Progress 01/01/14 to 12/31/18
Outputs
Target Audience:Food safety researchers, virologists, public health state and federal agencies involved in food safety, vegetable producers and processors, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for training one post-doc (Dr. Malak Esseili), one visiting scholar (Dr. Xiang Gao), one research associate (Ms. Susan Wang), one research assistant (Ms. Pat Boley), and one summer undergraduate student (Sarah Tegtmeiers). How have the results been disseminated to communities of interest?Two publications were published in Applied Environmental Microbiology and both were selected as the Spotlight. One more manuscript is in preparation. In addition, the results were presented at International Association for Food Protection (IAFP), USDA NIFA Food Safety PD meeting, S-1056 (Annual meeting of USDA multi-state food-safety project), NC1202 (Regional Research Technical Committee, Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety), Wayne county fair, annual Farm Review, and Ohio Produce Growers & Marketers Association (OPGMA). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Human noroviruses (NoVs) cause acute gastroenteritis in all age groups with more severe symptoms in children, elderly and immunocompromised patients, resulting in over 200,000 deaths worldwide annually. The global economic burden of human NoV infections has been estimated to be ~$64 billion in direct (healthcare) and indirect (loss of productivity) costs. In the US, human NoVs are the leading cause of foodborne outbreaks, with leafy greens being implicated in the majority of outbreaks. Knowledge regarding the mechanisms of human NoV contamination of leafy greens is important to develop better technologies to control human NoV contamination and prevent human NoV outbreaks. We focused on the most popular leafy green: lettuce. We found that human NoVs specifically bind to the histo-blood group antigen (HBGA)-like carbohydrates of lettuce cell wall. HBGAs are carbohydrates and are known as the attachment factors for human NoV infection in human intestines. Plant derived lectin that recognizes fucose blocked human NoV binding to lettuce. This result is vital since "better" inhibitors for human NoV can be designed. Although GI.1 human NoV binds significantly higher than GII.4, we did not find different binding levels or persistence to mature lettuce between them. Further studies are needed to investigate why GII.4 human NoVs that have lower binding to lettuce have been the predominant outbreak strains. Interestingly, the nanometer-sized infectious NoV particles can be transmitted from roots to edible leaves via contaminated water. Recently, we identified the critical lettuce-binding amino acid residue on the viral particles. This amino acid is located at the HBGA binding pocket and is also the essential binding site to human tissues. Our results suggest that specifically bound human NoVs cannot be removed by simple washing and NoV particles can enter the edible leaf tissue from roots. Since these leafy greens are consumed with minimal processing that only targets surface pathogens, the internalized human NoVs present an increased risk to consumers. Our findings provide new information needed to develop potential inhibitors to block binding and prevent initial contamination. Objective 1: To identify the capsid amino acids important in NoV binding to lettuce leaves. Human NoVs can be classified into at least three genogroups (GI, II, and IV) and into genotypes within each genogroup. We have identified carbohydrates, in particular fucose residues on the H-like HBGA on hemicelluloses, as specific binding moieties for GII.4 human NoVs on lettuce. Because human NoVs do not grow in routine cell cultures, we used the virus-like particles (VLPs) of each virus, expressed using baculovirus-expression system, for ELISA and immunofluorescence assays (IFA). We generated two VLP mutants (D374A, and G443A) of GII.4/HS194 strain and acquired one VLP mutant (W375A) of GI.1 strain, the prototype of human NoVs. The mutations were selected to be located within the HBGA-binding pocket. We aimed to test whether these critical residues for human NoVs binding to host HBGAs are also important for human NoV binding to lettuce leaves. The VLPs were examined by transmission electron microscopy (TEM). These mutant and wildtype (WT) VLPs were compared for binding to lettuce by immunofluorescent assay (IFA) and for their HBGA binding profile using porcine gastric mucin (containing HBGA type H, A and Lewis-Y) and HBGA-type B saliva by ELISA. We found that an amino acid mutation within the HBGA-binding pocket of GII.4 and GI.1 (amino acid residues 374 and 375, respectively) lost binding of the VLPs to lettuce leaves. Therefore, human NoVs utilize the same capsid sites to bind to HBGA in humans and HBGA-like carbohydrates in lettuce. We found that both GII.4/HS194 mutants (D374A and G443A) lost HBGA binding in ELISA. Interestingly, only one mutant (D374A) lost binding to lettuce tissues by IFA although both mutations were initially selected to be located in the P2 subdomain of the capsid protein that is responsible for human NoV-HBGA binding. Our results indicate that not all amino acids essential for HBGA binding are essential for lettuce binding. (manuscript in preparation) Objective 2. To identify the lettuce cell wall carbohydrate(s) involved in NoV binding and plant-derived lectin inhibitors for binding. We generated lettuce tissue paraffin slides and cell wall materials to which human NoV VLPs were tested for their binding using IFA and ELISA, respectively. In addition, the presence of HBGA-like carbohydrates in lettuce tissues was investigated by using monoclonal antibodies specific to different HBGAs. Lettuce xylan fraction of the cell wall materials was extracted separately and tested for binding to human NoV VLPs. We found that the VLPs of human NoV GII.4/HS194 strain bound to lettuce tissues with increased signal following digestion with cell-wall degrading enzymes. Also, the presence of HBGA-like antigens, in particular H-type, was detected in lettuce tissues. Both anti-HBGA monoclonal antibody, recognizing H type, and plant lectins, recognizing α-L-Fucose in H type, effectively inhibited VLP binding to lettuce tissues. VLP bound to commercial xylan as well as xylan extracted from lettuce cell wall materials. Objective 3. To investigate whether specific binding of NoV to lettuce carbohydrates enhances virus persistence and entry into the leaf. We did not find any difference at lettuce binding levels among the VLPs of historic GII.4 strains (containing natural mutations), but a significant difference between GII.4 and GI.1 human NoVs. Therefore, lettuce plants were grown under greenhouse conditions, inoculated with either GI.1 or GII.4 human NoV, and monitored for up to two weeks to investigate whether the two genotypes of human NoVs had difference persistence/internalization in lettuce plants. We did not found any difference between GI.1 and GII.4 human NoVs in persistence on lettuce plants. Therefore, the observed in vitro difference between GI.1 and GII.4 human NoVs may not translate into higher persistence of one genotype over the other on the actual lettuce plants. Further studies are needed to investigate why GII.4 human NoV strains that have lower binding to lettuce have been the predominant outbreak strains. Objective 4. To elucidate mechanisms of NoV (and infectious SaV) entry through the roots, their interaction with components of the vascular system, and their internal transport inside the lettuce plant. The purpose of this study was to assess the internalization and distribution of human NoV 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 day 1 and 6 post-root inoculation and on 16h and 72h post petiole-inoculation. Confocal Microscopy was used to visualize root-internalized human NoVs. In both lettuce and spinach: (i) human NoV internalized to 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) human NoV 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 a higher stability of TV compared with SaV. Human NoV was visualized inside the roots vascular bundle and the leaf mesophyll of both plants. In conclusion, using surrogate viruses may underestimate the level of human NoV internalization into edible leaves. The internalization of human NoV 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.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Esseili MA, Meulia T, Saif LJ, Wang Q. 2018. Tissue distribution and visualization of internalized human norovirus in leafy greens. Appl Environ Microbiol 84:e00292-18. doi: 10.1128/AEM.00292-18. [Epub ahead of print] (selected as Spotlight)
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Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Food safety researchers, virologists, public health state and federal agencies involved in food safety, vegetable producers and processors, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for training one research scientist (post-doc level) and one research assistant. How have the results been disseminated to communities of interest?The results were presented at IAFP (July 2017), USDA NIFA Food Safety PD meeting, andNC1202 (Regional Research Technical Committee, Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety) meeting. In addition, a manuscript describing results of objective 4 was submitted to AEM (control number AEM00292-18). What do you plan to do during the next reporting period to accomplish the goals?For objective 1, finish writing the manuscript and submit for publication.
Impacts
What was accomplished under these goals?
HuNoVs cause acute gastroenteritis in all age groups with more severe symptoms in children, elderly and immunocompromised patients, resulting in over 200,000 deaths worldwide annually. The global economic burden of HuNoV infections has been estimated to be ~$64 billion in direct (healthcare) and indirect (loss of productivity) costs. In the US, HuNoVs are the leading cause of foodborne outbreaks, with leafy greens being implicated in the majority of outbreaks. HuNoVs cause 23 million cases of illnesses, including an estimated 71,000 hospitalizations and 570-800 deaths annually, and over $700 million costs in health-care. Knowledge regarding the mechanisms of NoV binding, transport, and survival in leafy greens is important to develop better technologies to control HuNoV contamination and prevent HuNoV outbreaks. Our studies provide valuable insights on how HuNoVs interact with lettuce. We found that both GII.4 and GI.1 HuNoVs bind to the H-like HBGA in lettuce (which are similar to the H antigen in Humans). They also utilize the same capsid amino acid residues to bind to HBGA in humans as in lettuce. Plant derived lectin that recognizes fucose was able to block the GII.4 HuNoV binding to lettuce. The latter result is vital since "better" inhibitors for HuNoV can be designed. Our studies also confirm the ability of lettuce and spinach to internalize HuNoVs from contaminated water through the roots into the edible leaves. The virus was translocated through the roots' vascular bundle into the leaf mesophyll. Since these leafy greens are consumed with minimal processing that only targets surface pathogens, the internalized HuNoVs present an increased risk to consumers. Thus, preventive measures should be in place to limit contamination of irrigation water. In addition, better processing technologies are needed to inactivate bound viral pathogens. Objective 1. We previously reported that (i) the historic GII.4 HuNoV VLPs bound similarly to lettuce and (ii) that the binding of GI.1 HuNoV VLPs to lettuce tissue was ~2 folds higher than that of GII.4 HuNoV. Therefore, three VLP mutants were generated for GII.4/ HS194 strain (R345A, D374A and G443A) and one mutant for GI.1 (W375A) was obtained from our collaborator. Lettuce binding immunofluorescence assay revealed that an amino acid mutation within the HBGA-binding pocket of GII.4 and GI.1 (location 374 and 375, respectively) decreased binding of the virus VLPs to lettuce leaves. Therefore, HuNoV utilizes the same capsid sites to bind to HBGA in humans and HBGA-like carbohydrates in lettuce (manuscript in preparation) Objective 2 was completed and one paper was published (Gao et al., 2016). Objective 3. Our results for both experiments were inconclusive regarding which genotype, GI.1 or GII.4, persists longer on lettuce plants. Our ELISA and immunofluorescence results indicated that GI.1 binds higher (~2 folds) than GII.4. This difference may be too small to be picked up on the level of binding to mature lettuce. Therefore, the observed in vitro difference between GI.1 and GII.4 HuNoVs (ELISA and immunofluorescence) may not translate into higher persistence of one genotype over the other on the actual lettuce plants. Further studies are needed to investigate why GII.4 HuNoV strains that have lower binding (2 folds less than GI.1 HuNoV) have been the predominant outbreak strains. Objective 4. We previously reported on the transport of HuNoV from roots to leaves in both lettuce and spinach seedlings depending on the concentration of initial viral inoculum. Our developed assay based on LR embedding media and confocal analysis allowed us to visualize the presence of internalized HuNoV particles inside roots and leaves of both lettuce and spinach seedling plants that were root-inoculated with viruses. Although, based on RNA data, HuNoV internalized similarly between roots and leaves, our observation by confocal microscopy of ample HuNoV signal in roots as compared with less frequent detection of the virus in the leaves is in part due to the large surface area of leaves versus roots and the technical difficulty in embedding large sections of leaves. This leads to only one small section (~2 x 2 mm) from each leaf embedded in one capsule, from which semi-thin sections of 1µm are made, making the detection of the virus in the leaves more labor-intensive than the roots. Nevertheless, we were able to detect HuNoV inside the leaves of both plants. The observation of HuNoV inside the vascular bundle in roots of both lettuce and spinach, confirms that the virus is transported through the vasculature into the leaves. The detection of HuNoV inside leaf tissues by polyclonal antibody suggests that the antigenicity of the virus is preserved inside the leaves. This, in addition to the RNA extraction and detection of the viral RNA following RNase treatment suggests that internalized HuNoVs in our study were intact and potentially infectious. Therefore, human norovirus particles not only can internalize in both lettuce and spinach plants, but are also potentially infectious, emphasizing the importance of pre-harvesting prevention from initial HuNoV contamination (manuscript submitted to AEM)
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Esseili MA, Meulia T, Saif LJ and Wang Q. Tissue Distribution and Visualization of Internalized Human Norovirus in Leafy Greens. Applied and Environmental Microbiology (Submitted AEM00292-18).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
" Esseili MA, Tegtmeier S, Saif LJ, Farkas T and Wang Q. Differential Tissue Distribution of Internalized Human Norovirus, Porcine Sapovirus, and Tulane Virus in Lettuce and Spinach Plants. Presented at the International Association for Food Protection. Tampa, Florida. July9-12, 2017.
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Food safety researchers, virologists, public health state and federal agencies involved in food safety, vegetable producers and processors, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for training one post-doc researcher, one research scientist (post-doc level) and one research assistant. How have the results been disseminated to communities of interest?The results were presented at the Noro2016 in Lübeck, Germany (March 2016), the NoroCore 2016 in VA (April 2016), 2016 OARDC/OSU annual research conference (April 2016), Shanghai Jiaotong University at Shanghai, China (June 2016), the USDA Food Safety PD meeting in MO (July 2016), and the 6th international Calicivirus conference in GA (Oct 2016). In addition, a manuscript describing results of objective 4 is in preparation. What do you plan to do during the next reporting period to accomplish the goals?For objective 1, we will attempt to determine which molecules on lettuce are involved in the direct binding of GI.1/Norwalk to lettuce as we performed for GII.4. For objective 3, we will compare the persistence and entry into the leaf of GII.4 and GI.1 HuNoVs that have different binding properties. We will write and publish the manuscripts.
Impacts
What was accomplished under these goals?
Human norovirus (HuNoV) is the leading pathogen causing food- or water-borne gastroenteritis. Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. Our results suggest that (i) HBGA-H like antigens exist in the xylan portion of lettuce cell walls to which HuNoV VLP of GII.4 binds specifically. The binding can be inhibited by lectins that recognize the fucose of the H antigen. The latter result is vital since "better" inhibitors for HuNoV can be designed. (ii) Changes of the HBGA-binding sites on the surface of historical GII.4 strains over the years may not affect their binding to lettuce, in contrast to changes among genogroups. (iii) The transport of HuNoV from root to leaves and from petiole to leaf lamina in both lettuce and spinach was dependent on the initial inoculum. HuNoV was readily transported from the roots to the leaves without significant changes in the titers between tissues and across time. Both TV and SaV did not mimic this trend and may be more restricted to the roots. HuNoV was readily transported from the petiole/central veins to the lower and upper leaf lamina. In contrast, TV and SaV maybe more restricted than HuNoV to the central veins. Since current food processing technologies rely on washing pathogens adsorbed to the surface of leaves, bound and internalized viruses require more robust technologies to remove or inactivate them. Knowledge regarding the mechanisms of NoV binding, transport, and survival in leafy greens is important to develop better technologies to control NoV contamination and prevent HuNoV outbreaks. For objective 1, we found that the VLPs of all the historical pandemic GII.4 strains bound to lettuce by immunofluorescent assays, and showed positive binding results by ELISA. Interestingly, GII.4/Hunter/2004 strain that reportedly did not bind to either H-positive saliva samples or the synthetic HBGA oligosaccharides had positive binding results in this study. GI.1/Norwalk VLPs showed much stronger binding signal than GII.4 VLPs in ELISA. Also, GI.1 VLPs bound to lettuce tissue even without the cell wall degrading enzyme digestion. This differed from the binding pattern of GII.4 VLPs that bound to lettuce only after pretreatment with the enzymes to expose the binding sites. GII.4 strains generally have a broader HBGA binding pattern than GI.1/Norwalk strain. For objective 2), manuscript was completed and published. For Objective 3), we are currently growing the lettuce plants to test the persistence of HuNoV GI.1 in comparison to GII.4. For objective 4), the transport of HuNoV from root to leaves in both lettuce and spinach seedlings was dependent on the initial inoculum. At 108 GE/ml the virus was readily transported from the roots to the leaves without significant changes in the titers between tissues and across time. Both TV and SaV did not mimic this trend, since these surrogate viruses showed a decreasing trend from roots to leaves. TV maybe a better surrogate to HuNoV than SaV, as infectious TV, but not SaV viral particles were detected inside the leaves of both lettuce and spinach. Again, the transport of HuNoV from the petiole to the leaf lamina of mature lettuce and spinach leaves was dependent on the initial inoculum. At 108 GE/ml, the virus was readily transported from the petiole/central veins to the lower and upper leaf lamina with the ability to accumulate inside these tissues, depending on the plant. In contrast, TV and SaV may be more restricted to the central veins than HuNoV, as their RNA titers were highest in these tissues, even at 72 HPI, in comparison with upper lamina tissues. Again, since infectious TV but not SaV viral particles were detectable inside all leaf tissues of both lettuce and spinach, TV maybe a better surrogate to HuNoV than SaV.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Gao X, Esseili MA, Lu Z. Saif LJ, Wang Q. Recognizing HBGA-like carbohydrates in lettuce by human GII.4 norovirus. Appl Environ Microbiol. 2016 May 2;82(10):2966-74
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Xiang Gao, Malak A. Esseili, Zhongyan Lu, Linda J. Saif, Qiuhong Wang. Recognizing HBGA-like carbohydrates in lettuce by human GII.4 norovirus. Noro2016. L�beck, Germany. March 17-19, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Qiuhong Wang, Xiang Gao, Malak Esseili, Linda Saif, Lisa Lindesmith, Ralph S. Baric, Baijun Kou, Mary K. Estes, Robert L Atmar. Comparison of the binding of GII.4 and GI.1 human norovirus strains to lettuce. 6th International Calicivirus Conference. Savannah, GA. October 9-13, 2016.
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Progress 01/01/15 to 12/31/15
Outputs
Target Audience:Food safety researchers, virologist, public health state and federal agencies involved in food safety, vegetable producers and processors, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for training two post-doc researchers and one undergraduate student. How have the results been disseminated to communities of interest?The results of objectives 1 and 4 were presented at the IAFP director's meeting in Portland, Oregon, on July 2015. In addition, a manuscript describing results of objective 2 is currently under review. The results of other objectives will be presented in local and national scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?For objective 1) we will continue with testing the other VLPs of HuNoV GII.4 on lettuce tissue slides as well as we will use lettuce cell wall materials and ELISA to quantify the binding. We will obtain HuNoV GI.1 VLPs from our collaborators and test these as well. These natural mutant VLPs will be used to elucidate the nature of the capsid amino acids mediating the binding to lettuce carbohydrates. For Objective 2) we will address the reviewers' comments on the submitted manuscript. For objective 3) we will determine the persistence and potential internalization of bound HuNoVs on lettuce leaves over time. In addition, the involvement of carbohydrates in binding will be investigated. For objective 4) the experiments using HuNoVs will be repeated using higher HuNoV inoculum. Immunohistochemistry will be used to determine the location of HuNoV inside lettuce and spinach tissues which shows positive detection.
Impacts
What was accomplished under these goals?
For objective 1) our preliminary results obtained by screening some of the VLP mutants on lettuce slides showed that in comparison to the GII.4/HS194/2009 strain which showed the strongest signal, the GII.4/1997 strain had a weak binding signal while the GII.4/2004 strain showed a medium binding signal. While, as expected the VLPs of SaV and Hu/NoV/GII.12/HS206 showed no binding signal. For objective 2), our results showed that the VLPs of HuNoV GII.4/HS194 strain bound to lettuce tissues with increased signal following digestion with cell-wall degrading enzymes such as Cellulase R-10. In addition, the presence of HBGA-like antigens, in particular H-type, was detected in both lettuce tissues as well as porcine intestinal tissues which were used as positive controls. Competition assays showed that both anti-HBGA monoclonal antibody, recognizing H type, and plant lectins, recognizing α-L-Fucose in H type, effectively inhibited VLP binding to lettuce tissues. ELISA results showed that VLP bound to commercial xylan as well as xylan extracted from lettuce cell wall materials and porcine gastric mucins, which were used as a positive control. These results were written in a manuscript that was submitted for publication at Applied Environmental Microbiology and is in the revision step. For objective 3), our results showed that of an initial HuNoV titer of 6 log10 GE/g of lettuce leaves, approximately 4 log10 GE/g bound to lettuce leaves (per 4 cm2 area). Therefore, about 2 logs of HuNoV can be washed off lettuce following repeated flushing with sterile water. The amount of HuNoV bound was consistent between the four trials (Standard deviation <0.5 logs). Next, we will determine the persistence and potential internalization of bound HuNoV on lettuce leaves over time. In addition, the involvement of carbohydrates in binding will be investigated. For objective 4), our results showed that HuNoV RNA behaved differently than SaV RNA in the roots of lettuce and spinach. Specifically, while SaV RNA was found internalized inside the roots of spinach and lettuce and was transported into both plants' leaves, HuNoV RNA was only internalized inside lettuce and spinach roots, with no detection inside the leaves. This may be a result of the lower virus particles in the inoculum for HuNoV than SaV. Therefore, the petiole experiments were performed with 10x higher HuNoV in the inoculum. The latter showed that HuNoV RNA can be internalized into the bottom 2cm of both spinach and lettuce leaves, with infrequent detection inside the central veins and the leaf lamina. However, SaV RNA was detected in the bottom 2cm of the leaves, central veins and leaf lamina of both lettuce and spinach. This suggests that with higher virus titers, HuNoV may be transported through the petiole into the entire leaves. However, it remains to be investigated whether this holds true for transport of HuNoV from roots to leaves. No infectious SaV particles were detected in the internalized viruses from roots to leaves while only bottom 2cm of internalized SaV from petiole showed infectious SaV. Next, the survival of infiltrated viruses inside leaf lamina, showed again that HuNoV RNA behaved differently than SaV RNA over time, as the latter was detected on day 1 and with only slight decrease on day 7 while the former was rarely detected in both lettuce and spinach on both day 1 and 7. However, infectious SaV was detected on day 1, but significantly decreased on day 7 for both lettuce and spinach. These results suggests that SaV particles were restricted to the same area in which they were originally infiltrated and that compounds inside plant tissue affect the infectivity of SaV over time. It remains to be investigated whether infiltrated HuNoVs were transported to other parts of the plant or degraded inside leaf lamina.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Esseili, M.A. Xiang, G. Tegtmeiers, S. and Wang Q. Human norovirus binding to lettuce cell wall components and its internalization through the vascular system. Presented at the USDA NIFA Food Safety Project Directors Meeting, Portland, OR, July 2015.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Gao X, Esseili MA, Lu Z. Saif LJ, Wang Q. Recognizing HBGA-like carbohydrates in lettuce by human GII.4 norovirus. AEM (in revision).
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Progress 01/01/14 to 12/31/14
Outputs
Target Audience: Food safety researchers, virologist, public health state and federal agencies involved in food safety, vegetable producers and processors, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided opportunities for training one post-doc (50%), one visiting scholar with Ph.D (50%), and one undergraduate student. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? A team composed of two post-docs (50% effort each) and a technician (10-20% effort) has been built up to work on this project. We plan to continue Objectives 2 and 4 and initiate Objectives 1 and 3. For objective 1) we initiated a request from our collaborators for the VLPs of a number of HuNoV variants with various mutations. These VLPs will be used to elucidate the nature of the capsid amino acids mediating the binding to lettuce carbohydrates. For Objective 3) specific lectin inhibitors identified through our previously published manuscript, will utilized to study HuNoV persistence on lettuce leaf.
Impacts
What was accomplished under these goals?
Objective 2). Our results showed that when lettuce leaves were incubated with a mixture of glycosylases to digest the cells walls and release the protoplast (cytosolic contents), HuNoV VLPs bound the digested cell wall but not the cytosolic contents, confirming our previously published data that HuNoV VLPs bind specifically to lettuce cell wall materials. Next, the CWM was extracted from the lettuce leaf lamina and veins and thereafter subjected to cellulose R-10 digestion (Cellulase R10 is a multi-enzymatic system consisting of cellulase, hemicellulase, pectinase and a protease activity). Our results showed a significantly higher HuNoV VLP binding to leaf vein CWM than to leaf lamina CWM, only when the former was digested with cellulose R-10. This result suggested that cellulose R-10 exposes chemical structures within the CWM that are required for the binding. Third, the CWMs of leaf lamina and veins were fractionated into two fractions: fraction I constituted of the CWM without the ionically- bound cell wall proteins and pectins while fraction II constituted of the hemicelluloses. The VLP binding signal was lost from the leaf lamina and vein CWM only when fraction I was removed, suggesting that either pectins or the ionically bound cell wall proteins (CWP) mediate the binding, but not hemicelluloses. Next, the ionically bound proteins in the CWM were extracted using phenol: acetic acid standard method. This method isolates the structural CWP, however, it also extracts pectins, cross-linking glycans and cellulose. Again, HuNoV bound significantly to this fraction only after digestion with cellulose R-10, confirming that hemicelluloses are not required for the binding. To further explore the role of CWP in HuNoV VLP binding, we isolated a more enriched CWP fraction from both leaf vein and lamina CWMs and from whole lettuce leaves using the standard sequential salt extractants method. Again, the CWP from both leaf lamina and veins showed binding to HuNoV VLPs only after digestion with cellulose R-10. To further investigate the structure of the CWP that was involved in the VLP binding, complex sugar chains of glycoprotein of CWP should be digested into oligosaccharides by suitable glycosylases. Therefore, in addition to cellulose R-10, we used two single enzymes: cellulase and β-glucanases. Cellulase can hydrolyze β1,4-D-glycosidic linkages in cellulose, hemicellulose and cereal β-D-glucans, whereas β-glucanases hydrolyze β-1,4-glucans of cellulose, xyloglucan and β-1,4-xylan. Our preliminary trial on the cell wall glycoproteins obtained from the phenol: acetic acid fraction (containing the structural CWP, pectins, cross-linking glycans and cellulose) of leaf lamina and vein CWMs, showed that β-glucanases digestion had no effect on VLP binding to this fraction whereas cellulose digestion had a similar effect to cellulose R-10, i.e. significantly enhanced the binding of HuNoV VLP to this fraction. Therefore, these results suggest that removal of cellulose enhanced NoV VLP binding to this fraction, suggesting that the either pectin and/or CWP bind to HuNoV VLPs. Then, we sequentially extracted cell wall proteins, pectins from CWMs of leaf lamina and veins, respectively. Only pectins extracted by CDTA (cyclohexyl EDTA) solution rather than other components could bind to HuNoV VLP. Moreover pectins from leaf lamina but not from veins showed strong binding signal, consistent with our previously published results showing that CWMs from leaf lamina showed higher HuNoV VLP binding signal than those from veins. These results suggest that pectins from lettuce green lamina are the major carbohydrates in cell wall responsible for HuNoV VLP binding. To identify the sugar moieties in pectins that bind to HuNoV VLP, pectin carbohydrates were digested into oligosaccharides with a number of commercially available glycosidases including cellulase R10, cellulase, beta-glucanase and pectinase from Aspergillus niger. Porcine gastric mucin (PGM), reported to bind to HuNoV VLP was used as positive control. Our results showed that while PGM binding to HuNoV VLP decreased significantly following the digestion, lettuce pectins did not show any significant change in binding. While more detailed work is needed to decipher the nature of the pectin(s) binding to HuNoV VLPs, the latter result suggested that (i) digestion with these enzymes does not affect the pectin binding epitope and (ii) the nature of epitope in PGM binding to HuNoV VLP is different than that of lettuce pectins. Objective 4). Our results showed that both MNV and SaV internalized inside lettuce and spinach leaves. For both plants, there was no significant difference between MNV or SaV RNA titers at either 16h or 72h. Also, there was no significant difference between the RNA titers of the internalized viruses at 16 h or 72h for either the spinach or lettuce leaves. Next, we implemented an immunohistochemical staining for examining the viral particles inside the leaf tissues. Control and SaV/MNV-inoculated leaves were sectioned into 1 cm- sections starting from the upper edges of the leaves. The sections were incubated in a 40% sucrose solution (cyroprotectant) for 1h, before being transferred to cryo-molds containing tissue Tek OCT. The molds containing the leaves sections were immediately frozen at -80°C. Thin tissue sections (~10µm) were obtained using a cryotome. The tissue sections were adhered to superfrost plus Gold slides. The slides were subjected to immunohistochemiscal staining using SaV and MNV specific antisera. Our preliminary results showed that both SaV and MNV viral particles could be detected inside both spinach and lettuce. The viral particles were aggregated around the vascular bundles and were present in spaces between the cells. Our results showed that both MNV and SaV internalized inside the roots (at 6-7 log10 GE/g). Leaves also internalized both viruses, albeit with significantly less titers (at 5-6 log10 GE/g) than roots. Efforts are ongoing to determine the localization of both viruses using tissue slides prepared from both the roots and leaves.
Publications
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