Current therapy for hypervascular cancers, e.g., hepatocellular carcinoma, includes occlusion of the tumor blood supply by arterial infusion of embolic microspheres (beads) suspended in iodine-based contrast under fluoroscopic guidance. Available radiopaque, imageable beads use iodine as the radiopacifier and cannot be differentiated from contrast. This study aimed to synthesize and characterize imageable beads using bismuth as the radiopacifier that could be distinguished from iodine contrast based upon the difference in the binding energy of k-shell electrons (k-edge). Radiodense bismuth beads were successfully synthesized some with uniform bismuth distribution across the beads. The beads were spherical and could be infused through clinical microcatheters. The bismuth beads could be imaged with clinical dual-energy computed tomography (CT), where iodine-based contrast could be distinguished from the microspheres. The ability to separate iodine from bismuth may enhance the diagnostic information acquired on follow-up CT, identifying the distribution of the embolic beads separately from the contrast. Furthermore, with sequential use of iodine- and bismuth-based beads, the two radiopaque beads could be spatially distinguished on imaging, which may enable the development of dual drug delivery and dual tracking. Embolic beads are typically made of inert hydrophilic materials with elastic properties and spherical geometry. These beads are generally non-radiopaque but can be rendered radiopaque by incorporation of a radioabsorber, such as iodine, zinc, tantalum, bismuth, or barium6. The development and use of imageable beads can inform direct real-time feedback during the embolization procedure, or on follow-up imaging8.Embolic microspheres (beads) are used to occlude the vascular supply of tumors and may be loaded with chemotherapy drugs, which then elute from the microspheres and diffuse into the tissue. These drug-eluting beads are used for locoregional therapy, particularly for treating patients with unresectable hepatocellular carcinomaco-acrylamido-2-methylpropane sulfonate precursor microspheres10. The radiopacity of the beads during delivery or follow up can facilitate identification, localization, and distribution of undertreated tumor12. It may also enable estimation of doxorubicin dose and spatial distribution in the liver13. One limitation of iodinated beads, however, is the challenge in distinguishing them from iodinated contrast agents using imaging based on X-ray absorption, i.e., fluoroscopy, cone beam CT, or CT imaging. During embolization, embolic agents (both radiopaque and non-radiopaque beads) are mixed with contrast agents to improve suspension and handling, and to estimate dosimetry and delivery of the beads to the tumor. Contrast may be somewhat retained, although intravascular contrast washes out over time14. Similarly, a contrast-enhanced CT scan after bead delivery may not normally differentiate among intravascular liquid contrast, iodinated beads, and contrast in stained parenchyma.Recently, we have developed a radiopaque embolic bead through covalent attachment of iodinated groups onto the backbone of polyvinyl alcohol-15. In DECT, two CT scans are simultaneously acquired at different energies, a low-energy image, e.g., 80 keV, and a higher-energy image, e.g., 140–150 keV, that can be processed to generate additional datasets that enable material decomposition. Bismuth has a k-edge of 90.5 keV compared to 33.2 keV for iodine and thus has the potential to be differentiated from iodine using DECT.Attaching a radiopaque element with a higher atomic number, Z, and a different k-edge from iodine, may enable material decomposition and differentiation of a novel bead from iodine in contrast or from iodinated radiopaque beads using dual-energy CT (DECT)The purpose of this study was to synthesize and characterize novel imageable beads using bismuth as the radiopacifier. A secondary goal was to assess the ability to spatially differentiate the bismuth beads from iodine-based contrast agents using DECT imaging, as a requisite step towards dual tracked drug vectors in embolization therapies.1 with bifunctional linkers, as shown in Fig. 3SO3)3 to give radiopaque beads 6 and 7  and the sulfonated acylamido polyvinyl alcohol beads in DMSO solvent was a second-order reaction as the plot of the inverse concentration of (4) (M) versus time (min) showed a linear relationship with reaction rate constant, k = 8.5 × 10−5 L/mol min  and the sulfonated acylamido polyvinyl alcohol beads in DMF solvent is also second order, but the reaction rate constant was tenfold faster, k = 8.5 × 10−4 L/mol min , and energy-dispersive X-ray analysis (EDX) were performed for both beads 6 and 27.85% C, 3.87% H, 2.00% N, 3.18% S, 30.88% Bi and 32.22% O and Na (combined) for bead 7. The percent bismuth content from the elemental composition analysis indicated a slightly higher bismuth content for bead 6 (35.23%) compared to bead 7 (30.88%). Other physical characteristics of different sizes of bismuth beads are shown in Supplementary Table The elemental chemistry of a dry bead is 27.28% C, 4.16% H, 2.58% N, 3.3% S, 35.23% Bi and 27.45% O and Na (combined) for bead 7 are depicted in Fig. The synthesized bismuth beads appeared dark brown and opaque under optical microscopy and maintained their spherical morphology and smoothness. However, the beads had a gross white appearance to the naked eye. The shapes and optical appearances for beads 6 and 7 to be free of any visible pores  were deliverable through 2.0 Fr and 2.4 Fr catheters, while larger beads (250–355 µm) required a minimum of a 2.4 Fr catheter to enable delivery without clogging , while there was a poor linear correlation between the bismuth volume and DEI  as shown in Fig. The final material decomposition analysis was performed using the 80 kVp and Sn150 kVp paired acquisition, as the iodine extraction was favorable for this acquisition pair. There was a good linear correlation between the iodine concentration and dual energy index (DEI)  required a 2.4 Fr microcatheter. Both beads sizes are comparable to those in standard clinical use.For clinical delivery, beads should have a stable suspension in liquid contrast and must be able to pass through a microcatheter16. These novel bismuth beads have the potential to improve image-guided local drug delivery in patients with liver cancer by directly identifying either non-targeted delivery or undertreated tumor. The ability to choose from two bead formulations to load drugs opens the door to dual drug delivery and “dual drug dose tracking”. Two beads could be loaded with doxorubicin and a second agent, such as an immunomodulator. Two drugs could be selected and targeted with a mechanistic rationale that factors in the tumor microenvironment or molecular data. For example, doxorubicin intercalates in the nucleus and is more active for cell-cycle-specific regions of high mitosis, which may be reflected by high metabolic activity on FDG PET. Necrosis-targeting agents could be loaded and localized by injection into imaged regions of necrosis or high interstitial pressure, such as regions with restricted diffusion on MRI. Anti-vascular agents loaded on an image-able bead could be targeted to areas of high enhancement or vascular density, also easily localized on imaging. Fused imaging on top of procedural CBCT also allows exquisite mechanistic targeting, in such a paradigm17. Combining iodine-based and a bismuth-based beads loaded with different chemotherapy or immunotherapy drugs, could potentially enable dual drug delivery for a more rational, fusion image guided therapy grounded in mechanistic molecular reasoning. Such a paradigm could move image guided therapies into a new era after the field has delivered the same anthracycline for over four decades18.DECT imaging may be used for material classification by exploiting the differences in X-ray absorption of iodine and bismuth from high radiographic density. This model could also be imposed in the clinical setting of liver tumor embolization. A DEI threshold of 0.06 distinguished iodine from the background and from bismuth beads alone. However, there was an overlap in DEI values for the higher concentrations of bismuth beads and the lower concentrations of iodine. Therefore, the DEI as a single image feature could be used to identify if iodine was present, either alone or in combination with bismuth beads. The model dictates that densities (HU) above background must be either iodine or bismuth, while any tube with a DEI below the threshold that does not contain iodine and must be bismuth beads. For embolization, the only density would be iodine or bismuth beads. Refining and optimizing the synthesis process to increase the selection of only the bead populations with higher radiographic density could lead to an increase in the signal to noise ratio in CT scanning and material decomposition. The DEI could be used to define and follow the washout of iodine contrast after embolization. Post-embolization residual tumor enhancement may be hard to distinguish from high attenuation iodine beads, without a subtraction image in conventional CT. Bismuth beads would not present this problem19.The development of more advanced material decomposition-based algorithms, artificial intelligence-based CT image reconstruction, or artificial intelligence-based segmentation may further enable iodine/bismuth material classification and drug dose maps for treatment plans. Higher-resolution imaging and advanced material decomposition may be further explored in the next-generation CT scanners; photon counting CT, for example, can distinguish between bismuth, gadolinium, and iodine, in vivo21, in vivo safety studies are required, which may also include an assessment of the embolization or drug-eluting effect of the beads in tissues.Drug loading and elution profiles of the derived bismuth beads were not evaluated in this study. Based on the chemistry of the bismuth beads, it is expected that elution profiles will parallel its predecessor iodine-based radiopaque beads’ loading and elution profiles, but additional work will be required to confirm. The primary goal of the current study was to synthesize and characterize a radiopaque bismuth bead. In vitro evaluation of small agarose volumes containing beads may not reflect the full signal-to-noise levels observed in vivo with motion and in vivo environmental factors. In vivo preclinical studies were beyond the scope of this paper but are required to further explore the radiopacity and performance of the beads. However, the radiopacity of the beads on DECT was well above that of normal liver, so beads packed within hepatic arteries will be visible within the liver. While the bismuth is expected to remain bound to the chelation macrocycle6. The radiopacity of the bismuth beads was confirmed by microCT and DECT. Furthermore, DECT imaging permitted material classification (decomposition) that could distinguish bismuth beads alone from iodinated contrast or a mixture of iodine and bismuth beads, which should also extend to discrimination between iodinated beads and bismuth beads. Imaging of two distinct bead populations may enable and promote the development of dual drug delivery and dual drug tracking when the drugs are delivered by beads that can be distinguished on imaging. The combination of drugs, devices, and imaging-guidance with dual drug mapping is enabled by dual bead localization, which could stimulate the field to evolve beyond a four decade history of using the same drug, doxorubicin, delivered via similar catheters with similar x-ray guidance.In this study, we present facile methods for the synthesis of radiopaque polyvinyl alcohol-based microspheres, or beads, based on a marketed microsphere subsequently modified with a bifunctional macrocycle and chelated with bismuth. Bismuth distribution was uniform across the bead co-acrylamido-2-methylpropane sulfonate microspheres, dried to a free-flowing powder and untainted, were obtained from Biocompatibles UK Ltd, . Bi(CF3SO3)3 was chosen as bismuth salt and was obtained from Sigma-Aldrich . SEM and EDX were used to evaluate surface smoothness and bismuth distribution across the bead, respectively . Elemental composition was performed by Robertson Microlit Laboratories . Material decomposition techniques were used to determine whether bismuth and iodine could be distinguished using DECT.All reagents were purchased from commercial sources and used without further purifications. Acrylamido polyvinyl alcohol-18 column , mobile phase: (A) 90% (0.1% TFA in water); (B) 10% (0.1% TFA in acetonitrile); 0–5 min, 10–90% B, 5–15 min, 90% B, 15–25 min. The flow rate was set to 1 mL/min. TLC was carried out on Merck silica gel 60 TLC plates F254 and visualized by UV at 254 nm. Column chromatography was performed using silica gel 60 (70–230 mesh). 1H and 13C were NMR recorded on a Bruker Avance 300 instrument with a deuterated solvent. Mass spectra were recorded with a Waters  LCT Premiere ESI TOF mass spectrometer. The instrument was operated in positive ion ESI mode at a mass resolution of 10,000.Reaction progress was monitored by reverse-phase HPLC (Rp-HPLC) using a Beckman System Gold HPLC  equipped with a 126 solvent module and 168 UV detector (λ = 254 nm) controlled by 32 Karat software and Beckman Ultrasphere C2) was synthesized as described by Mondal et al.22 with slight modification. To a solution of DO3A-t-Bu-ester (1)  and 4-(bromomethyl)benzaldehyde  in anhydrous N, N-dimethylformamide (DMF) (60 mL) was added K2CO3 . The reaction was stirred at room temperature for 4 h. The reaction progress was monitored by Rp-HPLC as described in the general method. The reaction mixture was filtered and the solvent was removed. The product was purified by column chromatography  to give (2)  dull yellow powder. 1HNMR : δ ppm 1.5 , 2.2–2.9 , 3.0–3.1 , 3.2 , 7.7 , 7.9 , 10.0 ; 13C NMR : δ ppm 27.6, 49.7, 55.4, 55.7, 59.1, 82.2, 82.6, 129.7, 130.8, 135.3, 144.4, 172.4, 173.2, 191.5. MS–ESI: m/z (%) = 633.4 (100) [(M + H)+], HRMS[(M + H)+] = calcd. for: [C34H57N4O7]+ 663.4220; found 663.4227.The titled compound (t-Bu ester) (3) was made from 4-formylbenzoic acid (4-FBA) . 4-Formylbenzoyl chloride was synthesized by the method of Iserloh et al.23. 4-Formylbenzoyl chloride was coupled with the DO3A-tris-(t-Bu ester)  as described by Bhakuni et al. 24. The resulting reaction mixture was purified by column chromatography  with a gradient solvent system of ethyl acetate:methanol; 100%:0% to 95%:5% to give the desired product, 3 as pale yellow viscous syrup ; : δ ppm 1.41 , 1.45 , 1.47 , 2.70–2.68 , 2.80–2.73 , 2.91 , 3.03 , 3.07 , 3.31 , 3.32 , 3.62 , 3.79 , 7.55 , 7.90 , 10.04 ; 13C NMR : δ ppm; 28.16, 28.24, 44.67, 48.87, 52.00, 52.32, 52.82, 53.05, 53.52, 54.71, 54.98, 57.64, 58.16,, 80.89, 80.94, 80.98, 127.16, 129.81, 136.38, 143.32, 170.49, 170.64, 170.76, 170.80, 191.62.4-Formylbenzoyl-DO3A-tris- was treated with 4 N HCl in dioxane (450 mL) by the method described by Muhlemann et al.25 to give DOTA-4-formylbenzyl (4). 8 g  of dull white powder was obtained. MS–ESI: m/z (%) = 465.2 (100) [(M + H)+], HRMS[(M + 1)+]: calcd. for: [C22H33N4O7]+ 465.2345; found 465.2349.Compound 3 was treated using a similar method as described in compound 4 to give DOTA-4-formylbenzoyl (5) .Compound Method Ia DMSO as a reaction solvent4 (2.15 mmol). The resulting mixture was stirred for 30 min to distribute the macrocycle compound 4 into the swollen beads uniformly. Then, 2.2 mL of methanesulfonic acid was added dropwise from a syringe through a 30 G, 1/2-inch needle, and the reaction mixture was stirred at a lower stirrer setting under N2 at 50 °C for 26 h. The progress and the reaction rate were monitored with HPLC, every two hours for the first eight hours and then at 24 and 26 h, for the consumption of macrocyclic compound 4. The reaction mixture was filtered. The filtrate was washed with DMSO (10 mL), then with copious amount of saturated sodium bicarbonate to completely remove the acid (monitored with litmus paper) and finally with deionized water. The acetalated beads (6) were used for the subsequent reaction.1 g of beads , Farnham, UK) were swollen in 35 mL of anhydrous DMSO followed by adding 1 g of compound Method Ib DMF as a reaction solventAcetalation of the beads was repeated as described in (a), except that the DMSO solvent was replaced with DMF solvent.5 was performed in accordance with the method of Duran et al.10 with the following adaptations: NMP (N-methyl pyrrolidinone) was the solvent used, and the reactions were performed at 55–65 °C for up to 40 h, monitored with HPLC for the consumption of the macrocycle. The liquid phase was removed, and the filtered beads were washed with NMP (90 mL), then suspended in water and neutralized with 0.1 M sodium carbonate to pH 8.5. The beads were then washed with water (90 mL) and NMP (250 mL) followed by neat NMP (150 mL). The acetalated beads (7) were used directly in the subsequent reaction.Acetalation of the beads with compound 4 was performed with two different reaction solvents: dimethyl sulfoxide (DMSO) and dimethylformamide (DMF).Acetalation of the beads with compound 6) (Synthetic Scheme 1) were resuspended in deionized water at a pH of 8–9 using 0.1 N of sodium bicarbonate, and Bi(CF3SO3)3 (4 mmol) was added and stirred at 80 °C for 1 h. The final beads were filtered and washed with an aqueous solution of sodium bicarbonate (0.1 M) and finally with copious amounts of deionized water to remove residual bicarbonate (monitored by litmus paper). The complete removal of bismuth salt was confirmed using an aqueous solution of potassium iodide  as reported in the literature26.The acetalated beads (7) were suspended in anhydrous NMP (30 mL) under N2. Pyridine (0.54 mL) was added, then the mixture was warmed to 55 °C, followed by addition of bismuth trifluromethanesulfonate . The resulting suspension was stirred overnight at 55 °C under N2. The liquid portion was removed and the filtered beads were washed with NMP (250 mL). The beads were then suspended in phosphate buffered saline, neutralized to pH 7 with 0.1 M Na2CO3 and then washed with phosphate buffered saline (250 mL).Acetalated beads . Both dry bismuth beads (6 and 7) were analyzed for their elemental composition by Robertson Microlit Laboratories .The solid content of the hydrated bead, bismuth concentration of hydrated bead, and the bismuth content of the dry beads of 7) were sieved into four sizes ranging between 100–600 mm in diameter and evaluated under light microscopy for surface deformation and purity after the reaction process. The size and appearance of beads were examined9. Briefly, an aqueous suspension of 150 ml of beads in deionized water was placed on a chamber slide (Electron Microscopy Sciences). Bright-field images were acquired with a 5 × objective on an upright microscope  equipped with a color CCD camera .Beads  by using 20 keV energy, and the line scans were generated using Aztec software. Multiple line scans through the microsphere cross-sections of the bismuth elemental map were acquired for each bead population to show the distribution of bismuth from edge to edge.10. The catheters were laid on a bench with a 10 cm diameter coil mid-catheter to introduce curvature in the flow path. Any catheter clogging was recorded as a failure and denoted that bead size was not suitable to the respective catheter.Catheter deliverability of the bismuth beads through two clinical microcatheter sizes  was evaluated by administering a homogenous bead suspension in 100% iohexol  as a 1:10 bead dilution and using 1- or 3-mL syringesThe bead with the higher bismuth content and bismuth homogeneity across the bead surface was chosen for microCT and DECT evaluation.9. To assess radiopacity and material decomposition with DECT, iodine, bismuth beads, or a mixture of both were added to 1% agarose for a total volume of 1 mL For the iodine-agarose gels, four tubes were prepared using the clinical contrast agent Isovue-300 with 5, 10, 20, and 40 mg I/mL per tube. For the bismuth-agarose tubes, four tubes were prepared using suspensions of bismuth beads 6, with 62.5, 125, 250, and 500µL bismuth beads/mL per tube. Similarly, agarose tubes with mixtures of Isovue-300 and bismuth beads were prepared with the following ratios of mg iodine/mL and µL bismuthbeads/mL: 10:125, 10:250, and 20:125.A suspension of bead in agarose gel  was used to assess the radiopacity on microCT9. Homogeneously dispersed beads in an agarose phantom  were scanned with Bruker SkyScan1272  microCT at a nominal resolution (pixel size) of 5.0 microns with a 0.5 mm thick aluminum filter, a source current 125 mA, and an applied X-ray tube voltage of 80 kV. Reconstruction was carried out with a modified Feldkamp algorithm using the SkyScan NRecon software . Ring artifact reduction and beam hardening correction were applied. Image post-processing was performed . Beads within the 70 and 150 μm range were selected for post-analysis, while the beads in or near a saturated spot were excluded. The voxel values were converted to HU using a water calibration phantom. A 3D mask was created for each bead to enable the calculation of HU per bead and analyze the variation in mean density per bead size.MicroCT was performed to evaluate the radiopacity of individual bismuth beads suspended in agarose DECT imaging of the agarose gels was performed on a 192-slice DECT scanner . Clinical scanning protocols for the abdomen/pelvis were used with 80 kVp imaging paired with Sn150 kVp and 100 kVp paired with Sn150 kVp, where Sn150 kVp refers to the use of tin filtration of the X-ray beam. Images were reconstructed at 0.5 mm slice thickness. The tubes were inserted in a test tube rack  and the high energy acquisition (Sn150 kVp), respectively. The individual attenuations are shifted by 1000 HU to avoid negative values, which results in the factor of 2000 HU in the denominator in the reduced equation29. The acquisition pair that maximizes the difference in HU for iodine, while minimizing the difference in HU for bismuth was chosen for further evaluations.Linear regression was performed between measured CT numbers versus the known iodine concentration or bismuth bead volume for both pairs of imaging exposures. The dual-energy index (DEI) was calculated from the CT numbers using the low and high energy images of each pair as followshttps://www.R-project.org/). A DEI image was created, and values above the threshold value were displayed as a red color map superimposed on the original DEI image. A combined map of DEI and HU values above their respective thresholds was created by blending the two maps and then superimposing the results onto the low kVp acquisition.In addition, the threshold value of DEI that maximally separates the bismuth and iodine was determined, using an optimizer function (R Vienna, Austria. URL Supplementary Information.


Varroa destructor, has prompted intense efforts to manage honey bee health. However, little is known about the occurrence of honey bee viruses in bee predators, such as vespids. In this study, we characterized the occurrence of 11 honey bee viruses in five vespid species and one wasp from four provinces in China and two vespid species from four locations in France. The results showed that all the species from China carried certain honey bee viruses, notably Apis mellifera filamentous virus (AmFV), Deformed wing virus (DWV), and Israeli acute paralysis virus (IAPV); furthermore, in some vespid colonies, more than three different viruses were identified. In France, DWV was the most common virus; Sacbrood virus (SBV) and Black queen cell virus (BQCV) were observed in one and two samples, respectively. Phylogenetic analyses of IAPV and BQCV sequences indicated that most of the IAPV sequences belonged to a single group, while the BQCV sequences belonged to several groups. Additionally, our study is the first to detect Lake Sinai virus (LSV) in a hornet from China. Our findings can guide further research into the origin and transmission of honey bee viruses in Vespidae, a taxon of ecological, and potentially epidemiological, relevance. Since the discovery that honey bee viruses play a role in colony decline, researchers have made major breakthroughs in understanding viral pathology and infection processes in honey bees. Work on virus transmission patterns and virus vectors, such as the mite  Apis mellifera) make vital contributions to plant diversity and agricultural ecosystems [Israeli acute paralysis virus (IAPV), Deformed wing virus (DWV), Black queen cell virus (BQCV), Sacbrood virus (SBV), Chronic bee paralysis virus (CBPV), Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and Kakugo virus (DWV-A/KV) [Varroa destructor boosts viral titers and expands the transmission range of DWV [Honey bees  . In somee of DWV  and IAPVe of DWV . Vespa velutina nigrithorax)—an invasive species in Europe [V. velutina and its dramatic impact on honey bee survival in Europe [Virus transmission dynamics could also be affected by honey bee predators, a subject about which little is currently known. Hornets, for example, are major bee predators, and the yellow-legged hornet  lacks effective defensives against predators like the yellow-legged hornet. Beekeepers in Europe have reported that V. velutina preys on honey bee colonies—impacting populations [Apiculture is a major industry in China. Beekeepers manage more than 9 million hives and supply enough honey for the country’s consumers. However, the European honey bee  characterize the occurrence of major honey bee viruses in vespids and (2) explore the phylogenetic relationships among virus sequences from vespids and honey bees. Vespa bicolor, two colonies of Polistes rotheyi, two colonies of Vespa velutina, one colony of Vespa koreansis, one colony of Vespa affinia, one colony of Vespa sp. . A universal primer set (LCO-1490 and HCO-2198) was used to amplify a 709 bp fragment of cytochrome c oxidase subunit I (CO I) via polymerase chain reaction (PCR) ), and thFor the Chinese samples, DNA and RNA were extracted from a pool of approximately 20 workers or pupae per colony; queens were also used, if present. The samples were prepared using cold grinding (liquid nitrogen and a mortar/pestle). Total RNA was extracted using a TRIzol Kit  and TissuePrep homogenizer  in accordance with the manufacturers’ instructions. The resulting sample was then eluted in 20 µL of sterile water and stored at −80 °C. The quantity and purity of the RNA were measured using a Nanodrop spectrophotometer . We synthesized cDNA from 4 µg of the total RNA using M-MLV reverse transcriptase  and the oligo dT primer in accordance with the manufacturer’s instructions. Total DNA was extracted using a Wizard Genomic DNA Purification Kit  and TissuePrep homogenizer  following the manufacturers’ instructions. The resulting sample was then eluted into 20 μL of nuclease-free water and stored at −20 °C.g. Next, total RNA extraction was performed using 350 µL of the supernatant and a NucleoSpin RNA Isolation Kit  in accordance with the manufacturer’s instructions. The resulting sample was eluted into 40 µL of RNAse free water; this process was repeated three times with the same elute to increase final RNA concentration. RNA quality was visually controlled via electrophoresis (1% agarose gel), and RNA quantity was measured using a Qubit fluorometer. Reverse transcription was performed using a RevertAid First Strand cDNA Synthesis Kit  and random hexamer primers in accordance with the manufacturer’s instructions; in most cases, 1 µg of total RNA was employed.For the French samples, RNA extraction was performed as previously described . BrieflyThe primer sequences are provided in ® polymerase , 2.5 µL of 10× buffer , 1.5 mM of MgCl2, 0.2 mM of each dNTP, 0.4 µM of the forward and reverse primers, and 100 ng of cDNA; the total reaction volume was increased to 25 µL using nuclease-free water. The PCR conditions were as follows: an initial denaturation step at 95 °C for 2 min; four cycles of 95 °C for 1 min, 54 °C for 1 min, and 72 °C for 1 min; 30 cycles of 95 °C for 1 min, 49 °C for 1 min, and 72 °C for 1 min; and a final extension step at 72 °C for 10 min. The PCR products were separated using electrophoresis (2.5% agarose gel stained with Red DNA Stain) and visualized using a UV light.We used multiplex PCR (as previously described ) to testThe PCR products were purified and sequenced by Sango Biotech  and by GATC Biotech . The sequences from the Chinese samples were assembled and manually corrected using Lasergene 8 and Geneious (v. 9) software.http://www.atgc-montpellier.fr/phyml/) [For each virus, sequences were then aligned using the MAFFT algorithm  implemen/phyml/) . In each/phyml/) . Node su/phyml/) .The sequences of the viruses were submitted to GenBank under the accession numbers MF092814 to MF092832 for BQCV from China and MH133351 and MH133352 for BQCV from France; MF092819 to MF092825 for IAPV from China; MF092827 to MF092832 for DWV-A from China; MF092817 for AmFV from China; MF092826 for DWV-B from China; MF092818 for DWV-A/KV from China; and MH133361 for SBV from France. The DWV-A sequences VC-F1, VV-L2, VC-S2 were shorter than 200 bp long and were thus not attributed accession numbers.We screened the vespid colonies for the presence of 11 viruses . SeveralIn China, the co-occurrence of more than one virus in vespid colonies was fairly common for both adults and pupae . In adulPolistes rothneyi)   a. The BQ = 0.95) b. The Fr = 0.95) b.A. mellifera in South Korea . One SBV sequence was discovered in a hornet in France, and it was located within a clade formed by SBV sequences in A. mellifera in Australia and South Korea  , and the sister group of this clade was the cluster of the IAPV sequences found in th Korea c. The se = 0.75) d.V. destructor virus-1). Interestingly, both DWV genotypes were represented in the sequences obtained from vespids in China and France. Because different DWV loci were amplified for the Chinese and French samples, separate phylogenetic trees were reconstructed for each country  is a specialized predator of honey bee foragers and thus represents a serious threat to the apiculture industry and pollination ecosystem services [Vespids are widely distributed across the world, from tropical to temperate regions. While some species can help disperse plant seeds, such as those of tuberosa , other sservices . This spA. mellifera and A. cerana [Our study aimed to take a first look at the occurrence of 11 honey bee viruses in vespid species collected in China and France; it also examined phylogenetic relationships among virus sequences. The results show that several viruses were commonly present and broadly geographically distributed in a variety of vespid species. In addition, we found more viruses in pupae than in adults, which is not surprising given that adults feed larvae large quantities of masticated bees. While we did not investigate infection dynamics, this finding might suggest that pupae are more suitable or more susceptible hosts than adults, if the viruses are capable of replicating in vespids. In addition, we also did not detect the pupae from France vespids due to limited samples. Such stage-dependent susceptibility has been described for several viruses that infect both . cerana ,33. FurtBased on our phylogenetic analyses, the viruses infecting vespids do not form specific clades. This result could suggest that the honey bee viruses are not transmitted among vespids but rather from honey bees to vespids. It might also indicate that, if replication occurs in vespids, it primarily results from recent spillovers from honey bees. A recent study showed that such spillovers could result in DWV-B and KBV infections in the yellow-legged hornet, including a case in which an individual was symptomatic  . In our IAPV was first identified in Israel in 2004 but has since been found to have a worldwide distribution . It is aAlthough AmFV was first identified in the honey bee in 1963  and its To summarize, we characterized the occurrence of the 11 most important honey bee viruses in vespid species in China and France. We also identified differences in virus co-occurrence among different vespid species in China. All the virus sequences that we obtained from vespids were closely related to sequences obtained from honey bees, likely indicating that the vespids acquired the viruses while feeding on bees. However, further research is needed to explore infection dynamics in vespids, and the potential for virus transmission between vespids and honey bees.


Chronic idiopathic intestinal pseudo-obstruction (CIIPO) is a disease characterized by symptoms and signs of small bowel obstruction in the absence of displayable mechanical obstruction. Due to the known neuropathic capacity of several viruses, and their localization in the intestine, it has been hypothesized that such viruses could be involved in the pathogenesis of CIIPO. The most frequently involved viruses are John Cunningham virus, Herpesviridae, Flaviviruses, Epstein–Barr virus and Citomegalovirus. Therefore, the present narrative review aims to sum up some new perspectives in the etiology and pathophysiology of CIIPO. Chronic idiopathic intestinal pseudo-obstruction (CIIPO) is a disease characterized by symptoms and signs of small bowel obstruction in the absence of displayable mechanical obstruction ,2,3.In a Japanese survey, the prevalence of CIIPO was estimated to be 0.80 to 1.00 per 100,000, with an incidence of 0.21 to 0.24 per 100,000 [It represents one of the most frequent reasons of chronic intestinal failure both in children 15%) and adults (20%) ,5,6,7,8.% and aduCIIPO is an idiopathic condition in almost fifty percent of cases . HoweverCIIPO is usually sporadic, even if familial forms have also been described ,3,15,16.The most common clinical features of CIIPO are represented by bloating, abdominal pain and distension, which may be acute, recurrent, or chronic ,5.The diagnosis of CIIPO is usually based on the finding of longstanding symptoms of mechanical obstruction in the absence of an anatomic cause on radiologic examination and endoscopy, and evidence of impaired motility . We can Manometry is expected to represent the gold standard for diagnosis , even ifFull-thickness small bowel biopsies, acquired in surgical specimens, could help in identifying the site of damage and in guiding therapy ,19.In the diagnostic work-up of suspected CIIPO, laboratory tests can help to find secondary causes: complete blood count, electrolytes, liver tests, vitamin B12, folate, celiac serologies, thyrotropin, serologic test for herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein–Barr virus (EBV), markers of inflammation .In particular, searching for the aforementioned viral infections is mandatory because several systemic viral infections could involve the enteric nervous system with the consequence of an alterated peristaltic activity . The mosTherefore, the present narrative review aims to sum up some new perspectives in the etiology and pathophysiology of CIIPO.JCV belongs to human polyomaviruses and to the Papovaviridae family and is a non-enveloped, small (45 nm) virus with a circular double-stranded DNA genome ,22.JCV was firstly identified in 1952 in a patient  affected by Hodgkin’s lymphoma and progressive multifocal leukoencephalopathy (PML) ,23. It cJCV is most commonly known as being associated with PML in immunosuppressed patients  and in pIn fact, in literature, two possible mechanisms of cell infection by JCV are described . The patSelgrad et al., performed a case-control study comparing specimens from 10 patients with CIIPO with 61 control specimens. In this study, JCV proteins (TAg and VP1) were identified in glial cells of the myenteric plexus in seven out of 10 adult patients with idiopathic CIIPO and in none of the control specimens .TAg and VP1 were identified in the myenteric plexuses, indicating a lytic infection, potentially destroying the host cell; therefore, virus detection in enteroglial cells leads to a possible pathological role for JCV in enteric neuropathy.Our group also reported two cases of CIIPO associated with JCV infection  and viruHerpes viruses belong to a large family of viruses which present similar structural characteristics such as a linear double stranded DNA genome, envelope, capsid and tegument.Herpes viruses are divided into three subgroups: α  which includes herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2) and varicella zoster virus (VZV); β (beta) cytomegalovirus (CMV), human herpes type 6 (HHV-6) and human herpesvirus type 7 (HHV-7) and γ (gamma) Epstein-Barr virus (EBV) and human herpes type 8 (HHV-8) ,40.All herpes viruses are neurotropic and are able to cause motility disorders. However, to date, cases of colonic and ileal pseudo-obstruction have been reported in the literature only associated with VZV, CMV and EBV infection.Primary infection with VZV causes chickenpox which occurs with fever and diffuse pruritic rash usually affecting children.After the acute infection, the virus enters a latent phase in nerves, specifically in the dorsal ganglion cells of sensory nerves, the cranial nerves and in the enteric nervous system (ENS) ganglia ,42. ThisVZV can reach the ENS through transport by T lymphocytes during the acute infectious phase or with a retrograde transport from the epidermal projections of infected dorsal-root ganglion neurons to ENS ,42.Endogenous viral re-activation with VZV replication and ganglion inflammation occurs in about 30% of subjects also many years after the primary infection . FactorsDuring this phase, the virus moves antegrade from the ganglia along the axons of the neurons to the skin causing the so-called Herpes Zoster. In addition, in about 5% of subjects the virus reactivation can lead to peripheral somatic and visceral motor neuropathies. Moreover, based on the location of virus infection , patients can present variable clinical manifestations ,44,45.Gastrointestinal symptoms associated with VZV reactivation are very rare; however, some case reports have demonstrated the coexistence of VZV and colonic pseudo-obstruction ,59,60,61On the contrary, only a few cases are reported of paralytic ileus (PI) following VZV infection ,64,65,66Typical symptoms of ileal and colonic pseudo-obstruction  usually occur a few days or weeks before the rash, making a correct diagnosis very difficult. The most accurate test for the diagnosis of Herpes Zoster is PCR for VZV DNA in colonic mucosal biopsy samples . Only twDifferent hypotheses on pathogenesis of ileal and colonic pseudo-obstruction due to VZV have been formulated: (1) direct viral injury to colonic ENS and muscularis propria; (2) direct infection of celiac plexus ganglion and colonic autonomic nervous system (ANS) ; (3) virCytomegalovirus (CMV) is a member of herpes virus family and is widespread in the world population.CMV infection of the digestive tract is usually seen in immunodeficient patients and are characterized by ulcerations, erosions, and mucosal hemorrhage. A case of CIIPO was reported in an immunocompromised patient who had previously undergone a cardiac transplant . CMV incHowever, since 1964, several studies have reported GI disease due to CMV in patients without detectable immunodeficiency . To dateDèchelotte et al. reported three cases of paralytic ileus in fetuses caused by CMV infection. In these cases, CMV had been identified in the ganglion cells or in the myenteric and submucosal plexuses of the small bowel and colon.In one patient, paralytic ileus was transient and associated with a mild CMV infection not even identified at autopsy. In the two other cases reported, paralytic ileus persisted and was associated with severe CMV infection with inflammatory reaction and cell necrosis .Foucaud et al. reported a case of a 2-month-old boy affected by abdominal distension and inappetence leading to total parenteral feeding. Viruria and specific IgM and IgG antibodies confirmed CMV infection. The patient also underwent rectal biopsy with detection of hypoganglionosis, nerve thickening and CMV intranuclear inclusions .Moreover, a case of a 2-month-old girl affected by CMV-related CIIPO with abdominal distension and vomiting has been reported. At the small bowel biopsy, CMV was identified. Moreover, the patient’s trend of IgM and IgG and the absence of maternal IgG were indicative of infection within the first few weeks of life .Debinsky et al. evaluated the evidence of viral DNA in samples from 13 adult patients affected by CIIPO and 12 controls. Among CIIPO patients, three were positive; in particular, one patient, who presented visceral neuropathy and myopathy, had small bowel biopsies positive for CMV DNA. In control tissue, any virus was detected .Epstein-Barr virus (EBV) infection, the so-called infectious mononucleosis, frequently involves the nervous system  with casTwo pathogenetic mechanisms have been hypothesized: (1) viral injury of autonomic ganglia or postganglionic neurons ,79; (2) Vassallo et al. reported the first case of selective cholinergic dysautonomia that occurred following acute infectious mononucleosis. In particular, they reported the case of a young woman affected by nausea, vomiting, severe abdominal distention and constipation, insurgents about 3 years after an episode of EBV acute infection. Also, pupils, sweat glands, lacrimal and salivary glands, and urinary bladder were affected. Injury of sympathetic and parasympathetic postganglionic cholinergic nerves with the preservation of sympathetic adrenergic functions was identified at autonomic function tests. In addition, the ganglion cells of the myenteric plexus were normal at morphological and immunohistochemical evaluation and therefore the selective cholinergic dysnfuction was identified as the most likely cause of CIIPO .Besnard et al. published the case of a little boy affected by pharyngitis who subsequently developed intestinal obstruction, pandysautonomia and encephalomyelitis. After 3 months from the first onset of symptoms, the patient has undergone resection appendix and sigmoidoscopy and the tissue samples showed hypoganglionosis and a mononuclear inflammatory infiltrate in the myenteric neural plexus. EBV-RNA was detected in the blood and cerebrospinal fluid, and EBV-RNA was identified in myenteric appendix cells, in a mesenteric lymph nodeand in gastric biopsies. The serology for EBV showed previous infection but anti-early antigen antibodies were present .Two of the three CIIPO patients who tested positive in the Debinsky et al., series presented EBV DNA. Specifically, in a patient with visceral neuropathy, EBV DNA was isolated in the small intestine, while the other patient with visceral myopathy had EBV DNA in both the colon and the small bowel .Finally, De Giorgio et al., performed a PCR-based study of 13 patients with CIIPO diagnosticated clinically and manometrically. The study revealed an incidence of 31% of positive patients, with two affected by HSV-1 and two by EBV infections [Flavivirus belongs to a group of RNA virus that include the West Nile virus (WNV), Zika virus (ZIKV), Powassan virus (POWV), dengue virus, tick-borne encephalitis virus, yellow fever virus, and several other viruses. These viruses have some similar characteristics: size (40–65 nm), an icosahedralnucleocapsid, positive-sense, single-stranded RNA, and similar appearance in the electron microscope.In particular, WNV is a Flavivirus that is transmitted via mosquitoes and causes an acute fever . It is aZIKV is another virus belonging to this genus and mostly causes congenital malformations in fetuses during the period of pregnancy .To date, there has been a lack of data published in literature regarding the infection of Flavivirus on ENS neurons and consequent gastrointestinal symptoms.Some studies have been conducted on mice and have shown that mice infected can develop gastrointestinal pathology ,87. In c+T cell intestinal infiltration. The gradual clearance of viral RNA corresponds to an improvement of intestinal transit and an inflammatory infiltrate reduction [A recently published study showed that mice infected with WNV or Kunjin virus, during the acute phase, present elevated levels of viral RNA associated with intestinal dilation, damage of ENS neurons and CD8eduction .Moreover, mice with persistence of WNV or KUNVRNA in the intestine suffer from chronic GI transit delay that can last for weeks and months after the acute phase and are prone to GI symptom exacerbations .To date, the few data in humans are case reports of patients affected by Flavivirus acute infection and GI symptoms  ,90. The The management of individuals affected by CIIPO is often challenging .In its management, the most relevant diagnostic step is to exclude secondary causes susceptible to treatment.Due to the known neuropathic ability of the aforementioned virus, and their frequent identification in the intestine, it has been hypothesized that such viruses could be identifiable in tissues of patients with CIIPO, and be involved in the etiology of this condition. The studies reported in this review, although represented by case reports or case series, suggest that CIIPO may be caused by a viral infection through the alteration of the integrity of the ENS.In Despite the fact that serology could be helpful for diagnosis as it shows a predictive negative value, only full-thickness biopsy of the intestine, as shown for VZV ,47, CMV However, we should also consider that sequencing viral nucleic acids, whether from cultures or directly from clinical specimens, is complicated by the presence of contaminating host DNA . TherefoCurrently, genome sequencing of viruses can be achieved by ultra-deep sequencing or through the enrichment for viral nucleic acids before sequencing, either directly or by concentrating virus particles, but these approaches have their own costs and complexities .Finally, the pivotal action of the ENS in certain diseases of the gastrointestinal tract, and the identification of receptors on the enteric neuron for hormones and transmitters, suggests the rational basis for current and future targeted therapies potentially helping patients suffering from a broad range of gastrointestinal disorders .Some reports showed that immunomodulatory/immunosuppressive therapies were effective in CIIPO with an inflammatory neuropathy, if antineuronal antibodies are detected (ANNA-1 or anti-Hu), or in autoimmune-induced CIIPO ,95,96.Therefore, therapies promoting gastric and colonic motility will be the future optimal therapies to promote gut and small bowel transit. For example, neurotrophins could help to treat enteric neuronal degeneration, since these proteins stimulate the growth of nerve cells . In factIn spite of the fact that good evidence is scanty, it is intriguing to consider that CIIPO could be the consequence of an interaction with the several luminal microorganisms that populate the gut wall .


MC1R gene is associated with coat color phenotypes EM (melanistic mask), EG (grizzle/domino) and e1–3 (recessive red) in dogs. In addition, a previous study of archeological dog specimens over 10,000 years of age identified a variant p.R301C in the MC1R gene that may have influenced coat color of early dogs.The Melanocortin 1 Receptor (MC1R) plays a central role in regulation of coat color determination in various species and is commonly referred to as the “E (extension) Locus”. Allelic variation of the MC1R gene was present in 35 breeds or breed varieties, at an allele frequency of 1.5% in the tested population. We detected no linkage disequilibrium between R301C and other tested alleles of the E locus. Based on current convention we propose that R301C should be considered a novel allele of the E locus, which we have termed eA for “e ancient red”. Phenotype analysis of owner-provided dog pictures reveals that the eA allele has an impact on coat color and is recessive to wild type E and dominant to the e alleles. In dominant black (KB/*) dogs it can prevent the phenotypic expression of the K locus, and the expressed coat color is solely determined by the A locus. In the absence of dominant black, eA/eA and eA/e genotypes result in the coat color patterns referred to in their respective breed communities as domino in Alaskan Malamute and other Spitz breeds, grizzle in Chihuahua, and pied in Beagle.Commercial genotyping of 11,750 dog samples showed the R301C variant of the MC1R R301C variant, one of the earliest mutations captured by canine domestication, and citizen science empowered characterization of its impact on coat color.This study demonstrates a large genotype screening effort to identify the frequency and distribution of the The online version contains supplementary material available at 10.1186/s40575-020-00095-7. Melanocortin 1 Receptor (MC1R), known as the E locus, represents the key signaling molecule on melanocytes inducing expression of enzymes responsible for eumelanin synthesis. The alleles in order of dominance at the E locus are: EM (melanistic mask) > EG (grizzle/domino) > E (wild type) > e1–3 (recessive red) [e1–3 variants result in loss of gene function and consequently for dogs with the genotype e/e only pheomelanin (yellow/red) pigment is present. The allelic variant e1 is common and found in a wide variety of dog breeds [e2 and e3 alleles represent rare additional e variants found in Australian Cattle Dogs and white Alaskan and Siberian Huskies [EG allele is one of the rarest trait-associated alleles present in dogs, requiring specific genotype combinations at more than one locus in order to produce a domino or grizzle phenotype. This phenotype has been characterized in Afghan Hounds and Salukis only [Agouti Signaling Protein (ASIP) gene, known as the A locus, is an inverse agonist of MC1R inhibiting eumelanogenesis and promoting pheomelanogenesis. Four known alleles in order of dominance are ay (fawn) > aw (wolf sable) > at (tan point) > a (recessive black) [aw is considered the wild type ASIP allele. The most dominant allele ay represents a gain-of-function mutation causing an increased expression of pheomelanin, whereas the alleles recessive to wild type show reduced-function and increased expression of eumelanin [ay fawn dogs; when no dark hairs are present the ay fawn appears similar to recessive red, while abundant display of dark hairs produces an ay phenotype highly similar to the wild type. Such variable expression of ASIP expression and phenotype could be due to epigenetic mechanisms as shown in mice [canine beta-Defensin 103 (CBD103) gene, known as the K locus, is a neutral antagonist of MC1R. The dominant KB (dominant black) allele prevents ASIP inhibition enabling high levels of basal receptor activity resulting in solid eumelanin coat color. Similarly, a phenotypically intermediate kbr allele (brindle) of the K locus produces dominantly overlaying eumelanin stripes on A locus determined background [ky (wild type) allele of the K locus, the A locus is expressed normally. The phenotypic expression of the A and K loci is dependent on the presence of at least one functional MC1R allele, and are not expressed in the presence of an e/e genotype. Coat color variation in dogs and various other domesticated species is a result of domestication and intentional selection of novel phenotypes by humans and neither is explained by relaxation of natural selection [Coat color in dogs is determined by expression of two melanin pigments, eumelanin (black/brown) and pheomelanin (yellow/red) and by spatial and temporal regulation of these pigments’ expression in the body and in the individual hair shaft. ive red) . The e1–g breeds , 6, while black) –9; of thumelanin . There i in mice , 11. In  in mice . The canckground , 14. In election .MC1R and the dominant black KB allele of the CBD103 gene were both found in the DNA of over 10,000 years old Siberian and South-Eastern European dogs [KB allele suggesting that these variants could represent some of the first coat color variants present at the time of early dog domestication [alpha-Melanocyte Stimulating Hormone (α-MSH) [Coat color variation represent one of the first effects of domestication, where the selection for novel non-camouflage coat colors may have helped identification and tracking of animals in husbandry, differentiation of domesticated animals from wild herds, or be explained by human attraction for novelty . Experimean dogs . Only dotication . While ttication . Potenti (α-MSH) .MC1R found in prehistoric dogs to better understand its frequency and distribution in modern dog breeds. To further unravel the potential influence of R301C, coat color variant genotypes were correlated with the dog’s actual coat color phenotype from photos provided by dog owners.The aim of this study was to utilize the potential of commercial genetic panel screening to genotype large numbers of dogs for the presence or absence of the R301C variant of MC1R in today’s canine population, 11,750 dog samples were genotyped as a part of a custom-designed microarray panel test commercially available as MyDogDNA™/Optimal Selection™ Canine Genetic Breeding Analysis. The R301C variant was present in a total of 265 tested dogs representing 35 different breeds and breed varieties as well as mixed breed dogs. The allele frequency for R301C in all dogs representing 304 different breeds and mixed breeds was 1.5% , EG (grizzle/domino) and e1 (recessive red) alleles of the MC1R gene. Two rare additional recently characterized e allelic variants [e2 discovered in Australian Cattle Dog and e3 discovered in Siberian Husky were not genotyped as a part of this study. The R301C variant and the tested E locus variants showed no linkage disequilibrium in the 262 dogs with diverse breed background in which it was found present. The R301C variant was not present in dogs with two copies of the tested E locus variants; EM, EG or e1, while in dogs with two copies of the R301C variant no EM, EG or e1 variants were present. Also, no more than one copy of EM or e1 variants was present when one copy of R301C was found. The rarest MC1R coat color variant, the EG allele, is only found in one of the dog breeds, Kritikos Lagonikos, in which R301C was identified. However, in this study sample no individuals carrying both EG and R301C variants were identified.To elucidate the relationship of R301C and other known E locus variants, genotypes were obtained for E. As our findings suggested that R301C rather represents an independent alternative allele at the E locus, we refer to it as eA (for ancient red e) for clarity hereafter.Notably, using current conventional practices for calling of E locus genotypes at commercial genotyping laboratories, dogs carrying R301C would have been interpreted as carrying eA, on the dog’s coat color, also genotypes for Canine Beta-Defensin 103 (CBD103) and Agouti Signaling Protein (ASIP) were obtained for the analysis. Color phenotypes were available for 125 (47%) dogs of the 265 dogs identified with one or two copies of the eA allele in this study. Phenotyping using dog owner-provided photos initially focused on the Tamaskan Dog breed, which represented 35% (N = 43) of the dogs with phenotype information available. These dogs were carefully examined to elucidate the potential phenotypic effect of eA within a single breed. The expected coat colors for these dogs were aw wolf sable, at tan point or a recessive black determined by their A locus (due to only wild type variant ky being present on the K locus of these dogs). A maximum copy number of two allelic variants at the A locus was found in any of the Tamaskan Dogs and no ay variant, indicating that no A locus anomalies were present as recently observed in a small number of other dog breeds [EG domino in Afghan Hound and EG grizzle in Saluki. The aforementioned have been suggested to be dependent on the A locus at/at genotype for their manifestation [EG on divergent breed backgrounds , or compound heterozygosity of eA with e recessive red (N = 13), showed statistically significant association with domino phenotype (P = 2.37− 12)  with the eA allele present in homozygous form (N = 35) or in heterozygous form paired with the recessive red e1 allele (N = 35). These phenotypes manifested in dogs with eA/eA and eA/e1 genotypes as follows. All seven dogs with eA/eA or eA/e1 and KB  on the K locus express a non-solid and non-striped eumelanin shade phenotype (Table kbr brindle pattern is observed), the phenotype is clear fawn and virtually indistinguishable from recessive red e1/e1   or E/eA (N = 37) strongly suggesting an allelic hierarchy in which eA is recessive to EM and E and dominant to e, while further information on phenotypes produced by EG/eA genotype remains to be collected  the ability to produce eumelanin is reduced resulting in coat color patterns known by the names “domino”, “grizzle” and “pied” depending on the breed background, but may also result in phenotypes indistinguishable from recessive red (cream), tan point or wolf sable.Taken together, phenotype data available in 15 different breeds consistently shows that MC1R gene polymorphisms have been documented and associated with pigment variation in human and domestic animals, and several coat color associated MC1R polymorphisms have already been identified in dogs. The goal of this study was to screen for the presence of an ancient R301C variant in today’s dog population and determine if it has any effect on coat color phenotypes putatively selected for during the early stages of domestication. The R301C variant of MC1R was first identified in over 10,000-year-old prehistoric dog DNA samples as a variant absent from wolf DNA samples [Various  samples . Our stuMC1R, CBD103 and ASIP genes with phenotype information from photos provided by the owners of the tested dogs. R301C was not found to be linked with any of the tested E locus variants . Phenotype analysis further suggests that the R301C variant is a novel reduced-function allele at the E locus, recessive to EM and wild type E, but dominant to the e allele. Further support for genetic causality is provided by the association of the identical R301C mutation in MC1R with light coat color in two additional species; alpaca and Arabian camel [MC1R [eA, impacts phenotypes regulated by the K and A loci. We observed a loss of solid eumelanin shade in KB dogs and receding eumelanin expression in color patterns produced by the A locus, and note that eA behaves as a partially recessive red variant. The most recognizable eA produced phenotype pattern in aw wolf sable, at tan point and also in some ay fawn dogs has been referred to as ‘Husky domino’ seen in Alaskan Malamute or Siberian Husky, but the molecular cause for this phenotype has remained unknown until now. Also, we now demonstrate that eA is producing the coat color phenotype called “pied” in Beagle. For eA phenotype to be expressed this variant allele of E locus needs to be present in homozygous form or in heterozygous form with e as the second allele. We observed two Siberian Huskies manifesting a domino phenotype while carrying one copy of eA and no other tested E locus variant, which according to current conventions would be interpreted as wild type E being the second allele at the locus in these dogs. Although we did not have DNA sample availability to test for the presence of the recently discovered additional e alleles, e2 and especially e3 discovered in Huskies [eA/e3 genotype is the most likely explanation for the domino phenotype in these dogs.Phenotype analysis combined known coat color variant genotypes for an camel , 22. A san camel . The R30el [MC1R , 25. We eA, where “e” is chosen for its partial recessive red identity and “A” is denoting “Ancient” that is recessive to E wild type and dominant to the e1–3 alleles. We propose an updated dominance hierarchy at the E locus; EM > E > eA > e1–3, while acknowledging that we could not identify any dog with the EG/eA genotype and thus, the phenotype impact of this specific rare genotype combination remains unexplored. Interestingly, the previously identified domino variant EG is almost exclusively observed in breeds in which eA is not detected [EG and eA allele present in only one breed, Kritikos Lagonikos; a primitive hunting dog originating from the Greek island of Crete. Phenotype analysis of this rare breed might help to resolve the phenotypes presented by rare allele combinations, for which conclusions could not be made through this study.We propose that the newly characterized reduced-function variant R301C is designated detected . In thiseA associated patterning is an characteristically increased expression of pheomelanin and decrease in the level of eumelanin expression, indicating that eA represents a reduced-function variant of MC1R as postulated [e1–3 represent loss-of-function variants of MC1R enabling expression of pheomelanin only when present in two copies. Consequently, e/e dogs always only express pheomelanin pigment regardless of variants present at the K locus and A locus, whereas the R301C variant eA results in reduced MC1R function masking expression of the K locus in dominant black dogs while pigmentation phenotypes correlated with the expression pattern of the A locus. Logically on the other hand, increased pheomelanin expression is observed in dogs expressing the A locus due to the effects of eA. We propose that the eA variant is a partial loss-of-function variant not previously known to be present in dogs, which furthermore provides novel insights into the relationship of variants at the E, K and A loci. The dominant allele of the K locus  needs aeA/eA or eA/e1 genotypes, two copies of the reduced- function allele eA could allow a bit more eumelanin to be expressed than when the eA allele is present with a loss of function allele e1. We observed a Drever with eA/eA genotype expressing normal tan points with only reduced pigment in the centerline of the head (white star) and nose (dudley nose), respectively. Outside of this study sample, we have further observed full domino pattern present in this dog’s offspring with eA/e1 genotype. The most depleted eumelanin expression was observed in two Hungarian Hounds with eA/e1 genotype and at/at genotype for tan points in the A locus; these two dogs were essentially indistinguishable from recessive red individuals of the same breed. In addition, it should be noted that the domino pattern in all ay fawn dogs is not clearly visible and their phenotype is similar to recessive red.While partial recessive red phenotypes are produced by MC1R polymorphisms, where reduced-function variants are present in both Europeans and Asians. It is commonly believed that these variants represent adaptation to higher latitudes enabling sufficient synthesis of D-vitamin under lower solar radiation, while the population-genetic analysis suggests relaxed functional constraints out of Africa and South-Asia to explain the MC1R polymorphisms in human [MC1R polymorphisms are unlikely to associate with D-vitamin intake. A more plausible explanation for increase in frequency of a phenotype changing variant early in the domestication process is positive selection of novel traits applied by humans [eA being the major variant in some old Spitz and Hound type dogs bred mostly for utility suggests that R301C may have once been a common variant in the domesticated dog population that has become eradicated in more modern breeds to enable manifestation of other coat color phenotype associated variants.Substantial variation in pigment phenotypes in human populations is also explained by in human . In dogsin human , MC1R poy humans , 16. ConMC1R have also been investigated in the response to pain, analgesia and anesthetics [e1/e1reduced-function variants in human genotype has been shown to prevent clinical signs of disease in dogs carrying causal variant causing Squamous Cell Carcinoma of the Digit (SCCD) [MC1R is not only central to determination of pigment phenotype. Besides its role in stimulation of eumelanin synthesis to protect skin from UV radiation and DNA damage, MC1R has a physiological role in vascular homeostasis and cell migration , erythrosthetics –44. Moret (SCCD) . FurtherMC1R gene associated with reduced eumelanin pigment that potentially represents one of the earliest mutations enriched by canine domestication still present in the dog population. This ancient E locus variant (MC1R p. R301C), which we have designated as eA, is recessive to EM and E alleles of the E locus and dominant to the e allele. The genotypes eA/e and eA/eA result in phenotypically reduced expression of eumelanin, and these genotypes exhibit partial epistasis over the A locus expression pattern and epistasis over the K locus.In summary, genotype to phenotype correlation characterizes a novel allele of the E locus, caused by an old polymorphism in the This study represents a large genotype screening effort of pet dogs, aiming to identify the presence of and understand the potential effect of one of the earliest mutations captured by canine domestication. It underscores the crucial role of dog owners in citizen science and more specifically in supporting studies aiming to elucidate the genetic background of trait phenotypes. The present discoveries could only have been made by comprehensive screening of coat color variants across a large number of breeds and individuals, in combination with the openness of dog owners to submit pictures of their dogs for research purposes. In conclusion, our findings explain the non-eumelanin coat color phenotypes observed in some dogs despite presence of the dominant black allele on the K locus, and identify that the same molecular cause explains the coat color phenotype commonly referred to as “domino” in Alaskan Malamute and other Spitz breeds, “grizzle” in Chihuahua, and “pied” in Beagle.N = 11,750) consisted of non-invasive cheek swab samples collected by dog owners, and either blood or cheek swab samples collected at certified veterinary clinics in accordance with international standards for animal care and research as a part of voluntary submission of samples to commercial DNA testing. In addition, the dog owners provided consent for the use of their dog’s DNA information for research purposes. The samples were submitted for MyDogDNA / Optimal Selection analysis at Genoscoper Laboratories  and Wisdom Health (formerly Mars Veterinary) between April 3rd, 2015 and June 23th, 2020. Most of the tested dogs were from Finland  and the United States . The other major subgroups were formed by dogs from the Netherlands , Denmark , Austria , France , UK ,, Sweden  and Australia . Most of the tested dogs were from breeds recognized by Fédération Cynologique Internationale (FCI) or American Kennel Club (AKC), and the breed of the dog was reported by its owner with accompanying registration information. A few additional breeds not yet recognized by any major breed registry but with an established number of breed hobbyists, and mixed breed dogs, were also included in the study sample. Altogether, it amounted to 304 breeds and breed varieties, and 391 dogs representing the mixed breed population.The study sample  was evaluated with the Freeman-Halton extension of Fisher’s Exact Probability test for a 2 × 3 table [eA and an EM variant were combined with dogs carrying eA with an E variant due to only one individual present in the aforementioned category.The statistical significance of differences in the distribution of  3 table . Dogs wiAdditional file 1 Table S1. Allele frequency of R301C variant in the study sample composition.Additional file 2 Table S2. Phenotype analysis of 125 dogs with known genotype for MC1R, CBD103 and ASIP loci.


Complement, a part of the innate arm of the immune system, is integral to the frontline defense of the host against innumerable pathogens, which includes RNA viruses. Among the major groups of viruses, RNA viruses contribute significantly to the global mortality and morbidity index associated with viral infection. Despite multiple routes of entry adopted by these viruses, facing complement is inevitable. The initial interaction with complement and the nature of this interaction play an important role in determining host resistance versus susceptibility to the viral infection. Many RNA viruses are potent activators of complement, often resulting in virus neutralization. Yet, another facet of virus-induced activation is the exacerbation in pathogenesis contributing to the overall morbidity. The severity in disease and death associated with RNA virus infections shows a tip in the scale favoring viruses. Growing evidence suggest that like their DNA counterparts, RNA viruses have co-evolved to master ingenious strategies to remarkably restrict complement. Modulation of host genes involved in antiviral responses contributed prominently to the adoption of unique strategies to keep complement at bay, which included either down regulation of activation components  or up regulation of complement regulatory proteins. All this hints at a possible “hijacking” of the cross-talk mechanism of the host immune system. Enveloped RNA viruses have a selective advantage of not only modulating the host responses but also recruiting membrane-associated regulators of complement activation (RCAs). This review aims to highlight the significant progress in the understanding of RNA virus–complement interactions. The complement system (CS) is evolutionarily ancient and is a prime component of the innate immunity arsenal, capable of targeting a wide range of pathogens . Compris2O) by a mechanism known as the “tick-over” helps maintain basal levels of AP. C3(H2O) thus formed further binds to factor B (FB) and factor D (FD). FD, being a serine protease, cleaves FB into Ba and Bb, resulting in the formation of C3(H2O)Bb, which, like C4b2a, cleaves C3 to form C3b and C3a. The nascent C3b formed can associate with FB and, in the presence of FD, can form C3bBb, the AP C3 convertase  . These pInvestigation into virus-complement interactions had garnered much interest in the past, and this interest has only grown over the years to better comprehend the nature of these interactions. The knowledge acquired has enriched our insight into not only viral pathogenesis but also the underlying mechanisms of host immunity to these pathogens. Among animal viruses, RNA viruses constitute a large group of both veterinary and clinical significance. Based on the characteristics of the genetic material, they are classified into single- or double-stranded RNA viruses of positive or negative strand polarity that is either segmented or non-segmented. Human mortality of unprecedented proportions have been brought about by RNA viruses, the well documented being the pandemic H1N1 outbreak of 1918 (Spanish flu) that killed humans in millions . More reRNA viruses have limited genome yet are pathogenic enough to cause devastating infections in both humans and animals. The CS that acts as a frontline defense against several pathogens is a potent barrier faced by RNA viruses . Many ch–/– mice  compared to the pandemic virus suggest the preference of C3b for certain carbohydrates and its specificity to certain binding sites on them. The alteration in the C3b acceptor site on the surface glycoproteins, HA, and NA on the pandemic virus requires IgG to mediate the binding of C3b on them. Antagonists of C3a and C5a, and the corresponding receptor knock-out mice, showed that C5a played a less significant role in limiting the influenza A(H1N1)pdm09 virus infection (Orthomyxoviruses are enveloped viruses and the two major surface proteins hemagglutinin (HA) and neuraminidase (NA) activate complement to varying degrees, with HA being the better activator . Normal –/– mice . The rolnfection .Henipavirus of the Paramyxoviridae family are deadly BSL4 pathogens. Nipah virus has two major glycoproteins, the fusion protein (F), and the glycoprotein (G). Using VSV-based pseudoviruses expressing F and G glycoproteins, it was demonstrated that these glycoproteins activate the AP. However, when the pseudotypes were primed with specific antibodies, and more interestingly with the soluble EphrinB2 (the cognate receptor for NiV) linked to the Fc region of human IgG1, the CP was found to be predominantly activated  and Hendra (HeV) viruses belonging to the genus ctivated . Among octivated –28. Natuctivated . This clctivated . Newcastctivated . Apart fctivated , 31. Meactivated . Measlesctivated . An antictivated . Human rnfection . An antinfection , 35. HeLnfection , 37. Thenfection .flavivirus antibodies results in marked reduction of ADE in an IgG subclass-dependent manner  of WNV . This family of viruses is distributed globally and can cause a wide range of human illnesses including dengue hemorrhagic fever (DHF), yellow fever, and West Nile encephalitis. The LP plays a critical role in the neutralization of HCV, wherein MBL binds directly to the E1 and E2 surface glycoprotein and activate LP by engaging MASP-2 . The NS1t manner , 43. Amot manner . West Ni) of WNV . The neu) of WNV , 46. Whi) of WNV –49. Reco) of WNV , 51. Int) of WNV , 50, 52.nfection . Among tnfection .Togaviridae is a family of enveloped, positive-sense single-stranded RNA viruses distributed globally. This family consists of two genera, namely, Alphavirus, and Rubivirus. Extensive studies on Sindbis virus of the Alphavirus genus showed that while the virus was capable of activating both the AP and CP, the degree of activation of AP depended on the levels of sialic acid, with low levels being a better activator of AP , a prototypic member of the genus vesiculovirus, was found to activate the CP of complement either in an antibody-dependent or -independent manner. Natural IgM present in the serum was found to enhance the complement-mediated neutralization by opsonization , an enveloped retrovirus, is known to activate the CP and LP independent of antibody, with direct binding of C1q to gp41 acting as a trigger for CP activation –66. Intein vitro studies demonstrated that RRV could not independently activate the AP or CP  of SARS-CoV to the carbohydrate recognition domain of MBL resulted in complement activation and C4 deposition on virus-infected cells. The residue on the S protein that binds to MBL was identified as the N-linked glycosylation at N330 , complement activation caused significant damage to the host. Complement activation markers C3a and C5a (potent anaphylatoxins) were found to promote vascular leakage, contributing directly to DSS , 84–89. reversed . The memreversed .Human immunodeficiency virus-associated neurocognitive disorder (HAND) can be due to either HIV encephalitis (HIVE) or mild neurocognitive impairment (NCI), for which the neuropathological substrate has not been established. Earlier studies have suggested that the central nervous system (CNS) dysfunction in HIV infection is due to indirect effects rather than neuronal or glial infection . The expin vivo, and treatment with small-molecule inhibitors against C5aR exhibited antifibrotic effects  and serum from patients infected with H1N1 pandemic virus suggest their role in virus-induced acute lung injury (ALI). The increased expression of C5aR on dendritic cells (DCs) promoted CD8the lung . C5a-medthe lung , 112. C5the lung . Studiesthe lung . Treatmethe lung . Increasthe lung . The C3dnfection . The rolnfection .Increased viral load in the brain of mice infected with WNV, in the absence of C3 or CR1/CR2, suggested their importance in limiting viral infection. Deficiency in the components of the CP (C4 and C1q) was associated with decreased splenic infections and impaired B and T cell responses to WNV infection as C4 generated anti-WNV IgM responses, while C4 and C1q were required for anti-WNV IgG responses . In mousin vivo  coat protein with C1q, an upstream component of CP, mediated the dissociation of C1s from the protease tetramer. This arrested the C1s cleavage, limiting the progression of the pathway, resulting in the decreased levels of C3b and C4b. The coat proteins of at least two other serotypes, HAstV-2 and HAstV-4, were also found to inhibit complement activation, suggesting that this inhibition was not restricted to HAstV-1 alone . FurtherFlaviviridae has a unique mechanism of modulating the CP. The NS 3/4A, a non-structural protein of HCV with protease activity, could directly bind and cleave the γ-subunit of C4, abrogating the effect of complement on the virus  of influenza virus (A/WSN/33 strain) is an abundantly expressed protein in virus-infected cells and is released during necrosis to protect the newly formed virus particles from complement-mediated neutralization. The N-terminal domain of M1 binds to the globular head of C1q, blocking the interaction of IgG and C1q and thereby the CP .The main role of proteins associated with all the three complement pathways is to target pathogens including viruses, which, upon activation, facilitate virus neutralization. In many instances, these proteins have been found to be exploited by viruses to gain entry into cells, to replicate and utilize such cells as reservoirs, and to enhance virus spread. Many viruses preferentially exploit upstream components of the CP and LP like C1q and MBL to enhance infectivity and spread. The NS1 proteins of flaviviruses WNV, DENV, and yellow fever virus (YFV) have been shown to directly interact with C4, limiting the CP and LP. The NS1 protein directly binds to C4 and recruits C1s, the protease in the C1 complex, forming the C4–NS1–C1s complex that promotes active sequestration of C4, protecting the virus from the neutralizing effects of complement . InteracMannose binding lectins were found to enhance the infectivity of a range of RNA viruses including WNV, NiV, Hendra virus, and EBOV. Using EBOV glycoprotein pseudotyped lentivirus, it has been demonstrated that specific epitopes on the N-linked glycans of EBOV glycoproteins bound specifically to the carbohydrate recognition domain on MBL. Enhancement in virus uptake and infection due to MBL binding was found to be dependent on C1QBP (gC1q receptor) . The binRNA viruses have also been shown to down regulate the synthesis of complement proteins. HCV effectively puts a block in the progression of the complement pathway by transcriptionally regulating expression of critical components like C3, C4, C2, and C9 –153. AnaEnveloped RNA viruses acquire their envelope from the host during the process of virus egress. This, in many instances, turns out to be advantageous for the virus, as it also picks up the host proteins associated with the membrane, which can confer protection from the host complement. Widely reported host membrane-associated RCAs in the virus envelope include CD46, CD55, and CD59. RNA viruses including HIV-1, PIV5, MuV, NDV, and VSV are known to incorporate these RCAs into their envelope , 158. ExHuman immunodeficiency virus 1 incorporates CD46, CD55, and CD59 into the virus envelope during egress. The presence or absence of the RCAs in the virus envelope depended on the cell type in which the virus was generated. The levels of RCA incorporated into the virus envelope dictated the extent of neutralization. When sensitized with gp160 antibodies, only those viruses generated in cell lines underwent virolysis; however, primary HIV isolates required sensitization with anti-RCA antibodies to undergo virolysis by complement. Besides the primary isolates of HIV-1 and specific cell line-derived HIV, (SIV delta/B670) was also found to incorporate all three RCAs. The protective role of the RCAs in limiting the effect of complement on HIV is well evidenced from the findings that HIV cultured in CHO cells with individual RCAs was significantly protected from complement-mediated virolysis compared to those without the regulators , 161–163RNA viruses not only incorporate membrane bound RCAs but also recruit soluble RCAs. The NS1 protein of DENV, WNV, and YFV were all found to recruit C4BP, an RCA of the CP , while oMost of the evasion strategies described above involved recruitment of either membrane-associated or soluble RCAs and inhibitors of complement. Factor I is an important and unique serine protease involved in the regulatory arm of the complement. Factor I mediates the inactivation of C3b into iC3b or C3c and C3d, and C4b into C4c and C4d in the presence of specific cofactors. Earlier we had discussed that NiV exhibits an fI-like activity that could inactivate C3b but not C4b. Only the first order of C3b inactivation, which is the conversion of C3b into iC3b, was supported by the NiV-associated fI-like activity. Conversion of C3b into iC3b occurred with the incubation of C3b, fH, or sCR1 and wtNiV without the addition of fI. CD46 was also found to be associated with wtNiV, but interestingly, the associated CD46 was incapable of inactivating C3b into iC3b even with the addition of fI . More rePicornaviridae family. Many subtypes of echoviruses like echo-6, -7, -11, -12, -20, and -21 bind to CD55 to gain access into the cell  or the mucosal surface. It has been well established that the CS is active at these local sites of infection , 3. SurfThe big question that arises out of RNA virus–complement interactions is how these viruses productively infect and cause diseases in the host, overcoming complement. Both enveloped and non-enveloped RNA viruses have ingenious ways of limiting complement. Many DNA viruses including poxviruses and γ-herpes viruses have a large genome that supports expression of viral homologs that mimic the functions of complement regulatory proteins. RNA viruses might appear at a disadvantage with respect to its genome size; however, they are known to thwart complement in multiple ways. One well-studied mechanism is the recruitment of the host RCAs by enveloped RNA viruses. The protective effect of these RCAs on the virus is very significant, especially at the local sites of infection. Another area requiring significant investigation is the nature of incorporation of the RCAs. Detailed investigation is required to further understand the specificity or selectivity of incorporation of these RCAs.The evasion strategies adopted by RNA viruses bring the CS in the cross hairs of the virus. It is clear that the balance is quite delicate and can tip to any side, as is evidenced in the case of flaviviruses like DENV and more recently in SARS-CoV-2. Approaches to combat SARS-CoV-2, although not proven completely, include targeting complement activation to better manage the disease symptoms. Exploitation of RNA viruses as vaccine and oncolytic vectors is an emerging field. Viral vectors should be attenuated enough so as to not cause disease but yet be capable of productively infecting the target cells. An attenuated vector harboring RCAs would be a better candidate to bypass complement yet meet the requirements. Thus, a rational approach to develop therapeutics, inhibitors, or viral vectors should also take the role of complement into consideration. In conclusion, studies have highlighted the complexities in RNA virus–complement interactions, but with emerging viruses, a constant pursuit is required to better understand these interactions.NK, UK, and JJ conceived the concept for this review article. NK, UK, ST, and JJ wrote the manuscript. NK, UK, ST, and JJ read, edited, and reviewed the manuscript. All authors contributed to the article and approved the submitted version.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.