We report a case of atypical cowpox virus infection in France in 2016. The patient sought care for thoracic lesions after injury from the sharp end of a metallic guardrail previously stored in the ground. We isolated a cowpox virus from the lesions and sequenced its whole genome. The patient reported that he had been previously vaccinated against smallpox. We describe an alternative route of cowpox virus infection and raise questions about the immunological status of smallpox-vaccinated patients for circulating orthopoxviruses. Orthopoxvirus (family Poxviridae) is composed of 10 recognized viral species that infect vertebrates and cause serologic cross-reactions. Among the orthopoxviruses, variola virus, which causes smallpox in humans, was associated with the death of millions of persons. An extensive vaccination campaign promoted by the World Health Organization and using multiple vaccinia virus variants  Amiens-Picardie in Amiens, France. He was apyretic, with good general condition and normal vital signs. The whole cellulitis was painful, associated with multiple subcutaneous abscesses and axillary adenopathies. Relevant biologic exams showed increased lymphocyte count ; mild hepatitis ; and C-reactive protein (22 mg/L). Electrolytes, prothrombin ratio, partial thromboplastin time, hemoglobin, platelet count, creatinine, procalcitonin, and fibrinogen were normal. Skin biopsy showed a predominantly eosinophilic and neutrophilic necrotizing dermohypodermitis, with intravascular thrombi without vasculitis. Moreover, periodic acid–Schiff (PAS) and Grocott staining showed no pathogens.Ricketssia spp. Results of molecular detection of herpesviruses, herpes virus 1/2, and varicella zoster virus were negative, as were Bartonella henselae and Franciscela tularensis serologic test results.At this time, a disease by inoculation was suspected. Results of routine skin biopsy cultures for fungi, bacteria, and mycobacteria were negative, as were intracellular cultures performed on the scar biopsy for The apex of the disease occurred 8 weeks after the initial trauma. Cellulitis grew through the hemithoracic region with purulent discharge from open wounds because of severe delayed healing. The pain required morphine. No wound debridement was needed. Pain spontaneously ceased 4 months after the initial trauma, and the patient was declared healed after 9 months , panel BRickettsia spp. (intracellular bacteria), suspected by the presence of eschar. Nevertheless, we performed other PCR diagnostics at Centre Hospitalier Universitaire Amiens-Picardie. We performed biochemical, hematologic, and serologic examinations using Siemens analyzers . We used kits and reagents to detect Bartonella spp., Bartonella henselae, and Bartonella quintana  and the Virion ELISA classic kit  to detect Francisella tularensis.Similar to the process Ninove et al. described in 2009 (https://www.biomerieux-diagnostics.com). We ran PCR for DNA of the 16S RNA gene in parallel, adding to the specific PCR targeting Bartonella spp., Francisella tularensis, and Rickettsia spp. using primers previously reported  and resuspended it in Hanks’ solution . Afterward, we inoculated 200 μL of the sample containing 1 mL of Vero (ATCC CCL-81) African green monkey kidney cells at 106 cells/mL onto each of 2 shell vials using 7 mL TRAC bottle (Thermo Fisher Scientific). We placed one at 32°C and the other at 37°C under 5% CO2 atmosphere and observed the vials daily under an inverted microscope to detect any potential cytopathic effect.For culture, we macerated the biopsy sample in Potter-Elvehjem PTFE tissue grinder  (Thermo Fisher Scientific) with 5% of fetal bovine serum and 1% of glutamine. After the cells reached 80% confluence, we removed the medium and inoculated the monolayer with 5 mL of viral suspension with a multiplicity of infection of 0.01. We incubated the flasks at 37°C for 1 hour for adsorption, then added 20 mL of modified MEM to the flasks and incubated them for 3 days. On the third day, we discarded the supernatant, then washed the cell monolayer 3 times with phosphate buffered saline and removed it using a scraper. After all the flasks were scraped and washed twice to collect the cells, we transferred the contents to 50-mL falcon tubes that were kept on ice.2 1 mmol/L, Tris 10 mmol/L, pH 7.0 KCl 10 mmol/L). We incubated the suspension for 10 min on ice. We performed mechanical lysis using a sterile tissue grinder  (80 cycles on ice). We added 10 mL of 36% sucrose to a plastic centrifugation tube and transferred the viral mixture slowly, avoiding mixing with the sucrose solution . We centrifuged the tube at 14,000 rpm for 1 h at 4°C, collected the pellet, and stored it at −80°C in small aliquots.We then centrifuged the cells at 1,500 rpm for 10 min, discarded the supernatant, and re-suspended the pellet in 10 mL of a sterile lysis buffer (MgClhttps://www.agarscientific.com). In brief, we fixed cells for 1 h with 2.5% glutaraldehyde in a 0.1 mmol/L sodium cacodylate buffer and washed them with a mixture of 0.2 mmol/L saccharose and 0.1 mmol/L sodium cacodylate. Postfix was for 1 h with 1% OsO4 diluted in 0.2 mmol/L potassium hexa-cyanoferrate (III) and 0.1 mmol/L sodium cacodylate solution. After washing with distilled water, we gradually dehydrated the cells with ethanol, and then gradually replaced the ethanol with LR white resin. We performed polymerization for 24 h at 60°C. We used a UC7 ultramicrotome (Leica) to obtain ultrathin 70-nm sections and placed them onto HR25 300 mesh copper/rhodium grids . We colored sections with Reynolds solution and obtained electron micrographs on a Tecnai G2 TEM  operated at 200 keV. We used ImageJ software (https://imagej.nih.gov/ij) to determine particle size.Hep2 cells (ATCC accession no. CCL-23) were maintained in culture with MEM modified with 10% of fetal bovine serum. The virus inoculated the Hep2 cell monolayer at a multiplicity of infection of 0.01. We then collected the content after scraping the flask at 32 h postinfection. We followed the same protocol of cell embedding as described by Bou Khalil et al. (https://www.illumina.com) with the paired end strategy and barcoded samples to be mixed with 18 other genomic projects prepared with the Nextera XT DNA sample prep kit (Illumina). We quantified the DNAg by high-sensitivity Qubit assay  to 0.5 ng/µL and performed dilution requiring 1 ng of each genome as input to prepare the paired end library. The tagmentation step fragmented and tagged the DNA. Twelve cycles of limited-cycle PCR amplification completed the tag adapters and introduced dual-index barcodes. After purification on AMPure XP beads , we normalized the libraries on specific beads in accordance with the Nextera XT protocol (Illumina). We pooled normalized libraries into a single library for sequencing on the MiSeq, then loaded the pooled single-strand library onto the reagent cartridge and then onto the instrument, along with the flow cell. We performed automated cluster generation and paired-end sequencing with dual index reads in a single 39-hour run in 2 × 250-bp.We sequenced genomic cowpox virus DNA (DNAg) on MiSeq technology . Within this run, we determined the index representation for cowpox virus to be 9.74%. We filtered the 811,395 paired-end reads according to the read qualities.We obtained total information of 4.3 Gb from a cluster density of 343,000/mmhttps://www.clcbio.com/) using 64-world size. The genome’s extremities appeared incomplete in comparison to the reference strains. Mapping against cowpox virus France Nancy 2001 (GenBank accession no. HQ420894.1) as reference showed a missing part in the 2 ITRs. We completed the inverted terminal repeat (ITR) regions by PCR followed by sequencing using primers previously designed on primer-Blast (We assembled paired-end reads by using CLC genomics workbench version 7.5 (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) of all predicted proteins against the nonredundant database. To determine average nucleotide value, we compared close phylogenetic strains using the ANI online calculator (https://www.ezbiocloud.net/tools/ani) based on the OrthoANI algorithm (We computed gene prediction using Genemarks (https://www.viprbrc.org/brc/home.spg). Alignments were manually controlled on MEGA version 6.0  best-hit results were obtained for cowpox virus from various previously described strains, 9 for vaccinia virus, 4 for variola virus, 3 for monkeypoxvirus, 2 for ectromelia virus, and 1 each for horsepox, camelpox, and taterapox. The 2 other genes were considered as ORFan (Open Reading Frames without detectable homologues in other lineages), located in the ITR regions. We decided to explore the phylogeny of this new isolate. A central part of the orthopoxvirus genome is extremely conserved. Regarding the recent proposed classifying elements of cowpox virus . Using tAmong the orthopoxvirus genus, the genomes’ evolution seems to be driven by numerous deletions affecting the number of predicted proteins, which could lead to a reduction of the genome length . For theTo complete the description of this new isolate, we explored the morphological features in the viral replicative stage. Electron microscopy showed a typical A type inclusion  in the c,The story of orthopoxviruses seems to be clear, but many clues are still missing and ambiguous, where the literature shows much divergent data regarding traced sources, reservoirs, and contamination routes and tools. We are aware that rats, mice, raccoons, and field and bank voles are all recognized as susceptible to cowpox virus, and some of these animals can be associated with human cowpox virus infections. Moreover, serologic evidence highlights wide cowpox virus distribution in rodents and in cats (This transmission by fomites should be integrated into upcoming studies with the availability of improved tools, notably in molecular biology, cell culture, and genome sequencing. Nevertheless, the case we report highlights that cowpox virus infection can be misdiagnosed by an atypical clinical presentation, resembling varicella zoster lesions or even noninfectious related rashes. In addition, for this case, it was not possible to establish an epidemiologic link between the patient and the typical sources of infection. Because the tool that caused the wound was kept on the ground, it is possible that it had been contaminated by contact with rodent or cat urine or feces. As is the case in almost all reported infection cases where the diagnosis occurs months later, it becomes difficult to retrace and investigate the route, initial host, or reservoir at an early time of infection. Another scenario is contact between patient and cat after the patient injury, something that was not reported by the patient, but this second potential route of infection appeared doubtful when we examined the co-localization between the injury with the guardrail and the eschar.,Smallpox vaccination is known to confer cross-immunity against other orthopoxviruses (Finally, this case is the third reported in Europe within a year (. Additional information from the analysis of cowpox infection in a patient who had been vaccinated for smallpox. Appendix


As a consequence of unfavourable epidemiological trends and the development of disease management, the economic aspects of heart failure (HF) have become more and more important. The costs of treatment (direct costs) appear to be the most frequently addressed topic in the economic research on HF; however, less is known about productivity losses (indirect costs) and the public finance burden attributable to the disease. Therefore, the aim of this study was to estimate the indirect costs and public finance consequences of HF in Poland in the period 2012–2015.The study uses a societal perspective and a prevalence-based top-down approach to estimate the following components of HF indirect costs: absenteeism of the sick and their caregivers, presenteeism of the sick, disability, and premature mortality. The human capital method has been chosen to identify the value of productivity losses attributable to HF and the public finance consequences of the disease. Deterministic sensitivity analysis was performed to assess the robustness of the results.The total indirect costs of HF in Poland were €871.9 million in 2012, and they increased to €945.3 million in 2015. In the period investigated, these costs accounted for 0.212–0.224% of GDP, an equivalent of 22.63€–24.59€ per capita. Mortality proved to be the main driver of productivity losses, with 59.3–63.4% of the total costs depending on year, followed by presenteeism (21.1–22.5%), disability (11.1–14.2%) and the sick’s absenteeism (3.3–4.0%). The cost of caregivers’ absenteeism was unimportant. The social insurance expenditure for benefits associated with HF accounted for €40.7 million in 2012 and €45.6 million in 2015 . The potential public revenue losses associated with HF were €262.7–€287.9 million. Sensitivity analysis showed that the costs varied by − 12.1% to + 28.8% depending on the model parameter values.HF is a substantial burden on the economy and public finance in Poland. By confronting the disease more effectively, the length and quality of life for those affected by HF could be improved, but society as a whole could also benefit from the increased economic output. Heart failure (HF) is a cardiovascular syndrome that affects millions worldwide and is the subject of growing attention on the part of the health care community. HF is a rising concern particularly in developed countries where the disease is increasingly widespread as a result of population ageing and improvements in the survival of patients with cardiovascular conditions , 2. EpidDue to the unfavourable epidemiologic trends and the development of disease management, the economic aspects of HF have become more and more important. The costs of treatment (direct costs) appear to be the most widely addressed topic in the economic research on HF, as from the examples of Germany , the UniTherefore, the purpose of this study is to fill the gap in knowledge on the HF economic burden by estimating the productivity losses and public finance consequences resulting from the disease in Poland. This research’s contribution is as follows. It is the first study that attempts to estimate a wide range of indirect costs for a European country, including not only the sick’s absenteeism, disability and mortality costs but also losses caused by their presenteeism and informal carers’ absenteeism. Second, the estimates provide insight into the public finance significance of HF, both in terms of expenditure on social benefits and potential public revenue decline. Finally, based on a four-year period, the study gives a dynamic picture of the economic burden of HF.The study uses a societal perspective and prevalence-based top-down approach to estimate the indirect costs of HF in Poland in the years 2012–2015. The human capital method (HCM) is applied to assess the costs of absenteeism of the sick and their caregivers; presenteeism of the sick; and premature mortality and disability caused by HF (ICD-10 code: I50). For each of these categories, I estimated the total time not worked or worked with decreased productivity and the value of production lost because of the disease.Gross domestic product (GDP) per worker was used as a measure of productivity. For farmers’ population, I adopted this measure adjusted for lower productivity in the agricultural sector . The estimates accounted for decreasing marginal labour productivity. This was done by adjusting the productivity losses of each cost component with a 0.65 correction coefficient, which approximates the output elasticity of labour in the production function . A sex-sTable In this study, the sick’s absenteeism refers to short- and medium-term absence from work. It was approximated by a number of absence days recorded by two social insurance institutions that insure the general population  and farmers ; and by a number and duration of rehabilitation benefits issued in the case of an absence longer than 180 days but with an expected return to work afterwards. Estimating the magnitude of presenteeism required identifying the number of those affected by HF being of working age and professionally active. Because age-specific prevalence of HF in Poland is not available, I used US data  to approFor premature mortality costs, I used data on the number of HF sex-specific deaths in 5-year age groups  and assuThe public finance consequences of HF were identified in two areas. First, I summed up the expenditure of SII for social benefits related to the disease. Second, I estimated public revenue losses due to reduced economic output attributable to HF. To estimate these losses, I calculated the shares of four main taxes  and social insurance contributions in GDP and multiplied these shares by the indirect costs of HF; the sum of these products represents potential public revenue decline attributable to the disease. The public finance analysis did not account for direct costs of HF because data on cost of treatment was not available for the period investigated.using 0% and 3.5% discount rates;using minimum and maximum exchange rates from the period investigated instead of the mean rate;±0.05 variation in the coefficient, which accounts for decreasing labour productivity;±20% change in the rate of productivity reduction in presenteeism;±40% variation in the number of caregivers’ absence days;±20% in the number of people with HF at working age;using gross value added instead of GDP as a productivity measure;decreasing number of deaths due to HF by 20%.One-way deterministic sensitivity analysis was carried out to test the stability of the results. Variation in model parameters was restricted to year 2015 solely and included:Because this study does not involve any participants and relies only on data collected for other purposes, the ethics committee approval was not required.The total indirect cost (productivity losses) of HF in Poland was €871.9 million in 2012, and this increased to €945.3 million in 2015. In the period investigated, the dynamics of HF indirect costs (8.4–percent raise) was lower than the dynamics of GDP (10.4–percent increase). The highest costs were those of premature mortality, with the burden ranging from €517.4 million to €581.7 million depending on the year. Presenteeism of the sick generated substantial losses (€193.6 – €199.3 million), followed by disability (€96.5 to €126.3 million) and absenteeism of the sick (€28.8 – €37.9 million). The magnitude of caregivers’ absenteeism was negligible, with costs of €0.2 million at most. The production lost due to HF in Poland accounted for approximately 0.22% of GDP throughout the period; this share declined slightly in the years 2013–2014, followed by an increase in 2015. The indirect costs corresponded to 22.63€–24.59€ per capita  for benefits associated with HF accounted for €40.7 million in 2012 and €45.6 million in 2015, a change of 12.2%. These values corresponded to 0.56–0.59% of expenditure for all diseases. A majority of these expenses was disability pensions, which constituted more than 85% of the total HF benefits expenditure over the period. The other category, with a more than 10% share in total spending, was sickness benefits, and these payments increased by 35.1% from 2012 to 2015. The dynamics of other categories were even higher, although their magnitude in benefits structure was low, with less than €1 million spending per year for each one  budgets, and social insurance funds lost €262.7 million in 2012 and €287.9 million in 2015 because of the disease. The losses in social insurance contributions dominated the public finance burden, with €112.0 – €127.7 million of revenues lost; among taxes, decreased inflows in VAT and PIT were the highest Table .Table 4PThe results of sensitivity analysis show that the variation of the HF indirect costs in Poland differ depending on the parameters applied. With no discounting, the total costs proved to be 28.8% higher than in the base scenario, while using a 3.5% discount rate increased the costs by 6.5%. Variation in the exchange rate changed the results by − 7.2% to 5.2%, and a change in the value of the labour productivity correction coefficient by ±0.05 led to a ± 7.7% change in the overall loss. By a ± 20% change in the number of people with HF, I obtained a ± 4.5% change in the costs, and the effect of a one-fifth change in the rate of productivity reduction in presenteeism resulted in ±4.2% variation. Productivity losses proved to be lowest when using gross value added as a productivity measure (− 11.4%) and when decreasing the number of deaths by 20% (− 12.1%)  during the 4-year period, which is more than the rise of indirect costs (8.4%) and social spending for all diseases 11.2%). Expenditure for each benefit category increased year over year, including disability pensions, and the dynamics of this category were the opposite to that of breast cancer, wherein public finance expenditure decreased over time . Decreas.2%. ExpeSensitivity analysis showed that my estimates are fairly susceptible to the changes in model parameters. Varying model parameters led to estimates differing from the base scenario by − 12.1% to + 28.8%, and this variation is mainly attributable to a high share of premature mortality in the total costs. Because this cost category accounts for > 60% percent of the overall burden and, at the same time, death’s consequences extend into the future, with no discounting, we observe the relatively highest variation of the total costs (+ 28.8%). Similarly, when decreasing the number of deaths by 20%, to account for over-registration of HF deaths, the indirect costs estimate responds notably (− 12.1%).The estimates from this study are comparable with other findings to only a limited extent. In fact, it appears that only the American research , 16 repoThis study has certain limitations. First, although it comprises several cost categories not included in previous studies, it still lacks estimates for caregivers’ presenteeism, housekeeping activities and intangible costs. Because of data unavailability, I was not able to include these components in the productivity loss assessment, and for this reason, my results are underestimated. On the other hand, HCM, which is used here to identify costs, is sometimes thought to overestimate losses because it results in estimating the maximum burden of diseases. For this reason, HCM is subject to criticism , 27; howIn conclusion, HF is a substantial burden both for the whole economy and public finance in Poland. Although the disease largely affects older citizens who are no longer professionally active, the productivity losses associated with HF in the working population account for 0.22% of GDP, a high share compared to the US estimates discussed above , 16. Thi


BK virus-associated nephropathy develops in renal transplant patients with the main manifestation of tubulointerstitial nephritis or ureteral stenosis. Nephrotic syndrome is a rare manifestation of BK virus-associated nephropathy. Here we report a case of a 69-year-old female presenting with nephrotic and nephritic syndrome related to BK virus infection. Kidney biopsy revealed severe acute tubular injury, collapsing glomerulopathy, and focal microangiopathic changes. Four weeks of leflunomide and intravenous immunoglobulin (IVIG) resulted in the recovery of kidney function and improvement of proteinuria.  BK virus is a member of the polyomavirus family. Primary BK virus infections occur early in childhood via oral or respiratory exposure . Most prBK virus can replicate even when the body undergoes immunosuppression. BK virus replication is associated with hemorrhagic cystitis in bone marrow transplant recipients, ureteral stenosis and interstitial nephritis in patients with renal transplant , 6.We herein report a rare case of histologically-confirmed collapsing glomerulopathy associated with BK virus infection in the native kidneys of a patient with acute T cell leukemia/lymphoma syndrome. A 69-year-old African-American female presented with one month of worsening bilateral lower extremity swelling/edema. She was non-oliguric upon arrival. Her past medical history was significant for adult T-cell leukemia/lymphoma, which was diagnosed two months prior to admission. She was initially treated with interferon alfa-2b and lamivudine, which were discontinued due to prolonged neutropenia. Then, she underwent two cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen chemotherapy, which was complicated by an episode of acute kidney injury that quickly resolved. She has a family history of hypertension. On admission, her blood pressure was 135/78 mmHg. Physical examination was otherwise largely unremarkable except for bilateral lower extremities pitting edema up to the knees.Initial workup showed significantly elevated serum blood urea nitrogen (BUN) and creatinine at 78 mg/dL and 3.9 mg/dL respectively, and low serum albumin level of 1.7 g/dL. Hemoglobin was 8.2 g/dL and mean corpuscular volume (MCV) was 90.5 fL. Her urinalysis showed albumin was 3+, blood was 2+, red blood cells (RBCs) were 8/HP, white blood cells (WBCs) were 42/HP. She was found to have severe nephrotic range proteinuria with a urine protein/creatinine ratio of 10.7. High urine and serum BK viral load were detected. Autoimmune workup including anti-double-stranded DNA (anti dsDNA), antinuclear antibody (ANA), antineutrophil cytoplasmic antibodies (ANCA) and complements levels were within the normal range. Hepatitis panel and HIV 1/2 antibodies were negative. Thyroid-stimulating hormone (TSH) and parathyroid hormone (PTH) were intact. Ultrasound revealed increased echogenicity of both kidneys. Echocardiogram manifested acute declination of left ventricular systolic function with an ejection fraction of 25-30%, which was well-preserved for two months.For further investigation, a kidney biopsy was performed, demonstrating collapsing glomerulopathy Figure  and modeThe patient was diagnosed with nephropathy associated with BK virus infection in the kidneys. Interstitial nephritis related to Bactrim® may also be the cause but no interstitial nephritis was observed in this patient’s biopsy.The patient received intravenous immunoglobulin (IVIG) 2 g/Kg and Arava® (leflunomide) 100 mg p.o. daily for BK virus-associated nephropathy. A high-protein diet and protein supplements were offered as well. Three weeks of Arava® (leflunomide) and IVIG treatments resulted in resolution of her bilateral lower extremity edema, recovery of kidney function and improvement of proteinuria.BK virus is highly seroprevalent in humans, but it appears to cause problems only among immunocompromised patients. BK virus-associated nephropathy manifests as an asymptomatic gradual rise in creatinine, and an abnormal urinalysis revealing renal tubular cells and inflammatory cells with tubulointerstitial nephritis . CollapsIn our case, the patient presented as nephrotic/nephritic syndrome, with edema, azotemia, hypoalbuminemia, and heavy proteinuria. Kidney biopsy confirmed collapsing glomerulopathy and tubular injury as the cause. Tubular necrosis with lymphocytic invasion and nuclei were noted in tubules, which was suggestive of viral tubular invasion/nephropathy. Her urine and serum BK viral load were both found to be high. Two to three weeks after anti-BK virus therapy with IVIG and leflunomide, her kidney function recovered to her baseline and her proteinuria significantly decreased to 2 + on urinalysis. According to the time course and clinical scenario, a final diagnosis of BK virus associated-collapsing glomerulopathy and tubular necrosis was made. Although the patient had been taking Bactrim® for prophylaxis, no interstitial nephritis was observed in this patient’s biopsy. However, there might be a component of congestive nephropathy/cardiorenal syndrome in the backdrop of the acute decline of left ventricular systolic function, causing a lack of forwarding blood flow. And her primary hematological disease and race could also put her at risk for collapsing glomerulonephropathy. Minimization of immunosuppression upon detection of BK viremia may prevent clinically evident BK virus nephropathy. While there has been no FDA approved treatment for BK virus-associated nephropathy. Generally, it is treated with immunomodulant agent leflunomide, or IVIG together with the reduction of immunosuppression . For BK BK virus load should be examined on patients with nephrotic and or neprhitic syndrome, especially on these undergoing immunosuppression. Reduction of immunosuppression should be considered and carefully monitored if the nephrotic syndrome is associated with BK virus. Further studies are required to determine the effective treatment for BK virus-associated nephrotic syndrome.


P. oleracea L. contains high level of nutrients and biologically active compounds. Recently, lactic fermentation has been proposed as a biotechnological option to enrich the profile of biogenic compounds of Portulaca oleracea L. puree. This study investigated the capability of fermentation by selected lactic acid bacteria to enhance the restoring features of Portulaca oleracea juice towards intestinal inflammation and epithelial injury. Lactic acid fermentation markedly increased the total antioxidant capacity of P. oleracea juice, preserved the inherent levels of vitamins C, A, and E, and increased the bioavailability of the level of vitamin B2 and that of phenolics. The effects of fermented P. oleracea juice on a Caco-2 cell line were investigated using an in vitro model closest to the in vivo conditions. Fermented P. oleracea juice strongly decreased the levels of pro-inflammatory mediators and reactive oxygen species. It also counteracted the disruption of the Caco-2 cell monolayers treated with the inflammatory stimulus. We used a diversified spectrum of lactic acid bacteria species, and some effects appeared to be strains- or species-specific. Fermentation with Lactobacillus kunkeei B7 ensured the best combination for the content of bioactive compounds and the ability to counteract the intestinal inflammation and epithelial injury. P. oleracea L. has a huge potential as a raw substrate to develop novel functional foods, dietary supplements, or pharmaceutical preparations due to the high level of nutrients and biologically active compounds . Overlapping or complementary pharmacological effects  define the biogenic profile of this plant substrate [P. oleracea extracts [P. oleracea as a functional beverage [Portulaca oleracea L.) [P. oleracea juice towards intestinal inflammation and epithelial injury. Microbial metabolites may down-regulate the pro-inflammatory response by the intestinal epithelial cells attenuating the symptoms associated with the IBS [P. oleracea juice on a Caco-2 cell line have been investigated using an in vitro model closest to the in vivo conditions. After confluence, Caco-2 cells differentiate structurally and functionally into enterocyte-like cells representing a suitable model to assess the physiological response of intestinal mucosa to oxidative stress and inflammatory status [P. oleracea juice has been evaluated as the ability to modify the cellular redox status (production of reactive oxygen species), to modulate the secretion of pro-inflammatory mediators , and to counteract the disruption of epithelial integrity.Functional beverages represent the fastest-growing segment of the functional foods market . In partubstrate ,8. Ameliextracts . Althougextracts ,8, few sbeverage . Recentlacea L.) . Lactic  the IBS ,12. The y status ,14,15,16rRNA, recA, pheS, and rpoA genes. Cultures were maintained as stocks in 15% (v/v) glycerol at −80 °C and routinely propagated at 30 °C for 24 h in de Man Rogosa Sharp (MRS) broth . Sequences have been deposited in GenBank under the accession numbers given in Portulaca oleracea L. puree was also preliminarily verified by Filannino et al. [Eight strains of lactic acid bacteria, see o et al. .P. oleracea L. plant were collected in September 2017 from the North of the Apulia region . A botanist confirmed the identification. After harvesting, the aerial parts were washed with water, cut into small pieces, and blended with a Classic Blender 400 . Juice was collected from P. oleracea puree by centrifugation  and sterilized by filtration on 0.22 μm membrane filters . The sterility of the resulting P. oleracea juice (PJ) juice was confirmed through plate counts. PJ was fermented under a standardized protocol previously described by Filannino et al. [−1. PJ was incubated at 30 °C for 36 h. Growth was monitored by plating on MRS agar  at 30 °C. The pH was measured by a Foodtrode electrode . Samples were taken before (raw PJ) and after fermentation. PJ, without bacterial inoculum and chemically acidified with lactic acid , was incubated under the same conditions and used as control (CA–PJ). PJs were stored at −20 °C until analyses. The aerial parts of the ˙ and ABTS˙ radical scavenging activity assays and ferric reducing antioxidant power (FRAP) assay. All analyses were carried out on fermented PJ, Raw-PJ, and CA-PJ. DPPH˙ radical scavenging activity was measured by using the stable 1,1-diphenyl-2- picrylhydrazyl radical (DPPH˙) as reported by Brand-Williams et al. [˙) [Free radical scavenging capacity was determined through DPPHs et al. . A quant al. [˙) . The rea al. [˙)  with som1, B2, B3, A, and E followed the EN 14130:2003, EN 14122:2003, EN 14152:2003, EN 15652:2009, EN 12823-2:2000, and EN 12822:2000 standard methods, respectively [1, B2, B3, A, and E, respectively. HPLC determination of vitamin C, Bectively ,30,31,32Total phenolic compounds were assayed according to the Folin–Ciocalteu method ,34. Data2 culture flasks  in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% (v/v) heat-inactivated FBS, 1% (v/v) HEPES 1M, 1% (v/v) non-essential amino acids, 1% L-glutamine 200 mM, penicillin (100 U mL−1), and streptomycin (100 μg mL−1)  [2 and 95% air-humidified atmosphere. The medium was refreshed every 2–3 days and cells were split once a week. The cytotoxicity of PJ was examined by the use of the MTT assay, incubating Caco-2 cells with freeze-dried PJ powder (1–500 μg mL−1) for 24 h. Cytotoxicity was observed with a concentration ≥ 500 μg mL−1. Thus, 100 μg mL−1 of freeze-dried PJ powder was used as the maximum dose.Human colon carcinoma Caco-2 cells  were routinely cultured in 25 cmNY, USA) . Cells w−1, stock solution) and sterilized through 0.22 mm filter membrane  to remove lactic acid bacteria cells. Caco-2 cells were seeded onto 24 wells at a density of 10 × 10 3 cells and cultivated for 21 days to allow differentiation. Cells were pretreated for 6 h with freeze-dried PJ powder (100 μg mL−1) before the inflammatory stimulus was added and then incubated for others 24 h. For cell treatments, freeze-dried PJ powder was resuspended in DMEM . Interleukin-1β (IL-1β) was used at the concentration of 25 ng mL−1 for monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) assay. When added alone, cytomix was used as a positive control. After treatment, the culture supernatant was collected, and PGE2, MCP-1, and IL-8 were analyzed after centrifugation at 1500 rpm for 5 min and determined using a commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions [The inflammatory stimulus for prostaglandin E2 (PGE2) and nitric oxide was represented by a cytokine mix  was measured at an excitation and emission wavelength of 485 nm and 520 nm, respectively, using a spectrofluorimeter . The intracellular generation of ROS was assessed by measuring the oxidation of the probe 20,70-dichlorofluorescin diacetate (DCFH-DA)  . Briefly6 cells on polycarbonate inserts  and kept in the culture for up to 21 days to allow differentiation. The intestinal barrier function was evaluated by measuring the transepithelial electrical resistance (TEER) using a Millicell ERS device  [2 were used. Cells were exposed to PJ for 6 h before adding the inflammatory stimulus IL-1β (25 ng mL−1) and monitored for 24 h. DMEM containing IL-1β alone was used as a positive control. Cells were allowed to settle for 10 min at room temperature before each measurement to enable the electrodes to equilibrate with external temperature. The effect of each sample on the cell monolayer was expressed as the TEER value relative with respect to the value at time zero. The background resistance was determined by measuring the resistance from cell-free inserts. Caco-2 cells were seeded at a density of 2.5 × 10MA, USA) . Cells wp < 0.05), using the statistical software, Statistica 7.0 . Analyses were carried out in triplicate with three biological replicates for each condition. Data were subjected to an analysis of variance (ANOVA) test for multiple comparisons .−1. After 36 h of fermentation, the highest cell densities were found in PJ fermented with Lactobacillus plantarum strains (8.30–8.42 log cfu mL−1), whereas that of the other strains was slightly lower and ranged from 8.07 ± 0.12 to 8.25 ± 0.14 log cfu mL−1. Raw PJ had an initial pH value of 5.08 ± 0.03. After fermentation, the lowest (p < 0.05) value of pH were found with L. plantarum strains (4.10–4.15 pH units), followed by Pediococcus pentosaceus CILSWE5 (4.23 ± 0.02 pH units), and Lactobacillus rossiae 2MR8 (4.26 ± 0.03 pH units). For the other strains, only a slight variation of ca. 0.3 units was found. Eight lactic acid bacteria strains were singly used as starters to ferment PJ for 36 h at 30 °C. The aptitude of the strains, as well as the process parameters, were previously set up by Filannino et al. . The ini˙, ABTS˙, and FRAP assays. Raw-PJ showed a high inherent antioxidant activity, as estimated through the DPPH˙ (2.66 ± 0.02 Trolox mmol eq. per 100 mL of PJ), ABTS˙ (1.02 ± 0.01 Trolox mmol eq. per 100 mL of PJ), and FRAP assays (0.86 ± 0.03 Fe(II) mol eq. per 100 mL of PJ). Regardless of the assay and the bacterial starter, fermentation markedly increased (p < 0.05) the antioxidant activity of PJ, see p < 0.05) were mainly observed when PJ was fermented with P. pentosaceus CILSWE5 , L. kunkeei B7 , L. plantarum EnFIII3 , and L. rossiae 2MR8 , see Due to the many involved variables, the measurement of the antioxidant capacity of food cannot be evaluated satisfactorily using a single antioxidant assay. In this study, we determined the antioxidant activity of PJ by using DPPH3), riboflavin (vitamin B2), and thiamin (vitamin B1) were found at concentrations lower than 1 mg per 100 mL of PJ. Compared to Raw-PJ, reduced amounts of ascorbic acid were detected in CA-PJ and fermented PJs, except for those fermented with L. kunkeei B7, L. brevis POM4, and L. plantarum POM1 where the concentration remained unchanged, see L. kunkeei B7 and L. brevis POM4. Apart from PJs fermented with L. plantarum POM1 and EnFIII3, a decreasing trend (p < 0.05) for the niacin was found in almost all fermented PJs and CA-PJ, see p < 0.05) only in CA-PJ, whereas no significant change (p > 0.05) was found for thiamin, see p < 0.05) the riboflavin content, especially with L. plantarum POM1 and EnFIII3, and L. kunkeei B7 (67–56%), whereas no change (p > 0.05) was found in CA-PJ, see Ascorbic acid (vitamin C) was found as the most abundant vitamin in Raw–PJ (22 ± 2 mg per 100 mL of PJ), followed by α-tocopherol (vitamin E) (2.5 ± 0.1 mg per 100 mL of PJ), and β-carotene (vitamin A) (2.00 ± 0.02 mg per 100 mL of PJ), see  P. pentosaceus CILSWE5 (ca. 71%), whereas a significant (p < 0.05) decrease (ca. 18%) was found in CA-PJ, see The amount of total phenolics in Raw-PJ was 85 ± 6 gallic acid mg equivalents per 100 mL of PJ. Overall, an increasing trend was observed for the total phenolics in fermented PJs, especially with−1 did not significantly (p > 0.05) affect the Caco-2 cell proliferation.The cytotoxicity of PJs was checked using the MTT assay. Apart from the lactic acid bacterium used for fermentation, PJs behaved similarly to DMEM (negative controls). The concentration of freeze-dried PJ powder < 500 μg mLp < 0.05) the synthesis of PGE2 (734 ± 134 pg mg−1 of protein) and the release of NO (11.6 ± 1.7 µM) compared to the negative control (DMEM) , see p < 0.05) counteracted PGE2 accumulation in response to the cytomix. Raw-PJ and CA-PJ led to a decrease of PGE2 levels of ca. 30% and 20%, respectively. The highest (p < 0.05) decrease (47–66%) was observed with fermented PJs, see p < 0.05) of NO levels of 45–65%, whereas only a weak decrease (p < 0.05) was observed with Raw-PJ and CA-PJ , see Pro-inflammatory mediators play important roles in regulating the inflammatory response. The formation of prostaglandin E2 (PGE2), which is synthesized from arachidonic acid by cyclooxygenase enzymes, was determined. Treatment of Caco-2 cells with the cytomix solution including LPS, TNF-α, and IL-1β markedly stimulated (−1) in Caco-2 cells stimulated with Interleukin-1β (IL-1β)  compared to the negative control (DMEM) , see p < 0.05) decreased when Caco-2 cells were treated with the fermented PJs (53–71%), and to a lesser extent with Raw-PJ (37%), see p > 0.05) was found with CA-PJ, see p < 0.05) decrease (41–52%), see p > 0.05) were found with Raw-PJ and CA-PJ, see ELISA analysis revealed significant increases in levels of cytokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) (pg mLp < 0.05) increased when the cells were treated with H2O2 (631 ± 6 fluorescence units) compared to the negative control (DMEM) (17 ± 6 fluorescence units). When the cells were pretreated with fermented PJs and subsequently exposed to oxidative stress (+H2O2), ROS level was significantly (p < 0.05) decreased up to 70%, whereas a decrease of 36% and 19% was observed when the cells were pretreated with Raw-PJ and CA-PJ, respectively, see Intracellular ROS accumulation in Caco-2 cells is shown in p < 0.05) decrease of TEER (ca. 69%) in Caco-2 cells during 24 h of incubation, see p < 0.05) decrease of TEER was found when PJs was fermented with L. kunkeei B7 (ca. 16%), L. rossiae 2MR8 (ca. 17%), and Leuc. mesenteroides OP9 (ca. 20%), see TEER is a widely accepted method to quantitatively measure the dynamics of epithelial integrity through an in vitro model. Compared to the negative control (DMEM), IL-1β induced a significant  ,44,45,46reported , this stea juice . P. oleracea juice, mainly when Lactobacillus kunkeei B7 and Pediococcus pentosaceus CILSWE5 were used as starters. Reactive oxygen species (ROS) have been suggested as key molecules in mediating the tissue injuries promoted by the inflammatory processes occurring in IBS [2 and that of phenolics in P. oleracea juice. The protective effects of fermented P. oleracea juice against oxidative stress were investigated using Caco-2 cells line as a model to get information using an in vitro model closest to the in vivo conditions [P. oleracea juice to counteract such damage. In our study, we found a significant decreased level of ROS in Caco-2 cells pre-treated with fermented P. oleracea juice compared to raw and chemically acidified P. oleracea juice.First, lactic fermentation markedly increased the total antioxidant capacity of g in IBS ,52,53,54g in IBS ,56. Undenditions ,14,15,16P. oleracea juice strongly counteracted the inflammatory processes in Caco-2 cells treated with the inflammatory stimulus. The treatment with fermented juice limited the production of prostaglandin E2 by cyclooxygenase enzymes and nitric oxide by iNOS [P. oleracea juice. The role of MCP-1 and IL-8 in the pathophysiology of IBS has been widely recognized making them targets for anti-inflammatory treatments [P. oleracea juice preserved the integrity of the Caco-2 cell monolayers. Although the study was carried out in vitro on Caco-2 cells, this type of cells is widely used as a model for the intestinal epithelial barrier because they form a monolayer and tight junction complexes characteristic of enterocytes [P. oleracea juice might contribute to maintaining the integrity of the intestinal barrier in vivo in IBS subjects. Filannino et al. [P. oleracea puree dramatically increased the levels of linalool. This was shown to exert anti-inflammatory activity, thus might underlie the protective effects of fermented P. oleracea juice [P. oleracea puree. In light of these findings, P. oleracea fermentation might be exploited to develop dietary supplements and pharmaceutical preparations, in addition to functional beverages. On the other hand, inflammatory responses play a critical role in the pathogenesis of IBS. During the acute inflammatory reaction, clinical and experimental studies reported significant increases in the secretion of cyclooxygenases, inducible nitric oxide synthases (iNOS), and pro-inflammatory cytokines , interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1)), which promote inflammatory cell recruitment and activation ,59,60,61 by iNOS ,64,65,66eatments ,70,71,72eatments ,74,75. Teatments ,77. Gut eatments ,79,80. Uerocytes ,14,15,16o et al.  showed tea juice . Furtherea juice  speculatP. oleracea L. juice. Some effects appeared to be strains- or species-specific, see L. kunkeei B7, L. rossiae 2MR8, and Leuconostoc mesenteroides OP9. In conclusion, considering the totality of the descriptors that we analyzed, we can summarize that the fermentation of P. oleracea L. juice with L. kunkeei B7 guaranteed the best combination for the content of bioactive compounds (free phenolics and vitamins), the ability to preserve the integrity of the intestinal barrier and to counteract the intestinal inflammation and the oxidative damage, see P. oleracea L. juice will be further performed through controlled in vivo experiments. Our work represents a pioneering study on the exploitation of P. oleracea L. juice as a functional beverage. Further studies might continue along this track and employ fermentation-based biotechnologies to improve other nutritional and functional features, as well as technological and sensorial properties of P. oleracea L. juice [P. oleracea L. juice might be also proposed to develop probiotic beverages. Within this perspective, synergistic effects between juice and probiotic bacteria might come to light [Our study used a diversified spectrum of lactic acid bacteria species to investigate the effects of lactic fermentation on the restoring properties of L. juice ,82,83,84L. juice ,99,100, to light .


From its unexpected discovery as a bacterial adaptive immune system to its countless applications as one of the most versatile gene-editing tools, the CRISPR/Cas9 system has revolutionized every field of life science. Virology is no exception to this ever-growing list of CRISPR/Cas9-based applications. Direct manipulation of a virus genome by CRISPR/Cas9 has enabled a systematic study of cis-elements and trans-elements encoded in a virus genome. In addition, this virus genome-specific mutagenesis by CRISPR/Cas9 was further funneled into the development of a novel class of antiviral therapy targeting many incurable chronic viral infections. In this review, a general concept on the CRISPR/Cas9-based antiviral strategy will be described first. To understand the current status of the CRISPR/Cas9-based antiviral approach, a series of recently published antiviral studies involving CRISPR/Cas9-mediated control of several clinically-relevant viruses including human immunodeficiency virus, hepatitis B virus, herpesviruses, human papillomavirus, and other viruses will be presented. Lastly, the potential challenge and future prospect for successful clinical translation of this CRISPR/Cas9-based antiviral method will be discussed. Along with a proteinaceous structural component, a nucleic acid is an essential building block for assembly of an infectious virus particle. Therefore, efficient viral genome replication inside a host cell is one of the most important tasks for the successful completion of a virus life cycle. In theory, the ablation of viral genetic elements has been regarded as one of the most ideal antiviral strategies. However, the lack of a virus gene-specific destruction method has been a big hurdle for the realization of this virus genome-targeting antiviral strategy. Recently, a variety of sequence-specific endonucleases have been introduced and tested for their therapeutic potentials for direct manipulation of a viral genome in preclinical studies. They include zinc finger nucleases, transcription activator-like effectors nucleases (TALENS), and clustered regulatory interspaced short palindromic repeat (CRISPR)-associated (Cas) nucleases ,2. AmongType II CRISPR/Cas system, which is one of the most extensively characterized CRISPR/Cas systems, has a single effector DNA endonuclease called Cas9 . GuidancIn order to test the antiviral efficacy of the CRISPR/Cas9 system in any type of viral disease, candidate sgRNAs need to be designed and constructed based on a target virus genome sequence . Then, tMost of the current antiviral therapies for control of chronic viral infections by HIV, HPV, herpesviruses, and HPV failed to achieve a clinical cure due to their inherent inability to clear a virus genome from an infected host cell due to a latency state during which these viruses minimize its activity inside a host cell to avoid a host immune surveillance. The latency-related life cycle of these viruses plays a critical role in incurability of chronic infections induced by these viruses. Therefore, patients infected with these viruses need to take a life-long antiviral medication. In this regard, CRISPR/Cas9 technology holds great promise as a curative therapy for chronic infection. In order to understand the current status of this CRISPR/Cas9-based antiviral strategy, a series of recently published studies on CRISPR/Cas9-mediated control of HIV, HBV, herpesvirus, HPV, and other viruses will be presented as follows.Highly active antiretroviral therapy (HAART) transformed an acquired immunodeficiency syndrome (AIDS) from a form of the deadly and hard-to-treat disease to a treatable and manageable medical condition. However, its life-long duration and ultimate incurability have been a great burden on many AIDS patients . TherefoChromosomal integration-initiated HIV latency, subsequent minimal transcription of the HIV genome, and consequent preservation of HIV reservoirs are well-characterized hallmarks of HIV infection . In ordeStreptococcus pyogenes, which is the most widely utilized one .,34.33,34In spite of potent inhibition of HIV-1 replication by CRISPR/Cas9 system, HIV-1 has been shown to be able to generate escape mutants from a single antiviral gRNA by NHEJ-mediated modification of the target sequence  36,37,3,40,41,42More than 240 million people around the world still suffer from chronic HBV infection ,49. The Streptococcus pyogenes . In. In96]. Streptococcus pyogenes, one study employed a smaller version of Cas9 from Staphylococcus aureus [High-risk type HPV infection is responsible for the development of cervical cancers in women. In spite of the successful development of different kinds of prophylactic vaccines against HPV infection, HPV-specific antiviral drug is not yet available. Seven papers reported successful anti-HPV applications of CRISPR/Cas9 system through direct disruption of the HPV genome  97,98,9,102,103.s aureus ,101,102 s aureus ,100. Inas aureus ,101,102.The human neurotropic polyomavirus, JC (JCV), is responsible for the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) . As of nAfrican swine fever (ASF) is an economically important infectious disease of swine with high mortality rates. This disease is caused by an African swine fever virus (ASFV) with the double-stranded DNA genome . Hubner Pseudorabies virus (PRV), which is a swine herpes virus, causes significant morbidity and mortality in swine populations, which results in huge economic losses in the global swine industry . Peng etFrancisella novicida (FnCas9) to target a bacterial mRNA, which leads to the repression of a viral gene [Hepatitis C is an inflammatory liver disease caused by infection of hepatocytes with a hepatotropic single-stranded RNA virus known as the hepatitis C virus (HCV). Around 170 million people are estimated to be infected with HCV worldwide . Thanks ral gene . By takiral gene . They foral gene . Insteadral gene . Based oSo far, numerous successful applications of CRISPR/Cas9 technology to anti-viral manipulation of major viral diseases were presented. However, there seem to be many hurdles to overcome for its realization in human application. First, its potential off-target activity needs to be minimized to ensure safety in an in vivo application. Erroneous digestion of an essential host gene with a high degree of homology to the 20 bp seeding plus PAM sequence of the CRISPR/Cas9 target site could result in a catastrophic outcome in a host cell. For a tight control of this potential off-target activity of CRISPR/Cas9 system, a different version of Cas9, called Cas9nickase, which has a reduced off-target property due to its induction of a single strand DNA cleavage instead of a double strand one, is highly recommended for human use ,81. ReceTight linkage of a viral life cycle to a host cellular metabolism has made clearance of this invader out of our body a therapeutic challenge. Pharmacological intervention of viral infection by using a small molecule inhibitor against a virus-specific enzyme has been one of the best antiviral options so far. However, this conventional antiviral approach has been inappropriate for the control of most of the latency-associated chronic viral infections. In this regard, the introduction of the CRISPR/Cas technology with an unprecedented capability for direct targeting of a viral genome would contribute to the buildup of a new antiviral armamentarium aiming for a previously unthinkable, complete cure. In spite of many challenges ahead that need to be resolved for the full transition of this CRISPR/Cas technology from a preclinical study to a practical antiviral therapy, complete curative potential of the CRISPR/Cas-based antiviral strategy should provide continuous motivation for the development of novel antiviral therapeutics. Without a doubt, this new antiviral approach should help chronically infected patients stop taking a life-long medication in the near future.


Sweet potato vein clearing virus (SPVCV) isolate infecting a quarantined sweet potato accession from Fiji was determined. Sequence comparisons revealed the highest nucleotide sequence identity of 94.6% with that of the SPVCV type species, an isolate from the Dominican Republic. The virus was mechanically transmitted to Nicotiana bigelovii plants.The complete genome of a  Sweet potato vein clearing virus (SPVCV), a member of genus Solendovirus in the family Caulimoviridae, has been reported from Africa and the Americas (Ipomoea batata), mixed infections with other viruses cause disease. While undergoing quarantine testing in 2016, a sweet potato accession imported from Fiji displayed chlorotic and necrotic leaf spots. When the accession was grafted onto indicator I. setosa, vein clearing and necrosis were observed. To identify the causal agent, total RNA was isolated from the accession plant with the RNeasy plant minikit  and subjected to Illumina NextSeq 500 sequencing . Plant rRNA was removed using the Illumina Ribo-Zero rRNA removal kit before cDNA library construction. Analysis of total RNA reads was performed using CLC Genomics Workbench version 9.5.2. Contigs were annotated by BLASTx comparisons. De novo assembly of the RNA reads generated 25,070 contigs (>200 nucleotides [nt]) from the sample, and a BLASTx search of the contigs against the Viruses_NR database revealed the presence of a large contig  with high amino acid (aa) sequence identities (93 to 98%) to Sweet potato vein clearing virus (SPVCV) (GenBank accession number NC_015228). A total of 64,960,069 reads  mapped to this contig, supporting the infection of SPVCV in the accession. No other virus, viroid, or phytoplasma was identified. The virus genome (GenBank accession number MH188860) was resequenced using plasmid DNA cloned from PCR products that were amplified using virus-specific primers. The genome of this Fijian isolate shared 94.6% identity with Dom1, a SPVCV isolate from the Dominican Republic.Americas . Its icoNicotiana bigelovii plants, as confirmed by PCR using virus-specific primers. This is the first report and determination of a complete genome of a SPVCV isolate from the South Pacific islands.The genome of the Fijian SPVCV isolate  is 87 nt shorter than that of Dom1, mainly due to several deletions around the initial replication site, but its structure is virtually identical to that of Dom1. The aa sequence identities between the two isolates are 90.5% at the coat protein, 96.9% at the movement protein, 98.7% at the replicase polyprotein, and 94.8% at the inclusion body protein. The virus was mechanically transmitted to MH188860.The complete genomic sequence of the Fijian isolate of SPVCV has been deposited in GenBank under the accession number


Camelliasinensis. In recent decades, GTCs have been reported to provide various health benefits against numerous diseases. Studies have shown that GTCs, especially epigallocatechin-3-gallate (EGCG), have antiviral effects against diverse viruses. The aim of this review is to summarize the developments regarding the antiviral activities of GTCs, to discuss the mechanisms underlying these effects and to offer suggestions for future research directions and perspectives on the antiviral effects of EGCG.Over the centuries, infectious diseases caused by viruses have seriously threatened human health globally. Viruses are responsible not only for acute infections but also many chronic infectious diseases. To prevent diseases caused by viruses, the discovery of effective antiviral drugs, in addition to vaccine development, is important. Green tea catechins (GTCs) are polyphenolic compounds from the leaves of  Plant-derived natural products assist in the prevention and treatment of various diseases. Green tea and its major constituent polyphenols  are well known for their contributions to human health, including their antitumor, antioxidative, and antimicrobial activities ,2. AdditMany types of infection diseases caused by various viruses have a considerably negative effect on human health and lives. Viruses are responsible for not only acute infections, but also many chronic infectious diseases. Hepatitis B virus (HBV) and hepatitis C virus (HCV) play significant roles in chronic hepatitis, liver cirrhosis and liver carcinoma . The EpsInfection with hepatitis viruses is the main cause of hepatitis in the world. There are five types of hepatitis viruses: types A, B, C, D and E. Types B and C are the major cause of chronic liver diseases, such as liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV is a serious threat to international human public health, as hundreds of millions of people are infected. Despite vaccination programs, the possibility of and potential for outbreaks and epidemic spread cannot be excluded. Unfortunately, the most commonly used medications or therapies for HBV have success rates that are dependent on viral genotype and suffer from several limiting side effects . TherefoHepadnavirus family with a 3.2-kb genome of partially double-stranded DNA. In 2008, we reported the anti-HBV activity of GTCs in HepG2-N10, a stable cell line expressing HBV antigens [HBV is a member of the antigens . We founantigens ,19. Usinantigens . We founantigens . In 2014, Huang et al. found that different genotypes of HBV could be inhibited by EGCG in immortalized human primary hepatocytes and two constructed cell lines, DMSO-differentiated HuS-E/2 cells and HA-NTCP-expressing Huh7 cells (NTCP is the receptor of HBV) . FurtherHerpesvirus family. HSV-1 is commonly spread through mouth-to-mouth contact and causes cold sores and genital herpes. HSV-2 is usually spread by sexual contact and generally causes only genital herpes [Herpes simplex is a viral skin disease caused by infection with herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). Both HSV-1 and HSV-2 are enveloped viruses possessing a relatively large double-stranded, linear DNA genome and belong to the l herpes . The antl herpes . The autl herpes . A subsel herpes . The datl herpes . Anotherl herpes . The resl herpes . Palmitol herpes . An intel herpes . Moreovel herpes . The EBV is another member of the herpes family and infects humans ,70. EBV Adenovirus is an icosahedral non-enveloped DNA virus approximately 60–90 nm in diameter. Adenovirus infection can cause cold-like symptoms, sore throat, bronchitis, pneumonia and pink eye. People can contract an adenovirus infection at any age. Weber et al. found that EGCG at micromolar concentrations reduced the virus titers of adenovirus in two cell infection models and inactivated purified adenovirions . The resCamellia sinensis and demonstrated their potential as new inhibitors of HIV reverse transcriptase (RT). HIV, the pathogen of AIDS, was identified by Barré-Sinoussi and Luc Montagnier in 1983 [In 1994, Chang et al. reported for the first time the anti-HIV activities of polyphenolic catechins from Chinese green tea . The aut in 1983 . There a in 1983 . Therefo in 1983 ,75, moreEGCG is an inhibitor of HIV reverse transcriptase. Chang et al. have reported that three catechins, namely, EC, ECG and EGCG, demonstrate strong inhibitory action against HIVRT . KineticEGCG inhibits HIV entry into target cells. In 2002, Yamaguchi et al. reported that EGCG has a destructive effect on viral particles of HIV-1, causing a decrease in the ability of virions to infect cells and inhibiting viral attachment to cellular surfaces . The HIVEGCG attenuates neuronal damage mediated by HIV infection. HIV-associated dementia (HAD), previously referred to as AIDS dementia complex (ADC), is a condition caused by the action of HIV-1 infection on the central nervous system and leads to patients having difficulties with memory and learning. HAD is related to the activation of proinflammatory cytokines and neuropathology involving gp120 and Tat (a transactivating regulatory protein) of HIV. EGCG can mitigate neuron injury in the presence of IFN-gamma both in vitro and in vivo . Using aSemen-derived enhancer of virus infection (SEVI) is a fibrillar structure and an important HIV-1 sexual infection factor that captures virions and guides them to their targets. EGCG can inhibit SEVI activity by complex formation and degradation ,77. HartHCV is another major cause of chronic liver disease and can causes both acute and chronic infections. Timely antiviral therapy can cure more than 95% of persons with HCV infection, but access to diagnosis and treatment is currently low. According to the recommendations of the WHO, all patients with hepatitis C should be treated with sofosbuvir, daclatasvir and the sofosbuvir/ledipasvir combination based on direct-acting antivirals (DAA), except for a few specific groups of people in whom interferon-based regimens can still be used . AlthougFlaviviridae family member with a positive-sense single-stranded RNA genome and a small enveloped virus. In recent years, researchers have studied the anti-HCV activities of EGCG. Several independent research groups reported that EGCG was a potent inhibitor of the HCV entry pathway but had no effect on viral replication, viral RNA synthesis or virion secretion [HCV is a ecretion ,46. Moreecretion ,45. Besiecretion . Chen etecretion . Recentlecretion . Their recretion . The autecretion .Using HCV JFH-1 infectious systems, Lin et al. reported that HCV replication was inhibited significantly by epicatechin isomers. They revealed that the mechanism of the anti-HCV activity of the epicatechin isomers most likely operates through the downregulation of COX-2 . MoreoveInfluenza virus includes the pathogens of flu outbreak in birds and many mammals including human, pig, horse, whales, seals, bat and so on. There are four types of influenza virus: A, B, C and D, which are categorized according to the antigenicity of the nucleocapsid protein. Influenza A virus (IAV) is a member of the family of Orthomyxoviridae and has a segmented single-stranded, negative-sense RNA genome. IAV is the main virus of the flu pandemic because of its high mutation rate. As early as 1949, Green et al. reported the antiviral activity of tea extracts against influenza virus . The firTo determine the relationship between the structure and activity of different GTCs, Song et al. evaluated their capabilities to inhibit the replication of viruses. They found that EGCG exerted more inhibitory effects than ECG and EGC on the activity of both viral neuraminidase and viral genomic RNA synthesis, suggesting the 3-galloyl group of the catechin skeleton was more important for antiviral activity than the 5’-OH in the trihydroxy benzyl moiety at the 2-position ,54. SomeArbovirus refers to a group of viruses that are transmitted by insects, mostly commonly mosquitoes and ticks, sucking blood for nutrients or development . The mosFlaviviridae family, has a single-stranded RNA genome and is spread by mosquitoes. Since its outbreak in Brazil in 2015, ZIKV has spread rapidly on a global scale [Recently, molecular docking methods proposed that EGCG can dock in the same binding domain of different E proteins from DENV, JEV and TBEV . Furtheral scale . EGCG shal scale . Using VAdult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type I (HTLV-1) infection. In the 30 years since HTLV-1 was discovered, of ATL tend to yield poor results, and little progress has been made in the cure rate . TherefoRotaviruses and enteroviruses are viruses that can cause a series of intestinal symptoms. Enterovirus 71 (EV71) is the major pathogen of hand, foot and mouth disease. In addition, EV71 can lead to diarrhea, rashes and severe neurological disease. EV71 has done great harm to preschool children’s health in both developing and underdeveloped countries. As early as 1991, ECGC from green tea was reported to inhibit rotaviruses and enteroviruses in cultured rhesus monkey kidney cells by interfering with virus adsorption . Ho et aThe Ebola virus (EBOV) is among the most feared viruses and can cause Ebola hemorrhagic fever, a highly fatal disease. The recent WHO statistics showed that the 2014–2016 EBOV outbreak in West Africa had a high fatality rate of 28~75% . Reid etFish and other animals not only are good dietary sources of protein, but also have great natural value and can provide substantial economic and scientific benefits. However, diseases caused by fish virus infections can effect heavy economic losses and influence the development of fish farming. EGCG has been suggested to be able to inhibit infections of some novirhabdo viruses, such as hemorrhagic septicemia virus (VHSV), hematopoietic necrosis virus (IHNV) and spring viremia carpvirus (SVCV), specifically because of its ability as a low-molecular-weight inhibitor of serine protease inhibitor gene transcripts 1 (SERPINe1) . In 2016Over the past few decades, GTCs, especially EGCG, have been recognized as multifunctional bioactive molecules responsible for antitumorigenic, anti-inflammatory, antioxidative, anti-proliferative, antibacterial, and antiviral effects . In termEGCG can be regarded as a nucleophilic reagent because the phenolic hydroxyl groups in the pyrogallol and galloyl moieties provide more lone pair electrons than other catechins. This type of structural feature allows EGCG to react or combine with different molecules under appropriate conditions. From the antiviral effects reported, whether the viral genome is DNA or RNA, GTCs seem to be able to function in different stages of infections of both nuclear viruses  and cytoplasmic RNA viruses . The inhThe many previous investigations into the mechanism of action of EGCG indicate that the compound binds strongly to many molecules in cells, especially proteins, and then affects their original activities and functions ,94,95,96In the future, studies on the antiviral molecular mechanism, especially those on the molecules that bind with EGCG, will not only provide new insights into antiviral therapy but also contribute to the discovery of new targets. However, at least for now, the GTCs do not make good drugs, mainly because of their poor pharmacokinetic/pharmacodynamic (PK/PD) properties. Because of the unstable special structure and complexity of human physiological metabolism and cells, EGCG is easily oxidized or converted into other structural forms before entering its action targets. Therefore, structural transformation and modification and analog synthesis may subsequently become research emphases in EGCG studies.Scientists have described some derivatives through replacing the different hydrogens on the rings of A, B and D of EGCG structure, such as methylated ,98, acylIn recent years, this area of exploration has diversified into embedded forms of EGCG. For example, encapsulated nanosomes ,110,111


Cucurbita foetidissima , Cucurbita palmata (coyote gourd), and Datura wrightii (sacred thorn-apple). We used Illumina sequencing and network analyses to characterize viromes and viral links among species, using samples taken from multiple individuals at two different reserves. We also compared our Illumina workflow with targeted RT-PCR detection assays of varying costs. To make this process accessible to ecologists looking to incorporate virology into existing studies, we describe our approach in detail and discuss advantages and challenges of different protocols. We also provide a bioinformatics workflow based on open-access tools with graphical user interfaces. Our study provides evidence that dicot perennials in xeric habitats support multiple, asymptomatic infections by viruses known to be pathogenic in related crop hosts. Quantifying the impacts of these interactions on plant performance and virus epidemiology in our logistically tractable host systems will provide fundamental information about plant virus ecology outside of crop environments.Viruses are ubiquitous within all habitats that support cellular life and represent the most important emerging infectious diseases of plants. Despite this, it is only recently that we have begun to describe the ecological roles of plant viruses in unmanaged systems and the influence of ecosystem properties on virus evolution. We now know that wild plants frequently harbor infections by diverse virus species, but much remains to be learned about how viruses influence host traits and how hosts influence virus evolution and vector interactions. To identify knowledge gaps and suggest avenues for alleviating research deficits, we performed a quantitative synthesis of a representative sample of virus ecology literature, developed criteria for expanding the suite of pathosystems serving as models, and applied these criteria through a case study. We found significant gaps in the types of ecological systems studied, which merit more attention. In particular, there is a strong need for a greater diversity of logistically tractable, wild dicot perennial study systems suitable for experimental manipulations of infection status. Based on criteria developed from our quantitative synthesis, we evaluated three California native dicot perennials typically found in Mediterranean-climate plant communities as candidate models:  The integration of plant virology and ecology is recent, stimulated by the discovery that viruses are important but largely undescribed and understudied components of the ecology of wild plants. Since the inception of this field, ecologists and plant virologists have worked to illuminate two issues: (i) the ecological roles of plant viruses and vectors in unmanaged ecosystems, and (ii) the influence of ecosystem properties on the distribution and evolution of plant viruses and their vectors . Thanks The need for research in this area has never been greater. Among emerging plant diseases, viruses are the most common causal agents . NaturalAt present, systems-level understanding of plant virus dynamics across agro-ecological interfaces is hampered by a dearth of information from wild communities, including those that border agricultural and urban land cover. Historically, most monitoring of plant virus dynamics has focused on cropping systems , althougTo advance understanding in these critical research areas, it is essential that virus interactions within non-cultivated perennial systems be investigated. Perennials have potential to serve as modulators of viral virulence by exerting selection for longer-term host survival, or, in other cases, as sites for virus recombination through accumulation of co-infections or even as reservoirs for novel disease agents. In turn, non-cultivated perennial systems may be perturbed and affected by microbial movement from agricultural crops, which may also serve as reservoirs and exert selection pressures on viruses in ways that are not fully recognized. From an ecological perspective, viruses from crop environments may modify the expression of host plant functional traits in ways that alter stress tolerance, fitness , and comArabidopsis. In the study presented here, we conducted a review of plant virus ecology literature to quantify resource gaps in the field, then use these results to define criteria for selecting the most useful new systems for future work. We then present a case study of three potential model hosts to demonstrate how investigators with limited budgets might initially evaluate the viromes of candidate wild plant systems, and tractability of various detection assays, with the aim of incorporating virus dynamics into ecological studies.To advance work in this area, several hurdles need to be surmounted. One need is to simplify methods of virus detection and identify likely pitfalls so that researchers without backgrounds in molecular virology can properly examine the viromes of wild plant study systems. A second need is to identify promising wild plant–virus systems that can serve as useful models for short- and long-term manipulative work that goes beyond surveys and correlational studies. For example, we found that a large fraction of plant virus ecology work involving factor manipulation has been conducted on Poaceae hosts in a single ecosystem type: invaded California grasslands (see “Results” section). There is thus pressing need to identify new model systems involving dicot plant hosts beyond Cucurbita foetidissima and Cucurbita palmata (Cucurbitaceae), and Datura wrightii (Solanaceae)] that are summer-growing species within fragmented preserves of mixed grassland, xeric sage scrub and chaparral communities in the Southwestern United States , which is a Mediterranean-climate biodiversity hotspot . To expls Figure . Grasslas Figure , 2015. EFor our study, we targeted two agriculture and urban-adjacent preserves in Riverside County, CA, United States, that feature these Mediterranean climate communities Figure . Mediterprecursor papers) and subsequent papers that cited these precursor papers (product papers). Precursor papers consisted of all peer-reviewed publications listed in the NSF reporting website . A full description of the categories, as well as a complete list and categorization of the precursor papers, can be found in Supplementary Data Sheet Virus evolution, Virus discovery, Virus effects on host traits, Virus–vector interactions, Epidemiology, and Environmental virology) as core research categories of virus ecology based on previously defined criteria , which was established in 2007 by Ulrich Melcher and Carolyn Malmstrom with funding from the United States National Science Foundation (NSF). PVEN supported international workshops that were instrumental in connecting molecular virologists, epidemiologists, ecologists, entomologists, and other researchers with diverse skill sets relevant to plant virus ecology. NSF records indicate that PVEN stimulated the production of over 75 peer-reviewed publications between 2007 and 2011. To analyze the impact of PVEN on the field of virus ecology, and identify knowledge and resource gaps, we quantified the content of papers arising directly from PVEN support , the plant life history strategy , and the characteristics of the viruses studied . To evaluate impact, we used Google Scholar to obtain a list of all subsequent papers  citing each precursor paper since its publication. Each product paper was evaluated to determine if the content matched one or more of the six core virus ecology research areas. The ecological focus of product papers was determined in the same manner as for precursor papers. We assessed both literature sets for biases in favor of certain host and virus characteristics to identify knowledge gaps and resource needs within the field of virus ecology. We also evaluated characteristics of the most useful wild plant study systems in research categories that typically include experimental manipulations of infection status and other environmental conditions. This included all studies employing wild or crop/model + wild plants within the areas of Cucurbita foetidissima , Cucurbita palmata (coyote gourd), and Datura wrightii (sacred thorn-apple). We characterized individual viromes of representatives of these three species collected from two sites in southern California near the Los Angeles Urban Region in Riverside County: The Motte Rimrock Reserve  and the Shipley-Skinner Multispecies Reserve .Based on the quantitative literature synthesis, we evaluated California native plant species typically found in fragmented natural communities as candidate model species for basic and applied virus ecology research. We focused on perennial dicot species and identified three promising candidates: Salvia mellifera and Salvia apiana), Asteraceae (Artemisia spp.), Polygonaceae (Eriogonum spp.), and Poaceae  . As at Motte Rimrock, Cucurbitaceae and Solanaceae species are interspersed among the dominant vegetation. During the hot, dry summer months at both reserves (June–September), our target species are particularly green and apparent, as most other vegetation is senescent or dormant . These plants were distributed unevenly and so were sampled along a north-south transect through the reserve. In total, we collected tissue from 25 plants: nine C. foetidissima, four C. palmata, and two D. wrightii from Motte Rimrock Reserve, and six C. foetidissima, and four D. wrightii from the Shipley-Skinner Reserve  with a paired-end 75 bp read-length. This configuration can produce up to 260M paired-end reads. For a subset of viruses, we additionally used virus-specific RT-PCR and Sanger sequencing to characterize virus infections.Sequencing plant-infecting RNA viruses typically requires extraction of total RNA (host plus associated microbes), small RNA (21–24 nt), or double-stranded RNA (dsRNA) . There aBell pepper endornavirus  prior to dsRNA extraction (® (SPEX SamplePrep) by shaking at 1,700 rpm for 30 min (necessary for complete homogenization of 4 g of tissue). Samples were kept frozen during grinding by pre-cooling solid metal racks containing the tubes in liquid nitrogen for 5 min. We isolated double stranded RNA from 4 g of plant tissue using a low-cost extraction procedure involving binding and enrichment of dsRNA using Sigmacell cellulose type 101 powder  on an Illumina NextSeq 500 instrument using Mid Output v2 kit. We elected to use 75 bp paired end reads for cost savings. Sequencing, adapter removal and quality checks were performed by the UC Riverside genomics core facility. Sequences that could not be assigned an index based on the adapter sequence were discarded (see raw read statistics in Supplementary Data Sheet SRP149013). Reads were assembled into contigs with the Trinity assembly and analyzed with “NCBI BLAST+ blastn” using a Galaxy-based workflow (Supplementary Data Sheet Cucurbita maxima and Solanum lycopersicum). For a subset of samples, we compared this approach to a direct analysis without host genome filtering. Details of both approaches are available in Supplementary Data Sheet To construct libraries (one per sample), we denatured an aliquot of 5 μl of dsRNA solution from each extraction by incubating at 99°C for 5 min, then placed the solution on ice and used the NEBNext1. We chose this database since it includes complete nucleotide sequences for plant viruses, fungal viruses, and viruses infecting both plants and fungi. To minimize false or misleading hits, we did not include partial nucleotide or amino acid virus sequences available in GenBank.To identify known and novel virus sequences in our samples, we performed BlastN on the Illumina-based contigs using the “NCBI BLAST+ blastn” tool in Galaxy . We performed further phylogenetic analyses to identify possible origins and presence of host-specific variants. All sequences used for alignments and phylogenetics , we excluded GenBank sequences NC003885.1 and NC004018.1 in the alignment as they were highly dissimilar to other sequences returned in the BlastN analysis. Alignment trimming, manual checks for mis-alignments, and maximum likelihood phylogenetic analysis were performed as described above for known viruses. For all analyses, we used the WAG model of protein substitution.For putative novel viruses (PV1–4) we selected and aligned the longest coding sequence with a minimum of 400 bp matching the RNA-dependent RNA polymerase gene (RdRp) with corresponding RdRp coding sequences from viruses having a BlastN hit in the plant virus database. Alignments were performed using the ClustalW tool in Mega X software . We chosCucurbit aphid-borne yellows virus  and Tomato chlorosis virus ), we performed targeted detection assays (RT-PCR) using dsRNA and total RNA isolations. Tissue used for both isolations experienced only one freeze-thaw cycle. We isolated total RNA from ∼100 mg leaf tissue using a phenol-based method  and dissolved extracted RNA in 100 μl of nuclease-free water. Total RNA concentrations were 100–400 ng/μl; we repeated extractions until we obtained RNA from each sample with an A260/A280 ratio of greater than 1.5. Prior work with wild plant species demonstrated that low A260/A280 ratios are associated with inhibition of reverse transcription and PCR reactions . Sanger sequencing was performed by Retrogen Inc. and primer sequences were removed from contigs prior to analysis.Subsequent PCR reactions (20 μl reaction volume) consisted of 1 μl of template cDNA, 4 μl of 5X HF buffer (Thermo Fisher Scientific), 1 μl of each 10 μM primer, 2 μl of dNTP mix (2 mM each) and 0.2 μl of Phusion DNA polymerase (Thermo Fisher Scientific). Primers used for PCR detection were previously developed by others  , Virus effects on host traits (7/9 studies), and Virus–vector interactions (7/11 studies)  outnumber those focusing exclusively on annual hosts (1/9)  Figure . Studies) Figure . Virus d) Figure . Nearly s Figure .Virus discovery and Epidemiology categories – both of which are heavily based on surveys rather than manipulative studies. In contrast, studies on wild hosts are lacking in areas that typically employ experimental manipulations   including wild viruses in about 60% of studies  Figure . When ho) Figure . Nearly ) Figure . Solanac) Figure . About 8s Figure .Virus effects on host traits, Virus–vector interactions, and/or Environmental virology that included wild hosts, we detected overrepresentation of monocots , overrepresentation of annuals , a preference for uncomplicated seed-based propagation methods, and a tendency to prefer exotic/invasive hosts with weedy characteristics  growing in the same site as D. wrightii plants infected with ToCV  based on sequence similarity among isolates . ToCV is not known to be present in California and our detection represents the first instance of this virus being present in this state. To determine possible ToCV origins, we used sequences on GenBank for the major and minor coat proteins and the RNA-dependent RNA-polymerase. All trees suggest that our ToCV sequences are most similar to those of isolates collected within the continental United States (Florida and Colorado), and to those of isolates collected in China Figure . There ie Figure . Phylogee Figure . The fire Figure . Originse Figure . In conte Figure .Partitiviridae-like viruses revealed two possible virus lifestyles, most of which are consistent with patterns of detection among our three hosts and sampling sites. The PV1 group appears to contain sequences from a single virus species, as the sequences detected shared 100% identity. The PV1 group drew BlastN hits only for fungus-infecting Partitiviridae in the genus Gammapartitivirus, which does not include any plant-infecting viruses .Phylogenetic analysis of RdRp amino acid sequences from detected s Figure . A fungus Figure . Plants s Figure . Memberss Figure . Based os Figure . In conts Figure . The onlVirus discovery research area, several studies focused on detecting and describing viruses not associated with disease in crops   are preferred as hosts in the core research areas that are most likely to use manipulative experimental approaches to understand the roles of viruses in plant ecology   are those in which collection surveys dominate over experimental approaches  Figure . More tr) Figure . Within ) Figure , 4.Virus effects on host traits, Virus–vector interactions, and Environmental virology research areas  . We also found evidence of novel host family associations (CABYV in ) Figure . Along w Figures , 10D. Ou Figures . Surprisr Figure . Based or Figure , or toler Figure  in ways op hosts . We obseD. wrightii and C. foetidissima confirm that this virus is present in California, even if it is not currently reported infecting solanaceous crops. Our ToCV isolates group together in the coat protein nucleotide sequence tree, with good bootstrap support for divergence from other United States isolates  has diverged from isolates infecting solanaceous hosts  is not distinct from sequences of two other D. wrightii isolates collected at the same site (SSDW2B and SSDW2A). Deep sequencing also revealed instances of CABYV infecting D. wrightii , although two groups of CABYV are present in our populations, and only one contains an instance of infection in D. wrightii  was also infected with ZYMV (Genus Potyvirus). Synergistic interactions among criniviruses and potyviruses have been documented in crop systems (C. foetidissima and D. wrightii are both highly suitable wild hosts for a major ToCV vector (Bemisia tabaci MEAM1)   and, as Partitiviridae, which includes fungal, plant, and protozoan viruses  , even within a population. Future studies will leverage this natural variation, tractability of C. foetidissima for greenhouse research, and genomic resources for the genus Cucurbita  Figure  indicateucurbita , to begiCucurbita maxima) or subfamily (Solanum lycopersicum) significantly reduced the number of reads going into the BlastN analysis for both cucurbits (up to 83% of reads subtracted), but had little effect when applied to the more distantly related D. wrightii host (up to 14% of reads subtracted) , which is a Mediterranean-climate biodiversity hotspot .Mediterranean-climate regions like this experience a unique off-set of temperature and precipitation peaks  that offers advantages for agriculture  and human populations. As a result, these regions provide dramatic and clear examples of interfaces between annual crops and diverse native perennial vegetation, as likewise seen in South Africa and France  and AustTMS and KEM secured funding from the Shipley-Skinner Endowment Fund for this research, developed field sampling methods, collected plant and insect materials, and assisted with dsRNA extractions. TMS and KEM also assembled the literature databases and performed the quantitative synthesis. PS adapted molecular methods from published literature, performed dsRNA extraction, library preparation, and Illumina data analysis. CD assisted PS and TMS with alignments. CMM helped to develop molecular methods, assisted with bioinformatics workflow improvement, performed model selections for phylogenetic analyses, generated radial trees, and performed the network analysis. All authors discussed and interpreted the data and results, and collectively wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Glycogen storage diseases (GSDs) are genetic disorders that result from defects in the processing of glycogen synthesis or breakdown within muscles, liver, and other cell types. It also manifests with impaired neutrophil chemotaxis and neutropenic episodes which results in severe destruction of the supporting dental tissues, namely the periodontium. Although GSD Type Ib cannot be cured, associated symptoms and debilitating oral manifestations of the disease can be managed through collaborative medical and dental care where early detection and intervention is of key importance. This objective of the case report was to describe a child with GSD Ib and its associated oral manifestations with microbial, immunological and histological appearances. Case Presentation: An eight-year-old Hispanic male with a history of GSD type Ib presented with extensive intraoral generalized inflammation of the gingiva, ulcerations and bleeding, and intraoral radiographic evidence of bone loss. Tannerella forsythia was readily identifiable from the biofilm samples. Peripheral blood neutrophils were isolated and a deficient host response was observed by impaired neutrophil migration. Histological evaluation of the soft and hard tissues of the periodontally affected primary teeth showed unaffected dentin and cementum. Conclusions: This case illustrates the association between GSD Ib and oral manifestations of the disease. A multi-disciplinary treatment approach was developed in order to establish healthy intraoral conditions for the patient. Review of the literature identified several cases describing GSD and its clinical and radiographic oral manifestations; however, none was identified where also microbial, immunological, and histological appearances were described. Glycogen storage diseases (GSDs) are genetic disorders that result from defects in the processing of glycogen synthesis or breakdown within muscles, liver, and other cell types . It is eAn eight-year-old Hispanic male presented to the Stony Brook Dental Care Center with a history of GSD type Ib. Oral manifestations of the GSD Ib disease were observed and recorded upon the dental and radiographic examination. Overall, the patient presented with extensive generalized inflammation of the gingiva, erythema, ulceration, and generalized deep periodontal pocketing with bleeding on probing . General6 cells/mL, were loaded into the top chamber and allowed to migrate for 15 min at 37 °C. The filter was fixed in 100% 2-propanol, stained with Harris-type hematoxylin, clarified with xylene, and mounted for analysis. The distance that neutrophils traveled into the filter was measured using the leading-front method via bright-field microscopy. The microbial composition of the oral biofilm was characterized by multiplex PCR. 16S rRNA gene was used as the primers in PCR. Sterilized deionized water was used as negative control. Of the common putative periodontal pathogens, Tannerella forsythia was readily identifiable from the biofilm samples , Tanner


In vitro culture offers many advantages for yam germplasm conservation, propagation and international distribution. However, low virus titres in the generated tissues pose a challenge for reliable virus detection, which makes it difficult to ensure that planting material is virus-free. In this study, we evaluated next-generation sequencing (NGS) for virus detection following yam propagation using a robust tissue culture methodology. We detected and assembled the genomes of novel isolates of already characterised viral species of the genera Badnavirus and Potyvirus, confirming the utility of NGS in diagnosing yam viruses and contributing towards the safe distribution of germplasm. •in vitro growth of D. alata and D. rotundata.Robust tissue culture method boosts •Next-generation sequencing detected known and novel viruses in yam tissue culture.•Sequencing results enabled phylogenetic analysis of three novel yam virus isolates. In West and Central Africa, yams play a principal role in food and nutrition security and income generation for more than 60 million people and are important in cultural life ” and “Dioscorea bacilliform RT virus, isolate DBRTV3-[3RT]” for the two DBRTV3-like badnavirus transcripts. We reconstructed the 5′-ends of the DBRTV3-[2RT] and DBRTV3-[3RT] genomes by extending the mapped contigs with the raw RNA-seq reads using the Geneious  (MG711312), and YMV-NG (MG711313) were deposited in the NCBI GenBank database.The assembly of three full-length viral genomes derived from cv. Makakusa was achieved using Illumina HiSeq4000 RNA sequencing based on total RNA extracted from tissue culture leaves showing mild viral symptoms A. New me3.3.1Badnavirus recently detected in D. rotundata breeding line TDr 89/02475 and classified within the monophyletic species group K5  and DBRTV3-[3RT] were determined to be 7453 and 7448 bp in length, with a GC content of 43.4% and 43.6%, respectively. BLAST searches confirmed that both complete genomes were most similar to DBRTV3 , a new member of the genus group K5 . Pairwisordingly . A plantordingly . The tRNBadnavirus  and DBRTV3-[3RT] using NCBI ORF finder revealed three closely packed ORFs, arranged in tandem on the plus strand. Consistent with the genome organisation of DBRTV3 , start adnavirus . Analysi(RNaseH) . The CP (RNaseH)  were alsKF830000) and was found to belong to the monophyletic species group K5 described by Kenyon et al. , DBRTV3, DBALV, DBALV2, DBESV, DBRTV1, DBRTV2, DBTRV, DBSNV, and 19 additional yam badnavirus sequences available in the GenBank database with nucleotide identity values > 80% relative to DBRTV3-[2RT] in similarity searches with NCBI BLAST. DBRTV3-[2RT] is 93% identical to the sequence of an endogenous DBV described by Umber et al. , a member of the genus Potyvirus collected and characterised in 1977 from naturally infected yams in the Ivory Coast /DBRTV3-[3RT] and YMV-NG detected by RNA-seq in cv. Makakusa grown in tissue culture . One-ste44.1The use of virus-free, clonally propagated planting materials is the most effective method to control the spread of viruses infecting yam . Moleculin vitro propagation methodology for the two most important yam species, D. alata and D. rotundata. We compared two nutrient media compositions with or without the addition of AC. Different plant growth regulators present in a plant growth medium and their concentrations have a major influence on the success of in vitro propagation. Among plant growth regulators, auxin and cytokinins are the major determinants of root and shoot initiation in plantlets grown in vitro. Organogenesis (type and extent) in plant cell cultures is determined by the proportion of auxins to cytokinins  and DBRTV3-[3RT]) and a novel yam mosaic virus, YMV-NG. The RNA sequencing results support previous findings obtained using a combination of RCA and PCR for the detection of DBRTV3  and DBRTV3-[3RT], as well as a novel Yam mosaic virus isolate, YMV-NG, were detected in a cv. Makakusa sample from Nigeria, and complete genomes were assembled and characterised for these three viral isolates. The YMV and badnavirus infections were confirmed in RNA extracted from tissue-cultured plant material using one-step RT-PCR. This study presents a promising first step towards developing a robust in vitro propagation and NGS-based virus detection protocol, and confirms the value of NGS in safe movement of germplasm.We present a case study for sensitive NGS-based virus detection in yam plants grown using a robust tissue culture methodology. None.