Somatic Coliphage Profiles of Produce and Environmental Samples from Farms in Northern México

Somatic coliphages were quantified in 459 produce and environmental samples from 11 farms in Northern Mexico to compare amounts of somatic coliphages among different types of fresh produce and environmental samples across the production steps on farms. Rinsates from cantaloupe melons, jalapeño peppers, tomatoes, and the hands of workers, soil, and water were collected during 2011–2012 at four successive steps on each farm, from the field before harvest through the packing facility, and assayed by FastPhage MPN Quanti-tray method. Cantaloupe farm samples contained more coliphages than jalapeño or tomato (p range <0.01–0.03). Across production steps, jalapeños had higher coliphage percentages before harvest than during packing (p = 0.03), while tomatoes had higher coliphage concentrations at packing than all preceding production steps (p range <0.01–0.02). These findings support the use of targeted produce-specific interventions at multiple points in the process of growing and packing produce to reduce the risk of enteric virus contamination and improve food safety during fruit and vegetable production.     

Assessment of Disinfectant Performance in Chicken Cages Using Coliphages

To control the spread of avian flu (influenza) and other viruses of concern among commercial flocks, it is essential that proper disinfection procedures be developed along with methods for assessing their performance. Such methods must be rapid and inexpensive. Coliphages were used as indicators to demonstrate the efficacy of quaternary ammonium compounds and chlorine bleach for the inactivation of viruses in chicken cages. The concentration of indigenous coliphages in chicken litter was found to be 10⁴–10⁷ per gram and from 0 to 8,500 per 100 cm² of floor surface. To assess the effectiveness of the disinfectants, floor samples were collected pre and post disinfection. These results indicated that chlorine bleach was more effective than quaternary ammonium compounds in reducing the amount of indigenous coliphages. To obtain better quantitative data, MS-2 coliphage was sprayed onto cage floors, left overnight to dry, and then the surfaces disinfected. Similar results were obtained with both indigenous coliphages and MS-2. There appears to be no significant difference in coliphage reduction by increasing the contact time from 10 to 30 min. To ensure at least a 99.9% reduction of virus at least 236 ml of household bleach per 3.78 l should be used.     

Development and Evaluation of a Multiplexed Real-Time TaqMan RT-PCR Assay with a Sample Process Control for Detection of F-specific RNA Coliphage Genogroups I and IV

There are increasing concerns of zoonotic transmission of some animal enteric viruses, such as calicivirus, hepatitis E virus, and rotavirus, which are closely related to human pathogenic strains. Most enteric viruses are detected by molecular techniques because they cannot be cultured. Surrogates such as F-RNA coliphages are cultivable but few molecular methods exist. Individual real-time TaqMan RT-PCR assays for the replicase gene of F-RNA coliphage genogroups I and IV were developed and multiplexed with a real-time TaqMan RT-PCR assay for feline calicivirus as a sample process control for the simultaneous detection and enumeration of genogroup I and IV F-RNA coliphages. Genogroup IV were successfully detected with the multiplexed assay in 80% of fecal samples that contained F-RNA coliphage levels ≥3.2 log plaque forming units (pfu). F-RNA coliphage were at or below the limit of detection in most fecal samples when levels were ≤4 log pfu/g.     

Occurrence of <Emphasis Type="Italic">Pepper Mild Mottle Virus</Emphasis> (PMMoV) in Groundwater from a Karst Aquifer System in the Yucatan Peninsula,Mexico

The Yucatan Peninsula of Mexico hosts a karst aquifer system that is the only source of freshwater for the area; however, it is vulnerable to human-mediated contamination. Pepper mild mottle virus (PMMoV) is one of the most abundant RNA viruses associated with human feces, making it a viable indicator for tracking fecal pollution in aquatic environments, including groundwater. In this study, groundwater samples collected from a karst aquifer from fresh and brackish water locations were analyzed for fecal indicator bacteria, somatic and male F+ specific coliphages, and PMMoV during the rainy and dry seasons. Total coliform bacteria were detected at all sites, whereas Escherichia coli were found at relatively low levels <40 MPN/100 ml. The highest average concentrations of somatic and male F+ specific coliphages were 920 and 330 plaque forming units per 100 ml, respectively, detected in freshwater during the rainy season. PMMoV RNA was detected in 85% of the samples with gene sequences sharing 99–100% of nucleotide identity with PMMoV sequences available in GenBank. Quantification of PMMoV genome copies (GC) by quantitative real-time PCR indicated concentrations ranging from 1.7 × 10¹ to 1.0 × 10⁴ GC/L, with the highest number of GC detected during the rainy season. No significant correlation was observed between PMMoV occurrence by season or water type (p > 0.05). Physicochemical and indicator bacteria were not correlated with PMMoV concentrations. The abundance and prevalence of PMMoV in the karst aquifer may reflect its environmental persistence and its potential as a fecal indicator in this karst aquifer system.     

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.     

Impact of Milk Components on Recovery of Viral RNA from MS2 Bacteriophage

Noroviruses are responsible for approximately 44 % of outbreaks involving dairy products for which causative agents are reported. Recovery of viruses from milk and dairy products is a difficult task. The role of different components of milk in the recovery of viral RNA was evaluated in this study. Four model milk formulations (A–D) were prepared by mixing different combinations of lactose, whey protein, casein, and fat in water. Each model formulation was spiked with five concentrations of bacteriophage MS2. The phenol-guanidine thiocyanate-chloroform protocol was used for extracting viral RNA from the model milk formulations and then extracted RNA was measured by a nanodrop spectrophotometer in ng/μl. The results showed that casein and whey protein had the highest negative impact on RNA yield, especially when the number of MS2 was less than 1.3 pfu/ml. The highest RNA recovery was obtained from the model milk formulation containing all four components; lactose, whey protein, casein, and fat. The amount of extracted RNA was closely correlated with the dry matter content of each formulation and the spiked concentration of coliphage using response surface modeling (R²:0.93). It was determined that milk fat is the most effective component in facilitating RNA extraction and the highest RNA yield can be achieved via elimination of whey protein and casein from milk by centrifugation at 40,000×g for 60 min. To achieve the highest viral RNA recovery efficiency by the proposed method, milk fat must be recombined with the supernatant of the centrifuged sample and then homogenized before performing the extraction protocol.     

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.     

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.     

Environmental Surveillance of SARS-CoV-2 RNA in Wastewater and Groundwater in Quintana Roo,Mexico

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater has been reported as a result of fecal shedding of infected individuals. In this study, the occurrence of SARS-CoV-2 RNA was explored in primary-treated wastewater from two municipal wastewater treatment plants in Quintana Roo, Mexico, along with groundwater from sinkholes, a household well, and submarine groundwater discharges. Physicochemical variables were obtained in situ, and coliphage densities were determined. Three virus concentration methods based on adsorption-elution and sequential filtration were used followed by RNA isolation. Quantification of SARS-CoV-2 was done by RT-qPCR using the CDC 2020 assay, 2019-nCoV_N1 and 2019-nCoV_N2. The Pepper mild mottle virus, one of the most abundant RNA viruses in wastewater was quantified by RT-qPCR and compared to SARS-CoV-2 concentrations. The use of three combined virus concentration methods together with two qPCR assays allowed the detection of SARS-CoV-2 RNA in 58% of the wastewater samples analyzed, whereas none of the groundwater samples were positive for SARS-CoV-2 RNA. Concentrations of SARS-CoV-2 in wastewater were from 1.8 × 10³ to 7.5 × 10³ genome copies per liter (GC l⁻¹), using the N1 RT-qPCR assay, and from 2.4 × 10² to 5.9 × 10³ GC l⁻¹ using the N2 RT-qPCR assay. Based on PMMoV prevalence detected in all wastewater and groundwater samples tested, the three viral concentration methods used could be successfully applied for SARS-CoV-2 RNA detection in further studies. This study represents the first detection of SARS-CoV-2 RNA in wastewater in southeast Mexico and provides a baseline for developing a wastewater-based epidemiology approach in the area.

     

Distribution of Naturally Occurring Norovirus Genogroups I,II, and IV in Oyster Tissues

This study evaluated different tissues of naturally contaminated oysters (Crassostrea belcheri) for the presence of noroviruses. RNA from digestive tissues, gills, and mantle of the oysters was extracted and tested for norovirus genogroup (G) I, GII, and GIV using RT-nested PCR. In spiking experiments with a known norovirus, GII.4, the detection limits were 2.97 × 10² RNA copies/g of digestive tissues, 2.62 × 10² RNA copies/g of gills, and 1.61 × 10³ RNA copies/g of mantle. A total of 85 oyster samples were collected from a fresh market in Bangkok, Thailand. Noroviruses were found in the oyster samples (40/85, 47%): GI (29/85, 34.1%), GII (9/85, 10.5%), mixed GI and GII (1/85, 1.2%), and GIV (1/85, 1.2%). All three genogroups were found in the digestive tissues of oysters. Norovirus GI was present in all three tissues with the highest frequency in the mantle, and was additionally detected in multiple tissues in some oysters. GII was also detected in all three tissues, but was not detected in multiple tissues in the same oyster. For genogroup I, only GI.2 could be identified and it was found in all tissues. For genogroup II, three different genotypes were identified, namely GII.4 which was detected in the gills and the mantle, GII.17 which was detected in the digestive tissues, and GII.21 which was detected in the mantle. GIV.1 was identified in the digestive tissues of one oyster. This is the first report on the presence of human GIV.1 in oyster in Thailand, and the results indicate oyster as a possible vehicle for transmission of all norovirus genogroups in Thailand.     

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.     

Quantification of Human Adenoviruses in European Recreational Waters

The presence of human adenoviruses (HAdV) in recreational water might cause disease in the population upon exposure. HAdV detected by PCR could also serve as indicators of the virological water quality. In order to assess the applicability of HAdV to the evaluation of the faecal contamination in European bathing waters, a real-time quantitative PCR assay was used for the quantification of HAdV in 132 samples collected from 24 different recreational marine and freshwater sites in nine European countries. Selected samples presenting positive nested PCR results for HAdV were analyzed using quantitative PCR and 80 samples from a total of 132 produced quantitative results with mean values of 3.2 × 10² per 100 ml of water, being human adenovirus 41 the most prevalent serotype between the samples where adenovirus was typified. HAdV were quantified in samples from all 15 surveillance laboratories. Statistical analysis showed no homogeneous linear relation between HAdV and E. coli, intestinal enterococci or somatic coliphages concentrations in the tested samples when considering all the data together. Significant correlations between HAdV and at least one of the other indicators were observed only when data from individual laboratories were considered. The quantification of HAdV may provide complementary information in relation to the use of bacterial standards in the control of water quality in bathing water.     

Occurrence of Viruses and Protozoa in Drinking Water Sources of Japan and Their Relationship to Indicator Microorganisms

A nationwide survey of viruses, protozoa, and indicator microorganisms in drinking water sources of Japan was conducted. Among 64 surface water samples collected from 16 drinking water treatment plants, 51 (80?%) samples were positive for at least one of the 11 pathogen types tested, including noroviruses of genogroups I (positive rate, 13?%) and II (2?%), human sapoviruses (5?%), human adenoviruses of serotypes 40 and 41 (39?%), Cryptosporidium oocysts (41?%), and Giardia cysts (36?%). Total coliforms, Escherichia coli, and F-specific coliphages were detected in 63 (98?%), 33 (52?%), and 17 (27?%) samples, respectively, and E. coli was judged to be the most suitable indicator of pathogen contamination of drinking water sources. Genogroup-specific real-time PCR for F-specific coliphages revealed the presence of F-specific RNA coliphages of animal genogroup I and human genogroups II and III in 13 (41?%), 12 (39?%), and 1 (3?%), respectively, of 31 plaques isolated.     

Coxsackievirus B4 as a Causative Agent of Diabetes Mellitus Type 1: Is There a Role of Inefficiently Treated Drinking Water and Sewage in Virus Spreading?

This study proposed to detect the enterovirus (EV) infection in children with type 1 diabetes mellitus (T1D) and to assess the role of insufficiently treated water and sewage as sources of viral spreading. Three hundred and eighty-two serum specimens of children with T1D, one hundred serum specimens of children who did not suffer from T1D as control, and forty-eight water and sewage samples were screened for EV RNA using nested RT-PCR. The number of genome copies and infectious units of EVs in raw and treated sewage and water samples were investigated using real-time (RT)-PCR and plaque assay, respectively. T1D markers [Fasting blood glucose (FBG), HbA1c, and C-peptide], in addition to anti-Coxsackie A & B viruses (CVs A & B) IgG, were measured in control, T1D-negative EV (T1D–EV^?), and T1D-positive EV (T1D–EV⁺) children specimens. The prevalence of EV genome was significantly higher in diabetic children (26.2%, 100 out of 382) than the control children (0%, 0 out of 100). FBG and HbA1c in T1D–EV^? and T1D–EV⁺ children specimens were significantly higher than those in the control group, while c-peptide in T1D–EV^? and T1D–EV⁺ children specimens was significantly lower than that in the control (n = 100; p < 0.001). Positivity of anti-CVs A & B IgG was 70.7, 6.7, and 22.9% in T1D–EV⁺, T1D–EV^?, and control children specimens, respectively. The prevalence of EV genome in drinking water and treated sewage samples was 25 and 33.3%, respectively. The prevalence of EV infectious units in drinking water and treated sewage samples was 8.5 and 25%, respectively. Quantification assays were performed to assess the capabilities of both wastewater treatment plants (WWTPs) and water treatment plants (WTPs) to remove EV. The reduction of EV genome in Zenin WWTP ranged from 2 to 4 log₁₀, while the reduction of EV infectious units ranged from 1 to 4 log₁₀. The reduction of EV genome in El-Giza WTP ranged from 1 to 3 log₁₀, while the reduction of EV infectious units ranged from 1 to 2 log₁₀. This capability of reduction did not prevent the appearance of infectious EV in treated sewage and drinking water. Plaque purification was performed for isolation of separate EV isolates from treated and untreated water and sewage samples. Characterization of the EV amplicons by RT-PCR followed by sequencing of these isolates revealed high homology (97%) with human coxsackievirus B4 (CV B4) in 60% of the isolates, while the rest of the isolates belonged to poliovirus type 1 and type 2 vaccine strains. On the other hand, characterization of the EV amplicons by RT-PCR followed by sequencing for T1D–EV⁺ children specimens indicated that all samples contained CV B4 with the same sequence characterized in the environmental samples. CV B4-contaminated drinking water or treated sewage may play a role as a causative agent of T1D in children.     

Environmental Surveillance for Noroviruses in Selected South African Wastewaters 2015–2016: Emergence of the Novel GII.17

Norovirus (NoV) GII.4 is the predominant genotype associated with gastroenteritis pandemics and new strains emerge every 2–3 years. Between 2008 and 2011, environmental studies in South Africa (SA) reported NoVs in 63% of the sewage-polluted river water samples. The aim of this study was to assess whether wastewater samples could be used for routine surveillance of NoVs, including GII.4 variants. From April 2015 to March 2016, raw sewage and effluent water samples were collected monthly from five wastewater treatment plants in SA. A total of 108 samples were screened for NoV GI and GII using real-time RT-qPCR. Overall 72.2% (78/108) of samples tested positive for NoVs with 4.6% (5/108) GI, 31.5% (34/108) GII and 36.1% (39/108) GI + GII strains being detected. Norovirus concentrations ranged from 1.02 × 10² to 3.41 × 10⁶ genome copies/litre for GI and 5.00 × 10³ to 1.31 × 10⁶ genome copies/litre for GII. Sixteen NoV genotypes (GI.2, GI.3, GI.4, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GII.9, GII.10, GII.14, GII.16, GII.17, GII.20, and GII.21) were identified. Norovirus GII.2 and GII.17 co-dominated and the majority of GII.17 strains clustered with the novel Kawasaki 2014 variant. Sewage surveillance facilitated detection of Kawasaki 2014 in SA, which to date has not been detected with surveillance in children with gastroenteritis <5 years of age. Combined surveillance in the clinical setting and environment appears to be a valuable strategy to monitor emergence of NoV strains in countries that lack NoV outbreak surveillance.