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Hepatitis B virus (HBV) acts as a severe public health threat, causing chronic liver diseases. Although the quantified evaluation of HBV infection can be obtained by estimating the capacity of the HBV DNA genome, it still lacks an effective and robust detection method without using enzymes or chemical labeling. Herein, we have designed a binary split fluorescent DNA aptasensor (bsFDA) by rationally splitting the lettuce aptamer into two functional DNA short chains and utilizing the HBV DNA segment complementary sequences (HDs). In this strategy, the bsFDA has been investigated to specifically recognize the HDs, forming a triplex DNA with the lettuce aptamer structure. Meanwhile, the turn-on fluorescence of bsFDA is obtained upon formation of a fluorescent complex between DFHO and the triplex DNA structure, allowing the enzyme-free, label-free, fast-responsive, and reliable fluorescence readout for detecting HDs and the potential HDs mutants. Moreover, bsFDA has been applied for spiked HDs analysis in different real matrixes, including human serum and cell lysate. The satisfactory recovery rates and reproducibility of the bsFDA reveal its potential detection efficacy for HDs analysis in biological samples. Overall, bsFDA holds great potential in developing functionalized aptasensors and realizing viral genome analysis in biological research.

Using waste from sewage systems, particularly human excreta, could save resources and increase soil fertility, contributing to nutrient management. However, because of the pathogenic content in human feces, this resource can pose health risks to farmers and consumers. Therefore, this work analyzed the behavior of the microorganisms: Escherichia coli ATCC13706 and human adenovirus (HAdV-2) in the soil and the internal part of the plant tissue during the vegetative stage after applying spiked composted human feces as biofertilizer. In a greenhouse, we simulated the application of the biofertilizer in lettuce cultivation by spiking three concentrations of E. coli (6.58, 7.31, and 8.01 log 10 CFU.g -1 ) and HAdV-2 (3.81, 3.97, and 5.92 log 10 PFU.g -1 ). As a result, we achieved faster decay in soil at higher concentrations of E. coli. We estimated linear decay rates of -0.07279, -0.09092, and -0.115 days, corresponding to T 90 s of 13.7, 11.0, and 8.6 days from higher to smaller concentrations of E. coli, respectively. The estimated periods for the inactivation of 4 logarithmic units of E. coli bacteria in soil are longer than the cultivation period of lettuce for all concentrations studied. Concerning the bacterial contamination in plants, we found E. coli in the internal part of the leaves at the highest concentration tested during the first three weeks of the experiment. Furthermore, HAdV-2 was found in roots at a stable concentration of 2-2.3 log 10 PFU.g -1 in five of the six samples analyzed. Therefore, bacterial infection could pose a risk, even if fresh greens are washed before consumption, especially for short-term cultures. Regarding viral infection, a positive result in the roots after disinfection may pose a risk to root and tubercule vegetables. These discoveries highlight the importance of conducting comprehensive evaluations of hygiene practices in incorporating organic amendments in crops, explicitly aiming to minimize the risk of post-contamination., Competing Interests: Declaration of competing interest The authors declare that none of their known personal or financial conflicts could have possibly affected the research described in this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Controlled environment agriculture (CEA), or indoor agriculture, encompasses non-traditional farming methods that occur inside climate-controlled structures (e.g., greenhouses, warehouses, high tunnels) allowing for year-round production of fresh produce such as leaf lettuce. However, recent outbreaks and recalls associated with hydroponically grown lettuce contaminated with human pathogens have raised concerns. Few studies exist on the food safety risks during hydroponic cultivation of leaf lettuce; thus, it is important to identify contributing risk factors and potential mitigation strategies to prevent foodborne transmission via hydroponically grown produce. In this study, the concentration of infectious Tulane virus (TV), a human norovirus surrogate, in hydroponic nutrient solution at 15 °C, 25 °C, 30 °C, and 37 °C was determined over a duration of 21 days to mimic the time from seedling to mature lettuce. The mean log PFU reduction for TV was 0.86, 1.80, 2.87, and ≥ 3.77 log 10 at 15 °C, 25 °C, 30 °C, and 37 °C, respectively, at the end of the 21-day period. Similarly, average decimal reduction values (D-values) of TV at 15 °C, 25 °C, 30 °C, and 37 °C were 48.0, 11.3, 8.57, and 7.02 days, respectively. This study aids in the (i) identification of possible food safety risks associated with hydroponic systems specifically related to nutrient solution temperature and (ii) generation of data to perform risk assessments within CEA leaf lettuce operations to inform risk management strategies for the reduction of foodborne outbreaks, fresh produce recalls, and economic losses., (© 2024. The Author(s).)

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample., (Copyright © 2021. Published by Elsevier Ltd.)

Consumption of leafy greens and to a lesser extent fresh herbs has been associated with several foodborne outbreaks including human norovirus (HuNoV). However, the extraction and detection of viruses from these matrices present multiple challenges such as low recovery yields and relatively high PCR inhibition. A new magnetic silica bead based (MSB) extraction protocol was developed and used to recover norovirus from leafy greens and fresh herbs. The performance results were compared to the ISO 15216-1:2017 standard. The HuNoV GII.4 and GI.5 recovery yields from spiked lettuce using the MSB extraction protocol range from 33 to 82%. There was a good correlation between murine norovirus (MNV) and HuNoV recovery yields from fresh herbs and leafy greens. No reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) inhibition was detected from leafy green extracts using the MSB methodology. The selected commercial RT-qPCR detection kit had a major impact on RT-qPCR inhibition levels detected in the ISO 15216-1:2017 RNA extracts. RNase treatment was used to estimate genome recovery from HuNoV with intact capsids. This treatment resulted in similar HuNoV and MNV recovery yields. Between 2019 and 2020, the MSB protocol was used to conduct a survey of HuNoV in domestic and imported leafy greens and fresh herbs sold at retail in Canada. All of the 280 samples tested were negative. Overall, the use of MSB was shown to be an efficient approach to recover HuNoV from leafy greens and certain types of fresh herbs and to conduct surveys., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

To determine the effect of the serine/threonine protein kinase (STPK) gene on leaf lettuce bolting, we utilized virus-induced gene silencing (VIGS) using the TRV vector to silence the target gene. The 'GB30' leaf lettuce cultivar was the test material, and the methods included gene cloning, bioinformatics analysis, quantitative real-time PCR (qRT-PCR) and VIGS. LsSTPK, was cloned from the 'GB30' leaf lettuce cultivar via reverse transcription-polymerase chain reaction (RT-PCR). qRT-PCR analysis showed that the expression of LsSTPK in the stem of leaf lettuce was significantly greater than that in the roots and leaves, and after high-temperature treatment, the gene expression in the stems in the experimental group was markedly lower than that in the control groups. Following LsSTPK silencing via the VIGS method, the stem length in the treatment group was significantly greater than that in the blank and negative control groups, and the contents of auxin (IAA), GA3 and abscisic acid (ABA) in the treatment group were greater than those in the other two groups. Flower bud differentiation occurred in the treatment group but not in the control group. The above findings suggested that LsSTPK inhibits the bolting of leaf lettuce under high-temperature conditions.

The study was designed to develop a sensitive one-step duplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) to detect norovirus genogroup I and II (NoV GI and GII) in lettuce and strawberry. The specificity, sensitivity, repeatability and robustness of the assay was compared with RT-qPCR. The lowest concentration detected by RT-ddPCR for NoV GI and NoV GII were 4.68 and 8.47 copies/μL respectively, much lower than that of RT-qPCR with a number of 46.8 and 84.7 copies/μL, respectively. Lettuce and strawberry samples were artificially contaminated with NoV GI and GII suspensions, with inoculum size of 3.00 × 10 6 to 1.70 × 10 8 copies and 4.80 × 10 5 to 2.50 × 10 7 copies, respectively. Strawberry spiked with low inoculum size revealed positive results by RT-ddPCR, while recorded negative by RT-qPCR. Meanwhile, RT-ddPCR also showed a higher average recovery rate for NoV in lettuce and strawberry than RT-qPCR.The limit of detection (LoDs) of RT-ddPCR for NoVs in lettuce was 2.32 × 10 4 copies/25g (NoV GI) and 2.36 × 10 4 ciopies/25g (NoV GII), and that in strawberry was 2.56 × 10 4 copies/25g (NoV GI) and 2.64 × 10 4 ciopies/25g (NoV GII), which were 10 folds lower than that of RT-qPCR. The developed duplex RT-ddPCR assay exhibited stability and increased capacity to resist inhibitors in food samples with low concentration of NoV, making it a reliable method to avoid false negative result as opposed to RT-qPCR. In conclusion, one-step RT-ddPCR method developed in this study is pertinent in detecting foodborne virus such as NoV., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human noroviruses (HuNoVs) are the primary non-bacterial pathogens causing acute gastroenteritis worldwide. Attachment and invasion of HuNoVs are thought to involve histo-blood group antigens (HBGAs). Romaine lettuce, which is usually consumed raw, is a common food-related vehicle for HuNoVs transmission. This study investigated the possibility that bacteria resident on the surface of lettuce leaves contribute to norovirus adherence to this food. To test this hypothesis, bacteria were isolated from romaine lettuce and screened to evaluate whether they produced any polysaccharides with structures resembling HBGAs. Twenty-seven bacterial isolates were screened and 18, belonging to 13 different genera, were found to produce HBGAs-like polysaccharides that were recognized by monoclonal antibodies specific to type A, B, H and Lewis a, b, x and y. One bacterial isolate, belonging to the genus Pseudomonas was further investigated because it produced polysaccharides with the widest range of HBGA types, including type B, H and Lewis a, b and x. The Pseudomonas HBGAs-like polysaccharides were found to be extracellular and their production was enhanced when the bacteria were cultured in oligotrophic medium. HuNoVs capture assays revealed that GI.1, GI.8, and GII.2, GII.3, GII.4, GII.6, GII.12, GII.17 genotypes can be bind to Pseudomonas HBGAs-like polysaccharides. The direct evidence of bacterial production HBGAs-like polysaccharides demonstrates one possible mechanism driving accumulation of HuNoVs on lettuce., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human noroviruses (HuNoVs) are among the main pathogens causing acute nonbacterial gastroenteritis. Histo-blood group antigens (HBGAs) are widely accepted receptors for HuNoV specific binding. HBGA-like substances in produce are also considered as the critical ligands for capture of HuNoVs. However, the composition of viral ligands from food substrates remains unknown. In this study, an oligosaccharide (H2N2F2) was captured and isolated from romaine lettuce extract by a bacterial surface display system. Using electrospray ionization mass spectrometry and tandem mass spectrometry, it was shown that H2N2F2 was most likely to be a chimera of type A, H, and Lewis a HBGAs. The composition was consistent with our ELISA results using a panel of monoclonal antibodies against HBGAs. Our results revealed a possible interaction mechanism between HuNoVs and romaine lettuce. Better understanding of the interaction of HuNoVs with easily contaminated produce will ultimately aid in the control of and reduction in disease outbreaks.

Cold atmospheric-gaseous plasma (CAP) is an emerging non-thermal technology for decontamination of foodborne bacterial and viral pathogens. We obtained a >5 log 10 reduction in the titer (TCID 50 ) of feline calicivirus (FCV) on stainless steel discs and Romaine lettuce leaves after 3 min wet exposure to air plasma generated by a two-dimensional array of integrated coaxial-microhollow dielectric barrier discharge (2D-AICM-DBD). However, when human norovirus (HuNoV GII.4) was treated for 5 min under the same conditions, ~2.6 log 10 (>99.5%) reduction in genome copy number was observed as measured by ethidium monoazide-coupled RT-qPCR (EMA-RT-qPCR). To assess this discrepancy, we studied CAP's effect on FCV by the cell culture method and by the EMA-coupled RT-qPCR method. It was found that the molecular titration method (EMA-RT-qPCR) underestimates the level of virus reduction by CAP. Additionally, the fecal matter present in HuNoV samples partially suppressed virucidal activity of CAP. Assuming that the lower virus reduction measured by EMA-RT-qPCR method compared to cell culture method for FCV is the same as for HuNoV, we can conclude that FCV may be used as a surrogate for HuNoV to assess the virucidal effect of CAP. CAP is able to inactivate 3.5 Log 10 units of HuNoV at low titers after 2 min of exposure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Norovirus (NoV) is a major foodborne pathogen, and even low levels of virus can cause infection and gastroenteritis. We developed a supersensitive NoV sensor that detects NoV group-I capsid protein (NoVP) via three-dimensional (3D) total internal reflection scattering defocus microscopy (TIRSDM) with wavelength-dependent transmission grating (TG). The combination of evanescent wave scattering and TG significantly enhanced the detection sensitivity and selectivity of NoVP in first-order spectral images ( n = +1) by minimizing spectroscopic interference and background noise. In particular, wavelength-dependent 3D defocused TG imaging (3D TG-TIRSDM) separated silver nanotag and gold nanoplate signals on a NoVP immunoplasmon chip along the x , y , and z coordinates simultaneously. Additionally, the use of wavelength-dependent TG increased the spectral resolution by 5-fold along the xy -axis and 1.4-fold along the z -axis compared to conventional 3D TIRSDM at the subdiffraction limit. The NoVP sensor exhibited a lower limit of detection of 820 yM, which is 29 000 times better than the previous potentiometer method, and a wide dynamic detection range of 820 yM to 92.45 pM ( R = 0.9801). This new method could be applied to detect various pathogenic viruses during the initial stage of infection.

Lettuce is often implicated in human norovirus (HuNoV) foodborne outbreaks. We identified H-like histo-blood group antigens (HBGAs) on lettuce leaves as specific binding moieties for virus-like particles (VLPs) of HuNoV GII.4/HS194/2009 strain. The objective of this study was to determine whether HuNoV-lettuce binding is mediated through the virus HBGA binding sites (HBS). Toward this objective, VLPs of historical HuNoV GII.4 strains (1987, 1997, 2002, 2004 and 2006) with known natural mutations in their HBS, two newly generated VLP mutants of GII.4/HS194/2009 (D374A and G443A) and a VLP mutant (W375A) of GI.1/Norwalk/1968 along with its wild type VLPs, which displays distinct HBS, were investigated for their binding to lettuce. ELISA revealed that historical GII.4 strains binding to lettuce was dependent on their HBGAs profiles. The VLP mutants D374A and G443A lost binding to HBGAs and displayed no to minimal binding to lettuce, respectively. The VLPs of GI.1/Norwalk/1968 strain bound to lettuce through an H-like HBGA and the binding was inhibited by fucosidase digestion. Mutant W375A which was previously shown not to bind to HBGAs, displayed significantly reduced binding to lettuce. We conclude that the binding of HuNoV GII.4 and GI.1 strains to lettuce is mediated through the virus HBS.

Norovirus is a very contagious virus that causes acute gastroenteritis. Contaminated produce is a main vehicle for dissemination of human noroviruses (HuNoVs). As HuNoVs could bind to bacteria effectively, it is highly possible that produce could be contaminated by bacteria-HuNoVs complex. In this study, we used a bacterial-surface-display system to express genogroup I (GI) or genogroup II (GII) HuNoV capsid protein (P protein) on the surface of bacteria. The bacteria-P protein complex (BPC) was used to characterize the conditions for binding to Romaine lettuce extract and removal of the bound BPCs. We demonstrated both GI and GII BPCs could bind to extract from leaf (LE) and vein (VE) effectively. Carbohydrates in LE and VE were involved in GI BPCs binding, and both carbohydrates and proteins were involved in GII BPCs binding. Saliva from both type A and O secretors could completely block binding of both BPCs to LE and VE. Saliva from type B secretors only partially blocked binding of GII but not GI BPCs to LE and VE. However, LE- and VE-bound BPCs could not be reversely removed by washing solution containing free HBGAs from saliva. The binding of GI BPCs to LE and VE was enhanced when pH was below pI (6.1) of GI and reduced when pH was above pI of GI (p < 0.05). The optimal binding for GII BPCs to LE and VE occurred at pI (6.4) of GII. All LE- or VE-bound BPCs could be reversely removed by washing with low (3.0-5.0) or high (9.0-10.0) pH buffer. The effect of ionic strength (NaCl and MgCl 2 , from zero to 100 g/L) on binding of BPCs to LE and VE was tested. The optimal ionic strength for binding of BPCs to LE and VE was 10.0 g/L (GI) and 5.0 g/L (GII) for NaCl, and 5.0 g/L for MgCl 2 . LE- and VE-bound BPCs could be reversely removed by washing with high ionic solutions. All LE- or VE- bound BPCs could be released when washed with NaCl concentrations of above 75.0 g/L (GI) and 25.0 g/L (GII), or with MgCl 2 concentrations of above 75.0 g/L (GI) and 50.0 g/L (GII). Binding of BPCs to LE and VE was inhibited in the presence of Tween-80 (nonionic surfactant) as low as 0.05% (v/v). All LE- and VE-bound BPCs could be reversed by Tween-80 concentrations over 0.1% (v/v). The study provided important parameters for BPCs binding to and removal from lettuce extract., (Published by Elsevier B.V.)

To acquire data on contamination with Norovirus in berry fruit and salad vegetables in the United Kingdom, one thousand one hundred and fifty two samples of fresh produce sold at retail in the UK were analysed for Norovirus. Of 568 samples of lettuce, 30 (5.3%) were Norovirus-positive. Most (24/30) lettuce samples which tested positive for Norovirus were grown in the UK and 19 of those 24 samples contained NoV GI. Seven/310 (2.3%) samples of fresh raspberries were Norovirus-positive. Most (6/7) of the positively-testing fresh raspberry samples were imported, but no predominance of a genogroup, or any seasonality, was observed. Ten/274 (3.6%) samples of frozen raspberries were Norovirus-positive. The country of origin of the positively-testing frozen raspberry samples was not identified in most (7/10) instances. The collected data add to the currently limited body of prevalence information on Norovirus in fresh produce, and indicate the need for implementation of effective food safety management of foodborne viruses., (Copyright © 2018. Published by Elsevier Ltd.)

Protein intrinsic disorder is involved in many biological processes and good experimental models are valuable to investigate its functions. The potyvirus genome-linked protein, VPg, displays many features of an intrinsically disordered protein. The virus cycle requires the formation of a complex between VPg and eIF4E, one of the host translation initiation factors. An in-depth characterization of the hydrodynamic properties of VPg, eIF4E, and of their binary complex VPg-eIF4E was carried out. Two complementary experimental approaches, size-exclusion chromatography and fluorescence anisotropy, which is more resolving and revealed especially suitable when protein concentration is the limiting factor, allowed to estimate monomers compaction upon complex formation. VPg possesses a high degree of hydration which is in agreement with its classification as a partially folded protein in between a molten and pre-molten globule. The natively disordered first 46 amino acids of eIF4E contribute to modulate the protein hydrodynamic properties. The addition of an N-ter His tag decreased the conformational entropy of this intrinsically disordered region. A comparative study between the two tagged and untagged proteins revealed the His tag contribution to proteins hydrodynamic behavior.

Human norovirus is the major cause of non-bacterial epidemic gastroenteritis. Human norovirus binds to environmental solids via specific and non-specific interactions, and several specific receptors for human norovirus have been reported. Among them, histo-blood group antigens (HBGA) are the most studied specific receptor. Studies have identified the presence of HBGA-like substances in the extracellular polymeric substances (EPS) and lipopolysaccharides (LPS) of human enteric bacteria present in aquatic environments, gastrointestinal cells, gills, and palps of shellfish, and cell walls, leaves, and veins of lettuce. These HBGA-like substances also interact with human norovirus in a genotype-dependent manner. Specific interactions between human norovirus and environmental matrices can affect norovirus removal, infectivity, inactivation, persistence, and circulation. This review summarizes the current knowledge and future directions related to the specific interactions between human norovirus and HBGA-like substances in environmental matrices and their possible effects on the fate and circulation of human norovirus.

This study aimed to survey the environmental dissemination of enterovirus (EV) in a site of organic lettuce situated in the mountainous region of the state of Rio de Janeiro, Brazil. For this purpose, a total of 96 environmental samples, including water and lettuce samples obtained in different stages of the production chain (e.g., irrigation water, seedlings, lettuces grown, and washed lettuces ready-to-eat), were analyzed. EV genomes were detected in 12.5% (12/96) of the tested samples (eight from irrigation water and 4 from lettuce samples). Levels of viral concentration ranged from 3.37 × 10 3 to 4.72 × 10 6 genomic copies per liter (gc L -1 ) and from 2.14 × 10 4 to 5.56 × 10 4 genome copies per 25 grams (gc 25 g -1 ) for the water and lettuce samples, respectively. Such findings suggest that the use of viruses as human fecal contamination markers must be considered in order to improve food safety in organic supply chains., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Food production using recycled wastewater offers a sustainable way forward in light of limited freshwater resources. However, concerns of food safety should be addressed to protect public health. To this end, we developed a dynamic transport model to track norovirus from the irrigation water to the root and shoot of lettuce during the growth period. These processes were embodied in a system of ordinary differential equations that also incorporated plant growth, transpiration rate, viral attachment and detachment to culture media, viral decay, and plant barrier effects. Model parameters were either obtained from the literature or through fitting the model to experimental data from a study reporting human norovirus transport in hydroponically grown lettuce. The results showed that lettuce grown hydroponically resulted in a higher risk than lettuce grown in soil. In both cases, the risk predicted failed to meet the risk benchmarks established by the U.S. EPA and WHO. Viral attachment to growth media, such as the soil particles, was an important mechanism for risk reduction. A sensitivity analysis revealed that harvesting time and irrigation time are important factors influencing the viral loads in lettuce. Hence, this pathogen transport model provides a framework for investigating the effects of time and other factors on disease burdens from water reuse in agriculture, underscoring the utility of a dynamic model. In the absence of a routine monitoring of contaminants in the recycled irrigation water and food crops, a quantitative risk assessment based on objective scientific knowledge is the best approach to guide the policy decisions on water reuse practices., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Double-stranded RNAs purified from cultivated (Lactuca sativa) or wild (L. serriola) lettuce from southwest France were analyzed by high-throughput sequencing. For both samples, BLAST annotation revealed contigs with homology to Betaflexiviridae family members. The full genome sequence of the isolate from cultivated lettuce (JG1) was completed (8,536 nucleotides [nt], excluding the poly(A) tail). The sequence of the 3' half of the genome (4,800 nt) of a wild lettuce isolate (P22) was determined and found to share 95.1% nt sequence identity with the JG1 isolate. The JG1 genome contains four open reading frames, encoding a replicase, a movement protein, a capsid protein, and a protein of unknown function, respectively. Based on genome organization and phylogenetic relationships, the lettuce virus is most closely related to the recently described carrot chordoviruses 1 and 2 in the family Betaflexiviridae. Considering the species demarcation criteria in this family, the two lettuce viruses represent isolates of a new chordovirus species for which the name "lettuce chordovirus 1" (LeCV1) is proposed.

Application of bacteriophages to eliminate foodborne pathogens in food matrices is an emerging research field. In this study, a promising phage candidate specific for Salmonella strains was screened and its ability to decrease Salmonella counts in some food, such as milk, sausage, and lettuce, was investigated. A total of 58 Salmonella phages were isolated from a wastewater treatment plant, sewage near a river, farm ditch near a lake, and poultry house. Among them, phages LPST10, LPST18, and LPST23 were highly efficient in infecting Salmonella Typhimurium ATCC 14028. In particular, phage LPST10 could infect all the tested Salmonella Typhimurium and Salmonella Enteritidis strains with high efficiency. Bacterial challenge tests revealed that phage LPST10 and its combination with phages LPST18 and LPST23 could consistently inhibit the growth of multiple strains. Phage LPST10 presented a lysis time of about 50 min with a burst size of 101 PFU/CFU, exhibited two distinct phases in the one-step growth curve, and was stable at a pH range of 3-13 that corresponds to the pH of most of the foods (pH 3.5-7.5) and at temperatures between 30 °C and 60 °C. Transmission electron microscopy demonstrated that phage LPST10 belongs to the Siphoviridae family, with an icosahedral head with a diameter of 83.26 nm and tail length and width of approximately 144.89 nm and 10.9 nm, respectively. A significant decrease in the bacterial counts (0.92-5.12 log 10 CFU/sample) and an increase in phage titers (0-2.96 log 10 PFU/sample) were observed in different food matrices tested. These results demonstrated that phage LPST10 is a promising candidate for controlling Salmonella contamination in foods owing to its safety and effectiveness., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Genomic analysis of Lettuce infectious yellows virus (LIYV) has revealed two short open reading frames (ORFs) on LIYV RNA2, that are predicted to encode a 5-kDa (P5) and a 9-kDa (P9) protein. The P5 ORF is part of the conserved quintuple gene block in the family Closteroviridae , while P9 orthologs are found in all Criniviruses . In this study, the expression of LIYV P5 and P9 proteins was confirmed; P5 is further characterized as an endoplasmic reticulum (ER)-localized integral transmembrane protein and P9 is a soluble protein. The knockout LIYV mutants presented reduced symptom severity and virus accumulation in Nicotiana benthamiana or lettuce plants, indicating their importance in efficient virus infection. The P5 mutant was successfully complemented by a dislocated P5 in the LIYV genome. The structural regions of P5 were tested and all were found to be required for the appropriate functions of P5. In addition, P5, as well as its ortholog P6, encoded by Citrus tristeza virus (CTV) and another ER-localized protein encoded by LIYV RNA1, were found to cause cell death when expressed in N. benthamiana plants from a TMV vector, and induce ER stress and the unfolded protein response (UPR).

The hepatitis E virus (HEV), which is an increasing cause of acute viral hepatitis in Europe, is a zoonotic virus that is mainly transmitted through contaminated water, consumption of raw or undercooked meat from pigs or wild boar, blood transfusion, and organ transplantation. Although the role of HEV transmission through contaminated produce has not been confirmed, the presence of HEV has been reported in irrigation waters and in vegetables. The present study used a World Health Organization (WHO) international standard and clinical samples to evaluate the performance characteristics of three RT-qPCR assays for detection and quantification of HEV. Two of the evaluated assays provided good analytical sensitivity, as 250 international units (IU) per ml could be detected. Then, experiments focused on evaluating the elution conditions suitable for HEV release from vegetables, with the method proposed by the ISO 15216:2017 selected for evaluation in three types of fresh vegetables. The concentration method proposed by the ISO 15216:2017 combined with the RT-qPCR described by Schlosser et al. (2014) resulted in average HEV recoveries of 1.29%, 0.46%, and 3.95% in lettuce, spinach, and pepper, respectively, with an average detection limit of 1.47 × 10 5  IU/25 g. In naturally contaminated samples, HEV was detected in sewage only (10/14), while no detection was reported in lettuce (0/36) or in irrigation water samples (0/24)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Lettuce has been implicated in human norovirus (HuNoV) outbreaks. The virus is stable on the leaf surface for at least 2 weeks; however, the dynamics of virus internalization have not been fully investigated. The purpose of this study was to assess the internalization and distribution of HuNoV and two surrogate viruses, porcine sapovirus (SaV) and Tulane virus (TV), in lettuce and spinach. Viral inoculations through the roots of seedlings and the petiole of leaves from mature plants were performed, and the viruses were tracked on days 1 and 6 post-root inoculation and at 16 h and 72 h post-petiole inoculation. Confocal microscopy was used to visualize root-internalized HuNoV. In both lettuce and spinach, (i) HuNoV was internalized into the roots and leaves at similar RNA titers, whereas surrogate viruses were more restricted to the roots, (ii) all three viruses were stable inside the roots and leaves for at least 6 days, and (iii) HuNoV disseminated similarly inside the central veins and leaf lamina, whereas surrogate viruses were more restricted to the central veins. Infectious TV, but not SaV, was detectable in all tissues, suggesting that TV has greater stability than SaV. HuNoV was visualized inside the roots' vascular bundle and the leaf mesophyll of both plants. In conclusion, using surrogate viruses may underestimate the level of HuNoV internalization into edible leaves. The internalization of HuNoV through roots and cut leaves and the dissemination into various spinach and lettuce tissues raise concerns of internal contamination through irrigation and/or wash water. IMPORTANCE Human noroviruses are the leading cause of foodborne outbreaks, with lettuce being implicated in the majority of outbreaks. The virus causes acute gastroenteritis in all age groups, with more severe symptoms in children, the elderly, and immunocompromised patients, contributing to over 200,000 deaths worldwide annually. The majority of deaths due to HuNoV occur in the developing world, where limited sanitation exists along with poor wastewater treatment facilities, resulting in the contamination of water resources that are often used for irrigation. Our study confirms the ability of lettuce and spinach to internalize HuNoV from contaminated water through the roots into the edible leaves. Since these leafy greens are consumed with minimal processing that targets only surface pathogens, the internalized HuNoV presents an added risk to consumers. Thus, preventive measures should be in place to limit the contamination of irrigation water. In addition, better processing technologies are needed to inactivate internalized viral pathogens., (Copyright © 2018 American Society for Microbiology.)

Through sequencing and assembly of small RNAs, an orthotospovirus was identified from a celtuce plant (Lactuca sativa var. augustana) showing vein clearing and chlorotic spots in the Zhejiang province of China. The S, M, and L RNAs of this orthotospovirus were determined to be 3146, 4734, and 8934 nt, respectively, and shared 30.4-72.5%, 43.4-80.8%, and 29.84-82.9% nucleotide sequence identities with that of known orthotospoviruses. The full length nucleoprotein (N) of this orthotospovirus shared highest amino acid sequence identity (90.25%) with that of calla lily chlorotic spot virus isolated from calla lily (CCSV-calla) [China: Taiwan: 2001] and tobacco (CCSV-LJ1) [China: Lijiang: 2014]. Phylogenetic analyses showed that this orthotospovirus is phylogenetically associated with CCSV isolates and clustered with CCSV, tomato zonate spot virus (TZSV), and tomato necrotic spot-associated virus (TNSaV) in a separate sub-branch. These results suggest that this orthotospovirus is a divergent isolate of CCSV and was thus named CCSV-Cel [China: Zhejiang: 2017].

Aim: To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food., Methods and Results: Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT-qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT-qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx-Triton pretreatment resulted in complete removal of the RT-qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further sample dilution. In mussels and oysters, the developed viability RT-qPCR method reduced the signal of inactivated HAV between 1·7 and 2·2 logs at high inoculation level, and signal was completely removed at low inoculation level., Conclusions: This study showed that the use of PMAxx is an important improvement to assess HAV infectivity by RT-qPCR. It was shown that PMAxx-Triton pretreatment is suitable for the analysis of infectious HAV in complex food samples such as vegetables and shellfish., Significance and Impact of the Study: The PMAxx-Triton pretreatment can be easily incorporated to the ISO norm for infectious virus detection., (© 2017 The Society for Applied Microbiology.)

We determined the complete genome sequence of a lettuce chlorosis crinivirus (LCV) from China (LCV-NJ). The bipartite genome of LCV-NJ consists of RNA1 and RNA2 which are 8165 and 8454 nucleotides (nt) in length, respectively. The genomic structure of LCV-NJ RNA1 resembles that of LCV-California, an isolate with four open reading frames (ORFs) in RNA1. Although the amino acid sequences of ORF 1a and 1b have 92 and 99% identity between LCV-NJ and LCV-California, ORF 2 and ORF3 of LCV-NJ share only 63 and 71% identity with those of LCV-California, respectively. In addition LCV-NJ RNA2 contains 9 ORFs, compared to 10 ORFs in LCV-California. ORF10 was missing due to the deletion of a 173-nt sequence within the 3'-terminal region of LCV-NJ RNA2. Insertion or deletion of sequences of varying lengths was also observed in RNA1 and other regions of RNA2. Based on these findings, we propose that LCV-NJ/LCV-California may have undergone genome recombination and/or rearrangement in RNA1 and RNA2.

In recent years, fresh vegetables have frequently been associated with the foodborne transmission of enteric viruses, such as human norovirus (NoV). Therefore, several studies have focused on developing methods to inactivate foodborne viruses for preventing outbreaks of foodborne illnesses. Sodium hypochlorite (NaOCl) is commonly used as a disinfectant, but results in undesirable effects on the appearance and taste of foods and can generate toxic byproducts when it exceeds the allowable concentration. Here, we evaluated the efficacy of a range of NaOCl concentrations (50-1000 ppm) for reducing the amounts of human NoV (NoV GII.4) on lettuce (Lactuca sativa), celery (Apium graveolens L.), and white cabbage (Brassica oleracea ssp. capitata). In addition, the combination treatment of NaOCl and sodium metasilicate (SMS, 0.1-0.5%) pentahydrate was evaluated for its ability to decrease the populations of NoV GII.4 in the three food samples. An immunomagnetic separation procedure combined with reverse transcription quantitative polymerase chain reaction was used for virus detection. For lettuce, celery, and cabbage, the NoV GII.4 recovery rates were 57.3% ± 6.5%, 52.5% ± 1.7%, and 60.3% ± 3.9%, respectively, using a glycine/NaCl elution buffer (0.25 M glycine/0.14 M NaCl, pH 9.5). The reductions of NoV GII.4 were 3.17, 3.06, and 3.27 log 10 genomic copies/μL for lettuce, celery, and cabbage, respectively, at 1000 ppm NaOCl, while a reduction of ∼3 log 10 genomic copies/μL was obtained when the samples were treated with a combination of 100 ppm NaOCl and 0.4% SMS pentahydrate. Taken together, these results demonstrated that combined treatment with NaOCl and SMS pentahydrate was an efficient strategy to reduce the concentration of NaOCl for control of NoV GII.4 contamination in fresh vegetables.

Lettuce (Lactuca sativa L.) production in coastal California, one of the major lettuce-producing areas of the United States, is regularly affected by outbreaks of Impatiens necrotic spot virus (INSV), a member of the genus Orthotospovirus. Transmission of INSV among lettuce crops in this growing region has been attributed predominantly to the western flower thrips (Frankliniella occidentalis). INSV is acquired by first- or second-instar thrips nymphs feeding on infected host plants (not necessarily lettuce). The virus replicates within the insect vector, and is transmitted to new plants by adult thrips as they feed on epidermal and mesophyll cells of susceptible host plants. All currently grown cultivars of lettuce are susceptible to the disease. Screening lettuce for resistance to INSV under field conditions is problematic because natural infections appear sporadically and the virus is not evenly distributed across infected fields. We have developed a greenhouse-based assay that uses viruliferous thrips in combination with mechanical inoculation that allows dependable, year-round screening for resistance. In all, 89 cultivars, breeding lines, and plant introductions of cultivated lettuce, together with 53 accessions from 11 other Lactuca spp., 4 accessions from two dandelion (Taraxacum) species, and 4 tomato (Solanum lycopersicum L.) lines were evaluated for resistance to INSV. All tested material was susceptible to INSV to varying degrees, with the exception of two tomato lines that carry the Sw-5 gene that confers resistance to Tomato spotted wilt virus, a virus closely related to INSV. In cultivated lettuce, a partial resistance to INSV was observed in cultivars Amazona, Ancora, Antigua, Commodore, Eruption, Iceberg, La Brillante, Merlot, Telluride, and Tinto. Limited comparison of the greenhouse-based screening results with the data from opportunistic evaluations of resistance on 775 lettuce accessions from six field trials indicates consistency of results from both greenhouse and field environments. The most resistant lettuce accessions are being incorporated into our breeding program for introgression of resistance into lettuce breeding lines.

Human norovirus (NoV) is a leading cause of fresh produce associated outbreaks. Previous research indicates that the roots of growing leafy greens and berries internalize human NoV. However the effect of plant type and inoculum level on internalization rates has not been directly compared. In this study we compared the internalization and dissemination rates of human NoV and its surrogate, Tulane virus (TV) in green onion, radishes, and Romaine lettuce. We also evaluated the effect inoculum level and plant growth matrix on the rate of viral internalization. In the hydroponic growth system, we detected internalization and dissemination of human NoV RNA in green onions. In hydroponically growing green onions inoculated with high titer TV, we found higher rates of internalization and dissemination compared to green onions inoculated with low titer TV. In soil growth systems, no infectious TV was detected in either green onion or radishes. However, in Romaine lettuce plants grown in soil approximately 4 log 10  PFU/g was recovered from all tissues on day 14 p.i. Overall, we found that the type of plant, growth matrix, and the inoculum level influences the internalization and dissemination of human NoV and TV., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The development of rapid and sensitive detection methods for human noroviruses (HuNoV) in produce items is critical, especially with the recent rise in outbreaks associated with this food commodity. In this study, 50-g portions of various produce items linked to a norovirus outbreak (celery, cucumber, lettuce, grapes, and radish) were artificially inoculated with murine norovirus (MNV-1) and concentrated either by ultracentrifugation or polyethylene glycol (PEG) precipitation after elution with an alkaline Tris-glycine-beef extract buffer supplemented with pectinase. As a viral concentration step following virus elution and clarification, ultracentrifugation yielded a faster method (<8 h, including reverse transcription quantitative PCR), with MNV-1 recoveries similar to or better, than those obtained with PEG precipitation. The addition of polyvinylpyrrolidone to the elution buffer, to remove polyphenolic inhibitors, improved MNV-1 recoveries by over two- and fivefold for cucumber and grapes, respectively. However, despite MNV-1 recoveries ranging from 10 to 38% as calculated with 10-fold diluted RNA, contaminating HuNoV was not detected in any of the outbreak-associated samples tested. For store-bought produce samples, the limit of detection for artificially seeded HuNoV GII.4 was determined to be 10 3 copies per 50 g, with reproducible detection achieved in grapes, radish, and celery. The results support the use of ultracentrifugation as an alternative approach to PEG precipitation to concentrate norovirus from a variety of produce items.

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 log 10 (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84-2.5 log 10 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.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro . These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection., Competing Interests: Conflict of Interest: On behalf of my co-authors, I declare that there are no financial or other conflicts of interest in the present manuscript. This work was original research, and the data presented in this article have not been published in elsewhere., (Georg Thieme Verlag KG Stuttgart · New York.)

Agricultural reuse of wastewater is a common practice worldwide, especially in arid and semiarid area due to the freshwater scarcity. Wastewater irrigation in the Middle East, one of the most water-stressed regions in the world, could be a key factor for socio-economic development, but the microbial contamination of untreated or partially treated wastewater is a serious public health concern. Potential transmission of enteric viral infections through wastewater reuse in agricultural activities represents a true health risk for exposed individuals. Accordingly, it is important to assess the health risks associated with wastewater reuse. A quantitative microbial risk assessment (QMRA) with Monte-Carlo simulation was used to estimate the annual risk of enterovirus (EV) infection and disease burden for farmers and consumers of wastewater-irrigated lettuce in Iran, a semiarid country in the Middle East region. Risk analysis was performed based on the measured concentrations of EV in effluent of two activated sludge wastewater treatment plants (WWTP). Wastewater effluent sampling was carried out over a nine-month period, and the presence of total and fecal coliforms and EV was determined. Fecal coliform bacteria were found at a high level exceeded the guideline limit for wastewater reuse in agriculture. EVs were detected in 40% of samples with the highest frequency in summer with a mean of 12 and 16 pfu/ml for WWTP-A and B, respectively. Statistical analysis showed no correlation between the concentration of fecal coliforms and EV. The estimated infection risk for EVs was 8.8 × 10 -1 and 8.2 × 10 -1 per person per year (pppy) for farmers of WWTP-A and -B, respectively which was about 2 log higher than the tolerable infection risk of 2 × 10 -3 pppy. The estimated risk for lettuce consumers exhibited a lower level of infection and disease burden but higher than the guideline limits. The median disease burden for consumption of lettuce irrigated with activated sludge effluents was about 10 -3 Disability Adjusted Life Years (DALY) pppy which exceeded the WHO guideline threshold of 10 -4 DALY pppy. The results of study indicated that the activated sludge effluents require an additional reduction of EVs to achieve the acceptable level of risk for agricultural reuse of wastewater., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Human noroviruses (hNoVs) are a known public health concern associated with the consumption of leafy green vegetables. While a number of studies have investigated pathogen reduction on the surfaces of leafy greens during the postharvest washing process, there remains a paucity of data on the level of treatment needed to inactivate viruses in the wash water, which is critical for preventing cross-contamination. The objective of this study was to quantify the susceptibility of hNoV genotype I (GI), hNoV GII, murine norovirus (MNV), and bacteriophage MS2 to free chlorine in whole leaf, chopped romaine, and shredded iceberg lettuce industrial leafy green wash waters, each sampled three times over a 4-month period. A suite of kinetic inactivation models was fit to the viral reduction data to aid in quantification of concentration-time (CT) values. Results indicate that 3-log 10 infectivity reduction was achieved at CT values of less than 0.2 mg · min/liter for MNV and 2.5 mg · min/liter for MS2 in all wash water types. CT values for 2-log 10 molecular reduction of hNoV GI in whole leaf and chopped romaine wash waters were 1.5 and 0.9 mg · min/liter, respectively. For hNoV GII, CT values were 13.0 and 7.5 mg · min/liter, respectively. In shredded iceberg wash water, 3-log 10 molecular reduction was not observed for any virus over the time course of experiments. These findings demonstrate that noroviruses may exhibit genogroup-dependent resistance to free chlorine and emphasize the importance of distinguishing between genogroups in hNoV persistence studies. IMPORTANCE Postharvest washing of millions of pounds of leafy greens is performed daily in industrial processing facilities with the intention of removing dirt, debris, and pathogenic microorganisms prior to packaging. Modest inactivation of pathogenic microorganisms (less than 2 log 10 ) is known to occur on the surfaces of leafy greens during washing. Therefore, the primary purpose of the sanitizing agent is to maintain microbial quality of postharvest processing water in order to limit cross-contamination. This study modeled viral inactivation data and quantified the free-chlorine CT values that processing facilities must meet in order to achieve the desired level of hNoV GI and GII reduction. Disinfection experiments were conducted in industrial leafy green wash water collected from a full-scale fresh produce processing facility in the United States, and hNoV GI and GII results were compared with surrogate molecular and infectivity data., (Copyright © 2017 American Society for Microbiology.)

Lettuce necrotic leaf curl virus (LNLCV) was described as the first non-tomato-infecting member of the genus Torradovirus. Until today, the virus was found only in The Netherlands in two different areas in open field crops of lettuce. In 2015, LNLCV was accepted by the ICTV as a new member of the genus Torradovirus. The tomato-infecting (TI) torradoviruses Tomato torrado virus (ToTV), Tomato marchitez virus (ToMarV) and Tomato chocolàte virus (ToChV) are transmitted by at least three whitefly species in a semi-persistent and stylet-borne manner. As LNLCV was transmitted in open fields in The Netherlands, where whiteflies are present only in low incidence, transmission studies were set up to identify the natural vector of LNLCV. Whitefly species which survive Dutch open field conditions during summer, as well as lettuce colonizing aphid species, were tested for their ability to transmit LNLCV. Lengths of acquisition and inoculation periods were chosen in accordance with the conditions for TI torradoviruses. Transmission experiments involving whiteflies were never successful. Transmission with aphids was only successful in case of the lettuce-currant aphid, Nasonovia ribisnigri. Localization of LNLCV virions in N. ribisnigri with a nested RT-PCR indicated the stylets as possible retention sites. The willow-carrot aphid Cavariella aegopodii did not transmit LNLCV in our transmission experiment but the virus could be detected in the stylets of this aphid, leaving C. aegopodii as a possible vector for LNLCV., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

In this work, the effect of green tea extract (GTE) was assessed against murine norovirus (MNV) and hepatitis A virus (HAV) at different temperatures, exposure times and pH conditions. Initially, GTE at 0.5 and 5 mg/ml were individually mixed with each virus at 5 log TCID 50 /ml and incubated 2 h at 37 °C at different pHs (from 5.5 to 8.5). GTE affected both viruses depending on pH with higher reductions observed in alkaline conditions. Secondly, different concentrations of GTE (0.5 and 5 mg/ml) were mixed with viral suspensions and incubated for 2 or 16 h at 4, 25 and 37 °C at pH 7.2. A concentration-, temperature- and exposure time-dependent response was showed by GTE in suspension tests, where complete inactivation was achieved after overnight exposure at 37 °C for both viruses and also at 25 °C for HAV. In addition, antiviral effect of GTE proved efficient in the surface disinfection tests since 1.5 log reduction and complete inactivation were recorded for MNV and HAV on stainless steel and glass surfaces treated with 10 mg/ml GTE for 30 min, analyzed in accordance with ISO 13697:2001. GTE was also evaluated as a natural disinfectant of produce, showing 10 mg/ml GTE reduced MNV and HAV titers in lettuce and spinach by more than 1.5 log after 30 min treatment. The results show a potential of GTE as natural disinfectant able to limit enteric viral (cross-)contaminations conveyed by food and food-contact surfaces., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The viral protein genome-linked (VPg) of potyviruses is a protein covalently linked to the 5' end of viral RNA. It interacts with eIF4E, a component of the cellular translation initiation complex. It has been suggested that the 5' RNA-linked VPg could mimic the cellular mRNA cap, promoting synthesis of viral proteins. Here, we report evidence for recruitment of the plant eIF4E by Lettuce mosaic virus (LMV, potyvirus) particles via the 5' RNA-linked VPg. Analysis of the viral population was performed by enzyme-linked immunosorbent assay-based tests, either with crude extracts of LMV-infected tissues or purified viral particles. In both cases, LMV-VPg and LMV-eIF4E subpopulations could be detected. After reaching a maximum within the first 2 weeks postinoculation, these populations decreased and very few labeled particles were found later than 3 weeks postinoculation. The central domain of VPg (CD-VPg) was found to be exposed at the surface of the particles. Using a purified recombinant lettuce eIF4E and CD-VPg-specific antibodies, we demonstrate that the plant factor binds to the VPg via its central domain. Moreover, the plant eIF4E factor could be imaged at one end of the particles purified from LMV plant extracts, by immunoredox atomic force microscopy coupled to scanning electrochemical microscopy. We discuss the biological significance of these results.

During 2014 and 2015, 97 lettuce plants that showed big-vein-disease-like symptoms and seven weed plants were collected from the Riyadh region. DAS-ELISA revealed that 25% and 9% of the lettuce plants were singly infected with LBVaV and MiLBVV, respectively, whereas 63% had a mixed infection with both viruses. The results were confirmed by multiplex reverse transcription polymerase chain reaction using primers specific for LBVaV and MiLBVV. LBVaV and MiLBVV were also detected in Sonchus oleraceus and Eruca sativa, respectively. The nucleotide sequence of LBVaV and MiLBVV Saudi isolates ranged from 94.3-100%, and their similarities to isolates with sequences in the GenBank database ranged from 93.9 to 99.6% and 93.8 to 99.3%, respectively. Olpidium sp. was present in the roots of lettuce plants with big-vein disease and it was shown to facilitate transmission of both viruses.

Investigation of major viruses responsible for acute viral gastroenteritis, such as norovirus (NoV), rotavirus species A (RVA) and human adenovirus (HAdV), was conducted in the mountainous region of the state of Rio de Janeiro in a lettuce-producing area. Irrigation water and lettuce samples were collected at different production stages. Viruses were concentrated using an adsorption-elution method and detected by quantitative polymerase chain reaction (qPCR). We detected HAdV in all collection points, although no virus infectivity was shown. The RVA was the most prevalent virus from both water (16.7% [10/60]) and lettuce samples (11.1% [4/36]), with loads ranging from 2.97 × 102 to 6.88 × 103 genomic copies per litre (gc L-1) and 6.24 × 102 to 1.30 × 104 gc per 25 g, respectively. NoV was detected in 8.33% [8/96] in water and lettuce samples, with concentrations ranging from 7.29 × 101 to 1.92 × 103 gc L-1 and from 4.29 × 101 to 2.98 × 103 gc 25 g-1, respectively. Escherichia coli values also demonstrated poor quality of the irrigation and washing water. The presence of at least two different virus strains in all sites reveals the need to improve basic sanitation measures in order to increase food safety., (© Crown copyright 2017.)

Enteric viruses are pathogens associated with food- and waterborne outbreaks. The recovery of viruses from food or water samples is affected by the procedures applied to detect and concentrate them. The incorporation of an internal process control virus to the analyses allows monitoring the performance of the methodology. The aim of this study was to produce a recombinant adenovirus (rAdV) and apply it together with bacteriophage PP7 as process controls. The rAdV carries a DNA construction in its genome to differentiate it from wild-type adenovirus by qPCR. The stability of both control viruses was evaluated at different pH conditions. The rAdV was stable at pH 3, 7, and 10 for 18 h. PP7 infectious particles were stable at pH 7 and showed a 2.14 log reduction at pH 10 and total decay at pH 3 after 18 h. Three virus concentration methods were evaluated: hollow-fiber tap water ultrafiltration, wastewater ultracentrifugation, and elution-PEG precipitation from lettuce. Total and infectious viruses were quantified and their recoveries were calculated. Virus recovery for rAdV and PP7 by ultrafiltration showed a wide range (2.10-84.42 and 13.54-84.62%, respectively), whereas that by ultracentrifugation was 5.05-13.71 and 6.98-13.27%, respectively. The performance of ultracentrifugation to concentrate norovirus and enteroviruses present in sewage was not significantly different to the recovery of control viruses. For detection of viruses from lettuce, genomic copies of PP7 were significantly more highly recovered than adenovirus (14.74-18.82 and 0.00-3.44%, respectively). The recovery of infectious virus particles was significantly affected during sewage ultracentrifugation and concentration from lettuce. The simultaneous use of virus controls with dissimilar characteristics and behaviors might resemble different enteric viruses.

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 (10 2 , 10 4 , 10 6  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 10 6  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).

Lettuce big-vein disease caused by Mirafiori lettuce big-vein virus (MLBVV) is found in major lettuce production areas worldwide, but highly resistant cultivars have not yet been developed. To produce MLBVV-resistant marker-free transgenic lettuce that would have a transgene with a promoter and terminator of lettuce origin, we constructed a two T-DNA binary vector, in which the first T-DNA contained the selectable marker gene neomycin phosphotransferase II, and the second T-DNA contained the lettuce ubiquitin gene promoter and terminator and inverted repeats of the coat protein (CP) gene of MLBVV. This vector was introduced into lettuce cultivars 'Watson' and 'Fuyuhikari' by Agrobacterium tumefaciens-mediated transformation. Regenerated plants (T0 generation) that were CP gene-positive by PCR analysis were self-pollinated, and 312 T1 lines were analyzed for resistance to MLBVV. Virus-negative plants were checked for the CP gene and the marker gene, and nine lines were obtained which were marker-free and resistant to MLBVV. Southern blot analysis showed that three of the nine lines had two copies of the CP gene, whereas six lines had a single copy and were used for further analysis. Small interfering RNAs, which are indicative of RNA silencing, were detected in all six lines. MLBVV infection was inhibited in all six lines in resistance tests performed in a growth chamber and a greenhouse, resulting in a high degree of resistance to lettuce big-vein disease. Transgenic lettuce lines produced in this study could be used as resistant cultivars or parental lines for breeding.

A potyvirus causing necrosis and leaf distortion on lettuce was found in the Lazio region of Italy. Host range analysis showed its ability to infect only Chenopodium quinoa and C. amaranticolor in addition to some lettuce cultivars. The virus could be transmitted by aphids of the species Myzus persicae. The complete 9829-nt genome was characterized. BLAST analysis of sequence of the complete encoded polyprotein showed that the most closely related virus is asparagus virus 1, with 52 % amino acid sequence identity. These results suggest that this virus should be considered a member of a new species in the genus Potyvirus.

The consumption of organic fresh produce has increased in recent years due to consumer demand for healthy foods without chemical additives. However, the number of foodborne outbreaks associated with fresh produce has also increased. Contamination of food with enteric viruses is a major concern because the viruses have a low infectious dose and high persistence in the environment. Human adenovirus (HAdV) has been proposed as a good marker of faecal contamination. Therefore, the aim of this study was to evaluate the efficiency of the plaque assay (PA), real time PCR (qPCR) and integrated cell culture-RT-qPCR (ICC-RT-qPCR) for the recovery of HAdV from artificially and naturally contaminated fresh produce. Organic lettuce, strawberries and green onions were selected because these fresh products are frequently associated with foodborne outbreaks. The virus extraction efficiencies from artificially contaminated samples varied from 2.8% to 32.8% depending on the food matrix and the quantification method used. Although the HAdV recoveries determined by qPCR were higher than those determined by PA and ICC-RT-qPCR, PA was defined as the most reproducible method. The qPCR assays were more sensitive than the PA and ICC-RT-qPCR assays; however, this technique alone did not provide information about the viability of the pathogen. ICC-RT-qPCR was more sensitive than PA for detecting infectious particles in fresh produce samples. HAdV genome copies were detected in 93.3% of the analysed naturally contaminated samples, attesting to the common faecal contamination of the fresh produce tested. However, only 33.3% of the total samples were positive for infectious HAdV particles based on ICC-RT-qPCR. In conclusion, this study reported that HAdV can be an efficient viral marker for fresh produce contamination. Good detection of infectious HAdV was obtained with the ICC-RT-qPCR and PA assays. Thus, we suggest that the ICC-RT-qPCR and PA assays should be considered when quantitative microbial risk assessment (QMRA) studies are required and to establish reliable food safety guidelines., (Copyright © 2016 Elsevier B.V. All rights reserved.)

RNA silencing functions as an antivirus defense strategy in plants, one that plant viruses counter by producing viral suppressors of RNA silencing (VSRs). VSRs have been identified in three members of the genus Crinivirus but they do not all share identical suppression mechanisms. Here, we used Agrobacterium co-infiltration assays to investigate the suppressor activity of proteins encoded by Lettuce chlorosis virus (LCV). Of 7 LCV proteins (1b, P23, HSP70 homolog, P60, CP, CPm, and P27) tested for the suppression of silencing of green fluorescent protein (GFP) expression in wild-type Nicotiana benthamiana plants, only P23 suppressed the onset of local silencing. Small-interfering (si)RNA accumulation was reduced in leaves co-infiltrated with P23, suggesting that P23 inhibited the accumulation or enhanced the degradation of siRNA. P23 also inhibited the cell-to-cell and systemic movement of RNA silencing in GFP-expressing transgenic N. benthamiana plants. Expression of P23 via agroinfiltration of N. benthamiana leaves induced local necrosis that increased in severity at elevated temperatures, a novelty given that a direct temperature effect on necrosis severity has not been reported for the other crinivirus VSRs. These results further affirm the sophistication of crinivirus VSRs in mediating the evasion of host's antiviral defenses and in symptom modulation.

Unlabelled: Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall., Importance: Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The emergence of culture- and sequence-independent metagenomic methods has not only provided great insight into the microbial community structure in a wide range of clinical and environmental samples but has also proven to be powerful tools for pathogen detection. Recent studies of the food microbiome have revealed the vast genetic diversity of bacteria associated with fresh produce. However, no work has been done to apply metagenomic methods to tackle viruses associated with fresh produce for addressing food safety. Thus, there is a little knowledge about the presence and diversity of viruses associated with fresh produce from farm-to-fork. To address this knowledge gap, we assessed viruses on commercial romaine and iceberg lettuces in fields and a produce distribution center using a shotgun metagenomic sequencing targeting both RNA and DNA viruses. Commercial lettuce harbors an immense assemblage of viruses that infect a wide range of hosts. As expected, plant pathogenic viruses dominated these communities. Sequences of rotaviruses and picobirnaviruses were also identified in both field-harvest and retail lettuce samples, suggesting an emerging foodborne transmission threat that has yet to be fully recognized. The identification of human and animal viruses in lettuce samples in the field emphasizes the importance of preventing viral contamination on leafy greens starting at the field. Although there are still some inherent experimental and bioinformatics challenges in applying viral metagenomic approaches for food safety testing, this work will facilitate further application of this unprecedented deep sequencing method to food samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Over one-half of foodborne diseases are believed to be of viral origin. The ability of viruses to persist in the environment and fresh produce, as well as their low infectious dose, allows even a small amount of contamination to cause serious foodborne problems. Moreover, the consumer's demands for fresh, convenient, and safe foods have prompted research into alternative food disinfection technologies. Our study focuses on viral inactivation by both conventional and alternative nonthermal disinfection technologies on different fresh ready-to-eat food products. The use of chlorine, as well as that of nonthermal technologies such as UV light and ultrasound (US), was tested for different treatment times. UV nonthermal technology was found to be more effective for the disinfection of human adenoviruses (hAdVs) compared with US, achieving a log reduction of 2.13, 1.25, and 0.92 for lettuce, strawberries, and cherry tomatoes, respectively, when UV treatment was implemented for 30 min. US treatment for the same period achieved a log reduction of 0.85, 0.53, and 0.36, respectively. The sequential use of US and UV was found to be more effective compared with when the treatments were used separately, for the same treatment time, thus indicating a synergistic effect. In addition, human adenoviruses were inactivated sooner, when chlorine treatment was used. Therefore, the effect of each disinfection method was dependent upon the treatment time and the type of food.

Noroviruses are the leading cause of foodborne illness, and ready-to-eat foods are frequent vehicles of their transmission. Studies of the disinfection of fruits and vegetables are becoming numerous. It has been shown that strong oxidizing agents are more effective than other chemical disinfectants for inactivating enteric viruses. The aim of this study was to evaluate the efficacy of oxidizing disinfectants (sodium hypochlorite, chloride dioxide and peracetic acid) at inactivating noroviruses on fruits and vegetables, using a norovirus surrogate, namely murine norovirus 3, which replicates in cell culture. Based on plaque assay, solutions of peracetic acid (85 ppm) and chlorine dioxide (20 ppm) reduced the infectivity of the virus in suspension by at least 3 log10 units after 1 min, while sodium hypochlorite at 50 ppm produced a 2-log reduction. On the surface of blueberries, strawberries and lettuce, chlorine dioxide was less effective than peracetic acid and sodium hypochlorite, which reduced viral titers by approximately 4 logs. A surprising increase in the efficacy of sodium hypochlorite on surfaces fouled with artificial feces was noted., (Copyright © 2015 Elsevier B.V. All rights reserved.)