Often tumors are eventually observed at poultry slaughter, leading to condemnation, not of zoonotic, but because of esthetic reasons. Most avian tumors are caused by infection with oncogenic viruses, where the most prominent is a herpesvirus, Marek’s disease virus (MDV). MDV-infections are economically important in the poultry industry as they cause immunosuppression, decreases in productivity and profitability and increased morbidity, mortality, and condemnation at slaughter. MDV was discovered about 50 years ago, and still its study is ongoing until now. This review is presenting the MDV biology, and protection by vaccination, but also topics that were less brought to wider knowledge, like the vaccine and virulent virus presence in feathers, and their use in assessing the efficacy of vaccines application in commercial settings. In addition, two relatively novel topics are reviewed, like the meq MDV oncogene and its recent implication in molecular epidemiology and MDV virulence determination, and second, the importance of native, three-dimensional conformational epitopes of the immunodominant glycoprotein B and the molecular recombination between DNA-DNA and DNA-RNA viruses. Our studies were particular, as they were originally describing how the native virion is activating the chicken immune response to create virus-neutralizing antibodies. The topic might shed light on items to be focused on in future searches for effective vaccines. Marek’s disease virus is an economically important avian herpesvirus that causes tumors and immunosuppression in chickens and turkeys. The virus, disease, and vaccines have been known for more than 50 years, but as knowledge gaps still exists, intensive research is still ongoing. The understanding of MDV complexity can provide scientific insight in topics that cannot be experimented in humans, providing a unique model that is dually useful for the benefit of the poultry industry and for studying general herpesvirology. The present review presents the following topics: the MDV biology, the vaccine’s and virulent virus’ peculiar presence in feathers, protection by vaccination. In addition, two relatively behind the scenes topics are reviewed; first, the meq MDV oncogene and its recent implication in molecular epidemiology and in the MDV virulence determination, and second, the functionality of conformational epitopes of the MDV immunodominant protein, glycoprotein B. Our studies were particular, as they were the only ones describing three-dimensional MDV gB oligomers. MDV gB (glycoprotein B) continuous and discontinuous epitopes were shown to possess distinctive neutralization activities. In contrast, the significance of oligomerization of the viral membrane proteins for the creation of discontinuous epitopes in other herpesviruses was explored extensively. Viral induced tumors in poultry are economically important for the poultry industry because they cause decreased productivity and profitability, due to increased morbidity and mortality of birds during growth, and increased condemnation at slaughter. In the study of avian tumors, attention has been focused on those of viral etiology. Studying avian tumorigenesis is important not only from the standpoint of its economic importance, but also as a potential model for tumorigenesis in humans ,2. The aGallid alphaherpesvirus 2 , the avirulent Gallid alphaherpesvirus 3 (GaHV-3 or MDV-2) and Meleagrid alphaherpesvirus 1 (MeHV-1 or herpesvirus of turkey (HVT)), respectively, according to the latest ICTV nomenclature.The avian oncogenic viruses include the herpes virus, Marek’s disease virus, and four retroviruses. The herpes virus is the DNA double stranded Marek’s Disease Virus (MDV) that causes tumors in chickens and turkeys ,4,5,6,7.The IS in commercial poultry is economically important because of decreased productivity, increased mortality, fruitless investments in veterinary medical assistance and treatment, as well as increased susceptibility to infection by various pathogens and decreased efficacies of various vaccines . The MDVMDV was the first and most extensively studied virus regarding its presence in feathers ,13,14,15The feature of MDV replication in feather follicle epithelium motivated numerous studies that utilized the feathers to detect and isolate the virus. For first time we demonstrated the kinetics of MDV shedding by molecular amplification , later bMDV was the only poultry live vaccine whose vaccination uptake efficacy was studied by demonstrating its presence in feather tips. The presence of live vaccines was demonstrated in the feathers of experimentally infected and commercially vaccinated layers in Australia ,24,25. BThe worldwide intensification process of the poultry production, which occurred in the last 50 years, led to a dramatic increase in the incidence of MDV infections, where the tumors and mortality became a very costly component for the industry. As a result, researchers looked for effective vaccines that would protect poultry from mortality and could avoid condemnation at slaughter due to tumors. Avirulent viruses have been adapted to serve as effective vaccines against virulent MDVs, and prevention of disease. In that regard, the MD comprises a unique example in nature, where a malignant disease that is caused by a herpesvirus, can be effectively controlled by vaccination using naturally isolated avirulent MDVs.The first MDV vaccine, which was developed by Kawamura et al.  and WittSchat and Calnek  isolatedAlthough MDV vaccines have been successful in protecting chickens against tumors and mortality, the MDV vaccines do not provide sterilizing immunity, and vaccinated chickens still can support replication and shedding of virulent viruses ,15. The meq gene by Dr. H.-J. Kung and collaborators in 1992 [meq gene is an oncogene, encoding a leucine-zipper DNA-binding protein vvMDV and in vv+MDV pathotypes. The meq protein appears in two configurations, a 339 amino acid unspliced open reading frame and a larger form of meq of 398 amino acids, resulting from an in-frame insertion of 180 bp and having multiple duplications of a C-terminal proline rich repeat [meq leucine-zipper protein has a trans-activation N-terminal basic leucine-zipper domain and a C-terminal proline-rich trans-repression domain [meq gene activities are mediated by its dimerization with itself, as well as with c-Jun like proteins such as JunB, c-Jun, and c-Fos. The meq gene C-terminus binds also to cellular transcription factors that are involved in cell division and cycle regulation, such as SNF, ATF, CREB and HB-EGF [meq protein interacts also with proteins without a leucine zipper domain, such as the cellular tumor suppressors p53, retinoblastoma gene, the cyclin-dependent kinase 2, and the heat shock protein Hsp70 [meq N-terminal portion contains a “PLDLS” amino acid motif that interacts with CtBP co-repressor terminal binding protein-1, forming a meq-CtBP dimer [The innovative discovery of the MDV  in 1992  establish repeat . The meqn domain ,39,40. Tin Hsp70 ,42,43. TBP dimer ,42,43,44meq encoded oncoprotein, the meq encoded major oncoprotein, and transcription factor. Despite the rather low evolutionary rate of double-stranded DNA viruses [meq gene is evolving at a much faster rate than most genes in double-stranded DNA viruses [meq gene sequences not only evolved at a much faster rate than most dsDNA viruses, but its evolutionary rate was comparable to the evolutionary rate of RNA viruses, including the influenza virus. By analyzing the meq gene sequences of 84 MDV strains, Padhi and Parcells [−4 mutations per nucleotide per cycle of virus replication (mut/nt/rep), as compared to the range of 10−5–10−7 mut/nt/rep for herpesviruses. Moreover, the meq gene evolutionary rate is much higher than other viral genes in MDV.The strongest association with the observed increased virulence is the polymorphisms identified at the C-terminal domain of the  viruses ,46,47,48 viruses ,50, conc viruses ,50. Padh viruses  confirmeParcells  estimatemeq gene sequences enabled Shamblin et al. [meq gene sequence was confirmed by Renz et al. [meq gene sequences enabled the performance of epidemiological studies that proved useful in revealing epidemiological linkages between various field strains in numerous countries, like China [meq gene sequence in commercial flocks was provided in the recent studies on Italian commercial chicken flocks affected with MD [The great divergence of the n et al.  to charaz et al. . In addike China , India [ke China , Poland ke China  and Egypke China . Recent  with MD  and on M with MD .meq gene sequence revealed the geographical restriction and geographically dependent evolution, as demonstrated by Trimpert et al. [In addition to the epidemiological information, the t et al. , who expmeq gene is highly advantageous as compared to the classical gold standard method of pathotype determination [meq gene transactivation domain are an indicative marker for the pathogenicity of MDV strains isolated from chickens; the most virulent isolates showed the lowest number of PPPP repeats, unlike the attenuated and the low pathogenicity isolates, which showed a highest number of repeats, suggesting that the meq gene configuration is indicating the degree of GaHV-2 attenuation [meq gene is greatly influencing the viral telomerase RNA expression via the cellular myc gene.The nucleotide-based classification of MDV virulence based on their mination . The golmination  developeenuation ,52,57. Cenuation  providedmeq protein can physically, and potentially functionally, interact with the chicken anemia virus (CAV) apoptin protein transactivating the CAV transcription [meq protein interacts and inhibits the CAV apoptin protein, probably promoting cellular transformation and tumor formation.Recently Brown et al.  reportedcription . CAV is cription . The molThe biologically active membrane glycoproteins are recognized among the herpesviruses as the immunogenic proteins, which are important for the virus antigenicity, cell-attachment, virus-cell membrane fusion and other specific activities. The antigenic epitopes of the immunodominant antigens of MDV were characterized by immunoblotting using convalescent chicken sera from MDV-affected and tumor-bearing commercial birds and include the glycoproteins A (gA) and B (gB) . GlycoprThe similarity between MDV and HSV-1 gBs in their high molecular mass oligomeric conformation and the conservation of gB functions among the various herpesviruses prompted us to explore their antigenic relationship. Indeed, the MDV and HSV-1 gBs share two-ways cross-activities ,68. We dMore advanced studies unveiled the biological activity of MDV gB in its heat-stable and heat-labile configurations in inducing the production of virus-neutralizing antibodies. The virus neutralizing activity was demonstrated using monospecific antibodies to the heated and to the unheated oligomeric MDV and HVT gB ,69. The The demonstration that the MDV gB exists as heat-labile oligomers in its native form resembles the presence of herpes simplex virus 1 (HSV1) gB as a native conformational entity on the infected cell membrane. Membranal HSV gB was described to consist of heat-labile multiple oligomeric species in the 200–300 kDa range  or as diIn contrast to the vast body of studies on the biological properties of discontinuous conformational epitopes of human herpesviruses, the investigation of continuous and discontinuous antigenic epitopes important for viral infection and protection of veterinary herpesviruses is still limited. Our studies conveyed the only description of MDV native epitopes of the immunodominant proteins. In that respect, at present continuous and discontinuous epitopes were identified only on the bovine herpesvirus type 1 gB . SimilarIn retroviruses, a similar situation regarding the state-of the art of the contribution of 3D antigenic epitopes in virus biology was described only for the reticuloendotheliosis virus , in contMultiple viral infections of chickens involving MDV may enable genetic exchanges between the co-infecting viruses. The events involving MDV and an additional DNA virus include three studies in chickens. The first study describes molecular recombination between the fowlpox virus (FPV)  and MDV MDV is involved in molecular interactions between DNA and RNA viruses. Integration of retroviral sequences into the herpesvirus genome was documented in vitro by co-infecting CEF cultures with MDV and the retroviruses reticuloendotheliosis virus (REV) and avian leucosis virus (ALV) ,89,90,91Having experienced the relatively efficient creation of recombinant viruses in vitro, we questioned whether retroviruses also integrate into DNA viruses in vivo, in birds, during multiple viral infections. If such process would occur, serious consequences might follow; recombinant MDV might possess altered biological properties, and relatively known features associated with infection by these viruses may be altered resulting in unknown and unpredictable patterns of disease. Putative features, where changes might be of biological significance such as, pathogenicity, virus spread, antigenicity and immunogenicity. Changes in the latter two may lead to changes in the ability of specific vaccines to protect against diseases. We analyzed commercial birds that had acquired naturally mixed infections, chickens infected experimentally with prototype strains of MDV and ALV-J and commercial chickens infected experimentally with virus obtained from cases of double infection with MDV and ALV-J in the same commercial flock or the same bird from that flock . The maimeq gene diversity for the determination of the MDV virulence and molecular epidemiology is reviewed. For the first time, we review the relatively unknown studies showing the importance of the conformational epitopes of the immunodominant MDV gB in virus neutralization, as well as their serotype- and strain-specific activity, that is a topic that did not receive much attention regarding avian herpesviruses, in general, and particularly regarding GaHV-2. In addition, the molecular recombination between DNA-DNA and DNA-RNA viruses was also highlighted.In summary, we highlighted several aspects of the MDV biology, with the awareness that the ground is much wider, and that in each publication different angles are enlightening. Specifically, the recent development of the novel approaches to monitor the vaccine application efficacy using feathers and the use of the


The characterization of by FTIR spectroscopy, X-ray photoelectron spectroscopy (XRD), thermogravimetric analysis (TGA), energy-dispersive spectroscopy (EDS) and scanning electron microscopy (SEM), demonstrated an efficient preparation of nanocomposites, with nanoparticles well-dispersed in the polymer matrix. In vivo, subdermal implantation of the beads in Wistar rat′s tissue for 90 days showed a proper and complete healing process without any allergenic response to any of the formulations. Masson′s trichrome staining of the histological implanted tissues demonstrated the presence of a group of macrophage/histiocyte compatible cells, which indicates a high degree of biocompatibility of the beads. The materials were very stable under body conditions as the morphometry studies showed, but with low resorption percentages. These high stability beads could be used as biocompatible, resistant materials for long-term applications. The results presented in this study show the enormous potential of these chitosan nanocomposites in cell regeneration and biomedical applications.Scaffold development for cell regeneration has increased in recent years due to the high demand for more efficient and biocompatible materials. Nanomaterials have become a critical alternative for mechanical, thermal, and antimicrobial property reinforcement in several biopolymers. In this work, four different chitosan (CS) bead formulations crosslinked with glutaraldehyde (GLA), including titanium dioxide nanoparticles (TiO Due to many losses of organs in people during sickness or accident events, tissue engineering has gained significant ground as researchers seek alternatives for scaffolding design that promote cell adhesion, proliferation, and differentiation. Scaffold design (including porosity and interconnection) are of great importance due to their influence in cell migration, attachment, and proliferation . A largeThe use of biopolymers is preferred over synthetic versions, although they have a lower cost and more straightforward synthetic modification to improve biocompatibility and cell adhesion . DespiteAmong biopolymers, chitosan (CS) is one of the most used polysaccharides, which has gained much attention for biomedical applications due to its biocompatibility and biodegradability. There are several applications of chitosan and nanochitosan preparations in tissue engineering, thanks to their excellent cell adhesion and low toxicity ,9,10,11.Chitosan was used for wound healing applications with reinforcement using different nanoparticles . A comprNanotechnology offers solutions to the inconveniences of low durability of biopolymers , for exa2) has been synthesized for a wide variety of applications [Titanium-related composites are preferred for biomedical applications due to their excellent biocompatibility, resistance to body fluids, mechanical properties, anti-corrosion capacity, and flexibility ,21. Howeications . For exaications ,25. 2 structure [Likewise, the use of graphene has also skyrocketed in recent years thanks to the new properties it exhibits like low thickness (one to ten layers), high flexibility, and strong-resistance supported by the σ type bonds in the carbon two-dimensional crystal lattice sptructure . This pltructure ,28 and dtructure ,30.Graphene also presents high stability due to its flat aromatic polycyclic structure . However2-carbon network [Chemical oxidation to produce graphene oxide, introduces hydroxyl (OH), epoxy (COC), and carboxyl (COOH) functions on the surface, reinforcing the ability to interact with other molecules chemically (such as chitosan), especially by hydrogen bonding. In parallel hydrophilicity is simultaneously introduced to the substantial hydrophobic nature of the sp network ,34. Hydr network . At the  network .In comparison to other nanomaterials, GO offers several advantages for biomedical applications such as low-cost and safer preparation with lesser accumulation of toxic metallic impurities. Also, the extensive surface area is accessible for molecular interactions, significant for a highly-efficient drug loading . BesidesThe antibacterial properties of GO are based on the bacterial cells’ membrane rupture caused by the sharp structure of GO and destructive extraction of lipid molecules, but also on the multiple-cell functions affected by oxidative stress of proteins, enzymes, and DNA that react with reactive oxygen species .GO introduction to scaffolds and biomedical devices also provides thermal and mechanical reinforcement. For example, GO is introduced in bone-cements for mechanical support . HoweverCS beads have been prepared to investigate their in vivo and in vitro biodegradation ,45,46. L2 systems which could combine the biocompatibility of the three components and the reinforcement effect under physiological conditions, to produce biocompatible and resistant components for long-term applications, have not been reported.Although some studies have focused on the development of chitosan composites/nanocomposites for biomedical applications ,49,50, tThe requirement for improvement in the stability and biocompatibility under physiological conditions to prolong the durability of the nanocomposites with three-dimensional porous structures remains latent. In the literature, there is still a strong need to determine whether the GO functionalization or combination with other components improves the biocompatibility, especially with long-term in vivo and toxicity studies .2 and both nanomaterials were synthesized and chemically and thermally characterized. At the same time, the biocompatibility was assayed by subdermal implantations in Wistar rat tissues for 90 days. Based on their morphology, biocompatibility, and long-lasting durability the present beads could be promising in long-term applications like bone-tissue engineering.In the present work, beads of CS incorporating GO, TiO2 nanoparticles were prepared according to a previously reported methodology [2 nanoparticles by TEM  corresponds to the E2g mode of sp2 hybridized carbons. On the other side, the D band is related to the breathing mode of the sp2 carbon rings, which is activated by the introduction of carbon functionalization (oxidation) and a higher amount of sp3 hybridized carbons [Raman spectroscopy A shows t carbons . Moreover, X-ray diffraction patterns of the GO B showed In general, a smooth morphology with low thickness was the average observation . However, some aggregates of 20–30 nm were observed due to the cumulation of some graphene layers . In general, a smooth morphology with low thickness  was observed. However, some aggregates of 20–30 nm are apparent due to the cumulation of some graphene layers . Still, −1, due to the O-H and NH2 overlapped bands [−1. At 2924 cm−1, the -CH stretching vibration of -CH and -CH2 was observed, while the -CH symmetric bending vibrations of -CHOH- are also apparent at 1373 cm−1. At 1149 and 1026 cm−1, the -CO stretching vibration in -COH is present. The new band at 1570 cm−1 for CS-GLA beads is due to the new C=C bonds formed after the crosslinking reaction between CS-GLA [−1 band could indicate that primary amino groups are the main responsible for the crosslinking reaction with GLA [−1 due to unreacted aldehyde groups. For the CS-GO-GLA, the broadening of the O-H band is due to the presence of the GO. However, there is no presence of bands due to the C=O stretching bond of the low amount of GO added. Finally, CS-TiO2-GLA and CS-TiO2-GO-GLA spectra show related groups with O-H band shifting to 3331 cm−1 and the broadening of the bands due to the O-H (3331 cm−1) and C-O-C bonds of the oxygenated groups (1026 and 1035 cm−1). ed bands . Amino gwith GLA . It is a2-GLA diffractogram  was calculated to understand better the crystallinity of the beads . Althoug2, which has a higher crystallinity as compared to CS beads, probably due to the intermolecular hydrogen bonding, between CS and the nanomaterial giving a relatively ordered adjustment of the polymer chains along the GO nanosheets or TiO2 nanoparticles [2 leads to a higher crystallinity index than with GO, probably due to the fact that TiO2, having high crystallinity, might be more effective in achieving superior reinforcement for composite materials [2-GLA beads due to the lower crystallinity of the GO. More research is needed to understand why the two nanomaterials are not improving the crystallinity together and the thermal behavior, which could be due to the 3D morphology of the TiO2 and the 2D morphology of GO nanosheets in the tridimensional matrix of chitosan, decreasing the chitosan bindings.If the crystallinity is increasing with the introduction of the nanomaterials, it means that nanomaterials are well dispersed between the polymer matrix, especially for TiOarticles . The intaterials . However2 to the CS beads, the nanomaterial increases the roughness due to their texture and loss of the semi-crystalline chitosan structure [2-GLA beads with holes on the surface that support nutrient transportation and provide a waste outlet for cells [The microstructure of the CS composite beads was studied by the SEM technique. tructure . Figure or cells . Solventor cells .2 and -CHO groups [2 in the beads, there was an obvious darkening effect [The color of the CS-GLA beds ranged from yellow to light-red  due to tO groups . Howeverg effect .2 into the CS-GLA beads increased the thermal resistance, as observed in The thermogravimetric analysis, with four decomposition steps, is shown in 3%, 3% of mass loss) for all the composites are listed in 3% for CS-GLA shows higher thermal stability compared to pristine CS due to the crosslinking reactions. The Td3% dramatically increased with GO incorporation, demonstrating thermal stabilization with the nanofiller introduction, possible due to the higher crystallinity of the nanomaterial. From this table, we can conclude that the beads will not be thermally affected by body conditions (37 °C) since they are thermally degraded only between 215 °C and 500 °C. The decomposition temperatures at 3% , which could be explained by the higher compatibility shown by the TiO2 nanoparticles, which would favor the resorption process.A morphometric study from the 10× images was conducted to determine the percentage of resorption of the beads. Resorption percentages are presented in 2 are incorporated, the answer appears to be healthy for a typical foreign body reaction, possibly because the nanomaterials stabilize the CS by chemical interactions.It is also striking that the CS-GLA formulation showed the presence of skin histiocytes or macrophages, probably due to the presence of the CS producing a robust inflammatory response. However, when the GO and TiOFrom the H&E technique is not possible to directly determine the nature of the Fc tissue. However, Masson′s Trichromacy technique (MT) indicated that the fibrous capsule surrounding each bead corresponded to collagen type I because of the blue appearance after staining A–D.When examining the images at higher magnification, all the beads are surrounded by an inflammatory infiltrate (II). In general, all formulations A–F have 3), and distilled water were used for TiO2 nanoparticles synthesis. Graphene oxide was synthesized using graphite , sulfuric acid (H2SO4), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), and 2-propanol . CS beads were prepared using chitosan (from shrimp shells) with a molecular weight of Mv 144.000, measured by capillary viscometry . K and a constants for CS in the solvent used (acetic acid 0.3 M + sodium acetate 0.2 M) at 25 °C are 0.074 mL/g and 0.76, respectively [Titanium isopropoxide , 2-propanol, nitric acid  at a temperature of 300 K and elemental analysis of the CS was 89%. The sample was dissolved in D2O with two drops of trifluoroacetic and 3-(trimethylsilyl)propionic acid-d4 as reference salt. The elemental analysis was performed using a Flash EA 1112 system . The degree of deacetylation determined by 2 nanospheres were synthesized using a previously reported method [3) was prepared, while the precursor solution was added dropwise with vigorous stirring. After that, the hydrolysis of TTIP was produced at 60 °C and 20 h of reaction. After solvent evaporation, a yellow crystal precipitated. A yellow-white powder was obtained after ethanol washing and drying at 100 °C. The final step consisted of solid calcination at 400 °C for two hours. TiOd method ,67. Bried method . Then, 22 nanoparticle synthesis and characterization were already reported [2 nanoparticle morphology was studied by TEM  at 20 kV. Samples for TEM measurements were prepared by placing a drop of TiO2 on a carbon-coated standard copper grid (400 mesh) and evaporating the solvent. On the other hand, X-ray diffractometry (XRD) experiments for TiO2 were performed using a PANalytical X′Pert PRO diffractometer , with Cu Kα1 radiation (1.540598 Å) and Kα2 (1.544426 Å), in a 2θ range between 5° and 80°. FTIR experiments on TiO2 were performed in attenuated total reflectance mode using a diamond tip accessory .TiOreported . TiO2 naGO was prepared by the modified Hummers method ,83,84,85d is the lattice spacing, λ is the X-ray wavelength, and θ is the angle of incidence.X-ray diffractometry (XRD) experiments for GO was performed in a PANalytical X′Pert PRO diffractometer , using Cu Kα1 radiation (1.540598 Å) and Kα2 (1.544426 Å), in a 2θ range between 5° and 50°. Bragg′s law (Equation (2)) was used to calculate the interlayer distance in the GO: 2. AFM images were taken for each sample. Topography was examined by topographical AFM mode.The Raman spectrum recorded with an Invia Raman Microscope in a wavelength laser of 514.5 nm  was used for GO characterization. Atomic force microscopy (AFM) experiments were performed in tapping mode using a Multimode AFM  equipped with a Nanoscope Iva control system (software version 6.14r1). Silicon tapping probes  were used with a resonance frequency of ~300 kHz, scan rates of 0.4 Hz, 5 × 5, 2 × 2 μmv/v). Then, GO was added to this solution (10 mg) and stirred until a homogeneous dispersion was obtained. The procedure for obtaining TiO2 beads was similar, but 100 mg of TiO2 was added instead of GO (CS-TiO2-GLA). The above process was repeated, and 100 mg of TiO2 and 10 mg of GO were added for the chitosan/graphene oxide/TiO2 beads (CS-GO-TiO2-GLA) and stirred until a homogeneous dispersion was obtained.For the preparation of chitosan/graphene oxide beads and glutaraldehyde (CS-GO-GLA), 2 g of chitosan was dissolved in 100 mL of acetic acid  were obtained by dispersing the beads obtained previously in a 2.0% v/v solution of GLA in water (CS: GLA 1:1 molar ratio), under gentle stirring for 24 h at ambient temperature. Subsequently, they were washed with abundant distilled water to remove excess GLA and finally air-dry until constant dry weight and placed in a desiccator at 10% relative humidity (RH) until the time of the test [The formation of the different nanocomposite beads was carried out using the phase inversion technique , with a the test . C is the area under the peaks that represents the crystalline region, and AT is the total area of the crystalline and amorphous region.XRD experiments were run with the same procedure and apparatus than the previously reported range of 2θ from 0–80°, and time per step 304.390 s. According to Nara–Komiya methodology , the perThermogravimetric analysis was run on a Netzsch TG Libra 209 instrument  in a temperature range between 30–700 ± 2 °C. The melting temperature was determined by differential scanning calorimetry using a DSC2A-00181 system (TA Instruments) at 10 °C/min.FTIR analysis of the beads was performed in ATR mode  with a diamond tip on an IR Affinity-1 spectrometer . SEM analysis was performed on a scanning electron microscope  using an acceleration voltage of 20 kV, in which the samples were coated with a copper bath. The samples for SEM- energy-dispersive spectroscopy (EDS) were fixed in 75% alcohol for 48 h and dehydrated using an ascending alcohol gradient , after which they adhered on carbon tape. Then, an exterior gold coating was applied  to generate a conductive surface. Subsequently, a JEOL Model JSM 6490 LV microscope was used to inspect the samples in the secondary electron mode with an acceleration voltage of 20 kV to obtain electron microscope images. Additionally, chemical microanalysis was carried out on several inspection areas, employing the energy-dispersive spectroscopy (EDS) probe of a Model INCAPentaFETx3 instrument . The EDS probe which had a resolution of 137 eV to 5.9 keV, while SEM had a resolution of 15 nm with an acceleration voltage of 1 kV and a working distance of 6 mm in the secondary electron mode. 2 nanoparticles and their dispersion in the composite beads were analyzed by TEM (JEOL ARM 200 F) operating at 20 kV. Samples for TEM measurements were prepared by placing a drop of TiO2 on a carbon-coated standard copper grid (400 mesh) and evaporating the solvent.The morphology of the GO and TiO®, INVENT Laboratory, Santiago de Cali, Colombia) at a dose of 100 mg/kg. The samples were processed for histological analysis by the hematoxylin and eosin (H-E) and Masson′s trichrome stain (MT) techniques.Samples of 9.7 mg of the different types of beads were implanted in subdermal preparations of three adult male Wistar rats supplied and hosted by the LABBIO laboratory of the Universidad del Valle . Biomodels were sedated by the intramuscular application of ketamine 70 mg/kg and xylazine 30 mg/kg  to prepare the samples. Each preparation consisted of an incision one cm long and three cm deep on the dorsal surface of the biomodels. After 90 days of implantation the biomodel animals were euthanized via intraperitoneal injection of sodium pentobarbital 390 mg/mL and 50 mg/mL of sodium diphenylhydantoin , which was calibrated before measuring with the scale that was in each image. From this information, the resorption percentages of the areas corresponding to the pores were determined by applying the following equation: This research was reviewed, supported, and supervised by the Institutional Ethics Review Committee with experimental animals of the Universidad del Valle (CEAS 012-019).In vivo studies of the beads are presented as the mean value of at least three replicates ± SD. The Statgraphics Centurion XVI program  was used for these statistical analyses. 2 nanoparticles and GO nanosheets was evident from chemical, thermal, and biological tests. The chemical crosslinking of the CS using glutaraldehyde increased the decomposition temperature due to the increased crystallinity, especially for CS-GLA-GO-TiO2 beads. However, the introduction of the nanoparticles increased the thermal stability, as evidenced by TGA and DTGA results. The thermal results correlated with the crystallinity indexes and in vivo subdermal implantation studies during 90 days in Wistar rats, where remaining material was found after 90 days without the presence of pus or an immune response due to the high stability of the material and the reinforcing effect of the nanofillers. Graphene oxide and titanium dioxide nanoparticles were efficiently distributed in the polymer matrix as TEM results showed. The morphology of the beads become rougher with the introduction of the nanoparticles. The high balance between long-term stability, porosity, and surface irregularity observed by SEM could be useful for long-term applications, for example, bone tissue regeneration, where porosity and surface irregularities are useful for cell-adhesion and proliferation. Finally, examination of the beads using staining by the Masson′s Trichrome and hematoxylin and eosin (H-E) staining techniques after the in vivo studies showed collagen type I as the main constituent of the surrounding fibrous capsule on the surface of the beads, indicating the strong biocompatibility of the material. Very interestingly, CS-GLA beads displayed the presence of macrophage/histiocyte compatible cells, which demonstrates the beginning of a phagocytic process of the content. The successful preparation of very stable and biocompatible CS beads based on TiO


Turnip yellows virus (TuYV), which is strictly transmitted by aphids, and aphid’s life traits in six Arabidopsis thaliana mutants . We observed that mutations affecting the carbohydrate metabolism, the synthesis of a non-protein amino acid and the glucosinolate pathway had an effect on TuYV accumulation. However, the virus titer did not correlate with the virus transmission efficiency. Some mutations in A.thaliana affect the aphid feeding behavior but often only in infected plants. The duration of the phloem sap ingestion phase, together with the time preceding the first sap ingestion, affect the virus transmission rate more than the virus titer did. Our results also show that the aphids reared on infected mutant plants had a reduced biomass regardless of the mutation and the duration of the sap ingestion phase.During the process of virus acquisition by aphids, plants respond to both the virus and the aphids by mobilizing different metabolic pathways. It is conceivable that the plant metabolic responses to both aggressors may be conducive to virus acquisition. To address this question, we analyze the accumulation of the phloem-limited polerovirus  Myzus persicae, the green peach aphid, can transmit more than 100 different virus species to a variety of plant species including the model plant Arabidopsis thaliana [M. persicae are characterized in A. thaliana and involve ethylene, salicylic, jasmonic, and abscisic acid-signaling pathways [M. persicae [Brassicaceae that increase upon aphid feeding and reduce A. thaliana susceptibility to aphids [M. persicae [Aphids are a large group of Hemipteran insects that induce direct damage on plants but are, more importantly, major vectors of plant viruses. Among aphids, thaliana ,3,4,5. Tthaliana ,7,8,9. Spersicae ,12. Gluco aphids ,14,15. FArabidopsis thaliana can, among other plant viruses, be infected by poleroviruses  that are transmitted by aphids in a circulative and non-propagative mode [ive mode . Polerovive mode . Virus pive mode .Luteoviridae family. Polerovirus infection induces leaf yellowing, which may be visually attractive for aphids [Cucurbit aphid-borne virus (CABYV), Potato leafroll virus (PLRV) , and Barley yellow dwarf virus (BYDV)  [There is growing evidence that aphid-transmitted viruses can affect plant phenotypes and vector behaviors in ways that may ultimately facilitate virus acquisition and inoculation ,20. Thisr aphids . The majr aphids ,23. Thesr aphids ,25. In ar aphids ,27. Chan family) ,28,29. MCucumber mosaic virus, a non-persistently transmitted virus, drives accumulation of reactive oxygen species through the action of its 2b silencing suppressor, which consequently shortens aphid probes and increases virus acquisition [Turnip mosaic virus, which regulates ethylene production and ethylene-related defense mechanisms [Knowledge on the plant response to poleroviruses is lacking, but transcriptomic analyses on plants infected by other aphid-transmitted viruses identified several plant defense pathways induced by viral infection. In addition to the RNA silencing pathway, these include stress and defense responses characterized by the induction of heat shock proteins and the accumulation of pathogenesis-related proteins mediated by salicylic acid ,31,32. Guisition . Anotherchanisms ,37,38. Tchanisms .A. thaliana mutants known to be affected in carbohydrates and glucosinolates metabolisms, phloem functions, and defense signaling and investigated their potential incidence on aphid traits, on Turnip yellows virus  accumulation as well as on growth and feeding behavior of M. persicae, an efficient vector of TuYV. Mutants that influenced sap ingestion by aphids in TuYV-infected plants were further tested for their effect on virus transmission.Further functional studies are needed to identify metabolic pathways that are under virus control, influence aphid performance and/or feeding behavior, and ultimately impact virus acquisition and transmission. Consequently, we selected some Arabidopsis thaliana mutant seeds were obtained from stock centers for pad4-1 , xth33 , ss3-2 . quad (cyp79B2cyp79B3myb28myb29) and myc234 (myc2myc3myc4) were kindly provided by Philippe Reymond . atr1D was obtained from Judith Bender  and nata1 (GK-256F07) from Georg Jander . For nata-1, xth33-1, and ss3-2 A. thaliana mutants, homozygosis and T-DNA insertion were analyzed by PCR using primers described in pad4-1 mutant, the point mutation was confirmed by BsmF1 digestion of a PCR product amplified using the primers described in ATR1D point mutation was confirmed by sequencing the PCR product encompassing the mutation  (Hemiptera: Aphididae) clone originates from the Netherlands. Aphids were reared on Chinese cabbage (Brassica rapa) in a growth chamber under 20 ± 1 °C, and 16 h photoperiod.Arabidopsis thaliana mutants and the reference ecotype Col-0 were inoculated using viruliferous M. persicae. Non-viruliferous aphids were fed with an artificial medium [A. thaliana mutant. After 72 h inoculation, aphids were manually eliminated, and the plants were grown for 3 weeks before being tested by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for infection status.l medium  containil medium . After aA. thaliana by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with a TuYV-specific polyclonal antiserum (Loewe) 3 weeks post-inoculation [8 to 103 viral cDNA copies obtained from RNA extracted from purified virions were used for calibration. The comparison between the Ct values of the calibration standard and the samples provided an absolute quantification of TuYV genomes.TuYV was detected in non-inoculated leaves of culation . For qRT®, Certis Europe France, Guyancourt, France) and the test plants were assayed by DAS-ELISA 3 weeks later. In each virus transmission experiment, one to three virus source plants were used per condition and 20 to 60 test plants per condition.Non-viruliferous aphids (L4 and adults) were deposited onto TuYV-infected plants for virus acquisition during 24 h. After this acquisition access period, two aphids were deposited per Col-0 test plant for a 3-day inoculation period. Aphids were then eliminated by an insecticide treatment (PirimorCapsicum annuum) and deposited on 1.5% agar in Petri dishes during 24 h. Synchronized young adults were obtained 9–10 days later in the same device. The electrical penetration graph DC system [Pools of synchronized first-instar nymphs were obtained from parthenogenetic adult females placed on detached leaves of pepper plant (C system  was usedC system . The folC system : (Pr) stMyzus persicae performance was evaluated by measuring the weight of individual aphids. Synchronized M. persicae first instar (L1 stage) was obtained as described above and deposited on whole plants of A. thaliana mutant (50 aphids on three different plants). After 10 days, thirty aphids were randomly selected and weighted individually with an electronic precision scale  = 1 μg).Data on TuYV accumulation in mutants and Col-0 are not normally distributed, thus we carried out a generalized linear model (GLM using quasi-Poisson distribution (link:log) followed by pairwise comparisons using estimated marginal means (package R: ‘emmeans’).A. thaliana genome on M. persicae performance and feeding behavior. Data on aphid weight were analyzed by a GLM using Gaussian distribution. When a significant effect of one of the main factors was detected or when an interaction between factors was significant, a pairwise comparison using least-squares means (package R: “lsmeans”)  at the 0.05 significance level was used to test for differences between treatments. Data on aphid feeding behavior (s-E2) are not normally distributed, accordingly we carried out a GLM using a gamma (link = ”inverse”) distribution. When a significant effect of one of the main factors was detected or when an interaction between factors was significant, a pairwise comparison using least-squares means (package R: “lsmeans”)  at the 0.05 significance level was used to test for differences between treatments. Data on aphid feeding behavior (t1 < E2) were modeled using the Cox proportional hazards (CPH) model and we treated cases where the given event did not occur as censored. The assumption of validity of proportional hazards was checked using the functions “coxph” and “cox.zph”, respectively . The fit of all generalized linear models was controlled by inspecting residuals and QQ plots.We used a generalized linear model (GLM) with a likelihood ratio and Chi-squared test to assess whether there was an effect of TuYV infection, or an effect of the mutation introduced into p-value adjustment method: fdr) (package R: “RVAideMemoire”).Comparisons of transmission efficiencies were analyzed by Pearson’s Chi-squared tests and followed by pairwise comparisons using Pearson’s Chi-squared tests with Yates’ continuity correction (https://www.r-project.org/).All statistical analyses were performed using the R software version 3.3.2  [quad mutant contains mutations in two cytochrome P450s genes (CYP79B2 and CYP79B3) and two transcription factors, MYB28 and MYB29, resulting in very low glucosinolates containing plants [myc234 and quad mutants accumulate low levels of glucosinolates. In contrast, the overexpression of the MYB transcription factor ATR1, in the atr1D mutant, results in increased accumulation of indole glucosinolates [PAD4 gene is part of a defense regulatory pathway against various pests and pathogens including M. persicae and the mutation in the pad4-1 mutant results in a phytoalexin-deficient mutant, which positively affects aphid performance and phloem activity [We selected mutant lines of reported . Two mutpersicae ,45. The ng NATA1 . Glucosing NATA1 . We testnd MYC4)  and the g plants ,49. Theractivity ,52. Noneatr1D, quad, and pad4-1 and lower in xth33-1, ss3-2, nata1, and myc234 infected plants , the duration of sap ingestion (s_E2). Additionally, the time aphids took to perform the first phloem sap ingestion phase (t > 1E2), was analyzed as this parameter reflects plant palatability and might also affect TuYV acquisition.On non-infected plants, total duration of phloem sap ingestion on ss3-2 and myc234 mutants is longer relative to nata1 and the time until the first phloem sap ingestion is longer on nata1 when compared to myc234 . In TuYVThese results suggest that the reduced phloem sap ingestion phase on infected atr1D and pad4-1 during the 8 h of EPG recording could be responsible for the lower virus transmission efficiency observed when infected mutant plants are used as virus source . SimilarNo significant differences are observed in the total duration of the phloem sap ingestion between non-infected and infected plants (mutants and the reference Col-0) . In contA. thaliana mutations on aphid biomass, combined or not with TuYV infection. On non-infected plants and compared to Col-0, aphid biomass on ss3-2 and atr1D is not significantly different while the aphid weight is lower on xth33 and nata1 and higher on quad, myc234, and pad4-1 . In contrast, in all mutants, the combined effect of the mutation and the infection effect aphid biomass, and in some of them, an additional effect on the feeding activity. This indicates that the virus infection interferes with several metabolic pathways that may impact aphid growth and feeding activity, as developed below. In the wild-type context (Col-0), the impact of the virus infection on several metabolic pathways can induce opposite effects on the aphids, which ultimately can result in a neutralization of these effects and no visible consequences on insect feeding or fitness parameters.XTH33 gene encodes a xyloglucan:xyloglucosyl transferase involved in cell wall remodeling. In a previous report, M. persicae preferred settling on A. thaliana xth33 mutant [xth33, but only when the plants were infected. This suggests that in a non-mutated context (Col-0), and upon TuYV infection, this metabolic pathway could hinder aphid access to the phloem and reduce virus acquisition. We postulate that knocking out this pathway alleviates its negative effect on aphid feeding in infected plants. The second mutant in the carbohydrate metabolism is ss3-2, which overaccumulates starch and reduces M. persicae populations [The 3 mutant . In our ulations . Reared A. thaliana nata1 mutant, which is unable to synthesize the non-protein amino acid Nδ-acetylornithine [M. persicae infestation and has an in vitro toxic effect on sap-feeding insects including M. persicae [nata1 because of the absence of this aphid defensive compound, but no such effect was observed. Surprisingly, in the infected nata1, the time to perform the first phloem phase increased, revealing a beneficial effect of this component on aphids in Col-0.In rnithine , the phlpersicae . We expeA. thaliana mutants, which accumulate different levels of these compounds. Glucosinolates are secondary metabolites that are hydrolyzed into toxic compounds after tissue damage of generalist herbivores. They affect not only herbivores by also their natural enemies [atr1D accumulates higher amounts of indole glucosinolates [myc234 and quad mutants sustain very low levels of glucosinolates [myc234 mutant is practically devoid of glucosinolates and only traces of two indole glucosinolates remain [myc234 mutant increases aphid attraction by releasing different volatiles and potentially also by modifying composition of plant metabolites [quad mutant, the synthesis of both indole and aliphatic glucosinolates is impaired, but no impact on M. persicae population growth was reported [M. persicae should not be in contact with vacuolar glucosinolates-hydrolyzing myrosinases when ingesting sap, indole glucosinolates are nonetheless degraded in the aphids and this degradation may be responsible for the deterrent effect of these compounds on aphids [myc234 plants in which aphids reach the phloem faster and feed longer both in non-infected and infected conditions. In accordance with the overaccumulation of indole glucosinolates in atr1D, access to the phloem and sap ingestion are both negatively impacted but only in infected plants.The effect of glucosinolates is addressed in this study using three  enemies ,59. The inolates  while myinolates ,49. The s remain . It was abolites . In the reported . Althougn aphids . Supportn aphids ,15. Our PAD4 is a central player in the signaling defense pathway against pathogens including aphids, and PAD4 expression in A. thaliana is stimulated by M. persicae infestation [pad4-1 background, the aphid-induced senescence is inhibited resulting in a better aphid settling, feeding, and fecundity [pad4-1. The discrepancy in the results may be due to intrinsic differences in the aphid clones, but it is also conceivable that the increased sap ingestion observed on pad4-1 by Pegadaraju et al. [M. persicae on the reference Col-0 in this study. In our study, we observed an opposite effect of the mutation (reduced sap ingestion phase) but only upon virus infection.estation . In the ecundity ,52. In cu et al.  is due tA. thaliana mutants was not addressed before. As expected, M. persicae’s growth increases on non-infected pad4-1, which accumulates a low content of phytoalexins, and on quad and myc234, which are substantially deprived of glucosinolates. The aphid biomass on the other mutants is not related to the biological function of the mutated genes. However, regardless of the mutation in the A. thaliana genome, we always observe a reduction of the aphid biomass on infected mutant plants when compared to the non-infected mutants. This effect on aphid biomass was not clearly observed on Col-0 despite a slight, but not significant, reduction in aphid size. One hypothesis to explain this result is an additive effect of the virus infection and the mutations on aphid development. TuYV infection, together with the mutations in the different metabolic pathways, likely affect sap nutrient value and therefore aphid biomass. In this regard, the constitutive expression of the P4 protein from Potato leafroll virus  is reported to induce changes in carbohydrate status which could have consequences on the aphid biomass [Aphid biomass on the  biomass .It is interesting to point out that the aphid weight measured on non-infected or infected mutants is not correlated to the duration of the sap ingestion. Indeed, no statistical differences in the sap ingestion phase were observed between non-infected and infected mutants whereas a significant reduction in the aphid biomass was visible with all infected mutated plants. This strongly suggests that nutritional quality of phloem sap contributes more to aphid biomass than the volume of sap ingested by aphids.atr1D, quad, myc234, and pad4-1 on TuYV transmission efficiency is addressed. Only the atr1D mutation has a reproducible negative effect on TuYV transmission by aphids regardless of the time of virus acquisition (8 or 24 h), while it has no effect on virus accumulation. A slight decrease in the virus transmission rate is also observed with pad4-1 but only when the acquisition time is 8 h. The reduced sap ingestion phase observed during the 8 h of recording on infected atr1D and pad4-1 could be responsible for a lower virus uptake, and subsequently, a reduced TuYV transmission. It is conceivable that the decreased sap ingestion observed on pad4-1 during the first eight hours of feeding is transient and that the expected beneficial effect of the absence of phytoalexins in this mutant sets in only later. This delay would explain why an effect on virus transmission was observed after a virus acquisition period of 8 h but not 24 h. In the case of atr1D, it is also possible that the higher content of indole glucosinolates influences virus uptake, internalization into intestinal cells or even virus inoculation into test plants. Detection of plant glucosinolates in aphids [The impact of the mutations in n aphids  suggestsatr1D, could be a more important factor for virus uptake than expected. Hindrance during phloem access could discourage aphids to ingest sap as they normally do, and this could limit virus acquisition.Lastly, our study also suggests that the time required to perform the first phloem sap ingestion phase, which is extended in myc234 result in an increased phloem sap ingestion and a faster phloem access, but no beneficial effect on virus transmission was observed in our experimental conditions, maybe because of the low virus accumulation in this mutant. A completely adverse situation was observed in atr1D mutant with reduced sap ingestion and a delayed phloem access leading to a decreased virus transmission . Such gene downregulation in the condition of virus infection and aphid infestation could indicate a plant manipulation by the virus upon aphid infestation. However, this assumption requires extensive studies to be confirmed. This work points the way towards the identification of metabolic pathways which could be potential targets to reduce virus acquisition by aphids, and as a consequence, virus propagation among plants.In this work, we addressed the effect of different mutations affecting several plant biological pathways known to influence aphid performance and behavior. We observed that virus infection and most mutations have cumulative effects and induce a biomass reduction of aphids suggesting that the deregulations triggered by TuYV infection also impact the biological pathways targeted by the mutations. It is however conceivable that the infection activates plant defense responses which, in combination with the mutations, affects aphid traits. The mutations in smission . This re


Emerging infectious diseases (EIDs) are an increasing threat to public health on a global scale. In recent times, the most prominent outbreaks have constituted RNA viruses, spreading via droplets (COVID-19 and Influenza A H1N1), directly between humans (Ebola and Marburg), via arthropod vectors  and zoonotically . However, specific approved antiviral therapies and vaccine availability are scarce, and public health measures remain critical. Patients can present with a spectrum of ocular manifestations. Emerging infectious diseases should therefore be considered in the differential diagnosis of ocular inflammatory conditions in patients inhabiting or returning from endemic territories, and more general vigilance is advisable in the context of a global pandemic. Eye specialists are in a position to facilitate swift diagnosis, improve clinical outcomes, and contribute to wider public health efforts during outbreaks. This article reviews those emerging viral diseases associated with reports of ocular manifestations and summarizes details pertinent to practicing eye specialists. Emerging infectious diseases (EIDs) pose a considerable ongoing threat to humanity. The World Health Organization (WHO) define EIDs as those that are either novel and severely affect a population for the first time, or those that have existed previously but have a rapidly increasing incidence or degree of spread to new geographical areas . GlobaliRecent outbreaks combined with the development of diagnostic techniques have increasingly allowed characterisation of the ocular manifestations of EIDs . The mosEmerging viral diseases were identified for inclusion using the WHO list of designated ‘priority’ pathogens as well as an initial scoping literature search for emerging viral diseases with ocular manifestations . For eacEbola virus (EBOV) is an enveloped, filamentous, negative-sense, single-stranded RNA virus of the Filoviridae family. EBOV is the cause of the frequently lethal Ebola virus disease (EVD), which has a documented case fatality rate of around 50% . EVD outAfter contact with the virus, the asymptomatic incubation period ranges from 2 to 21 days with an average of 8 to 10 days. EVD is typically characterized by an initial fever, myalgias and fatigue, progressing to gastro-intestinal symptoms (diarrhoea and vomiting) and eventually a fatal multiple organ dysfunction syndrome. During acute infection, ocular manifestations include conjunctival injection , subconjunctival haemorrhages and vision loss of unclear origin [An important clinical sequel has surfaced in survivors, termed the ‘post-Ebola virus disease syndrome’. This commonly manifests with ocular disease (60%), and may be accompanied by arthritis, hearing loss, abdominal pain and neuropsychiatric disorders as well as viral persistence in immune-privileged organs . UveitisDiagnosis requires a combination of case definition and laboratory tests, typically real-time RT-PCR or enzyme-linked immunosorbent assay (ELISA). This permits the swift initiation of appropriate cycloplegic and anti-inflammatory treatment  for uveitis and recognition and management of complications. Notably, the finding of replicating EBOV in the aqueous humour following recovery from EVD (and for at least 100 days post acute EVD diagnosis) highlights possible infectivity of ocular fluid posing risk to ophthalmologists performing invasive procedures on EVD survivors , 21.Timely diagnosis can help avoid long-term visual disability. Specifically, whilst USA healthcare workers swiftly treated recovered vision following therapy, nearly 40% of eyes in a Liberian cohort in whom treatment was comparatively delayed had vision worse than 20/400 than their initial presentation, sometimes because of structural complications including cataract and dense vitreous opacity . This ilThe mainstay of treatment for the underlying EBOV infection is currently supportive and symptomatic; however, antiviral drugs are emerging . An expeRousettus aegyptiacus bats, the natural reservoir of MARV, with outbreaks mostly endemic to East and Central Africa [Marburg virus (MARV) is a negative-sense RNA virus of the Filoviridae family. Zoonotic infection occurs following prolonged exposure to l Africa . Secondal Africa , 27. Aftl Africa . Ocular l Africa , 30. MARl Africa . Diagnosl Africa . There il Africa .HHV-6 is a double-stranded DNA virus of the β-herpes virus subfamily found worldwide, with two variant species HHV-6A and HHV-6B. Little is known about the acquisition or prevalence of HHV-6A and it is less associated with disease . In contThere are typically three stages in the natural history of HHV-6 infection . Acute pOcular conditions of the posterior segment are more commonly associated with HHV-6, though the exact role of HHV-6 in these conditions remains unclear. Posterior segment inflammation associated with HHV-6 includes AIDS-associated retinitis –41, uveiIn addition, reactivation of HHV-6 frequently accompanies CMV reactivation . In nineAnterior segment disease associated with HHV-6 includes corneal inflammation , 52, 53.Diagnosis of clinically relevant HHV-6 is challenging due to the high prevalence of infection and need to distinguish between active and latent infections . DetectiLassa virus (LASV) is an enveloped, negative-sense, single-stranded, bi-segmented RNA virus of the Arenaviridae family and is endemic to West Africa. Infection often occurs through exposure to food or household items contaminated with urine or faeces of infected Mastomys rats. Person-to-person transmission may occur after exposure to virus in bodily fluids, particularly in healthcare settings in the absence of control measures. Following a 7–21-day incubation period, LASV can cause Lassa fever (LF), an acute viral haemorrhagic illness with an overall case fatality rate of 1%, though most-affected individuals harbour the infection asymptomatically (80%) . Ocular Paramyxoviridae of the genus Henipavirus. It was first isolated during the 1999 outbreak in Malaysia and Singapore. Repeated outbreaks have since occurred in India and Bangladesh. Transmission is primarily zoonotic from contact with infected pigs or bats, though person-to-person transmission has also been reported in Bangladesh and India, highlighting its pandemic potential [Nipah virus is an enveloped, negative-sense, single-stranded RNA virus in the family otential . There aThe disease has a high mortality rate of 40%  to over 70% (India) . VariatiRegarding ocular manifestations, one small study from Singapore of 13 patients reported nystagmus, VI nerve palsy and transient blindness during the acute phase of the illness . ResiduaDiagnosis focuses on RT-PCR of throat swabs, blood, urine and CSF . There iRVF virus is an enveloped, negative-sense, single-stranded RNA virus, of the family Phenuviridae . It is tIn its most common mild form, RVF is characterised by fever and non-specific flu-like symptoms. In a small percentage of cases, a severe form develops, categorised into three syndromic presentations: ocular disease with risk of permanent loss of vision; meningoencephalitis with confusion and potentially coma; and haemorrhagic fever with liver involvement and jaundice, this being the most fatal form .Ocular manifestations are well-documented, and present symptomatically with blurred vision, decreased vision, floaters, scotomatous areas or periocular pain –82. The RVF and its ocular manifestations may mimic other viral diseases, and so diagnosis relies on laboratory testing with either RT-PCR or serology . No posiHantaviruses are enveloped, single-stranded, negative-sense RNA viruses of the Bunyaviridae family. The viruses are rodent-borne and transmission is via aerosols of excreta, saliva and urine, leading to an initially non-specific viral presentation of pyrexia, chills and myalgias following an incubation period (10–25 days). Person-to-person transmission is generally rare for hantaviruses, with the notable exception of Andes virus (ANDV) following reports of transmission of ANDV in the hantavirus cardiopulmonary syndrome (HCPS) outbreak in Argentina in 1996 , 85. NowTransient ocular involvement in hantavirus infection is now increasingly recognised, and to date, has mainly been described in the context of HFRS caused by Puumala virus. An acute, transient myopia may be the first and most cardinal ocular symptom of a hantavirus infection, and its incidence has been variably reported (12–53%) in Puumala virus infections in Europe –91. The At present, there are currently no approved treatments or vaccines available for hantavirus infections . SupportDengue viruses are enveloped, positive-sense, single-stranded, RNA viruses of the Flaviviridae family. They are the cause of dengue fever. There are four viral serotypes 1–4 with no cross-immunity, and spread is via Aedes mosquito vectors . IncidenDengue fever occurs 3–14 days after a mosquito bite, with development of a non-specific febrile phase lasting 3–6 days . Upon feTrue incidence of ocular manifestations is poorly understood, with studies only including cases that reach hospital specialists. In such studies incidence has been reported between 7.1 and 40.3%, a range that likely reflects differing disease severities and differing ocular workups in each study , 104. OcPosterior segment manifestations most commonly include maculopathy and posterior uveitis, and less commonly vascular occlusions, panuveitis and retinal and vitreous haemorrhage . MaculopDiagnosis relies upon dengue symptomatology, and confirmation by laboratory testing. In the first 5 days, real-time RT-PCR or NS1 antigen detection allow confirmation. More commonly confirmation occurs after 5 days, with ELISA-based detection of anti-Dengue IgM or seroconversion in paired acute and convalescent serum samples . Workup Zika virus (ZIKV) is an enveloped, positive-sense, single-stranded, RNA virus with an icosahedral capsid, and is a member of the Flaviviridae family , 114. FiZika is principally transmitted via the bite of an infected Aedes mosquito , usually during daytime . Human-tInfected patients are typically asymptomatic, however up to 20% may develop a mild, self-limiting symptom triad of generalized maculopapular rash, arthritis or arthralgia and non-purulent conjunctivitis following a 3–14 day incubation period , 121. ThOcular involvement related to Zika virus may be acquired in the acute phase or present congenitally. In the acute phase, non-purulent conjunctivitis and retro-orbital pain are the most frequent ocular signs, both reported in 40% in a Brazilian case series of 57 ZIKV positive patients . ReportsOcular manifestations of Zika have been described in up to 55% of infants with CZS , 140. TwConfirmative diagnosis of ZIKV infection requires laboratory testing of whole blood or urine, and is also possible in other bodily fluids . For insWest Nile virus (WNV) is an enveloped, positive-sense, single-stranded RNA virus of the Flaviviridae family, and a member of the Japanese encephalitis serocomplex of viruses . WNV hasOf patients who contract the virus, ~75% are asymptomatic, 25% develop West Nile fever (WNF) and <1% a neuroinvasive disease fatal in 10% , 153–155Multifocal chorioretinitis Fig.  is the mDiagnosis is typically confirmed by laboratory testing using ELISA to detect IgM in serum of cerebrospinal fluid, and in limited scenarios supported by RT-PCR. There is no vaccine, treatment is supportive, with vector control as the mainstay of prevention .The yellow fever virus (YFV) is an enveloped, positive-sense, single-stranded RNA virus of the Flaviviridae family. It is an arbovirus transmitted to humans primarily through the bite of infected Aedes or Haemagogus species mosquitoes, and principally maintained by a sylvatic (jungle) transmission cycle between mosquitoes and non-human primates and mosquito–human transmission cycles in urban areas, the latter leading to outbreaks of yellow fever (YF) in human populations. Dozens of countries in both Africa and South and Central America are endemic for YF, leading to an estimated 30,000–60,000 fatalities per year .The majority infected with YFV are asymptomatic or exhibit an initial phase of non-specific viral symptoms after 3–6 days , which then resolves within days. After less than a day of resolution, 15–25% of patients develop a toxic form of the disease, with fever, a haemorrhagic diathesis and multi-organ failure  leading to a 50% mortality rate , 166.A common ocular finding in the initial phase is conjunctivitis, whilst scleral icterus is often a feature of the toxic phase , 166. A Neisseria Meningitidis, Hepatitis A/B or Typhoid. The reported complications are varied and include uveitis, unilateral optic neuropathy, evanescent white dot syndrome, multifocal choroiditis, arteriolar occlusion and even a Vogt–Koyanagi–Harada-like disease  [There also exist case reports of ocular disorders associated with YFV vaccination, especially when the live YFV vaccine is combined with other vaccines, such as ckening) , 169. ThDiagnosis of YFV relies on PCR of blood or urine samples in the early stages, and ELISA or PRNT to detect antibodies in later stages. Treatment is mainly supportive, with a strong emphasis on prevention through public health measures and vaccination , 170.Aedes genus—Aedes aegypti and Aedes albopictus are the primary vectors [Chikungunya virus (CHIKV) is an enveloped, positive-sense, single-stranded RNA virus belonging to the genus Alphavirus of the family Togaviridae. CHIKV has been identified in over 60 countries in Asia, Africa, Europe and the Americas, and several epidemics have been reported in the past 20 years commonly during monsoon season. CHIKV is principally transmitted by the bite of the mosquitoes of the  vectors . Transpl vectors .CHIKV infection results primarily in an acute fever with a severe polyarthralgia predominating in the distal extremities that may persist for several weeks or months . CervicaCHIKV has a variety of ocular manifestations ranging from conjunctivitis to retinitis. Photophobia, conjunctival hyperaemia and retro-orbital pain are frequent in the acute phase, and may be isolated without other ocular involvement. Anterior uveitis is the most frequently reported ocular complication (one in three ocular cases), appearing 4–12 weeks after disease onset . The antPosterior uveitis often presents weeks after the acute symptoms, as retinitis, multifocal choroiditis or neuroretinitis. Neuroretinitis involves exudative haemorrhagic lesions essentially localized to the posterior pole, which can be associated with intra-/sub-retinal macular oedema . The lesOptic neuropathy is evident in 10% of cases with ocular involvement, developing typically 1 month after disease onset . In one Formal diagnosis of CHIKV in the initial 8 days of acute infection is done by RT-PCR or direct isolation of virus/viral antigens, following which serological testing with ELISA is recommended . Case reCrimean-Congo haemorrhagic fever virus (CCHFV) is an enveloped, circular, negative-sense, single-stranded RNA virus in the Nairoviridae family, which causes an acute viral haemorrhagic fever. Transmission of the virus is primarily through tick bites belonging to the genus Hyalomma, which are widely distributed in Asia, Africa and Eastern Europe . TransmiAn estimated 90% of patients are oligo- or asymptomatic whilst the remaining 10% can present with the more severe form of disease described in many studies . The disOnly one study has formally characterised the ocular findings in CCHFV patients after previous studies reported conjunctival injection and photophobia . In a caDiagnosis is via serum RT-PCR in the early stages of disease or ELISA late in disease and treatment is largely supportive with some evidence that ribavirin shows some benefit , 193. AfSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, single-stranded, positive-sense, RNA virus of the family coronaviridae . SARS-CoCOVID-19 has a viral pneumonia picture. Early symptoms occur after a median incubation period of 4 days, most commonly fever, cough and fatigue . In a prViral RNA has been detected in ocular secretions , 198. SeRT-PCR of oropharyngeal and nasopharyngeal swabs is the mainstay of testing, with other sources sampled less commonly. However, false negatives present a problem and so clinical suspicion based on symptomatology, biochemistry and imaging findings play an important role. Many clinical trials are already underway to assess potential treatment options and trials have begun for candidate vaccines .Influenza A H1N1 is an enveloped, negative-sense, single-stranded RNA virus of the orthomyxoviridae family. The subtype of Influenza A relates to cell surface glycoproteins. Influenza A undergoes continuous antigenic drift causing seasonal influenza, and sporadic antigenic shifts causing pandemics . NotableCommon symptoms include fever, cough, sore throat, coryza, myalgia, headaches and fatigue, and the disease is usually self-limiting . The mosOcular involvement in influenza A virus disease is common often involving mild conjunctivitis to reports of more severe eye involvement. True prevalence of ocular manifestations is difficult to ascertain from isolated studies due to lack of thorough ophthalmoscopic examinations during influenza outbreaks, and the benign nature of the disease in most patients. This accounts for the paucity of literature and detailed retinal findings .A growing body of evidence highlights the potential ocular findings in influenza A H1N1 disease. A case series of 89 patients with H1N1 infection found that acute conjunctivitis was the most common finding occurring bilaterally in 65.17% of patients and unilaterally in 12.36% patients . FeatureInvolvement of the posterior segment is less frequent. However, there have been reports of uveal effusion syndrome, retinopathy, retinitis and optic neuritis –216. UveDiagnosis is made clinically except in certain scenarios where testing may influence clinical decisions, such as whether to initiate antiviral treatment, perform other diagnostic testing or to implement infection prevention and control measures . RecommeThese emerging viruses pose a major public health risk due to their known pandemic potential and/or insufficient counter measures. Notably, these pathogens are all RNA viruses, whose error prone RNA-dependent RNA polymerase enzymes result in inherently high mutability and capacity for host immune evasion, which may underlie their pandemic potential . ContinuIn the absence of specific antiviral drugs or immunisation, precautionary strategies are the mainstay for curbing transmission. These include vector control and exposure limitation, screening and prophylaxis of exposed individuals, isolation of infected individuals and potential contacts, precautions depending on transmission route, and both personal and environmental hygiene and decontamination. Strategies for pandemic preparedness also need to be capable of mitigating risks posed by yet to emerge, unknown viruses, as reflected by inclusion of “Disease X” on WHO’s shortlist of blueprint priority diseases.Many of these viruses confer potential risk to eye care providers. Isolation of viral RNA from ocular secretions has been confirmed in humans for Ebola, Marburg, Zika, Chikungunya, COVID and influenza A H1N1, as well as in animal models of LF , 226. ThThe threat of emerging viral disease has been made all the more clear in the midst of the current COVID-19 pandemic. Further, the possibility that a yet unknown pathogen could cause a future epidemic has also been acknowledged by the WHO on their shortlist of blueprint priority diseases as ‘Disease X’. It has been estimated that there are 1.67 million unknown viruses circulating in animal reservoirs and there remains the potential for them to evolve and transmit to humans . It is iOphthalmologists should therefore be mindful of the variation of presentations of eye diseases in this context. The COVID-19 pandemic and studies of other viral diseases highlight that globalisation and modernisation play a significant role in the spread of viral disease and consequently returning travellers could present with manifestations of both current known and novel viral diseases. An awareness of the ocular manifestations of emerging viral diseases can therefore enable eye specialists to facilitate swift diagnosis, improve clinical outcomes and contribute to wider public health efforts during outbreaks.


WNV) lineage 2 was detected in Catalonia (Northern Spain) in northern goshawks by passive surveillance. The phylogenetic analyses showed that it was related to the Central/Southern European strains, evidencing WNV lineage 2 spread to Western Europe. WNV local transmission was later detected in bearded vultures housed at the Wildlife Recovery center where the goshawk was transferred to. Further studies, before the following period of high mosquito activity, indicated that WNV had circulated intensively in poultry and horses but only surrounding of the area where the virus was detected. In other areas of Catalonia, circulation of flaviviruses different to WNV was identified. Public Health investigations failed to detect WNV infection in humans.In September 2017, West Nile virus ( Its transmission cycle involves mosquitoes, mainly Culex spp. and birds. Horses and humans are considered dead‐end hosts, and their infection is usually asymptomatic or mild, but sporadically severe disease with neurological symptoms or even death may occur.West Nile virus (WNV), within the genus Accipiter gentilis) with neurological symptoms, the first report of this lineage outside Africa , which were caused by a WNV lineage 1 strain  was found with dehydration, apathy and low weight near an urban area of Lleida province , and was transferred to the Wildlife Recovery Center (WRC) of Vallcalent. Five days later it developed nervous symptoms , and was euthanized. Samples of nervous tissue were positive to WNV infection by RT‐qPCR  and was confirmed by SNT at the CVL (titer of 1/40).In September 2017, a northern goshawk  from Northern‐Eastern Europe. Interestingly, in Europe, WNV lineage 2 has been repeatedly isolated from northern goshawks , a near threatened‐species housed in Vallcalent‐WRC, showed neurological symptoms compatible with WNV infection. The animal tested positive by cELISA and negative by SNT, but 1‐month later antibodies against WNV were detected by SNT, evidencing seroconversion, and WNV transmission within Vallcalent‐WRC. Serum samples from other 13 bearded vultures were positive by cELISA, and two were confirmed by SNT at the CVL (titers between 1/20 and 1/40).By the same time, one bearded vulture .Detection of WNV in birds by molecular techniques implied the activation of the Emergency Program for WNV in Catalonia. Public Health authorities and people in contact with horses  in the area were informed. Vector surveillance was conducted in Vallcalent‐WRC at the end of October using three BG traps baited with COTo evaluate the extent of WNV circulation, in October 2017, a surveillance zone of 10 km around the first positive goshawk and Vallcalent‐WRC was defined, and within that area, a cross‐sectional survey in poultry and horses was carried out. Chickens and horses were first tested by cELISA, and positive samples were confirmed by SNT at the CVL. The SNT performed included Bagaza virus (BAGV) and WNV in the virus panel, but no other flaviviruses such as Usutu virus (USUV) which was detected in another area of Catalonia in 2006  for more than 12 years. In fact, since 2010, WNV positivity by SNT has been detected in wild birds (mainly raptors) in this same area, which was not included among the high‐risk areas for WNV in Catalonia (Alba et al., Given the ability of WNV to overwinter (Rudolf et al., Furthermore, WNV lineage 2 recurrence combined with the northward expansion of WNV lineage 1, the emergence of novel WNV genotypes and the circulation of other flaviviruses, e.g., USUV (Busquets et al.,


In this review, encouraged by the dictum of Theodosius Dobzhansky that “Nothing in biology makes sense except in the light of evolution”, we outline the likely evolutionary pathways that have resulted in the observed similarities and differences of the extant molecules, biology, distribution, etc. of the potyvirids and, especially, its largest genus, the potyviruses. The potyvirids are a family of plant-infecting RNA-genome viruses. They had a single polyphyletic origin, and all share at least three of their genes  with other viruses which are otherwise unrelated. Potyvirids fall into 11 genera of which the potyviruses, the largest, include more than 150 distinct viruses found worldwide. The first potyvirus probably originated 15,000–30,000 years ago, in a Eurasian grass host, by acquiring crucial changes to its coat protein and HC-Pro protein, which enabled it to be transmitted by migrating host-seeking aphids. All potyviruses are aphid-borne and, in nature, infect discreet sets of monocotyledonous or eudicotyledonous angiosperms. All potyvirus genomes are under negative selection; the HC-Pro, CP, Nia, and NIb genes are most strongly selected, and the PIPO gene least, but there are overriding virus specific differences; for example, all turnip mosaic virus genes are more strongly conserved than those of potato virus Y. Estimates of dN/dS (ω) indicate whether potyvirus populations have been evolving as one or more subpopulations and could be used to help define species boundaries. Recombinants are common in many potyvirus populations (20%–64% in five examined), but recombination seems to be an uncommon speciation mechanism as, of 149 distinct potyviruses, only two were clear recombinants. Human activities, especially trade and farming, have fostered and spread both potyviruses and their aphid vectors throughout the world, especially over the past five centuries. The world distribution of potyviruses, especially those found on islands, indicates that potyviruses may be more frequently or effectively transmitted by seed than experimental tests suggest. Only two meta-genomic potyviruses have been recorded from animal samples, and both are probably contaminants. At a meeting of the Fellows of the National Institute of Agricultural Botany in Cambridge, UK, on 14 November 1924, Dr. Redcliffe Salaman gave a lecture entitled “Degeneration of the Potato—An Urgent Problem” ,,24,19,24nfestans  and two nfestans . None ofhttps://pfam.xfam.org/ (accessed July 2019)) found only the C-terminal four motifs of the polyprotein in the complete polyproteins of barley yellow mosaic bymovirus, cardamom mosaic macluravirus, sweet potato mild mottle ipomovirus, and wheat streak mosaic poaceaevirus. A Pfam analysis of celery latent celavirus (CLV) found only the helicase C and RdRp genes found in other potyvirids. Thus, the RdRp or the helicase proteins are probably most appropriate for inferring the phylogeny of the potyvirids.Most of the genome of all potyvirids encodes a single polyprotein, which is post-translationally hydrolyzed into ten proteins ,27. It aThe phylogeny of RdRp genes of all named potyvirids shows that they form at least eleven genera, of which by far the largest is the potyviruses . The RdRIn summary, although the potyviruses and astroviruses share an RdRp ancestor , none ofThe potyviruses and rymoviruses are sister taxa ; they diAgropyron ), Hordeum ), and Lolium ), and many of the basal potyviruses are too . Most viruses of Lineages 1 and 9 were first isolated from lilioids, most of Lineage 2 from commelinids, Lineage 4 and 7 from rosids, and Lineage 5 and 8 from asterids, whereas the hosts of some large lineages were from two plant clades, such as Lineage 3 which was from monocots  or rosids, but rarely asterids, and Lineage 6, which was from a mixture of lilioids or asterids, but not rosids. These specificities are interesting as early attempts “to find some logic in the confusing issues of experimental host ranges of plant viruses” [Welwitschia mirabilis . Two ponosperm; .Genetic recombination is common in potyvirus populations  and in pThe ORF phylogeny in The patristic distances graph  also shoAs mentioned above in Solanum tuberosum) were domesticated in South America and first taken to Europe in the 16th century. They became a major international vegetatively propagated crop after the mid-19th century. PVY, which is mostly spread by locally migrating aphids and planting infected seed tubers, is now common in the potato crops of the world, where recombinant PVYs have become particularly damaging.PVY, which originated in South America  from a lBrassicaceae [TuMV, which is the only dicotyledon-infecting member of a lineage of potyviruses of lilioid monocotyledons b. It divsicaceae . TuMV hasicaceae ,81, but Prunus fruit trees and shrubs. Its host populations are therefore much smaller and longer lived than the host populations of PVY and TuMV. PPV is usually considered to be a single species , which is a potyvirus of long-lived woody domesticated and wild  species b, even tIpomoea batatas) crops in all continents, except Antarctica. The SwPVs are usually considered to be five species [The SwPVs, or sweet potato potyviruses  is a potyvirus found in the wild only in southwestern Australia where it is widespread in the perennial native legume ptoniana . It is aptoniana , and mayA summary of the results of the popgen analyses of the non-recombinant (n-rec) ORF sequences of these five viruses is shown in The popgen comparisons of the different phylogroups/groups/strains of PVY, TuMV, SwPVs, and HarMV isolates are given in Comparisons of the popgen results for the five viruses are instructive. There are five major phylogroups in the world PVY population , three oTuMV, which is common in cultivated and weed brassicas worldwide, has four major groups: world-B, Iranian, BRs , and basal-B. It also has the orchid outgroup found only in Europe, as mentioned above in The PPV population has a smaller mean nt diversity than the PVY and TuMV populations; π for PPV is 0.121  and a meThe popgen results for the SwPVs indicate that they are a lineage of separate viruses found in a single host species, not a single megaspecies. The mean nt diversity  of the SThe HarMV population was represented by a single population of 10 ORF sequences. It is the only ‘wild plant’ potyvirus which we examined. The results are remarkably similar to those of the other four viruses we checked. The HarMV sequences have a nt diversity (π) similar to that of TuMV and a ω value (0.078) similar to those of PVY and PPV but less than that of the SwPVs . The ω vIn summary, the results of the popgen analyses complement and confirm inferences obtained from phylogenetic analyses of the same ORF sequences  of the zucchini yellow mosaic virus (ZYMV) population , the papPhytophthora infestans)-resistant potatoes and a significant increase in European potato cropping.External corroborative evidence for the PVY and TuMV dating studies is imprecise. TuMV studies  estimateThe estimated TMRCA of the population of one potyvirus can be extrapolated to those of other potyviruses, if their gene sequences align, and the alignment is used to calculate and compare them in a single ML tree; Fuentes et al.  in theirhttps://en.wikipedia.org/wiki/Trade#Prehistory (accessed Dec 6 2019)), and seed-borne pathogens, such as potyviruses, are likely to have been carried along the ‘Spice Trade’ routes of the Indian Ocean, the Silk Road of Central Asia for many centuries. However, arguably the most important factor contributing to the success of the potyviruses and geminiviruses has been worldwide marine trade, which started with the exploration of the Americas by Columbus in the 15th century and resulted in the major exchange of plants and animals between the Americas and other regions of the world known as the Columbian Exchange (https://en.wikipedia.org/wiki/Columbian_exchange (accessed 6 December 2019)). Most recently, travel and trade by air has become important in the worldwide movement of viruses and their hosts despite an increased awareness of the importance of quarantine.The conclusion that the potyviruses originated in Eurasia only a few tens of thousands of years ago is important, as it helps define the conditions that have allowed potyviruses to migrate and become the important and speciose genus it is nowadays. The hallmark property of potyviruses seems to be their ability to infect and maintain populations in a wide range of plants and to switch hosts often, but be constrained to particular clades of eudicotyledons. The divergence of potyviruses has occurred during the period when humans and their activities have come, increasingly, to dominate the world, and the ability of potyviruses to exploit those conditions have allowed them to become numerous and often damaging. Although humans originated in Africa around 300,000 YBP, they did not migrate beyond Africa’s immediate environs until c. 80,000 YBP, then migrating to Asia and Australia, and eventually the Americas. Trade is an ancient human activity ). Australia was first colonized by the Aborigines at least 40,000–65,000 years ago [https://en.wikipedia.org/wiki/History_of_Indigenous_Australians (accessed 10 December 2019)). It was linked to Papua New Guinea by a land bridge during the last Ice Age, forming the continent of Sahul, until this bridge was flooded 6500–8000 years ago [Aphidini, which, although much more ancient [Aphidoideae), with no more than 20 endemic species; however, there are now around 200 other species of the 4700 mostly recorded in the temperate Holarctic regions of the world [A large number of potyviruses have been found on the isolated continent of Australia, with many of them being found only there. They provide insights into the timing of potyvirus migration, as Australia was biologically isolated for many millions of years , donkey orchid virus A (DOVA), and caladenia virus A  ,124,125.Carica papaya) [Robinia pseudoacacia [The spread of PRSV around the world is also an informative story. It was first recorded in Hawaii, by Jensen , and has papaya)  and Robidoacacia . In 1984doacacia . These vdoacacia ,131. PRSdoacacia ,103,118)doacacia . PRSV prdoacacia . An isoldoacacia ,134 groudoacacia  questiondoacacia  to make doacacia , where pdoacacia  found evIt seems that the PRSV-W to PRSV-P conversion occurs very infrequently. It was suggested  that thiIt is clear that the host ranges and distribution of potyviruses have been greatly affected by the Columbian Exchange, mentioned above, in https://en.wikipedia.org/wiki/Sweet_potato (accessed on 10 December 2019)). There is evidence that the five related potyviruses  from central America, passion fruit (Passiflora edulis) from South America, cowpea (Vigna unguiculata) from central Africa, only soybean (Soja max) being from SE Asia. By contrast, most of the primary hosts of the PVY lineage are asterids, and Fribourg et al. [https://en.wikipedia.org/wiki/Settlement_of_the_Americas (accessed 17 December 2019); https://en.wikipedia.org/wiki/Indigenous_peoples_of_the_Americas (accessed 23 Dec 2019)) from Beringia, which was, at that time, at the eastern end of the mammoth steppe biome. This biome stretched from Iberia, across the north of Eurasia to Beringia (https://en.wikipedia.org/wiki/Mammoth_steppe (accessed 11 December 2019)). Around 16 thousand YBP, ice retreated enough to open one, possibly two, routes from Beringia into the Americas [Artemisia spp. [Artemisia yet, and to do so might be very instructive.The BCMV and PVY lineages are the largest in the potyvirus phylogeny  to cause widespread infection in recently introduced crop, forage and naturalized weed legumes and Passiflora spp., which originated in South and Central America, or East Asia [Clitoria and siratro (Macroptilium atropurpureum) have so far only been isolated from naturalized introduced weeds but presumably spread from unknown native plant hosts to introduced species after European colonisation [There is evidence of agriculture being practiced by Australia’s aboriginal population well before the island continent was first colonized by Europeans in 1788. The crops they grew consisted of Australian native plants, such as wild yams and grasses grown for their storage roots and seeds, respectively . Interfa species ,154. PWVast Asia ,140,155.nisation ,118,155.H. comptoniana. PWV and PaVY occur most commonly in the warmer tropical and subtropical regions of the Australian continent, where, as described above in In situations where a stable mixed plant population infected with viruses has co-evolved with native wild plants over a significant period in a given world region, populations of its isolates collected over a limited geographic range are likely to be diverse . IsolatHistorical collections of plant viruses are proving extremely valuable in linking the pre- and post-sequencing eras of potyvirus research. This applies not only to providing sequence data for dating studies described above in Studies on historical isolates preserved since the 1970s and 1980s from the Andean region of South America and Europe provide examples. With PVY, a dating study without these historical isolates suggested the “time to most common ancestor” (TMRCA) of PVY was the 16th century , whereasTropaeolum tuberosum) sample was sequenced and compared with other potyvirus sequences, it was found to be a distinct potyvirus to which the name mashua virus Y (MasVY) was given; MasVY’s relationships to three other potyviruses , described previously from the same host, are unknown, as the genomes of these viruses are still not sequenced [Solanum muricatum) in Peru in 1976, and kept in a historical collection thereafter, have been recently shown, along with five new Peruvian pepino potyvirus isolates, to be isolates of wild potato mosaic virus, a virus previously known only from the wild potato Solanum chancayense [Historical samples were also valuable when the sequence of a historical arracacha virus Y (ArVY) isolate collected in Peru in 1976 was compared with other potyvirus sequences, as it had 79% nucleotide identity with a 2013 Brazilian isolate of the subsequently described arracacha mottle virus, showing the latter was actually ArVY . Similarequenced . Finallyncayense .Recently, new methods of gene sequencing have permitted surveys of virus-like genes and genomes in environmental samples  and in bulk cellular material . The resulting metagenomic sequences have greatly expanded the size of the known virosphere ,162.Mastigeulota kiangsinensis), collected in Wuhan, and has 73% ID with 95% cover of the genome of broad-leaved dock virus A (NC_038560) from Rumex obtusifolius from New Zealand. It is more distantly related to yam chlorotic necrosis virus (MH341583) from Dioscorea alata from India. Thus, Wuhan poty-like virus is more likely to be a constituent of the snails’ last supper than a virus infecting the snails. The other metagenomic potyvirid is Hubei poty-like virus 1 (NC_032912), which was obtained from a “dipteran mix” of insects collected around Hubei. It is a recombinant with a major parent  closest to an unusual isolate of sugarcane mosaic virus from Canna sp., in China (KY548507), and a minor parent (nts 7631-9094) closest to an unusual isolate of bean yellow mosaic virus  also isolated from “naturally planted” Canna sp., in China. This again suggests a contaminant virus, not an infection of the dipterans in the source material. Thus, both potyvirus-like metagenomes are more likely to be from contaminating plant materials than from the tissue of the animals being tested. Furthermore, Hubei poty-like virus 1 is probably an in silico recombinant, as its likely recombination site is adjacent to the region encoding the -GNNSGQP- motif, which is a conserved potyvirid motif [Two metagenomes appeared in the genomic sequences downloaded from GenBank, using the search term “Potyviridae”, and both were reported from China. One is of Wuhan poty-like virus 1 (KX884573), which groups with the macluraviruses . It was id motif ,12, and The major value of the esoteric studies of potyviruses described in this review is to provide a coherent evolutionary framework in time and space within which it is possible to plan how best to minimize the damage done by potyviruses to plants valued by humankind. Such a framework also contributes to the understanding of how potyvirus infections interact with plant populations within managed and natural ecosystems. Moreover, knowledge of the relationships of each virus, especially a newly emerged one, can provide valuable predictions of its likely behavior by extrapolating the known properties of near relatives and adding a timescale to those inferences greatly enhances their value and likely accuracy.The story in our review started in 1931, with PVY, one of the first two described viruses of potato, but now, nine decades later, at least 49 more viruses of potato have been recorded . The gro