The obtained materials were utilized as nano-adsorbents for the elimination of methylene blue (MB), a hazardous water-soluble cationic dye, from aqueous solutions, and showed excellent regeneration capacity. The formation of the nanocomposites was followed by high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM) equipped with energy dispersive X-ray spectrometry (EDX), Fourier-transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM), X-ray diffraction (XRD) and adsorption of N2 at −196 °C. The rate of adsorption was investigated varying several factors, namely contact time, pH, amount of adsorbent and MB concentration on the adsorption process. Studies dealing with equilibrium and kinetics were carried out in batch conditions. The obtained results indicated that the removal rate of MB was 99.53% in 90 min. Langmuir’s isotherm fitted better to the equilibrium data of MB. Fe3O4/AC/CD/Alg polymer beads shows amazing adsorption capacities in the elimination of cationic dyes (2.079 mg/g for polymer gel beads and 10.63 mg g−1 for dry powder beads), in comparison to other adsorbent materials. The obtained adsorbent is spherical with hydrophobic cross-linked surface properties that enable an easy recovery without any significant weight loss of in the adsorbent used.New magnetic iron oxide (Fe Balkizetal. [Ceibapentandra seeds. Natural polymeric materials are presently gaining more attraction, being used as adsorbents in wastewater treatment, as they were found to be non-toxic and biodegradable [Several contaminants with organic and inorganic nature end up often in water sources. Some of them are dyes, which have harmful effects on the environment, animals and humans. Even when present in small amounts, dye compounds discharged into water sources can affect the aquatic life. Synthetic dyes are often utilized for different purposes, like food coloring, textiles, paper, leather, cosmetics, medicines, inks for printing, paints, lacquers, soaps and lubricants . Therefokizetal.  used quakizetal.  used a pkizetal.  removed kizetal.  used comkizetal.  used phygradable . Activatgradable .β-Cyclodextrin (often named as β-CD) is an oligosaccharide with cyclic structure, containing seven glucopyranose units connected by α- linkages. β-CD has an inner cavity with hydrophobic nature, while the exterior is hydrophilic . Such stThe β-cyclodextrin can form complexes with several molecules, like aromatic compounds by means of intermolecular weak forces. Particularly, β-CD based polymers are quite effective in the elimination of reactive and disperse dyes from water solutions . Hence, 2+, Ca2+ and Fe3+) and is easily adsorbs heavy metals [Functionalized polymer nanocomposites attracted considerable interest due to their physical and chemical features, availability, easy separation and different reactive groups on the backbone chain ,31. Alg y metals , dyes [3y metals  and antiy metals ,35. At ty metals .In recent years, the polymer nanocomposites have been acquiring a great interest due to the not so high costs of production and good removal efficiency of some dyes ,38. Hydr3O4)/activated charcoal (AC)/β-cyclodextrin (CD)/sodium alginate (Alg) polymer composite gel beads. The sodium alginate-based material has high mechanical properties and high MB adsorption capacity, excellent regeneration ability and good separation properties [The present work aims to continue to explore the advantages of both polymeric and nanoparticle-based materials. In particular, our aim is to synthesize and characterize magnetic n , FeCl3·6H2O (ferric chloride), FeCl2·4H2O (ferrous chloride), CaCl2 , NH3 (ammonia) solution, NaOH (sodium hydroxide) and HCl (hydrochloric acid) were obtained from Merck . In the experiment, 0.1 M solutions of NaOH and HCl were used for pH adjustment. Activated charcoal, β-cyclodextrin, MB and ethanol were received from HIMEDIA . The structure of MB is illustrated in , dropwise, with strong stirring. A dark precipitate was immediately obtained, which was heated to 80 °C for 30 min. The formed black nanoparticles were removed with the help of an external magnetic field and three times washed with double distilled water and ethanol, heated at 250 °C in air for 2 h. The finely prepared 1.0 g Fe3O4 nanoparticles were dispersed in 10 mL double distilled water and then 1.0 g AC was added to the solution (1:1) and continuously stirred for 2 h. The liquid was removed from the resulting black slurry by decantation and filtration, and the solid was washed with double distilled water several times. The obtained precipitate was dried in an oven at 90 °C for 12 h and 500 °C for 2 h in a muffle furnace.Feterature , with sl3O4-AC nanoparticles were mixed with 10 mL double distilled water to obtain a colloidal solution that was mixed with 1.0 g β-CD under intense stirring at 40 °C. The formed nanoparticles were collected by applying an external magnetic field. The particles were washed with double distilled water several times and dried in an oven at 90 °C for 24 h.The obtained 1.0 g Fe3O4/AC/CD nanoparticles were well mixed with the solution. 2% CaCl2 solution was added to the gel beads with a syringe with non-stop stirring (300 rpm) and stored overnight to complete gelation and form stable beads. The latter were repeatedly washed with double distilled water and kept in an aqueous medium for later use. A scheme outlining all the steps involved in the synthesis of Fe3O4/AC, Fe3O4/AC/CD and Fe3O4/AC/CD/Alg nanocomposites, from preparation to analysis, is shown in 2.0 g of powder sodium alginate were dissolved into 75 mL double distilled water with strong stirring until a viscous solution was obtained. Then 1.5 g of Fe2 adsorption and desorption analysis of Fe3O4/AC/CD/Alg polymer composite gel beads were carried out at −196 °C in a BELSORP-miniII, BEL, Japan apparatus. The samples were heated at 120 °C for 3 h in N2 atmosphere, to eliminate any water or gas adsorbed on the surface, on a pretreatment unit . The surface area was determined by the Brunauer–Emmett–Teller (BET) method with data from the N2 adsorption and desorption isotherms. The total pore volume (totV) was determined from the quantity of N2 adsorbed at a P/P0 = 0.99. The pore size distribution was obtained by the Barrett–Joyner–Halenda (BJH) method. The prepared Fe3O4-AC, Fe3O4/AC/CD and Fe3O4/AC/CD/Alg polymer composite gel beads were characterized by X-ray diffraction (XRD) using a Philips PW3064/60 . Morphology was studied on a JEOL  analytical scanning electron microscope (SEM). A Fourier transform infrared spectrometer  was utilized to record the infrared spectra of Fe3O4-AC, Fe3O4/(AC)/β-(CD) and Fe3O4/AC/CD/Alg polymer composite gel beads in the range of 400–4000 cm−1, with KBr pellets. The microstructure of the prepared nanocomposite beads was analyzed by energy dispersive X-ray spectrometry . The magnetic properties were recorded for the Fe3O4/AC/CD/Alg polymer composite gel beads with a vibrating sample magnetometer . High resolution transmission electron microscopy (HR-TEM) images were obtained in a JEOL .N3O4/AC/CD/Alg polymer composite gel beads. A standard MB stock solution was used and diluted with deionized water in order to obtain different concentrations. The obtained MB solutions was kept in a flask with fixed volume (10 mL of 5 ppm) and the Fe3O4/AC/CD/Alg polymer gel beads were added (in different amounts) to the MB solutions. The flask was placed in a shaker (150 rpm) for 90 min, at pH 6, in an orbital incubator shaker, at room temperatures. The beads were removed with the help of an external magnetic field, when the experiment was over. The upper layer liquid was analyzed by UV-Vis , at a wavelength of 664 nm, after a certain time. The synthesis of Fe3O4/AC/CD/Alg polymer composite gel beads and image of gel beads being attracted by the external magnetic field are shown in R, %) was calculated from Equation (1) and adsorption capacity  from Equation (2), as follows:R, tq (kinetic adsorption capacity of MB dye) were calculated by Equations (1)–(3), respectively [oC and eC are the initial and equilibrium concentrations of MB (mg L−1), respectively, Ct is the concentration of MB at a given time t, m is the adsorbent mass (g) and V is the dye solution volume (mL). The equilibrium adsorptions of MB dye from aqueous solutions were investigated using the Langmuir and Freundlich models for isotherms. Furthermore, the kinetics of MB dye adsorption was examined using pseudo-first order and pseudo-second order kinetics. The Fe3O4/AC/CD/Alg beads were desorbed using 0.1 mol L−1HCl solutions after adsorption of MB until saturation. The flask containing 0.1 mol L−1 HCl and the saturated Fe3O4/AC/CD/Alg beads was placed in an orbital incubator shaker at room temperature, for 1 h, to full desorption. Subsequently, beads were filtered and three times washed until neutrality was reached before the following adsorption experiment.The MB adsorption experiments were conducted in batch conditions using the Feectively , where C3O4/AC/CD/Alg polymer gel beads (0.2 g) were placed in a conical flask containing MB solution  and the mixture was shaken in an orbital incubator shaker for 90 min at room temperature. The mixture was separated with a magnet and the final dye concentration was found. The Fe3O4/AC/CD/Alg polymer gel beads were recycled by washing with 0.1 N HCl and water under constant stirring for three times, respectively. Then the gel beads were added into another MB solution in order to begin a new adsorption. The recyclability of the material was followed up to five consecutive adsorption–desorption cycles.Fe3O4/AC/CD/Alg in more detail, and the results were shown in 3O4/AC/CD/Alg is a highly mesoporous material with particle sizes around 20 nm, and grain sizes around 2–15 nm. This decrease in size is mainly due to β-CD [High resolution Transmission Electron Microscopy (HR-TEM) was used to investigate the Fe to β-CD .3O4/AC/CD/Alg polymer beads before (A) and after adsorption (B), indicating that the surface of Fe3O4/AC/CD/Alg polymer beads had rough porous structure and large surface area. A 1000× magnification was used. 3O4/AC/CD/Alg showed irregular structure and large surface area, which improved the material adsorption capacity. The elemental analysis of the Fe3O4/AC/CD/Alg polymer beads are shown in 3O4/AC (A), Fe3O4/AC/CD (B) and Fe3O4/AC/CD/Alg (C) polymer nanocomposite beads, showing the presence of magnetic Fe3O4 particles, which can be utilized for magnetic separation D= nanocomposite were 11.04, 2.71 and 5.63 nm, respectively, as shown in In the present case, the mean crystallite size of the Fe3O4-AC, Fe3O4/AC/CD and Fe3O4/AC/CD/Alg polymer composite beads were analyzed by FTIR and results are in 3O4-AC, the bands at 3562.77, 3392.93, 3060.08, 2953.49, 1540.31, 1126.89, 632.92 and 563.79 cm−1 were observed in −1 were ascribed to Fe–O vibration modes, just like for Fe3O4/AC/CD and Fe3O4/AC/CD/Alg. The bands at 3562.77 and 3392.93 cm−1 were due to –OH and –NH2 stretching vibrations. The band at 3060.08 cm−1 was from the (=C–H) stretching vibration. The band at 2953.49 cm−1 was assigned to the antisymmetric stretching vibrations of the methylene group. The peaks for the C–N stretching vibration were found at 1126.89 cm−1. For Fe3O4/AC/CD, bands at 3415.26, 3021.36, 1713.47, 1563.97, 1140.92, 1032.28, 633.92 and 561.74 cm−1 were observed  stretching vibration. The peak at 1713.47 cm−1 was related with the stretching vibrations of the –C=O carbonyl groups and 1563.97 cm−1 was from the N–H bending of –NH2. Peaks at 1140.92, and 1032.28 cm−1  related to the glycosidic (C–O–C) antisymmetric vibrations and ʋ(C–C/C–O) coupled stretching vibration. Therefore, it could be concluded that Fe3O4/AC/CD was present [3O4/AC/CD/Alg (−1 were found. The bands at 570.97 cm−1 (characteristic peak of Fe3O4) and at 3427.52, 2923.88, 2856.61, 1625.96, 1411.60 and 1033.27 cm−1  were ascribed to –OH (stretching vibration), –CH2 (stretching) from CH3,–COO−(asymmetric stretching), –CH2 (bending) and C–O–C (stretching) vibrations [−1 were related to the carboxylic groups –C–O stretching vibrations. The absorption bands at 1320.90, 1247.16 and 808.28 cm−1 could be ascribed to the C–OH stretching vibrations, C–O–C stretching vibrations and C–H bending vibrations, respectively.The adsorption capacity of an adsorbent is related with its molecular structure and functional groups. For the identification of the functional groups, the Feobserved B. The ba polymer . The ban present . For Fe3C/CD/Alg C, bands brations , respect3O4/AC/CD/Alg nanocomposite, the magnetic-hysteresis (MH) loops were recorded at room temperature. Results can be found in 3O4/AC/CD/Alg nanocomposite exhibited hysteresis, which confirmed the usual ferromagnetic behavior of nanoparticles with negligible coercivities and moderate saturation magnetization. From MH loops, the saturation magnetization value (MS) of Fe3O4/AC/CD/Alg was found to be 0.12849 emu g−1. Since the composite was composed of a mixture of three non-magnetic materials, viz. activated charcoal, β-(CD) and sodium alginate, the MS obtained for Fe3O4/AC/CD/Alg had a lower value. The results were in conformity with those reported for the β-(CD) coated Fe3O4/carbon nanocomposite in literature, for which the MS was 1.2 emu g−1 [To determine the magnetic properties of the obtained Fe emu g−1 . The ads2 adsorption and desorption isotherms for Fe3O4/AC/CD/Alg at −196 °C. The Brunauer–Emmett–Teller (BET) analysis was utilized for determining the surface area of the material. The BET specific surface area of was 8 m2 g−1, and 3 g−1 with a mean pore diameter of 1.47 nm. This shows that the surface area of the material is larger than of other adsorbents [3O4/AC/CD/Alg nanocomposite are shown in sorbents . The tex3O4/AC/CD/Alg polymer beads = 0.2 g/10 mL dye solution, agitation speed = 150 rpm, contact time = 90 min, room temperature). These dye sorption behaviors could be explained the following way: at low pH, the lower dye adsorption onto Fe3O4/AC/CD/Alg can be linked to the large amount of H+ ions present, which compete with the dye positive groups for the adsorption sites of the beads. With the increase of the pH of the solution (above 3), the active sites of the surface of the Fe3O4/AC/CD/Alg onto the hybrid beads were deprotonated and the competition for the adsorption sites between the dye cations and H+ decreased, which increased the amount of dye adsorbed [3O4/AC/CD/Alg polymer beads and the positively charged dye ions. Similar results were observed for contact times of 60 min  and dry powder beads (0.06 g) were agitated for 90 min. The obtained results are shown in The adsorption capacity was determined as a function of time to evaluate the maximum time of contact during the adsorption process. −1, pH 6 and contact time of 90 min. As shown in To determine the effect of temperature on adsorption, tests were carried out at four different temperatures, i.e., 35 °C, 45 °C, 55 °C and 65 °C, at an initial dye concentration of 5 mg L3O4/AC/CD/Alg polymer gel beads and dry powder beads for removal of MB. The Langmuir model is applicable for monolayer adsorption and this isotherm assumes that the adsorbent surface contains active sites with uniform monolayer formation energy. The linearized Langmuir isotherm model is represented as:LK (L/mg) is the Langmuir adsorption constant, −1) is the equilibrium concentration and Adsorption isotherm models have been often used to explain the adsorption processes and mechanisms. They describe the interaction between adsorbate and adsorbent, under propitious conditions. The obtained equilibrium data were fitted to several theoretical models, as Langmuir  and FreuLR can be calculated by the equation below:−1) is the initial dye concentration. The LR = 1) or unfavorable (The main characteristics of the Langmuir adsorption can be expressed in terms of a constant (that is dimensionless), called separation factor or equilibrium parameter, used to predict if an adsorption system would be favorable or unfavorable. FK (mg g−1) is the adsorption capacity and n is an empirical parameter that varies with the intensity and heterogeneity of the adsorption. The values of 1/n and FK are calculated from the intercept and slope of the log logThe multilayer adsorption heterogeneous surface energy is described by the Freundlich isotherm that can be linearized as:The adsorption parameters derived from the isotherm models are in 2 = 0.992) model showed a better fitting than the Freundlich model (R2 = 0.971), proving the surface homogeneity of the adsorbent and showing that the monolayer adsorption capacity  is the rate constant, determined from the slope. t plot.The effect of contact time on MB removal by Fek2 (g/mg min−1) is the rate constant. The values of t/t shown in 3O4/AC/CD/Alg polymer beads.The pseudo second order kinetic model has this equation:The pseudo first and second order rate constants and correlation coefficients are listed in The high correlation coefficient obtained for pseudo second order kinetics indicated that the model nicely represented the experimental data.H°), entropy (ΔS°) and Gibbs free energy (ΔG°) change were calculated by the Equations (10)–(12), respectively [−1) is the distribution coefficient, ΔG° (kJ mol−1), ΔH°(kJ mol−1) and ΔS°(kJ mol−1) are the changes in Gibbs free energy, enthalpy and entropy, respectively, T is the temperature (in K). The adsorption capacity of the Fe3O4/AC/CD/Alg polymer composite beads increased with a temperature increase from 35 to 55 °C. Van’t Hoff Equation (11) related the distribution coefficient with S° at constant temperature (1/T). The values of entropy and enthalpy were calculated from the intercepts and slopes of T linear regression. ΔH° had a positive value suggesting the reaction to be endothermic, favoring the adsorption of the MB at higher temperature. The positive value of ΔS° indicated high randomness of adsorption, favoring the constancy of adsorption. The negative values of (ΔG°) confirmed the viability and spontaneity of the adsorption. The adsorption thermodynamic parameters, like enthalpy (Δectively .(10)ΔG°The reusability was tested up to five consecutive adsorption-desorption cycles and results are shown in 3O4/AC/CD/Alg polymer beads was considerable, compared to other biological adsorbents found in literature  or inorganic  layer.In this work, novel FeThe effects of several parameters on the adsorption process  were discussed. The versatility in surface modifications of magnetic nanoparticles offers advantages for diverse surface modifications. They could be easily functionalized using many different kinds of modification agents, including organic acids and polymers, based on their potential applications.Magnetic separation shows advantages, like easy separation from solutions and possibility to treat a large amount of wastewater within a short time. Moreover, enhanced performances were achieved, due to cooperative interactions between the constituents.Furthermore, a MB removal percentage of 99.53% at pH 6 was found, and the adsorbent could easily be regenerated and recycled, being reused up to five adsorption–desorption cycles, without any significant weight loss. The use of dry powder beads considerably enhanced the adsorption of MB, compared to gel beads, in terms of time and dosage.3O4/AC/CD/Alg polymer nanocomposite is a good adsorbent for large scale wastewater purification since it has high adsorption capacity and easy separation.In conclusion, this work opened a new path for effective elimination of cationic dyes from wastewater using an environmentally friendly biodegradable adsorbent. The Fe


Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ultraviolet-induced damage to the tumor genome from excessive sunlight exposure. An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors. However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC.In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes. To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure.Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA. Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival. Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival.These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC. Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors. Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin. Risk factors for developing MCC include advanced age, light skin color with excessive sunlight exposure, and a variety of immunocompromised conditions . In 2008RB1, TP53, NOTCH1, and FAT1 . As. As22]. For further advancements to be made in understanding MCC, especially for patients not responsive to current therapies, clear and accurate determination of the MCPyV virus status and actionable variants in these tumors are required. In this study, we developed a viral hybrid capture next-generation sequencing (NGS) method to detect the presence of integrated MCPyV DNA in FFPE clinical specimens for routine use in a clinical setting. This approach was combined with targeted sequencing of several hundred cancer-related genes to assess oncogenic changes in the tumor genome. Lastly, we compared the sensitivity and accuracy of this viral hybrid capture approach to more traditional approaches, PCR detection of viral DNA, IHC for detection of MCPyV LT, and synoptic assessment of MCC pathology.n = 71) with a reported diagnosis of MCC at Dana-Farber/Brigham and Women’s Cancer Center who underwent comprehensive genomic profiling by OncoPanel between May 2013 and April 2018. OncoPanel version 3 (POPv3) is a custom hybrid capture assay targeting the exons of 447 genes and 191 regions across 60 genes commonly rearranged in cancer . As. AshttpsBased on previous MCC WGS, MCPyV integration sites frequently coincide with focal amplifications in the human genome. We can therefore infer that the regions between distant (> 10 kb) viral integration sites were amplified; however, no targeted exon were within these regions . BecauseUsing automated computational methods, we could not confidently determine an integration site for case MCC037 with the highest viral genome copy number in this study. Manually interrogating the human sequence hits from the assembly revealed that it matched a tandem repeat sequence flanked by MLT1H2 ERVL-MaLR elements. Based on the estimated copy number and the assembly graph, the viral component of this fusion DNA structure is likely larger than 10 Mbp , higher TMB, and absence of integrated MCPyV compared to virus-positive MCC. The virus-negative MCC without a UV signature (MCC007) originally presented as a subcutaneous breast mass . A totalFFPE sections were available for 28 of the 71 cases to assess for MCPyV LT by IHC with antibodies CM2B4 and Ab3. For 8 of the virus-negative MCC, all were negative by IHC with both antibodies. For 20 virus-positive MCC cases, we observed 16 stained positive with both antibodies and 4 were negative , primary biopsy with further recurrence (N = 22), and recurrence biopsy (N = 19). For all biopsies annotated as a recurrence, the first recurrence occurred before the biopsy was obtained. Among the 19 recurrence biopsies, 15 were distant metastatic biopsies, one local recurrence (MCC027), one unspecified recurrence (MCC063), one second recurrence (MCC057), and one local recurrence with no prior chemo/XRT (MCC026). Regardless of the biopsy type, all patients and sequencing data were grouped into either no relapse (N = 30) or relapse (N = 41). Table Overall, 28 patients remained disease free after initial therapy and 43 developed one or more relapses or persisted as stage IV  , respectively. We acknowledge that some cases have relatively short follow-up times that may impact survival analysis; however, the association of OS and immunosuppression only shifts slightly after keeping patients with follow-up times greater than 6 months (n = 63) remaining statistically significant. Under this criterion, the median OS for immunocompetent and immunosuppressed cases were 48.5 months (95% CI 35.4 to 113.3 months) and 21.6 months , respectively.Consistent with known risk factors of MCC, 10 of the 71 cases had immunosuppression diagnosed prior to developing MCC. Remarkably, 8 of the 10 (80%) of the immunosuppressed cases were identified as virus-negative MCC with relatively high TMB compared to the 28% virus-negative MCC in immunocompetent patients Fig. . Virus-n01) Fig. . ImmunosWe undertook this study to develop an assay to more accurately distinguish between virus-positive and virus-negative MCC by genetic features. We built upon an NGS platform that has been instituted as a routine part of clinical care at the Dana-Farber Cancer Institute, Brigham and Women’s Hospital, and Boston Children’s Hospital. The viral hybrid capture assay, ViroPanel, acquired a high number of MCPyV reads for many samples. Importantly, evidence for specific integration was associated with all cases with a high number of reads (> 6000). Spurious MCPyV reads were also detected in 19 of 20 MCC cases that were deemed to be virus-negative by TMB and UV mutations. There was no evidence for integration in these cases; rather, these reads could be traced to be extremely low-level contamination from MCC037 during library preparation or sequencing. In contrast, true virus-positive MCCs have low TMB with clear assemblies of virus-host junctions with MCC-hallmark deletions in the MCPyV genome.Integration sites were observed in 12 different chromosomes with the most occurring on chromosome 5. In addition, two fully overlapping integration sites from two different tumors were observed on chromosome 1 separated by only 10–20 kb. Based on the clonality of deletions and point mutations in the MCPyV genome, these events most likely occurred before or during integration as was similarly determined from another study on MCC cell lines . For botCase MCC026 has three apparently separate integration events occurring on different chromosomes. The integration event on chromosome 16 only contains a small section of the viral genome from positions 2853–3521, which would only encode the helicase domain of LT and therefore is unlikely to contribute to tumor survival. Conversely, the event on chromosome 18 has a full copy of the viral genome while the event on chromosome 9 contains the NCCR, ST, and a truncated LT, likely sufficient to contribute to oncogenesis. Based on the assembly graphs and coverage, one or both of these integration events have more than one copy of ST and LT  involving MYCL was observed more commonly in virus-negative cases, but was identified in a few virus-positive cases. Interestingly, in virus-positive MCC, MCPyV ST binds MYCL and the EP400 chromatin modifying complex to activate transcription of several hundred target genes . AmplifiVEGFA, which promotes angiogenesis and has been observed to be expressed at higher levels in distant ovarian cancer metastases [q = 2.47 × 10− 7) pathway enrichment for angiogenesis as well as EGFR signaling and p53 dysregulation. FANCE is also carried on this chromosome and as a DNA damage response gene act to limit number of point mutations and copy number changes observed in virus-positive MCC, hindering tumor evolution. Together, this amplification co-occurring with MCPyV may represent a less fatal, but more metastatic subtype of MCC. Additionally, this result could be impacted by diagnosis, treatment, or study recruitment of metastatic MCC.A recurrent amplification of chromosome 6 has previously been observed for MCC; however, this observation predated the discovery of MCPyV and was not associated with morphology or outcome . In othetastases . InteresUnexpectedly, we observed that 8 of 10 cases with immunosuppression were virus-negative MCC. While it was recognized in the early 1990s that individuals with hematologic malignancies that developed MCC had a poor prognosis , it was In the present report, three solid organ transplant recipients, three with chronic autoimmune diseases, and two with hematologic malignancies developed virus-negative MCC. It is well established that the risk for developing MCC is increased in patients with chronic inflammatory disorders such as rheumatoid arthritis or medically induced immunosuppression for solid organ transplantation –61. WithDespite the significant differences in the TMB between virus-positive and virus-negative MCC, there were few phenotypic differences in the two types of MCC. Based on histopathological features alone, two subtypes of MCC can be recognized: pure neuroendocrine tumors and combined tumors with neuroendocrine and divergent (mainly squamous) differentiation. Most pure tumors are MCPyV-positive and CK20-positive while combined tumors are uniformly MCPyV-negative and occasionally CK-20 negative , 65. VirWhile genomic sequencing has revealed that virus-negative MCC has evidence for a high degree of UV damage, this does not exclude a role for UV exposure in the development of virus-positive MCC. The relative lack of UV damaged DNA in virus-positive MCC indicates that the etiologies are clearly different, suggesting that the precursor to virus-negative MCC was a recipient of lifelong intense UV exposure while the virus-positive MCC were not exposed to sunlight for the same degree or for as long. It was reported that the early promoter of MCPyV responds to UV exposure and that levels of ST mRNA increased in UV exposed skin from a healthy human volunteer . TransieHere we present a comprehensive characterization of the Merkel cell carcinoma genetics using a clinically implemented sequencing platform. This platform was augmented using a hybrid capture baitset against Merkel cell polyomavirus. From our analyses, we identified CNV clusters unique to and common to virus-negative and virus-positive, which reflect the evolutionary mechanisms of the tumors. We also accurately reconstructed the viral integration events providing clear evidence for a circular host-fusion DNA intermediate initiated by recombination at 4+ bp microhomology enriched at low-complexity regions in the human genome. Lastly, we observed a surprising number of virus-negative tumors in immunosuppressed patients in our cohort potentially reflecting a previously misunderstood risk population.Additional file 1: Table S1. SNV data for all patients. Table S2. CNV data for all patients. Table S3. CNV cluster definitions. Table S4. Synoptic review of dermatopathology. Table S5. Association between relapse and genomic sequencing (N = 52).Additional file 2: Fig. S1. Oncoprint for all genes in this study. Oncoprint summarizing point mutations and CNVs for all genes and patients in this study. Sample are in order of descending TMB and genes are in order of highest point mutations to least. Fig. S2. Network graph for recurrent CNVs. Network graph with all significantly (q < 0.05) co-occurrent CNVs showing how they cluster into reoccurring groups. Fig. S3. Additional file 4.pdf: CNV frequency by cluster for all patients. Cluster number is shown in gray bars above the bar plots representing amplifications/gains (red) and deletions/losses (blue). Below the bar plots is a heat map of all CNVs   across all samples (y-axis) annotated by cluster and chromosome. On the left side pRB, p53 shown in gray and black for 1 or 2 copy loss/mutant, respectively. Presence of UV mutations are shown in black. Fig. S4. RFS survival divided by CNV cluster 5. Kaplan-meier plot for MCC patients split by presence or absence of CNV cluster 5 showing no difference in regression free survival (RFS). Fig. S5: Plot of tumor sampling by patient for sequencing. Plot of all patients along the y axis and time since diagnosis on the x axis. Initial diagnosis, primary biopsy sequencing, recurrence biopsy sequencing, death, and last contact times are plotted as applicable.Additional file 3: Fig. S6. Assembly graphs for all integration events. 28 assembly graphs annotated by MCPyV genome position (colors labeling each segment are under the header “as.factor(V9)”). The “V3” variable represents the coverage of each contig as determined by SPAdes and contigs are scaled to reflect this value.


The coronavirus SARS-CoV-2 has turned our own health and the world economy upside down. While several vaccine candidates are currently under development, antivirals with the potential to limit virus transmission or block infection are also being explored. Plant production platforms are being used to generate vaccines and antiviral proteins inexpensively and at mass scale. The following review discusses the biology and origins of the current coronavirus pandemic, and describes some of the conventional, synthetic, and plant-based approaches to address the challenge that it presents to our way of life. Coronaviruses (CoVs) constitute a very diverse group of positive-sense enveloped RNA viruses with genomes ranging between 27–32 kb . The virAlphacoronaviruses, Betacoronaviruses, Gammacoronaviruses, and Deltacoronaviruses [Coronaviruses belong to the order Nidovirales, which is classified into four families, including the Coronaviridae, Mesoniviridae, Arteriviridae and Roniviridae. Coronaviridae is categorized into two sub-families: the Coronavirinae and Torovirinae. The sub-family Coronavirinae is further divided into four genera based on serological and phylogenetic findings: aviruses . The Alpaviruses . In the aviruses . The earaviruses . Coronavaviruses ,10 and Saviruses ,12. Amonaviruses . SARS-Coaviruses . A novel Coronavirus that recently emerged in Wuhan City, China was initially detected on 29 December, 2019, from four human cases who were all linked to the Huanan seafood market in Southern China. Patients displayed “pneumonia of unknown etiology” similar to the 2003 SARS. Deep sequencing of samples obtained from the patients’ lower respiratory tract revealed a novel strain of coronavirus that was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,17. Scientists quickly identified the causative agent as betacoronavirus ,18,19. PThe SARS-CoV-2 virus particle has a spherical shape that exhibits some degree of pleomorphism with a diameter ranging from 60 to 140 nm, and distinct spikes 8–12 nm long . The recThe virus primarily spreads through the respiratory secretions of an infected person when he sneezes, coughs or talks . DropletImportant similarities exist between the symptoms of COVID-19 and previous betacoronavirus infections including fever, dyspnea, dry cough and ground-glass opacity in CT scans of the chest . HoweverThere is a lack of precise antiviral treatment and vaccines for COVID-19 currently . HoweverRemdesivir is currently recognized as a proficient antiviral drug with promising potential against a broad range of RNA virus infections (including SARS/MERS-CoV) in cell cultures, nonhuman primate (NHP) models, and mice. Clinical development of Remdesivir to treat Ebola virus infections is underway . RemdesiRdRp gene as found in SARS-CoV and MERS-CoV [Favipiravir is a pyrazine carboxamide derivative anti-viral drug that has been approved to treat influenza in Japan. It is a prodrug, intracellularly phosphorylated and ribosylated to give rise to the active favipiravir ibofuranosyl-5′-triphosphate (T-705-RTP) metabolite . T-705-RMERS-CoV ,66,67. TMERS-CoV . ResultsMERS-CoV  Another MERS-CoV .A study carried out by Jian-ya et al.  on 51 paConvalescent plasma (CP) therapy is a classic approach of immunotherapy that has been in use for over a century to treat various infectious diseases. CP proved successful in treating SARS, MERS and pandemic 2009 H1N1 over the last two decades with satisfactory results ,73,74,75A recent study by Shen et al.  was carrArtemisia kermanensis, Eucalyptus caesia, Mentha spp., Rosmarinus officinalis, Satureja hortensis, Thymus spp., and Zataria multiflora are typical examples of rich sources of phenolic compound [Torreya nucifera inhibited the replication of SARS-CoV 3CLpro [The application of phytomedicines has increased due to their therapeutic value when compared to allopathic medicines as these bio-compounds exhibit fewer side effects . The poscompound ,82. BiflV 3CLpro .Senna L. is large genus of flowering plants in the legume family (Fabaceae) of subfamily Caesalpinioideae, comprised of 300–350 species [Senna are commonly used in foods and herbal medicine [Senna alata plant is currently being used in the treatment of flu, fever, malaria, and large number of other medical conditions due to the presence of bioactive compounds in the plant, including quinones, alkaloids, and terpenes [Senna alata considerably inhibited 3D7 strain of the Plasmodium falciparum parasite in vitro [Senna leaves in 500 mL water for 10 min provided relief from virus symptoms in COVID-19 patients (unpublished data). After boiling, it is recommended that half of the aqueous leaf extract can be used immediately while the second half can be used after 8–10 h to avoid toxicity. In the intervening time, water taken frequently can avoid toxicity. In order to avoid the bitter taste of aqueous leaf extract, honey can be added, which has also an antimicrobial effect. As Senna alata plants are currently being used to treat different medical conditions in different parts of world, including Africa and Asia, there is a need to confirm Senna leaf aqueous extracts in COVID-19 patients as a herbal therapy.  species . It is amedicine . In diffterpenes ,88,89,90in vitro ,88. ReceLianhuaqingwen (LH), a Traditional Chinese Medicine formula composed of a combination of 13 herbs was shown to suppress SARS-CoV-2 replication, reduced pro-inflammatory cytokine production, and changed the morphology of SARS-CoV-2 cells . These hRadix astragali (Huangqi) and Glycyrrhizae radix Et Rhizoma (Gancao) [Toona sinensis Roem, a popular Chinese vegetable that is already used safely. In vitro studies show that the tender leaf of Toona sinensis Roem, can inhibit SARS-CoV [Chinese health authorities in 23 out of 31 provinces have issued herbal remediy programs to prevent the spread of COVID-19. The top two herbal formulas used were (Gancao) . AnotherSARS-CoV . It is eAs mentioned above, licorice root has some bioactive properties, including antitumoral, antiinflammatory, and antiviral effects on health. Glycyrrhizic acid  and its aglycone glycyrrhetinic acid (GLA) are active against a broad spectrum of viruses, including herpes viruses, flaviviruses, Hepatitis C virus, human immunodeficiency virus, and SARS coronavirus (SARS-CoV) in vitro. Virus inhibition has been demonstrated in Vero cells and in patients ,94,95,96Rhizoma cibotii (gǒu jǐ), Gentianae radix (lóng dǎn), Dioscoreae rhizoma (shān yào), Cassiae semen (jué míng zǐ), and Loranthi ramus (sāng jì shēng); all of them inhibited SARS-CoV replication, and two inhibited virus protease activity.Earlier studies using herbs to block SARS could also successfully inhibit SARS-CoV2 infection. For example, Wen et al., 2011  examinedEcklonia cava found that several of these bioactive compounds were able to inhibit SARS-CoV activity by functioning as protease inhibitors. One of these, dieckol, had the most potent antiviral activity; this took place through competitive binding at the catalytic site of the protease [Studies using phlorotannins isolated from the edible brown algae protease . It is fPlants are being used for the production of recombinant vaccines and drugs for more than 30 years and the whole process is described under term ‘molecular farming’ ,100. SecVaccines generated in plants have been shown to elicit a robust immune response in humans and animals . Plants Agrobacterium, a common soil bacterium that is taken up by plants [Nicotiana benthamiana, a close relative of the tobacco plant, to produce VLPs of the SARS-CoV2 virus (COVID-19: Medicago’s Development Programs). The VLPs are similar in size and shape to actual coronavirus but are lacking in nucleic acid and are, thus, noninfectious. Medicago has successfully completed its Phase 1 clinical trials and is currently working on Phase 2 clinical trials [Medicago, a biopharmaceutical company based in Canada, has successfully developed a virus-like particle (VLP) of the coronavirus 20 days after obtaining the SARS-CoV-2 genetic sequence. Instead of using egg-based methods to develop vaccines, this technology inserts a genetic sequence encoding the spike protein of COVID-19 into y plants . The resl trials . Previoul trials . The cosl trials .In Canada, the University of Western Ontario and Suncor are developing diagnostic test kits for COVID-19 using algae as a production factory to make the viral spike proteins . Algae hNicotiana benthamiana plants for production. The vaccine has elicited a positive immune response by pre-clinical testing and will be onto Phase 1 human clinical trials soon [British American Tobacco, through its biotech subsidiary in the US, Kentucky BioProcessing (KBP), is developing a potential vaccine for COVID-19 and is currently undergoing pre-clinical testing . Expertsals soon . BAT couals soon . South African company Cape Bio Pharms (CBP) is also responding to the SARS-CoV-2 pandemic through the production of reagents in plants, which could be used for diagnostic kits . CBP is Another example of a plant molecular pharmed solution to COVID-19 is taking place in the department of nanoengineering at the University of California, San Diego. Researchers in Nicole Steinmetz’ lab have been using Cowpea mosaic virus like particles, with B- and T-cell epitopes from the S protein of SARS-CoV-2 displayed on their icosahedral surfaces . The recThe Steinmetz research group have recently developed positive control probes, composed of Cowpea mosaic virus-like particles, to be used as COVID-19 diagnostics and improve the accuracy of COVID-19 tests. The researchers hope that these positive controls, which are stable at room temperature for prolonged periods of time and cheap to generate, could be useful in resource-poor settings . Another collaboration between two research groups in Toronto, Canada, has brought about a novel way to fight COVID-19 using a synthetic peptide that binds to the viral deubiquitinase (DUB) and is carried by a plant virus. The work initially began by examining the role of the virus protease, located in ORF 1a of the coronavirus genome responsible for the related MERS virus. This protease contains a deubiquitinase activity as a means of protecting the virus from the innate immune system of the cell. Ubiquitin is a protein found in eukaryotic cells that play an important role in the regulation of proteins. It labels unwanted proteins  to be degraded by the proteasome into shorter fragments or amino acids that can be recycled for cellular metabolism. Some viruses, such as coronavirus, express deubiquitinases (DUBs) to prevent destruction by the cell. A synthetic peptide of approximately 80 amino acids and known as a ubiquitin variant (UbV) was created by phage display library design and shown to bind tightly to MERS DUB at its ubiquitin binding site, thus blocking its deubiquitinase activity as well as its proteolytic activity . This sySenna leaf extracts are the best available against COVID-19, until we have vaccine candidates in hand which have successfully undergone laboratory experiments, animal trials, and all phases (1–3) of clinical trials. Possible targets including the spike, nucleocapsid, membrane, envelope, viral RNA polymerase, and 3-chymotrypsin-like protease (3CLpro), which cleaves the virus polyprotein at 11 distinct sites to generate various non-structural proteins that are important for viral replication, are all being used to develop potential vaccines and antiviral drugs. Virus like particles (VLPs) of SARS-CoV-2 may act as promising vaccines because they have the potential to activate the human immune response in a fashion similar to the original virus.The COVID-19 pandemic is a challenge for us all. As a result of the current COVID-19 pandemic, therapies such as Remdesivir, convalescent plasma (CP), and There is need to explore plant-based systems to check whether VLPs with retained structure and in sufficient quantity can be generated in these systems . On the


Geminiviridae) can cause severe tomato yield losses in the neotropics. Here, next-generation sequencing was employed for large-scale assessment of single-stranded (ss)DNA virus diversity in tomatoes either harboring or lacking the large-spectrum begomovirus tolerance Ty-1 gene. Individual leaf samples exhibiting begomovirus-like symptoms (n = 107) were field-collected, circular DNA-enriched, subdivided into pools (with and without Ty-1), and Illumina-sequenced. Virus-specific PCR and Sanger dideoxy sequencing validations confirmed 15 distinct ssDNA virus/subviral agents (occurring mainly in mixed infections), which highlight the potential drawbacks of employing virus-specific resistance in tomato breeding. More viruses (14 versus 6 species) were observed in tomatoes without the Ty-1 gene. A gemycircularvirus (Genomoviridae), a new alpha-satellite, and two novel Begomovirus species were identified exclusively in samples without the Ty-1 gene. A novel begomovirus was found only in the Ty-1 pool, being the only species associated with severe symptoms in Ty-1 plants in our survey. Our work is the first step towards the elucidation of the potential begomovirus adaptation to Ty-1 and its specific filtering effects on a subset of ssDNA viral/subviral agents.A complex of begomoviruses ( Geminiviridae is the largest family of plant-infecting viruses, and is currently organized into nine genera: Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, and Turncurtovirus [-enveloped, circular, single-stranded DNA (ssDNA) genomes with sizes ranging from 2.0 to 2.4 kb were created and named as Pleolipoviridae and Genomoviridae. The Genomoviridae family also comprises nine genera: Gemycircularvirus, Gemyduguivirus, Gemygorvirus, Gemykibivirus, Gemykolovirus, Gemykrogvirus, Gemykroznavirus, Gemytondvirus, and Gemyvongvirus [rtovirus . The clartovirus ,2,3. In ongvirus .Begomovirus is composed of whitefly-transmitted species with one (=monopartite) or two (=bipartite) circular, ssDNA genomic component(s) with ≈2.6 kb that are encapsidated separately into twinned particles formed by two incomplete icosahedrons [Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species complex [The genus ahedrons . The beg complex . The beg complex ,8.Solanum lycopersicum L.) crop is grown year-round across major tropical and subtropical regions [Begomovirus species in tomatoes became more intensively reported after the invasion of B. tabaci Middle East–Asia Minor 1 (MEAM 1 = biotype B) in the early 1990s [B. tabaci MEAM 1  facilitated the rapid dispersal of tomato-infecting begomoviruses across all major producing areas of the country [Begomovirus species (composed mainly by bipartite viruses), occurring in all Brazilian biomes. Currently, over 21 tomato-infecting Begomovirus species have been characterized in Brazil (Sida mottle virus (SiMoV) and Sida micrantha mosaic virus (SiMMV) [Tomato severe rugose virus  and Tomato mottle leaf curl virus  are the most widespread and economically important begomoviruses, with occurrence reported across all major tomato-producing regions, including central Brazil. The remaining viral species have an overall more restricted (sometimes endemic) geographic distribution [The tomato  ,13. Currribution .Solanum (section Lycopersicon) germplasm: Ty-1 [Ty-1 gene/locus introgressed from Solanum chilense LA 1969  is by far the most employed genetic factor in tomato breeding programs across the globe. In Brazil, cultivars carrying the Ty-1 gene have been widely used, mainly across producing regions in central Brazil [Ty-1 gene is located on chromosome 6 in a genomic region in repulsion phase linkage with resistance genes against other pathogens, including the Mi-1.2 gene that confers resistance in tomato to the three most important root-knot nematode species: Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne arenaria [Ty-1 gene/locus in tomato cultivars are now available [The preferential strategy for begomovirus management in tomatoes is the employment of cultivars with genetic resistance/tolerance, since the use of insecticides for controlling viruliferous vector populations is neither efficient nor economically and environmentally sustainable ,16. Currsm: Ty-1 , Ty-2 [1sm: Ty-1 , Ty-3 [1sm: Ty-1 , Ty-4 [2sm: Ty-1 , ty-5 [2sm: Ty-1 , Ty-6 [2sm: Ty-1 , tcm-1 [sm: Ty-1 , and tgrsm: Ty-1 . The Ty-l Brazil ,26. The arenaria ,28. Molevailable ,30,31.Ty-1 gene is best described as a tolerance response [Ty-1 gene encodes an RNA-dependent RNA polymerase. Therefore, the Ty-1 gene is representing an entirely new class of disease resistance/tolerance genes that operates by intensifying the levels of transcriptional silencing of viral genes. More recent studies have shown that the Ty-1 gene can also confer resistance to Beet curly top virus  in genetically transformed Nicotiana benthamiana plants [Ty-1 gene on ssDNA viruses and subviral agents described in association with tomatoes in neotropical areas.The phenotypic expression of the response , since presponse . This toresponse ,33. Genea plants . HoweverNext-generation sequencing (NGS) technologies have intensified the advances in elucidating many aspects of plant–microbe interactions by enabling the generation of a huge amount of low-cost sequence data of both hosts and pathogens . CurrentTy-1 gene may represent a relevant selection factor on viral populations that could make them either more adapted or even capable of entirely overcoming this tolerance factor [Ty-1 gene and other tomato resistance/tolerance factors have not yet been extensively studied. The complete sequence information of the DNA-A and DNA-B genomic segments generated by NGS provides large-scale assessment tools to study viral population diversity in the tomato–begomovirus pathosystem. In this context, the objective of the present work was to carry out metagenomic analyses aiming to reveal the diversity of Begomovirus species as well as other ssDNA viruses and subviral agents in tomato cultivars either lacking or harboring the Ty-1 gene in central Brazil.Due to the extreme variability of the neotropical tomato-infecting begomoviruses, it is possible that species and strains not yet identified can be emerging in this region. The increase in the crop acreage with tomato varieties and hybrids harboring the e factor ,26. Howen = 107) of field-grown tomato cultivars/hybrids (with and without the Ty-1 tolerance gene) showing distinct degrees of begomovirus-like symptoms  were collected from 2001 to 2016 across three geographic regions . In our survey, the majority (over 95%) of the samples collected from plants displaying begomovirus-like symptoms (in both pools) were confirmed to be infected by one or more begomovirus species  from the susceptible recessive allele (ty-1) after cleavage with the restriction enzyme Taq I [Ty-1 gene/locus, PCR products (amplicons) were cleaved with the enzyme Taq I for 2 h at a constant temperature of 65 °C. The products obtained after cleavage were analyzed in 1% agarose gels, stained in ethidium bromide, and visualized under ultraviolet light. In order to confirm the presence of the c region . This come Taq I . In ordeEach individual sample was subjected to total DNA extraction using a modified (high pH buffer) 2X CTAB + organic solvent protocol . These s-derived circular DNA molecules in the samples were selectively enriched by rolling circle amplification (RCA) assays [-characterized samples were then subdivided into two pools: one composed of DNAs of tomato plants without the Ty-1 (n = 64) and one composed of DNAs of tomato samples with the Ty-1 (n = 43).The virus) assays . After gthe Ty-1  gene (n the Ty-1  gene (n https://www.ncbi.nlm.nih.gov/). The viral contigs were annotated and the trimmed reads were mapped back to the annotated genome using the tool “Map to reference” available in the Geneious 11.0 program [The sample pools (with and without Ty-1 gene) were subjected to high-performance sequencing in an Illumina platform with the HiSeq 2500 system . The analyses were performed in paired ends reads of 100 nucleotides in length. The number of raw reads (filtered reads in parentheses) obtained for each pool were 16,227.547  and 16,345.987  for resistant and susceptible pools, respectively. The NGS-derived sequences were analyzed according to the following workflow: (1) elimination of low-quality reads, (2) assembly of the sequences using the program CLC Genomics Workbench 10, and (3) validation of the contigs via BLASTx and BLASTn algorithms by comparing with the ssDNA virus database of GenBank  were designed in opposite and overlapping directions. Primer design was carried out on the basis of the consensus contigs obtained with the Geneious 11.0 program . Virus sTaq polymerase 10× buffer, 50 mM of MgCl2, 2.5 mM of dNTPs, 10 µM of each primer (forward and reverse), 100 ng of DNA, 8.0 µL of Milli-Q water, and 0.5 U Taq DNA polymerase. The reactions were amplified in a thermal cycler  programmed for 35 cycles with the following conditions: initial denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing , aiming to verify the sample-associated viral species. We adopted the current pairwise identities of 91% and 94% as the demarcation thresholds to identify Begomovirus species and strains, respectively [Direct Sanger dideoxy sequencing reactions of positive virus-derived amplicons were carried out to double-check the viral diversity observed in a subset of individual samples. Sequencing reactions were performed at the Genomic Analysis Laboratory (at CNPH), employing the same virus-specific primer pairs  in one Aectively .Ty-1 gene is presented in Ty-1 gene is presented in Ty-1 gene and 7045 contigs in the sample pool with the Ty-1 gene. ToSRV and Tomato rugose mosaic virus (ToRMV) displayed the two highest numbers of reads, indicating their relative predominance in the tomato samples with the Ty-1 gene. Ten begomoviruses were found in the pool without the Ty-1 gene. Interestingly, only the DNA-A component was recovered from well-characterized bipartite species, including Bean golden mosaic virus (BGMV), Tomato common mosaic virus (ToCmMV), and SiMMV. In the pool harboring the Ty-1 gene, the complete genomes of four previously described bipartite Begomovirus species were recovered viz. ToSRV, Tomato chlorotic mottle virus (ToCMoV), Tomato golden vein virus (TGVV), and ToRMV. The monopartite species ToMoLCV was also recovered in both pools. Some of the neotropical tomato-infecting Begomovirus species (included on the RefSeq database) displayed overall high identity levels . The NGS sequences were then realigned against the viral genomes found using SeqMan NGene (99% stringency parameter) in order to minimize assembling artifacts. This allowed a more precise evaluation of the representation of each virus and subviral agents in the sequenced pools. The validation of the NGS results via PCR assays with virus-specific primers coupled with Sanger dideoxy sequencing was also a very important tool to verify the presence of each individual virus species described here.The assembled contig sequences of both pools that were characterized here and their corresponding GenBank accessions are presented in Begomovirus species (one in the pool with the Ty-1 gene and two species in the pool without the Ty-1 gene). In the pool of samples with the Ty-1 gene, a putative new virus (named here as species #1 = isolate DF-640) displayed a bipartite genome organization, having a DNA-A component with 2605 nts and a DNA-B component with 2625 nts. The putative new species #1 displayed the highest level of identity (85%) with Tomato rugose yellow leaf curl virus (TRYLCV) isolates. The isolate DF-640 was recovered from a field-grown tomato plant in the vicinities of Gama city , with severe symptoms, indicating a putative increase in virulence in relation to the Ty-1 gene. However, we cannot exclude that these severe symptoms might have been induced by synergisms involving DF-640 and RNA viruses (since they were not investigated in the present study). Two putative new species were detected in the pool lacking the Ty-1 gene. The first one was tentatively named here as new species #2 (=isolate MG-378) and displayed only the DNA-A component with 2649 nts. Tomato bright yellow mottle virus (ToBYMoV) was the begomovirus with the highest identity level (84%) to the new species #2. Additional PCR assays were carried out using the isolate MG-378 as a template, but no amplicon for the putative cognate DNA-B component was recovered (data not shown), indicating that it is more likely a monopartite virus. The new species #3 is also more likely a monopartite begomovirus, having a DNA-A genome with 2636 nts. The new species #3 displayed the highest identity level (84%) to Abutilon mosaic Brazil virus (AbMV). In addition, a four genetically identical isolates of a novel alpha-satellite species and two isolates of a gemycircularvirus (Family: Genomoviridae) species were also detected exclusively in the pool of tomato samples without the Ty-1 gene . The simultaneous presence of distinct virus species detected in single plants ranged from two to up to five .Over 286 viral species have been reported to infect tomatoes in Brazil and worldwide ,49,50,51 to five . HoweverBegomovirus species already reported in the neotropics, we were able to detect two putative new Begomovirus species in the samples without the Ty-1 and one novel Begomovirus species in a sample with the Ty-1 gene. The putative new species #1 (DF-640) displays all typical features of the New World bipartite begomoviruses, having a DNA-A with a size of 2605 nts and a DNA-B component with 2625 nts. The new species #1 was the only begomovirus found to be associated with severe (dwarf) symptoms in plants harboring the Ty-1 gene in our survey. The new species #1 displayed the highest identity level (85%) with Tomato rugose yellow leaf curl virus (TRYLCV). Only the DNA-A components were found in the putative new species #2 (=isolate MG-378) and in the new species #3 (=isolate GO-169), suggesting that both might be novel monopartite viruses. The new species #2 (2649 nts) displayed the highest identity (84%) with the Tomato bright yellow mottle virus (ToBYMoV), and the new species #3 (2636 nts) displayed the highest identity level (84%) with Abutilon mosaic Brazil virus (AbMBV). According to the current criteria for species demarcation in the genus Begomovirus, nucleotide identities of the DNA-A component that are less than 91% with the complete DNA-A genome of any other known begomovirus sequence will correspond to a new species [In our study, in addition to the detection of Ty-1 gene. Alpha-satellite DNA molecules are subviral agents classified in the family Alphasatellitidae that have been found in association with Begomovirus [Geminialphasatellitinae, viz. Ageyesisatellite, Clecrusatellite, Colecusatellite, and Gosmusatellite. Nucleotide identity less than 88%  is the criterion currently used for the classification of a new species within the family Geminialphasatellitinae [Clecrusatellite, which is composed of species found in association with bipartite Begomovirus from the New World [Begomovirus species is able to transreplicate this novel alpha-satellite.A single alpha-satellite isolate with 1321 nts was detected in four individual samples collected in distinct areas of the Federal District in plants lacking the omovirus . The genllitinae . The alpllitinae ,55. Thusew World ,55. ThisGemycircularvirus (family Genomoviridae), was also detected in two tomato samples from the pool without the Ty-1 gene. Both isolates were collected in Leopoldo de Bulhões, Goiás (GO) State in 2004. These isolates displayed 98% identity to Capybara genomovirus 9 isolate cap1_561 (MK483081.1) from Brazil [Momorcadia charantia and Euphorbia heterophylla [A plant-associated genomovirus 12, classified into the genus m Brazil ,58,59. Irophylla . HoweverIn the present study, the complete DNA-A sequences of the begomoviruses BGMV, SiMMV, and ToCmMV were detected and subsequently confirmed in the individual samples via PCR assays and Sanger sequencing. BGMV was found in two samples collected in the Gama (DF) region in 2003 (isolates DF-045 and DF-046) and in one sample collected in Leopoldo de Bulhões (GO) (isolate GO-142). In fact, BGMV has been previously found in association with tomatoes in the Submédio São Francisco River valley in northeast Brazil . HoweverTy-1 gene  and in the Federal District (three samples). SiMMV was already reported infecting tomatoes in Brazil . All SiMy-1 gene , suggestTy-1 gene (GO-023 and MG-388) collected in Luziânia (GO) and Viçosa (MG), respectively. The isolate GO-023 is a mixed infection with ToCMoV, and the isolate MG-388 is a mixed infection with ToSRV. The absence of the DNA-B component of ToCmMV also suggests that these isolates might be using this component of these co-infecting species. This hypothesis remains to be investigated. ToCmMV was initially reported infecting tomato plants in southeast Brazil [Ty-1 gene, there are reports indicating that this virus can replicate and cause mild symptoms in tomato plants carrying this tolerance factor (manuscript in preparation).Somewhat surprising, only the DNA-A component of the bipartite species ToCmMV was detected in two samples of the pool without the t Brazil ,63. HoweEuphorbia yellow mosaic virus (EuYMV) and Cleome leaf crumple virus (CILCrV) were also recovered in our NGS analyses only from samples without the Ty-1 gene. EuYMV and CILCrV were detected in samples from Minas Gerais State. EuYMV was first characterized as infecting the weed E. heterophylla [Cleome affinis [The DNA-A and DNA-B genome sequences of rophylla , and CIL affinis . HoweverBegomovirus species in distinct geographic areas across central Brazil. ToSRV has been described as the predominant begomovirus species, as indicated by independent surveys carried out across all tomato-producing regions in Brazil [Ty-1 gene and in 26 samples in the pool harboring the Ty-1 gene. This ability of ToSRV isolates replicate in plants with the Ty-1 gene could also be considered as an additional factor that explains the overall predominance of this virus under Brazilian conditions.We found that the NGS analyses in combination with PCR assays with virus-specific primers and Sanger sequencing to be powerful tools that allow us to assess the relative prevalence of the predominant n Brazil ,65,66. ITomato chlorotic mottle virus (ToCMoV) has already been reported across the northeast, southeast, and central Brazil [Tomato golden vein virus (TGVV) is commonly found in central Brazil [Tomato rugose mosaic virus (ToRMV) is a recombinant viral species with genomic contributions of ToSRV and ToCMoV [ToSRV is the prevalent begomovirus in the central and meridional regions of Brazil ,67, wherl Brazil . Howeverl Brazil , and ourd ToCMoV . In accod ToCMoV .Ty-1 gene on the diversity of neotropical Begomovirus species. The majority of the samples obtained from tomato plants carrying the Ty-1 gene displayed mild symptoms restricted to the apical leaves. It was possible to observe putative filtering effects as well as gene-specific viral selection in samples with the Ty-1 gene, indicating a potential evolution of viral populations more adapted to this genetic factor. It would be interesting to know if the viruses detected in the apical mild symptoms in plants carrying the Ty-1 gene are indeed able to escape its effects or if the occurrence of multiple infections on these plants makes a more permissive cellular environment. However, an illustrative example is the isolate DF-640 that was the only one found in association with severe disease symptoms in a field-grown Ty-1 tomato plant. This strong susceptible-like reaction associated with the isolate DF-640 may indicate its potential ability to overcome the Ty-1 gene. Another possibility is that the isolate DF-640 may represent a singular “host switch” event that is not necessary associated with viral adaptation to the Ty-1 gene. The production of infectious DF-640 clones is now underway, and they will be used to verify this hypothesis. Nevertheless, it is well documented that the increase in the acreage of cultivars harboring resistance genes such as Ty-1 can result in strong selection forces towards more aggressive viral isolates, accelerating the change in the composition of the viral population and potentially culminating with the loss of effectivity of the source of resistance/tolerance [Ty-2 gene caused by a strain of Tomato yellow leaf curl virus (TYLCV) [Tomato leaf curl Bangalore virus (ToLCBV) in India [The present work is the first exploratory study on the potential impact of the olerance ,73,74,75 (TYLCV)  and by ain India .Ty-1 gene. Thus, another plausible explanation for some of the reported field events of Ty-1-mediated resistance/tolerance “breakdown” under Brazilian conditions could be related to some natural synergistic interactions with distinct group of viruses. In fact, it has been demonstrated that the Ty-1 gene does not confer resistance to major tomato-infecting RNA viruses such as Tomato spotted wilt virus (TSWV) and Cucumber mosaic virus (CMV) [Our preliminary set of analyses showed no unique  polymorphisms (data not shown). Several point mutations were found, but none of them were specific to the viruses present in the pool with or without the us (CMV) . Howeverus (CMV) ,79,80. ITy-1. However, in order to carry out a more precise study on the selective impact of the Ty-1 locus on begomovirus diversity and evolution, a distinct experimental strategy would probably be more appropriate, since our analysis was conducted on samples collected in different regions of a large country, in different years and from tomato plants grown in different microenvironmental situations. Therefore, it is possible that these variables  can generate some biases that may not allow us to estimate the actual effect of the Ty-1 gene. For this purpose, the analysis could be more appropriately conducted on samples collected from experimental plots cultivated with tomato isolines with and without the Ty-1 gene. On the other side, our ecologically oriented approach allowed us to carry out a more ample exploration of an array of environments, which may enhance the opportunity to detect a larger number of yet undescribed viral species associated with the tomato crop. Even though with a slightly different number of evaluated samples in the pools with (n = 43) and without (n = 64) the Ty-1 gene, virus-specific PCR assays and Sanger sequencing validations of NGS-derived data indicated greater diversity (14 versus 6 species) in samples lacking this gene. Moreover, two novel Begomovirus species, one gemycircularvirus (Genomoviridae) and one alpha-satellite were identified exclusively in samples without Ty-1, whereas a novel begomovirus was found exclusively in the Ty-1 gene pool. These results indicated a potential viral adaptation to this tolerance factor, as well as virus-specific filtering effects of the Ty-1 on a subset of single-stranded DNA viruses and subviral agents. However, these hypotheses will be better tested with tomato isolines (with and without the Ty-1 gene) after controlled experiments employing infectious clones.The results reported here provide useful information about the population dynamics of begomoviruses associated with tomato crops across three major tomato-producing regions of central Brazil in the last decade. Our work is in agreement with the notion that NGS is a powerful strategy to identify not only known viruses but also novel viruses and subviral agents. The present work is the first step towards the elucidation of the potential begomovirus adaptation to the tolerance factor


Phaseolus vulgaris–tobacco necrosis virus (TNV), we demonstrated that PD-L1 and PD-L4, type-1 ribosome inactivating proteins (RIPs) from leaves of Phytolacca dioica L., possess a strong antiviral activity. This activity was exerted both when the RIPs and the virus were inoculated together in the same leaf and when they were inoculated or applied separately in the adaxial and abaxial leaf surfaces. This suggests that virus inhibition would mainly occur inside plant cells at the onset of infection. Histochemical studies showed that both PD-L1 and PD-L4 were not able to induce oxidative burst and cell death in treated leaves, which were instead elicited by inoculation of the virus alone. Furthermore, when RIPs and TNV were inoculated together, no sign of H2O2 deposits and cell death were detectable, indicating that the virus could have been inactivated in a very early stage of infection, before the elicitation of a hypersensitivity reaction. In conclusion, the strong antiviral activity is likely exerted inside host cells as soon the virus disassembles to start translation of the viral genome. This activity is likely directed towards both viral and ribosomal RNA, explaining the almost complete abolition of infection when virus and RIP enter together into the cells.Using the pathosystem  Their mop (SRL) ,6).Structurally, RIPs are divided into two groups considering the presence or absence of a quaternary structure. Classically, these enzymes are categorized in monomeric RIPs (type 1) and dimeric RIPs (type 2). Type-1 RIPs consist in a single polypeptide chain with toxic N-glycosylase activity, while type 2 RIPs are constituted by a polypeptide chain exhibiting N-glycosylase activity (A-chain) linked to a lectin chain (B-chain) through a disulphide bond able to recognize carbohydrate  moieties of mammalian cell surface . Alterna50 0.0003–1.7 nM on Hela cell lines) with respect to type 1 (IC50 170–3300 nM on Hela cell lines), while in acellular systems the toxicity of the two groups is comparable [Sambucus [Type 2 RIPs are more toxic in cellular systems (ICmparable . In addiSambucus  or proteSambucus ) were alAt the cellular level, the inhibition of protein synthesis by RIPs promotes cell death by apoptosis pathway considering that many studies report the activation of various caspases, caspase-like and serine proteases and poly(ADP-ribose) cleavage ,14. MoreRIPs have also been used in agriculture to protect crops from diseases caused by viruses and fungi, and from insect pests. Genes encoding for PAP, trichosanthin, and maize RIP are the most commonly used, while the main noted host plants are tobacco, potato, and tomato. Thus, transgenic plants carrying genes of some RIPs have been obtained with different degrees of resistance to viruses, fungi, and insects .Despite the large number of works on RIPs field, the biological role of RIPs in plants has not been completely unveiled; although all researchers agree that RIPs are involved in plants defense or physiology ,21. IndePhytolacca americana L., was isolated given its pronounced antiviral activity [Phytolacca dodecandra L’Herit [Phytolacca heterotepala H. Walter [Phytolacca insularis [Phytolacca dioica L. [P. dioica plant, three , fouPD-S1-3; ,34), andPD-S1-3; ,36,37) tPD-S1-3; ,39 have P. dioica very few information on their antiviral activity are available with respect to type-1 RIPs from P. americana. Therefore, in this work we report the antiviral activity of both PD-L1 and PD-L4, the major isoforms of type-1 RIPs expressed in leaves of P. dioica adult plant, by using the pathosystem tobacco necrosis virus (TNV)-Phaseolus vulgaris L. TNV is a member of the genus Necrovirus in the family Tombusviridae with unsegmented and uncapped TNV genome consisting of a single stranded linear positive sense RNA of 3.8 kb that lacks a poly A tail and replicates itself with the aid of its own RNA-dependent RNA polymerase [Despite the numerous type-1 RIPs isolated and studied from leaves of lymerase . In beanlymerase . These llymerase ,42.Phytolaccaceae family.In this framework, the new information emerged by this work can be useful for deepening the knowledge on type-1 RIPs in P. dioica as described previously using a general protocol for the preparation of basic proteins [Native PD-L1 and PD-L4 were purified from fully expanded leaves of proteins ,35. Fromproteins .Homogenous preparations of both PD-L1 and PD-L4, verified by SDS-PAGE  when applied together with the virus suspension in the same bean leaf surface, as assessed by the reduction of lesion number with respect to bean plants inoculated only with the virus suspension in water . HoweverTo verify if this inhibition was due to a direct effect of the RIPs on viral RNA and not to ribosomes inactivation, TNV was inoculated separately from RIPs. For this purpose, the virus suspension was rubbed either on the adaxial or abaxial leaf surface and PD-L4 on the opposite leaf surface. In these experiments, only PD-L4 was utilized being the most effective at the lowest concentration of 2 µg/mL. Results showed that the inhibitory activity of this RIP against TNV was partially weakened, but still high, with a reduction of lesion number over 70% with respect to controls . Though To shed further light on the antiviral activity of these RIPs, bean leaves only treated with PD-L1 or PD-L4 at 2 µg/mL or 10 µg/mL were stained with Evans blue for the detection of cell death at 24, 48, and 72 h after treatment. Dead cells were detected only occasionally after PD-L1 and PD-L4 treatments at both concentrations and at any tested time point a–c sugge2O2 deposits were present in the tissues, except for veins that were stained in brown because they normally contain H2O2 for cell wall lignification (2O2 deposition was instead elicited by TNV infection due to the hypersensitive reaction (HR) caused by the virus on the onset of infection, when lesions were not yet visible at naked eyes  staining of bean leaf disks 48 h after treatment with PD-L1 2 µg/mL or PD-L4 2 µg/mL showed that no Hfication a–c. Thisked eyes d.Leaf inoculated simultaneously with a mixture of RIP and TNV showed no DAB staining except for the veins e; arrow,2O2 deposition in The above microscopic observations confirm that the strongest antiviral activity is exerted when virus and RIP are inoculated together into the leaves, reducing almost completely cell damages. Considering that RIPs should not have access to viral RNA in assembled virus particles , it is lP. dioica are extensively characterized from a structural and enzymatic point of view .Type-1 RIPs from mary see ). Vice vigitatum . Thus inP. vulgaris leaves in the same leaf surface compared to leaves inoculated with the virus alone. In particular, the decrease in lesions is particularly evident in the presence of PD-L4 with respect to PD-L1. The different action of the two toxins could justify their different expression, considering that PD-L4 is more expressed in the leaves growing during spring and summer and in minor amount in the autumn and winter [Data show that both toxins have the ability to reduce the number of lesions when applied on d winter . On the d winter  shows a d winter .Moreover, a decrease in antiviral activity for PD-L4 occurred when the virus suspension was inoculated either on the adaxial or abaxial leaf surface and PD-L4 on the opposite leaf surface.Therefore, it is likely that PD-Ls antiviral activity is fully expressed only when both RIPs and virus are present in the same cells, whether they had entered together or separately. At this regard, the absence of dead cells in leaves only treated with PD-L1 and PD-L4 suggests that these proteins cannot be internalized, at least in a sufficient amount to cause cell death. It is more likely that they enter easily in those cells already damaged by the virus, thus accelerating their death and hampering virus spreading. Whether these RIPs, once present in the virus infected cells, inhibit viral RNA translation and replication by depurination of host ribosomal RNA, or directly depurinate viral RNA, as recently demonstrate for PAP , it is nMaterials for chromatography were described elsewhere ,46. All Phytolacca dioica, growing in the Botanical Garden of the University of Naples “Federico II” . Phaseolus vulgaris plants, cv. Borlotto Nano Lingua di Fuoco (BLF) were grown in a greenhouse at 24 ± 2 °C, RH 60 ± 5%, 16 h/8 h light/dark period and used when primary leaves were completely expanded.Type-1 RIPs, namely PD-L1 and PD-L4 were from fully expanded leaves, collected from a single female adult tree plant of PD-L1 and PD-L4 were purified according to the procedure previously reported . BrieflyGeneral methodology using for analytical biochemical characterization (SDS-PAGE and protein concentration by bicinchoninic acid (BCA) assay) are reported in detail in previously publisher paper . As moleTM SDS 14–200 kit in a 47 cm capillary, monitoring at 214 nm following manufacturer’s instructions , Italy).Capillary Electrophoresis (CE) in sodium dodecyl sulphate (SDS) was carried out on a Beckman P/ACE System 5550, using the eCAPNicotiana benthamiana plants, previously inoculated with a TNV-D strain gently supplied by Institute for Sustainable Plant Protection (IPSP), Turin, Italy, were used for virus purification. Frozen material was ground in cold 0.1 M ammonium citrate buffer (1:3 w/v), filtered, clarified by differential centrifugation as described by [g for 40 min. A light scattering virus band was recovered from the gradients and concentrated by centrifugation at 150,000× g for 4 h. Finally, virus sediment was resuspended in distilled water at the final concentration of 40 µg/mL and stored at −80 °C until utilization.Symptomatic ribed by  and furtPlants were inoculated on the first developed leaf by rubbing with 1 mL/leaf mixture of PD-L1 or PD-L4 (2 µg/mL or 10 µg/mL) and purified virus (1:1), using a 600 mesh carborundum as an abrasive. The final virus concentration in the inoculum was of 100 ng/mL. In some other experiments, PD-L4 and virus were inoculated separately on the abaxial and adaxial surface of the same leaf. As controls, some plants were inoculated respectively with: (1) PD-L1; (2) PD-L4; (3) TNV; (4) water.Plants were observed for the development of lesions for 4–5 days. When lesions were fully developed, infected leaves were detached and immediately scanned at 300 DPI resolution. Images were analyzed with Global Lab  to determine the number of lesions and PD-Ls inhibitory activity was calculated as percent reduction of this number in respect to controls.2O2 deposits, following a previously reported protocol [Five leaf disks, 1 cm in diameter, were randomly punched with a cork-bore from PD-Ls or TNV or PD-Ls + TNV inoculated bean leaves at two days post-inoculation (dpi) and stained with Evans blue, to identify dead cell in the tissues and with 3,3′- Diaminobenzidine (DAB) to localize Hprotocol . Samplesp < 0.05.For the antiviral assay on bean plants, results are expressed as mean ± standard deviation of data collected from at least three independent experiments, with 5 bean plants and 10 treated/inoculated leaves for each treatment. Data were subjected to one-way analysis of variance, and comparison among means was determined according to Fisher’s least significant difference test. Significant differences were determined at


Antimicrobial peptides (AMPs) with antiviral activity  have become a research hotspot and already show immense potential to become pharmaceutically available antiviral drugs. AVPs have exhibited huge potential in inhibiting viruses by targeting various stages of their life cycle. Insects are the most speciose group of animals that inhabit almost all ecosystems and habitats on the land and are a rich source of natural AMPs. However, insect AVP mining, functional research, and drug development are still in their infancy. This review aims to summarize the currently validated insect AVPs, explore potential new insect AVPs and to discuss their possible mechanism of synthesis and action, with a view to providing clues to unravel the mechanisms of insect antiviral immunity and to develop insect AVP-derived antiviral drugs. Drosophila melanogaster emerged as an important paradigm of genetic analysis of innate immunity. Outstanding pioneering achievements were awarded the Nobel Prize, which has since greatly stimulated interest in this field  gp83 inhibits Toll signaling through the regulation of NF-κB transcription factors  are a group of immune proteins that mainly function against bacteria and fungi , 17. A cbyx mori –20. Howeosophila , 22. Int B. mori , 24. On  factors . The imm factors . Because factors . AMPs an factors . InsectsHerein, we will summarize the AMPs with antiviral activity reported in the database and literature and we will predict the antiviral activity of insect AMPs through AVP prediction software. This article aims to compile relevant information from insect AVPs as important components of insect antiviral innate immunity and to inspire the development of effective antiviral drugs.AVPs are considered as a subset of AMPs which act as the first line of defense in many organisms as an innate immune response to viral infection. Compared to a hot field such as the development of antiviral and antitumor drugs in human medicine, the concept of AVP has not appeared often in the field of insect research, although the idea appeared more than 10 years ago , 29. WitB. mori, Galleria mellonella, Aedes aegypti, Pachycondyla goeldii (Ponerine ant), Manduca sexta, D. melanogaster, Danaus plexippus, Anopheles gambiae, Apis mellifera and others , herpes simplex virus 1 and 2  and against the arenavirus Junin virus (JV)  , 40.A. mellifera (Picornaviridae), Influenza A viruses (Orthomyxoviridae), Respiratory Syncytial Virus , Vesicular Stomatitis Virus  and the plant virus tobacco mosaic virus   . More inviridae) .Calliphora vicina, showed antiviral activity against influenza virus A and influenza virus B (Herpesviridae) and analogs were active against coxsackievirus in vitro using cell lines  of Autographa californica multiple nucleopolyhedrovirus   . The antviridae) .Drosophila AMP coding genes, diptericin B (dptB) and attacin C (attC), are upregulated in transgenic flies expressing a Sindbis virus (SINV) replicon. Silencing their expression led to a significant increase in SINV titers, suggesting that dptB and attC involved in Drosophila antiviral response to SINV  production and progeny virus virulence in vivo and in vitro  and their corresponding peptides from the NCBI database, their antiviral activity was predicted using Meta-iAVP  genes has been found to be essential for effective Drosophila Toll-mediated immune responses , Sacbrood virus (SBV), black queen cell virus (BQCV), and Israeli acute paralysis virus (IAPV) in transcriptome data (A. mellifera while little related research has been conducted on A. cerana. Recent research found that in A. cerana the predicted AVP genes abaecin and hymenoptaecin were significantly upregulated by Chinese Sacbrood virus (CSBV) infection  overexpression could activate the Imd pathway and induce AMP upregulation, enhancing silkworm antiviral resistance , but expression levels were too low to be considered as biologically important (B. mori bidensovirus (BmBDV), BmNPV and BmCPV infection  such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), NOD-like receptors (12), interferon-γ-inducible protein 16 (IFI16), AIM2 (absent in melanoma 2) and cyclic GMP-AMP synthase (cGAS) that subsequently lead to the activation of inflammatory cytokines and chemokines as well as interferon (IFN) and ISG production through a cascade reaction . HoweverB. mori cGAMP and PGRP2 were confirmed to be involved in host responses to BmNPV , Gram-negative bacteria-binding proteins (GNBPs), scavenger receptors (SRs), thioester-containing proteins (TEPs) and lectins have been identified , 108. Unto BmNPV , 109. Inimmunity , 65, 110immunity . The claimmunity . Howeverimmunity .The AVPs possess diverse structures as well as might act according to different mechanisms. Based on the antiviral peptide database AVPdb , a totalMany scientific questions about the identities of insect AVPs and their modes of action remain unresolved. Besides, viruses are the causative agents of various dreadful diseases in humans and animals. Recently, the testing and discovery of AVPs was accelerated because extraordinary advantages. Insects are considered an important source of natural AMPs, and their potential to act as AVPs is worthy of in-depth studies. In future research, the research on insect AVPs can mainly focus on the following key issues: (1) Identification of insect AVPs; (2) Recognition by PRRs and downstream cascade reactions involved in insect AVPs production; (3) Molecular mechanism of action of AVPs against insect viruses and vertebrate viruses; (4) AVP counter defense (immune escape) mechanisms by viruses; (5) Evaluation and application of insect AVPs as antiviral drugs.MF participated in the design, collected and analyzed data, and drafted the manuscript. SF and JX helped with data collection. VL, LS, and JS participated in the design and coordination of the study, and revised the manuscript. All authors read and approved the final 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.


The present report describes a case of pseudocowpox virus (PCPV) infection in a seven-year-old female bison euthanized due to a history of declining condition and sores on the vulva and udder.Parapoxvirus (PPV) particles, which were also confirmed by real-time PCR (Ct =18.6). Metagenomic analysis of the skin samples revealed only poxviruses. The bison parapox B2L envelope gene clustered with other parapox sequences identified from ruminants.External examination revealed multifocal, raised, keratinized plaques (0.5–2 cm) covering the skin of the ventral surface of the tail, perineum, caudoventral abdomen, udder, both inguinal recesses, and the medial aspects of both thighs. No significant gross lesions were present in the reminder of the tissues examined. Histopathological examination of the affected skin showed moderate epidermal hyperplasia with rete pegs, marked parakeratotic hyperkeratosis with crusts of degenerate neutrophils and cell debris, and few epithelial cells undergoing ballooning degeneration with occasional eosinophilic intracytoplasmic inclusion bodies (3–5 μm Bollinger body). Negative staining electron microscopy from skin revealed typical This is the first report of PCPV virus infection in an American bison. Identification of novel susceptible hosts of parapox viruses sheds light on the viral evolution and highlights the importance of potential economic impact of this disease to the bison industry. Parapoxvirus (PPV) within the family Poxviridae is composed of four viruses: bovine popular stomatitis virus, orf virus, pseudocoxpox virus (PCPV) and PPV of red deer in New Zealand [The genus  Zealand . PPVs ar Zealand . The com Zealand . Lesions Zealand , 5. In uPPVs are of zoonotic importance and cause cutaneous lesions in humans from direct contact with infected animals commonly cows and sheep .PPVs are epitheliotropic viruses with varying severity of infections been reported in several ungulate species such as red deer , reindeeA seven-year-old female intact American bison from the Konza Prairie Biological Station (KPBS) was presented for necropsy examination at Kansas State Veterinary Diagnostic Laboratory (KSVDL). The bison was humanely euthanized by KPBS following a clinical history of poor and declining condition prior to submission to KSVDL. Humane euthanasia was performed by KPBS using the gunshot method according to the American Veterinary Medical Association (AVMA) guidelines. During the euthanasia, multiple cutaneous nodules were noticed in the vulva and udder, and a mucus discharge was seen from the nose. A complete postmortem examination revealed lesions limited to the skin. There were multiple wart-like proliferative nodules on the ventral tail, perineum, caudoventral abdomen, udder, both inguinal recesses, and the medial aspects of both thighs, and axillary regions Fig. a and b. Multiple skin sections were fixed in 10% neutral buffered formalin immediately after necropsy, and routinely processed for histopathologic examination in an automated tissue processor and embedded in paraffin. Slide-mounted tissue sections (~ 4 um thick) were stained with hematoxylin and eosin and evaluated by a certified veterinary anatomic pathologist. Histopathology of multiple skin nodules . A representative section of the skin nodule submitted to Texas Veterinary Medical Diagnostic Laboratory (TVMDL) was positive for PPV by real-time PCR with a Ct value of 18.6, confirming strong positivity for PPV.Skin nodules were processed for visualizing viral particles by negative staining. The suspensions obtained from ground skin nodules were mixed with 2% phosphotungstic acid and nebulized onto EM grids. The negatively-stained grids examined by electron microscopy revealed characteristic parapox viral particles that were oval in shape (approx. 260 nm long and 160 nm wide) with a spirally organized surface tubules . Of the poxvirus related reads, 96% of the reads were identified as PCPV and 2% were identified as orf virus  using previously described primers and methods [B2L gene included representative sequences previously described [own PPVs , 8, 19. escribed , Maximumescribed . PhylogeBison bison) that was euthanized for declining condition and sores on the vulva and udder. Gross necropsy and histopathological examination confirmed the presence of pox viral infection. Further, real-time PCR and electron microscopy characterized the causative agent as a PPV. Phylogenetic analysis based on the B2L gene confirmed the causative agent as PCPV. The presence of PCPV and absence of other infectious agents in skin lesions was confirmed by metagenomic sequencing.The present study reports the first case of PCPV virus infection in American bison  with other PCPV of cattle origin.PCPV infections are generally reported as mild infections with only a few animals affected in a herd, and commonly seen in milking cows as skin infections of teats, udder and foot. However, an unusual presentation of pseudocowpox associated outbreak with pustular ulcerative vulvovaginits was reported in a Swedish dairy herd of approximately 80 cows. Among the affected cows, 90% of the cows had vesicles, papules and scabs affecting the vulval and vaginal mucosa . An outbs a PCPV . SimilarMetagenomic sequence analysis revealed a majority of the reads were related to viruses, of which 87% corresponded to poxviruses and 5% of the viral reads were papillomavirus related. The small percentage of reads matching papillomavirus were found to be the conserved gene sequences that are common to poxviruses and papillomaviruses .Cases of severe atypical PPV infections were previously reported in sheep from the United States . These aB2L gene, showed that the PPV outbreak of 1999–2000 was closely related to bovine PCPV whereas the PPV outbreak of 1992–93 was closely related to sheep orf virus [B2L gene of the PPV from red deer in Italy showed that the virus was closely related to New Zealand PPV of red deer [Investigating the PPV outbreaks in Finnish reindeer using phylogenetic analysis based on the rf virus . PPV outrf virus  and fromrf virus . PhylogeTransmission of poxvirus to a new species can have detrimental effects on the new population. When squirrel pox virus was transmitted from grey squirrels to red squirrels in Great Britain, it led to significant losses in the red squirrel population .Although the bison PPV from the current study is closely related to bovine PCPV, the pathological lesions seen in the bison were very distinct from PCPV lesions seen in cattle. The source of the PPV infection in bison is not known. PCPV virus infection in cattle is generally a mild infection characterized by small “ring” or “horseshoe”- shaped papules on the teats and udder that progress to form vesicles or pustules and heal by forming scabs . In compB2L gene. Further investigation to determine the pathogenicity and zoonotic potential of this bison PCPV is warranted.In conclusion, the present report describes an atypical and severe form of PCPV infection in a new host species. The infection was characterized by multifocal raised nodules, extending over large cutaneous areas in an American bison. Definitive identification of the PCPV was made by gross and histopathological examination, real-time PCR, electron microscopy, metagenomic sequencing and phylogenetic analysis based on the