to 60 °C appeared to be irreversible for inactivated particles only, which may provide a means to discriminate infectious from inactivated murine noroviruses. When exposed to a temperature of 72 °C or to free chlorine at a concentration of 50 mg/L, the genome became available for RNases.     

Assessment and Evaluation of an Integrated Hybrid Anaerobic–Aerobic Sewage Treatment System for the Removal of Enteric Viruses

The capability of a cost-effective and a small size decentralized pilot wastewater treatment plant (WWTP) to remove enteric viruses such as rotavirus, norovirus genogroup I (GGI), norovirus genogroup II (GGII), Hepatitis E virus (HEV), and adenovirus was studied. This pilot plant is an integrated hybrid anaerobic/aerobic setup which consisted of anaerobic sludge blanket (UASB), biological aerated filter (BAF), and inclined plate settler (IPS). Both the UASB and BAF are packed with a non-woven polyester fabric (NWPF). Results indicated that the overall log₁₀ reductions of enteric viruses’ genome copies through the whole system were 3.1 ± 1, 3.3 ± 0.5, and 2.6 ± 0.9 log₁₀ for rotavirus, norovirus GGI, and adenovirus, respectively. Reduction efficiency for both norovirus GGII and HEV after the different treatment steps could not be calculated because there were no significant numbers of positive samples for both viruses. The overall reduction of rotavirus infectious units through the whole system was 2.2 ± 0.8 log₁₀ reduction which is very close to the overall log₁₀ reduction of adenovirus infectious units through the whole system which was 2.1 ± 0.8 log₁₀ reduction. There was no considerable difference in the removal efficiency for different rotavirus G and P types. Adenovirus 41 was the only type detected in the all positive samples. Although the pilot WWTP investigated is cost effective, has a small footprint, does not need a long distance network pipes, and easy to operate, its efficiency to remove enteric viruses is comparable with the conventional centralized WWTPs.     

Design and Application of Nucleic Acid Standards for Quantitative Detection of Enteric Viruses by Real-Time PCR

Synthetic multiple-target RNA and DNA oligonucleotides were constructed for use as quantification standards for nucleic acid amplification assays for human norovirus genogroup I and II, hepatitis E virus, murine norovirus, human adenovirus, porcine adenovirus and bovine polyomavirus. This approach overcomes the problems related to the difficulty of obtaining practical quantities of viral RNA and DNA from these viruses. The quantification capacity of assays using the standards was excellent in each case (R ² > 0.998 and PCR efficiency > 0.89). The copy numbers of the standards were equivalent to the genome equivalents of representative viruses (murine norovirus and human adenovirus), ensuring an accurate determination of virus presence. The availability of these standards should facilitate the implementation of nucleic acid amplification-based methods for quantitative virus detection.     

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.

     

Minimum Infective Dose of the Major Human Respiratory and Enteric Viruses Transmitted Through Food and the Environment

Viruses are a significant cause of morbidity and mortality around the world. Determining the minimum dose of virus particles that can initiate infection, termed the minimum infective dose (MID), is important for the development of risk assessment models in the fields of food and water treatment and the implementation of appropriate infection control strategies in healthcare settings. Both respiratory and enteric viruses can be shed at high titers from infected individuals even when the infection is asymptomatic. Presence of pre-existing antibodies has been shown to affect the infectious dose and to be protective against reinfection for many, but not all viruses. Most respiratory viruses appear to be as infective in humans as in tissue culture. Doses of <1 TCID₅₀ of influenza virus, rhinovirus, and adenovirus were reported to infect 50% of the tested population. Similarly, low doses of the enteric viruses, norovirus, rotavirus, echovirus, poliovirus, and hepatitis A virus, caused infection in at least some of the volunteers tested. A number of factors may influence viruses’ infectivity in experimentally infected human volunteers. These include host and pathogen factors as well as the experimental methodology. As a result, the reported infective doses of human viruses have to be interpreted with caution.     

Critical Review of Norovirus Surrogates in Food Safety Research: Rationale for Considering Volunteer Studies

The inability to propagate human norovirus (NoV) or to clearly differentiate infectious from noninfectious virus particles has led to the use of surrogate viruses, like feline calicivirus (FCV) and murine norovirus-1 (MNV), which are propagatable in cell culture. The use of surrogates is predicated on the assumption that they generally mimic the viruses they represent; however, studies are proving this concept invalid. In direct comparisons between FCV and MNV, their susceptibility to temperatures, environmental and food processing conditions, and disinfectants are dramatically different. Differences have also been noted between the inactivation of NoV and its surrogates, thus questioning the validity of surrogates. Considerable research funding is provided globally each year to conduct surrogate studies on NoVs; however, there is little demonstrated benefit derived from these studies in regard to the development of virus inactivation techniques or food processing strategies. Human challenge studies are needed to determine which processing techniques are effective in reducing NoVs in foods. A major obstacle to clinical trials on NoVs is the perception that such trials are too costly and risky, but in reality, there is far more cost and risk in allowing millions of unsuspecting consumers to contract NoV illness each year, when practical interventions are only a few volunteer studies away. A number of clinical trials have been conducted, providing important insights into NoV inactivation. A shift in research priorities from surrogate research to volunteer studies is essential if we are to identify realistic, practical, and scientifically valid processing approaches to improve food safety.     

Human Enteric Viruses Occurrence in Shellfish from European Markets

Different sources were consulted to obtain information on the occurrence of viruses in bivalve molluscs on the European market. Twenty-six peer-reviewed articles were identified reporting on the molecular detection of viral RNA in 4,260 samples in total. The data obtained will be presented geographically on virus types detected, the origin and treatment of the shellfish, and the detection technique applied. The data demonstrate that viral RNA can be detected in shellfish from polluted areas, in depurated shellfish as well as those for human consumption. The European Rapid Alert System for Food and Feed (RASFF) database was consulted as another source. Twenty-eight notifications were identified on the presence of hepatitis A virus or norovirus in shellfish on the European market. The most recent report of the European laboratory network was referred to, to gain insight into the laboratory capability at present for the analyses of shellfish on the presence of viruses. Approximately 67% of 27 participating laboratories obtained intended results for all samples, consisting of lenticules loaded with 10³ copies norovirus (genogroup I (GGI) and/or genogroup II (GGII)) and/or 1 × 10⁵–8 × 10⁴ copies of hepatitis A virus. From 1993, there has been a continuous development of molecular detection techniques and tools have been described to ensure quality assurance. End product testing will, however, not be achievable. As depuration has been shown not to be effective for the complete elimination of viruses, shellfish should not be in contact with faecal contaminated water in order to minimise the risk of shellfish-transmittable viral diseases.     

International Standardisation of a Method for Detection of Human Pathogenic Viruses in Molluscan Shellfish

The viruses primarily associated with shellfish-borne illness are norovirus, causing gastroenteritis and hepatitis A virus (HAV). Recent years have seen a proliferation of publications on methods for detection of these viruses in shellfish using polymerase chain reaction (PCR). However, currently no standard harmonised procedures have been published. Standardisation is necessary before virus methods can be considered for adoption within a regulatory framework. A European standardisation working group is developing a two-part (quantitative and qualitative) standard method for virus detection in foodstuffs, including shellfish, which has the potential to be incorporated into EU legislation as a reference method. This article describes the development of the standard method and outlines the key methodology principles adopted, the controls and other quality assurance measures supporting the method and future necessary developments in the area.     

Potential Risk of Norovirus Infection Due to the Consumption of “Ready to Eat” Food

In this study, we investigated the presence of enteric viruses such as norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and adenovirus (HAdV), in vegetables available on the Italian markets. For this aim, 110 national and international ??ready to eat?? samples were collected and analyzed by biomolecular tests and positive samples were confirmed by sequencing. All samples (100?%) were negative for HAV, HEV, and HAdV, while 13.6?% (15/110) were positive for NoV. Actually there is not a formal surveillance system for NoV infections in Italy but we clearly demonstrated a potential risk associated with the consumption of ??ready to eat?? vegetables. This study confirmed for the first time in Italy the presence of norovirus in semi-dried tomatoes by PCR technique.     

Detection and Typing of Norovirus from Frozen Strawberries Involved in a Large-Scale Gastroenteritis Outbreak in Germany

During September/October 2012, a norovirus gastroenteritis outbreak affecting about 11,000 people occurred in Germany. Epidemiological studies suggested that frozen strawberries represented the vehicle of infection. We describe here the analysis of frozen strawberries for the presence of norovirus. Samples were taken by applying a stratified subsampling scheme. Two different methods for virus extraction from strawberries were compared. First, viruses were eluted from strawberries under alkaline conditions and concentrated using a polyethylene glycol precipitation. Second, ultrafiltration was applied for concentration of viruses rinsed off of the berries. In both cases, RNA was extracted and analyzed by real-time RT-PCR. Application of the ultrafiltration method generally resulted in a lower detection rate. Noroviruses were detected in 7/11 samples derived from the lot of strawberries implicated in the outbreak using the precipitation method. Typing of norovirus revealed three different genotypes including a combination of norovirus genotype II.16 (viral polymerase) and II.13 (viral capsid). This genotype combination was also found in some of the patients that were involved in the outbreak, but that had not been reported in Germany so far. In conclusion, heterogeneously distributed noroviruses in frozen strawberries can be detected by applying an optimized combination of sampling procedures, virus extraction methods, and real-time RT-PCR protocols. The detection of several different genotypes in the strawberries may suggest contamination from sewage rather than from a single infected food handler.     

Virus Type-Specific Removal in a Full-Scale Membrane Bioreactor Treatment Process

We investigated removal of noroviruses, sapoviruses, and rotaviruses in a full-scale membrane bioreactor (MBR) plant by monitoring virus concentrations in wastewater samples during two gastroenteritis seasons and evaluating the adsorption of viruses to mixed liquor suspended solids (MLSS). Sapoviruses and rotaviruses were detected in 25% of MBR effluent samples with log reduction values of 3- and 2-logs in geometric mean concentrations, respectively, while noroviruses were detected in only 6% of the samples. We found that norovirus and sapovirus concentrations in the solid phase of mixed liquor samples were significantly higher than in the liquid phase (P < 0.01, t test), while the concentration of rotaviruses was similar in both phases. The efficiency of adsorption of the rotavirus G1P[8] strain to MLSS was significantly less than norovirus GI.1 and GII.4 and sapovirus GI.2 strains (P < 0.01, t test). Differences in the adsorption of viruses to MLSS may cause virus type-specific removal during the MBR treatment process as shown by this study.     

Persistence of Viruses by qPCR Downstream of Three Effluent-Dominated Rivers in the Western United States

This study was designed to determine the quantitative polymerase chain reaction (qPCR) signal persistence of viruses in three effluent-dominated streams. Samples were collected from the effluent outfall of three wastewater treatment plants in the Western United States and downstream at different locations. All samples were tested for the presence of pepper mild mottle virus (PMMoV), adenoviruses, norovirus GI and GII, Aichi virus, and enteroviruses using qPCR. PMMoV was detected most frequently in 54/57 (94.7%) samples, followed by adenoviruses which was detected in 21/57 (36.8%) samples. PMMoV was detected at all locations downstream and up to 32 km from the discharge point. This study demonstrated that the detection signal of PMMoV was able to persist in wastewater discharges to a greater degree than human enteric viruses in effluent-dominated rivers.     

Natural Plant Essential Oils Do Not Inactivate Non-enveloped Enteric Viruses

The application of plant essential oils (EOs) (hyssop and marjoram) was evaluated for inactivation of non-enveloped viruses using murine norovirus and human adenovirus as models. No significant reduction of virus titres (TCID₅₀) was observed when EOs were used at different temperatures and times.