Preliminary Source Tracking of Male-Specific (F<Superscript>+</Superscript>) RNA Coliphage on Lettuce as a Surrogate of Enteric Viruses Using Reverse Transcription-PCR

The aim of this research was to preliminary track fecal source male-specific F⁺RNA coliphages including human and animals in lettuce. At first, two published virus extraction procedures of ultracentrifugation and PEG precipitation were compared using DAL assay for determining the recovery efficiency in lettuce spiked artificially with three concentrations (10², 10⁴, 10⁶ pfu/100 ml) of MS2 coliphage. The results showed that PEG precipitation had the highest recovery in which the recovery efficiency at the spiked level of 10⁶ pfu/100 ml was 16.63 %. Aqueous phase obtained from the final step of PEG method was applied for enumeration of coliphage and viral RNA extraction in naturally contaminated lettuce samples (N = 30) collected from two sources (market and farm). The samples were then analyzed based on (I, II, III, and IV primer sets) using RT-PCR method. Coliphages were detected in 9 (60 %) and 12 (80 %) out of 15 market and farm samples, respectively, using DAL assay, whereas male-specific F⁺RNA coliphages were detected using the RT-PCR method in 9 (60 %) and 13 (86.6 %) out of 15 samples of market and farm, respectively. Based on the results, only genotype I of male-specific F⁺RNA coliphages was detected in lettuce samples and no sample tested was positive for other genotypes (II, III, and IV).     

Impact of Sodium Chloride, Sucrose and Milk on Heat Stability of the Murine Norovirus and the MS2 Phage

Until now, little is known about the influence of food additives on heat inactivation of noroviruses. Only a few studies have shown a protective or inhibiting effect on virus infectivity caused by the food matrix. Therefore, the aim of this study was to examine the influence of sodium chloride, sucrose and milk on heat stability of the surrogates murine norovirus (MNV) and MS2 phage at 60 °C for 1–5 min in PBS for MNV and for 5–120 min in suspension medium buffer for MS2 phage. Different concentrations of sodium chloride (5, 10 %) and sucrose (5, 50 %) were added to the respective buffers. In addition, commercially available milk with different fat concentrations (0.3, 1.5, 3.5 %) was investigated in this study. In general, a linear titre reduction for MNV and MS2 phage could be observed, except for the heat treatment of MNV in PBS with 50 % sucrose. A protective effect of PBS with 50 % sucrose and of the matrix milk on MNV could be concluded. All other tested conditions did not show any influence on virus inactivation. However, MS2 phage did show a higher heat resistance throughout the experiments compared to MNV. In future investigations, it should be tested, whether the achieved data may be considered in risk assessments of heat-treated food products with high concentrations of sugar. Furthermore, it should be clarified, whether these results can also be referred to complex food matrices.     

Evaluation of Virus Reduction by Ultrafiltration with Coagulation–Sedimentation in Water Reclamation

The evaluation of virus reduction in water reclamation processes is essential for proper assessment and management of the risk of infection by enteric viruses. Ultrafiltration (UF) with coagulation–sedimentation (CS) is potentially effective for efficient virus removal. However, its performance at removing indigenous viruses has not been evaluated. In this study, we evaluated the reduction of indigenous viruses by UF with and without CS in a pilot-scale water reclamation plant in Okinawa, Japan, by measuring the concentration of viruses using the real-time polymerase chain reaction (qPCR). Aichi virus (AiV) and pepper mild mottle virus (PMMoV) were targeted in addition to the main enteric viruses of concern for risk management, namely, norovirus (NoV) genogroups I and II (GI and GII) and rotavirus (RoV). PMMoV, which is a plant pathogenic virus and is present at high concentrations in water contaminated by human feces, has been suggested as a useful viral indicator. We also investigated the reduction of a spiked model virus (F-specific RNA bacteriophage MS2) to measure the effect of viral inactivation by both qPCR and plaque assay. Efficiencies of removal of NoV GI, NoV GII, RoV, and AiV by UF with and without CS were >0.5 to 3.7 log₁₀, although concentrations were below the detection limit in permeate water. PMMoV was the most prevalent virus in both feed and permeate water following UF, but CS pretreatment could not significantly improve its removal efficiency (mean removal efficiency: UF, 3.1 log₁₀; CS + UF, 3.4 log₁₀; t test, P > 0.05). CS increased the mean removal efficiency of spiked MS2 by only 0.3 log₁₀ by qPCR (t-test, P > 0.05), but by 2.8 log₁₀ by plaque assay (t-test, P < 0.01). This difference indicates that the virus was inactivated during CS + UF. Our results suggest that PMMoV could be used as an indicator of removal efficiency in water reclamation processes, but cultural assay is essential to understanding viral fate.     

Biodegradation by bioaugmentation of dairy wastewater by fungal consortium on a bioreactor lab-scale and on a pilot-scale

A fungal consortium including Aspergillus niger, Mucor hiemalis and Galactomyces geotrichum was tested for the treatment of dairy wastewater. The bio-augmentation method was tested at lab-scale (4 L), at pilot scale (110 L) and at an industrial scale in Wastewater Treatment Plants (WWTP). The positive impact of fungal addition was confirmed when fungi was beforehand accelerated by pre-culture on whey (5 g/L lactose) or on the dairy effluent. Indeed, chemical oxygen demand (COD) removal yields increased from 55% to 75% for model medium, diluted milk. While after inoculation of an industrial biological tank from a dairy factory with the fungal consortium accelerated by pre-cultivation in a 1000 L pilot plant, the outlet COD values decreased from values above the standard one (100 mg/L) to values in the range of 50-70 mg/L. In addition, there was a clear impact of fungal addition on the ’hard’ or non-biodegradable COD owing to the significant reduction of the increase of the COD on BOD 5 ratio between the inlet and the outlet of the biological tank of WWTP. It was in the range of 451%-1111% before adding fungal consortium, and in the range of 257%-153% after bio-augmentation with fungi. An inoculated bioreactor with fungal consortium was developed at lab-scale and demonstrated successfully at pilot scale in WWTP.     

Prevalence of Human Parainfluenza Viruses and Noroviruses Genomes on Office Fomites

The aim of this study was to evaluate the potential role of office fomites in respiratory (human parainfluenza virus 1—HPIV1, human parainfluenza virus 3—HPIV3) and enteric (norovirus GI—NoV GI, norovirus GII—NoV GII) viruses transmission by assessing the occurrence of these viruses on surfaces in office buildings. Between 2016 and 2017, a total of 130 surfaces from open-space and non-open-space rooms in office buildings located in one city were evaluated for HPIV1, HPIV3, NoV GI, and NoV GII viral RNA presence. Detection of viruses was performed by RT-qPCR method. Study revealed 27 positive samples, among them 59.3% were HPIV3-positive, 25.9% HPIV1-positive, and 14.8% NoV GII-positive. All tested surfaces were NoV GI-negative. Statistical analysis of obtained data showed that the surfaces of office equipment including computer keyboards and mice, telephones, and desktops were significantly more contaminated with respiratory viruses than the surfaces of building equipment elements such as door handles, light switches, or ventilation tracts (χ ² p = 0.006; Fisher’s Exact p = 0.004). All examined surfaces were significantly more contaminated with HPIVs than NoVs (χ ² p = 0.002; Fisher’s Exact p = 0.003). Office fomites in open-space rooms were more often contaminated with HPIVs than with NoVs (χ ² p = 0.016; Fisher’s Exact p = 0.013). The highest average concentration of HPIVs RNA copies was observed on telephones (1.66 × 10² copies/100 cm²), while NoVs on the light switches (1.40 × 10² copies/100 cm²). However, the Kruskal–Wallis test did not show statistically significant differences in concentration levels of viral RNA copies on surfaces between the all tested samples. This study unequivocally showed that individuals in office environment may have contact with both respiratory and enteric viral particles present on frequently touched surfaces.     

Effectiveness of Poliovirus Concentration and Recovery from Treated Wastewater by Two Electropositive Filter Methods

Enteric viruses are often present in low numbers in various water matrices. Virus sampling therefore involves multiple concentration steps to condense large samples down to small volumes for detection by cell culture or molecular assays. The NanoCeram^® Virus Sampler has been demonstrated to be effective for the recovery of viruses from tap water, surface waters, and seawater. The goal of this study was to evaluate a new method using NanoCeram^® filters for the recovery of poliovirus 1 (PV-1) from treated wastewater. Activated sludge effluent samples were spiked with PV-1 and concentrated in side-by-side tests by two methods: (1) NanoCeram^® filtration, elution with sodium polyphosphate buffer, secondary concentration via centrifugal ultrafiltration; and (2) 1MDS filtration, elution with beef extract, secondary concentration via organic flocculation. The virus retention and elution efficiencies did not differ significantly between the two methods. In contrast, the secondary concentrate volume was smaller for the NanoCeram^® method (8.4 vs. 30 mL) and the secondary concentration efficiencies were different between the two methods with 98 % for centrifugal ultrafiltration (NanoCeram^®) and 45 % for organic flocculation (1MDS). The overall method efficiencies were significantly different (P ≤ 0.05) with the NanoCeram^® method yielding a 57 % and the 1MDS a 23 % virus recovery. In addition, there appeared to be less interference with viral detection via polymerase chain reaction with the NanoCeram^® concentrates. This NanoCeram^® method therefore is able to efficiently recover PV-1 from large volumes of wastewater and may serve as an inexpensive alternative to the standard 1MDS filter method for such applications.     

预酸化＋UASB＋CASS工艺处理乳业废水

乳业废水中乳蛋白、乳脂肪的生物降解比较慢,转化不毫全时容易引起污泥上浮,脂肪及其中间产物（尤其是游离的长链脂肪酸）会对厌氧微生物产生抑制作用。这里介绍一种乳业废水处理技术-预酸化＋UASB＋CASS工艺,经过2年多实际工程运行表明,此工艺有效的克服以上难题,并且运行稳定,处理费用低,操作管理方便。     

The Effect of Heat on the Physicochemical Properties of Bacteriophage MS2

The differences in physicochemical characteristics between infectious and non-infectious viral particles are poorly known. Even for heat, which is known as one of the most efficient treatments to inactivate enteric viruses, the global inactivation mechanisms have not been described yet. Such knowledge would help distinguish between both types of particles and therefore clarify the interpretation of the presence of viral genomes in food after heat treatment. In this study, we examined in particular the differences in electrostatic charge and hydrophobicity between the two particle types. MS2 phage, a common surrogate for enteric viruses, was used as a model virus. The heat-induced inactivation process of the infectious phages caused hydrophobic domains to be transiently exposed and their charge to become less negative. The particles also became progressively permeable to small molecules such as SYPRO Orange dye. The presence of non-infectious phage particles in which the genome was not accessible to RNases has been clearly demonstrated. These observations were done for MS2 phages exposed to a temperature of 60 °C. When exposed to a temperature higher than their critical temperature (72 °C), the particles were disrupted and the genome became available for RNases. At lower temperatures, 60 °C in this study, the transient expression of hydrophobic domains of remaining infectious phages appeared as an interesting parameter for improving their specific detection.     

Seasonal Occurrence of Human Enteric Viruses in River Water Samples Collected from Rural Areas of South-East Poland

The study was carried out in 2007, and its main aim was 1 year monitoring of surface water of the River Wieprz in Poland for the presence of human adenoviruses group F and noroviruses (NoVs). In total, 60 water samples were collected from four sampling sites situated along the river. The viruses were concentrated from water samples using glass wool, followed by elution with a glycine buffer containing skimmed milk powder. Subsequently, the viral nucleic acids were extracted and purified from water concentrates using a NucliSENS^® kit and a QIAamp Viral RNA Mini Kit^®. The presence of viral nucleic acids was confirmed by applying traditional PCR-based methods with incorporated internal amplification controls. Human pathogenic viruses were detected in 35% of analysed water samples. Adenoviruses were detected in 28.3% of analysed samples, and were present at all seasons of the year. 11.6% of the samples were positive for NoVs; they were present only during summer, in contrast to conventional findings. Molecular identification of norovirus strains revealed that they belong to genogroup I and II.     

Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR

Several foodborne norovirus gastroenteritis outbreaks have been linked to fresh produce. Rapid and sensitive detection can help prevent the release of contaminated produce items in the market. The objectives of this study were to apply a relatively inexpensive SYBR Green I-based real-time RT-PCR assay for the rapid detection of human norovirus (NoV) GI and GII on the surfaces of lettuce, cherry tomatoes, and green onions. Each washed produce commodity (25 g) was spiked with serial dilutions of NoV GI and GII stool samples. RNA was eluted from the produce surface and extracted using the TRIzol^? method. This was followed by detection using SYBR Green I real-time RT-PCR with primers specific for NoV GI (COG1F-COG1R) and GII (COG2F-COG2R) along with an internal RNA amplification control. End-point detection limits from lettuce and tomatoes were found to be 10 RT-PCR units/25 g for GI and GII and 1 RT-PCR unit/25 g for GI and 10 RT-PCR units/25 g for GII from green onions. These results were confirmed by Tm analysis (showing peaks at 81.5 and 84°C for GI and GII, respectively; and 83°C for the IAC) as well as agarose gel electrophoresis that confirmed products of ~95 bp for GI and GII and ~155 bp for the RNA IAC. Results could be obtained within one working day, showing potential for routine use in diagnostics and monitoring of NoV contamination by the produce industry.     

Survival of Human Adenovirus 41 in Land-Applied Manure and Biosolids

Human Adenovirus 41 (Ad41) is an important human enteric pathogen and widely prevalent in the environment. The aim of this study was to assess the survival of Ad41 based on genome stability and infectivity in different types of manure and three types of biosolids. For viral survival studies, Ad41 was added to pelletized poultry litter (PL), alum-treated poultry litter (AL), raw poultry litter (RPL), liquid dairy manure (DM), swine manure (SM), and three types of biosolids 1, 2, 3. All samples were stored at 20 or 4°C and analyzed every 10 days for up to 60 days. Quantification PCR (qPCR) standard curves were generated for PL, AL, biosolids 1, and DM to measure the number of viral genomic copies remaining in the samples. To study the infectivity, all contaminated manure/biosolids samples were added to mammalian cell culture and viral mRNA was detected using one-step RT–PCR. Overall, Ad41 viral genomes were stable at both 20 and 4°C and there was no significant loss of viral DNA after 60 days in PL, AL, biosolids type 1, and DM. However, infectivity was lost almost immediately in high pH biosolids type 2 and 3, and infectivity decreased quickly in DM, with estimated T90 of 4.3 and 8.7 days at 20 and 4°C, respectively. Ad41 had ~1.9 log loss of infectivity after added in SM and biosolids type 1 at day 0, and estimated T90 was 12.5 and 28.6 days for biosolids type 1, and 19.1 and 51.0 days for SM at 20 and 4°C, respectively. Ad41 maintained infectivity in all three poultry litter, and after 60 days incubation, there were significantly more infectious virus in PL, AL, and RPL than biosolids 1, SM, and DM at 20°C.     

Minimizing Bias in Virally Seeded Water Treatment Studies: Evaluation of Optimal Bacteriophage and Mammalian Virus Preparation Methodologies

One key assumption impacting data quality in viral inactivation studies is that reduction estimates are not altered by the virus seeding process. However, seeding viruses often involves the inadvertent addition of co-constituents such as cell culture components or additives used during preparation steps which can impact viral reduction estimates by inducing non-representative oxidant demand in disinfection studies and fouling in membrane assessments. The objective of this study was therefore to characterize a mammalian norovirus surrogate, murine norovirus (MNV), and bacteriophage MS2 at sequential stages of viral purification and to quantify their potential contribution to artificial oxidant demand and non-representative membrane fouling. Our results demonstrate that seeding solvent extracted and 0.1 micron filtered MNV to ~10⁵ PFU/mL in an experimental water matrix will result in additional total organic carbon (TOC) and 30 min chlorine demand of 39.2 mg/L and 53.5 mg/L as Cl₂, respectively. Performing sucrose cushion purification on the MNV stock prior to seeding reduces the impacts of TOC and chlorine demand to 1.6 and 0.15 mg/L as Cl₂, respectively. The findings for MNV are likely relevant for other mammalian viruses propagated in serum-based media. Thus, advanced purification of mammalian virus stocks by sucrose cushion purification (or equivalent density-based separation approach) is warranted prior to seeding in water treatment assessments. Studies employing bacteriophage MS2 as a surrogate virus may not need virus purification, since seeding MS2 at a concentration of ~10⁶ PFU/mL will introduce only ~1 mg/L of TOC and ~1 mg/L as Cl₂ of chlorine demand to experimental water matrices.     

Detection and Characterization of Hepatitis A Virus and Norovirus in Mussels from Galicia (NW Spain)

Shellfish are recognized as a potential vehicle of viral disease and despite the control measures for shellfish safety there is periodic emergence of viral outbreaks associated with shellfish consumption. In this study a total of 81 mussel samples from Ría do Burgo, A Coruña (NW Spain) were analysed. Samples were collected in seven different harvesting areas with the aim to establish a correlation between the prevalence of norovirus (NoV) and hepatitis A virus (HAV) in mussel samples and the water quality. In addition, the genogroup of the detected HAV and NoV strains was also determined. The HAV presence was detected in 18.5 % of the samples. Contamination levels for this virus ranged from 1.1 × 10² to 4.1 × 10⁶ RNA copies/g digestive tissue. NoV were detected in 49.4 % of the cases reaching contamination levels from 5.9 × 10³ to 1.6 × 10⁹ RNA copies/g digestive tissue for NoV GI and from 6.1 × 10³ to 5.4 × 10⁶ RNA copies/g digestive tissue for NoV GII. The χ²-test showed no statistical correlation between the number of positive samples and the classification of molluscan harvesting area based on the E. coli number. All the detected HAV strains belong to genogroup IB. NoV strains were assigned to genotype I.4, II.4 and II.6.     

Hepatitis A Virus Disinfection in Water by Solar Photo–Fenton Systems

This study evaluates and compares the effectiveness of solar photo–Fenton systems for the inactivation of hepatitis A virus (HAV) in water. The effect of solar irradiance, dark- Fenton reaction and three different reactant concentrations (2.5/5, 5/10 and 10/20 mg/L of Fe²⁺/H₂O₂) on the photo–Fenton process were tested in glass bottle reactors (200 mL) during 6 h under natural sunlight. Disinfection kinetics were determined both by RT-qPCR and infectivity assays. Mean water temperatures ranged from 25 to 27.3 °C, with a maximum local noon UV irradiances of 22.36 W/m². Photo–Fenton systems yielded increased viral reduction rates in comparison with the isolated effect under the Fenton reaction in darkness (negligible viral reduction) or the solar radiation (0.25 Log of RNA reduction). With the highest concentration employed (10–20 mg/L Fe²⁺–H₂O₂), an average RNA reduction rate of ~ 1.8 Log (initial concentration of 10⁵ pfu/mL) and a reduction of 80% in the infectivity capacity were reached. Results showed a strong synergistic effect between Fe²⁺/H₂O₂ and sunlight, demonstrating that significant disinfection rates of HAV under photo–Fenton systems may occur with relatively higher efficiency at middle environmental temperatures and without the need for an energy-intensive light source.     

Use of Preservative Agents and Antibiotics for Increased Poliovirus Survival on Positively Charged Filters

Environmental surveillance of poliovirus (PV) and other non-enveloped viruses can help identify silent circulation and is necessary to certify eradication. The bag-mediated filtration system is an efficient method to filter large volumes of environmental waters at field sites for monitoring the presence of viruses. As filters may require long transit times to off-site laboratories for processing, viral inactivation or overgrowth of bacteria and fungi can interfere with virus detection and quantification (Miki and Jacquet in Aquatic Microb Ecol 51(2):195–208, 2008). To evaluate virus survival over time on ViroCap^? filters, the filters were seeded with PV type 1 (PV1) and/or MS2 and then dosed with preservatives or antibiotics prior to storage and elution. These filters were stored at various temperatures and time periods, and then eluted for PV1 and MS2 recovery quantification. Filters dosed with the preservative combination of 2% sodium benzoate and 0.2% calcium propionate had increased virus survival over time when stored at 25 °C, compared to samples stored at 25 °C with no preservatives. While elution within 24 h of filtration is recommended, if storage or shipping is required then this preservative mixture can help preserve sample integrity. Addition of an antibiotic cocktail containing cephapirin, gentamicin, and Proclin^? 300 increased recovery after storage at 4 and 25 °C, when compared to storage with no antibiotics. The antibiotic cocktail can aid sample preservation if access to appropriate antibiotics storage is available and sample cold chain is unreliable. This study demonstrated that the use of preservatives or antibiotics is a simple, cost-effective method to improve virus detection from ViroCap cartridge filters over time.     

Simultaneous Concentration of Enteric Viruses and Protozoan Parasites: A Protocol Based on Tangential Flow Filtration and Adapted to Large Volumes of Surface and Drinking Waters

A filtration system, based on tangential flow filtration (TFF) followed by ultracentrifugation was developed in order to concentrate simultaneously viruses and parasites from large volumes of water. For TFF, no pre-treatment of the membrane is performed but a post-rinsing step using high pH-beef extract-based eluant. Applying our protocol to 20 l of surface waters spiked with vaccinal poliovirus-1, ϕX174 and MS2 bacteriophages resulted in an averaged viral recovery of 75% by TFF and 91% by ultracentrifugation (total viral recovery of 70%). Our protocol was further applied to 31 environmental samples including surface, ground and drinking waters from the Grand Duchy of Luxembourg in order to assess the occurrence of protozoan parasites (Cryptosporidium parvum and Giardia lamblia (oo)cysts), pathogenic viruses (enterovirus, norovirus and adenovirus) and infectious bacteriophages (somatic coliphages and F-specific phages) in these samples. High viral recovery rates of > 70% were confirmed concentrating environmental strains of somatic and F-specific coliphages from non-spiked surface waters. Parasites and enteric viruses were detected in 86 and 40% of the surface waters used for drinking water production, respectively. Infectious bacteriophages were isolated from all surface waters and in two out of seven (29%) groundwaters revealing a susceptibility of the corresponding wells to viral pollution. TFF-based method proved to be efficient for surveying the occurrence of non-bacterial pathogens such as enteric viruses and protozoan parasites in large volumes of environmental waters.     

乳制品废水中蛋白质回收利用途径的研究

本文以某乳制品企业产生的废水为研究对象,对乳制品工业废水的处理和资源回收应用进行研究,为乳制品废水中蛋白质的综合利用,为实现乳制品废水的资源化、减量化提供技术方法和理论基础.     

Diet-dependent heat emission reveals costs of post-diapause recovery from different nutritional sources in a carnivorous beetle

Restoration of fat stores is metabolic first priority for many insects that emerge from hibernation with depleted fat bodies. To some extent, the animals must be flexible and use whatever foods available irrespective of their nutrient composition. Previously, the carabid beetles Anchomenus dorsalis have been found to refill their fat stores to the same extent over 9 days irrespective of the nutrient composition of their food. However, a higher cost of fat deposition when the food was rich in sugar or protein rather than lipid was indicated by higher total energy consumption. Here, we test the hypothesis of increased metabolic costs of building fat stores from sugar- or protein-rich food than from lipid-rich food by microcalorimetry. We measured the heat emitted from beetles that had fed on sugar-, protein-, or lipid-rich food for 0 (common control), 2, 5, or 10 days. As predicted, heat emission was increased in beetles getting sugar- and protein-rich food compared with those getting lipid-rich food. However, we did not confirm the beetles’ ability to rebuild fat stores from protein-rich food; instead, they increased in lean mass. Overall, sugar-rich food seems to be optimal for post-winter recovery, because it is better than lipid-rich food that allows concurrent rebuilding of fat stores and lean mass, which may benefit preparation for spring migration and reproduction. We propose that overwintered fruits may be highly preferred post-diapause food for these otherwise mostly carnivorous beetles.