Recycling composted human feces as biofertilizer for crop production: Assessment of soil and lettuce plant tissue contamination by Escherichia coli and human adenovirus.

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 log10 CFU.g-1) and HAdV-2 (3.81, 3.97, and 5.92 log10 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 T90s 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 log10 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.

Two Crinivirus-Conserved Small Proteins, P5 and P9, Are Indispensable for Efficient Lettuce infectious yellows virus Infectivity in Plants.

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).

Molecular characterization of a divergent strain of calla lily chlorotic spot virus infecting celtuce (Lactuca sativa var. augustana) in China.

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].

Testing for Human Norovirus and Recovery of Process Control in Outbreak-Associated Produce Items.

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 103 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.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

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.

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

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

Dissemination of gastroenteric viruses in the production of lettuce in developing countries: a public health concern.

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.

Comparison of internal process control viruses for detection of food and waterborne viruses.

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.

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

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

Molecular identification and biological characterization of a new potyvirus in lettuce.

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.

Detection of human adenoviruses in organic fresh produce using molecular and cell culture-based methods.

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.

Effect of Nonthermal, Conventional, and Combined Disinfection Technologies on the Stability of Human Adenoviruses as Fecal Contaminants on Surfaces of Fresh Ready-to-Eat Products.

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.

Lettuce chlorosis virus P23 Suppresses RNA Silencing and Induces Local Necrosis with Increased Severity at Raised Temperatures.

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.

Diversity and evolutionary history of lettuce necrotic yellows virus in Australia and New Zealand.

Lettuce necrotic yellows virus (LNYV) is the type member of the genus Cytorhabdovirus, family Rhabdoviridae, and causes a severe disease of lettuce (Lactuca sativa L.). This virus has been described as endemic to Australia and New Zealand, with sporadic reports of a similar virus in Europe. Genetic variability studies of plant-infecting rhabdoviruses are scarce. We have extended a previous study on the variability of the LNYV nucleocapsid gene, comparing sequences from isolates sampled from both Australia and New Zealand, as well as analysing symptom expression on Nicotiana glutinosa. Phylogenetic and BEAST analyses confirm separation of LNYV isolates into two subgroups (I and II) and suggest that subgroup I is slightly older than subgroup II. No correlation was observed between isolate subgroup and disease symptoms on N. glutinosa. The origin of LNYV remains unclear; LNYV may have moved between native and weed hosts within Australia or New Zealand before infecting lettuce or may have appeared as a result of at least two incursions, with the first coinciding with the beginning of European agriculture in the region. The apparent extinction of subgroup I in Australia may have been due to less-efficient dispersal than that which has occurred for subgroup II - possibly a consequence of suboptimal interactions with plant and/or insect hosts. Introduction of subgroup II to New Zealand appears to be more recent. More-detailed epidemiological studies using molecular tools are needed to fully understand how LNYV interacts with its hosts and to determine where the virus originated.

Quantitative farm-to-fork risk assessment model for norovirus and hepatitis A virus in European leafy green vegetable and berry fruit supply chains.

Fresh produce that is contaminated with viruses may lead to infection and viral gastroenteritis or hepatitis when consumed raw. It is thus important to reduce virus numbers on these foods. Prevention of virus contamination in fresh produce production and processing may be more effective than treatment, as sufficient virus removal or inactivation by post-harvest treatment requires high doses that may adversely affect food quality. To date knowledge of the contribution of various potential contamination routes is lacking. A risk assessment model was developed for human norovirus, hepatitis A virus and human adenovirus in raspberry and salad vegetable supply chains to quantify contributions of potential contamination sources to the contamination of produce at retail. These models were used to estimate public health risks. Model parameterization was based on monitoring data from European supply chains and literature data. No human pathogenic viruses were found in the soft fruit supply chains; human adenovirus (hAdV) was detected, which was additionally monitored as an indicator of fecal pollution to assess the contribution of potential contamination points. Estimated risks per serving of lettuce based on the models were 3×10(-4) (6×10(-6)-5×10(-3)) for NoV infection and 3×10(-8) (7×10(-10)-3×10(-6)) for hepatitis A jaundice. The contribution to virus contamination of hand-contact was larger as compared with the contribution of irrigation, the conveyor belt or the water used for produce rinsing. In conclusion, viral contamination in the lettuce and soft fruit supply chains occurred and estimated health risks were generally low. Nevertheless, the 97.5% upper limit for the estimated NoV contamination of lettuce suggested that infection risks up to 50% per serving might occur. Our study suggests that attention to full compliance for hand hygiene will improve fresh produce safety related to virus risks most as compared to the other examined sources, given the monitoring results. This effect will be further aided by compliance with other hygiene and water quality regulations in production and processing facilities.

Electron beam inactivation of Tulane virus on fresh produce, and mechanism of inactivation of human norovirus surrogates by electron beam irradiation.

Ionizing radiation, whether by electron beams or gamma rays, is a non-thermal processing technique used to improve the microbial safety and shelf-life of many different food products. This technology is highly effective against bacterial pathogens, but data on its effect against foodborne viruses is limited. A mechanism of viral inactivation has been proposed with gamma irradiation, but no published study discloses a mechanism for electron beam (e-beam). This study had three distinct goals: 1) evaluate the sensitivity of a human norovirus surrogate, Tulane virus (TV), to e-beam irradiation in foods, 2) compare the difference in sensitivity of TV and murine norovirus (MNV-1) to e-beam irradiation, and 3) determine the mechanism of inactivation of these two viruses by e-beam irradiation. TV was reduced from 7 log10 units to undetectable levels at target doses of 16 kGy or higher in two food matrices (strawberries and lettuce). MNV-1 was more resistant to e-beam treatment than TV. At target doses of 4 kGy, e-beam provided a 1.6 and 1.2 log reduction of MNV-1 in phosphate buffered saline (PBS) and Dulbecco's Modified Eagle Medium (DMEM), compared to a 1.5 and 1.8 log reduction of TV in PBS and Opti-MEM, respectively. Transmission electron microscopy revealed that increased e-beam doses negatively affected the structure of both viruses. Analysis of viral proteins by SDS-PAGE found that irradiation also degraded viral proteins. Using RT-PCR, irradiation was shown to degrade viral genomic RNA. This suggests that the mechanism of inactivation of e-beam was likely the same as gamma irradiation as the damage to viral constituents led to inactivation.

Virus transfer proportions between gloved fingertips, soft berries, and lettuce, and associated health risks.

Multiple outbreaks of human norovirus (hNoV) have been associated with fresh produce, such as soft berries and lettuce. Even though food handlers are considered an important source for the introduction of hNoV into food chains, their contribution to public health risks associated with hNoV remains unknown. To assess to which extent food handlers contribute to the introduction and spread of hNoV in fresh produce chains quantitative virus transfer data are needed. We estimated transfer proportions of hNoV GI.4, GII.4, murine norovirus (MNV-1), a culturable surrogate of hNoV, and human adenovirus (hAdV-2), a human pathogen proposed as an indicator for human faecal pollution, between gloved fingertips and raspberries, strawberries, and lettuce, by quantitative RT-PCR and cell culture if applicable. Virus transfer proportions were corrected for virus-matrix specific recoveries, and variability and uncertainty of the parameters were estimated. Virus transfer from gloves to soft berries was generally lower as compared to lettuce, with mean transfer proportions ranging between 0.1 to 2.3% and 9 to 10% for infectious MNV-1 and hAdV-2, respectively. Transfer from produce to glove was mostly greater than transfer from glove to produce, adding to the likelihood of virus transfer due to cross contamination from contaminated produce via food handlers. HNoV GI.4 and hNoV GII.4 showed no significant difference between their mean transfer proportions. Using the estimated transfer proportions, we studied the impact of low and high transfer proportions on the public health risk, based on a scenario in which a food handler picked raspberries with contaminated fingertips. Given the made assumptions, we could show that for a pathogen as infectious as hNoV, low transfer proportions may pose a greater public health risk than high transfer proportions, due to a greater viral spread. We demonstrated the potential of food handlers in spreading hNoV in food chains, showing that prevention of virus contamination on food handlers' hands is crucial for food safety. Nevertheless, complete prevention of virus contamination on fresh produce cannot be achieved in reality, and reliable and effective intervention measures are consequently required. We estimated that, especially for low transfer proportions, a robust one log10-unit reduction of infectious hNoV on contaminated produce, and on food handlers' hands, could lower the public health risk substantially. Using the obtained data in quantitative risk assessment will aid in elucidating the contribution of food handlers in hNoV transmission.

Structure of the C-terminal domain of lettuce necrotic yellows virus phosphoprotein.

Lettuce necrotic yellows virus (LNYV) is a prototype of the plant-adapted cytorhabdoviruses. Through a meta-prediction of disorder, we localized a folded C-terminal domain in the amino acid sequence of its phosphoprotein. This domain consists of an autonomous folding unit that is monomeric in solution. Its structure, solved by X-ray crystallography, reveals a lollipop-shaped structure comprising five helices. The structure is different from that of the corresponding domains of other Rhabdoviridae, Filoviridae, and Paramyxovirinae; only the overall topology of the polypeptide chain seems to be conserved, suggesting that this domain evolved under weak selective pressure and varied in size by the acquisition or loss of functional modules.

Effect of temperature and sunlight on the stability of human adenoviruses and MS2 as fecal contaminants on fresh produce surfaces.

Determining the stability, or persistence in an infectious state, of foodborne viral pathogens attached to surfaces of soft fruits and salad vegetables is essential to underpin risk assessment studies in food safety. Here, we evaluate the effect of temperature and sunlight on the stability of infectious human adenoviruses type 2 and MS2 bacteriophages on lettuce and strawberry surfaces as representative fresh products. Human adenoviruses have been selected because of their double role as viral pathogens and viral indicators of human fecal contamination. Stability assays were performed with artificially contaminated fresh samples kept in the dark or under sunlight exposure at 4 and 30°C over 24h. The results indicate that temperature is the major factor affecting HAdV stability in fresh produce surfaces, effecting decay between 3 and 4 log after 24h at 30°C. The inactivation times to achieve a reduction between 1 and 4-log are calculated for each experimental condition. This work provides useful information to be considered for improving food safety regarding the transmission of foodborne viruses through supply chains.

Identification of a movement protein of Mirafiori lettuce big-vein ophiovirus.

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.