Filoviruses, categorized as World Health Organization (WHO) Risk Group 4 (RG-4) pathogens, represent significant global health risks due to theirextraordinary virulence. The Filoviridae family encompasses Ebola strains such as Sudan, Zaire, Bundibugyo, Tai Forest (formerly known as Ivory Coast),Reston, and Bombali, in addition to the closely related Marburg and Ravn virus strains. Filoviruses originated from a common ancestor about 10,000 yearsago and displayed remarkable consistency in genetic heterogeneity until the 20th century. However, they overcame a genetic bottleneck by mid-century.Paradoxically, this resulted in the emergence of boosted virulent strains from the 1970's onward. Filovirus research is included in the NIAID BiodefenseProgram and utilizes the highest level specialized protective laboratories, Biosafety Laboratory (BSL)-4. The spread of Filoviruses as well as other RG-4pathogens within Africa poses a significant health threat increasingly both in Africa and out of Africa. Viruses classified under the World Health Organization's (WHO) Risk Group 4 (RG-4) pose a substantial threat to global health, as their rate of spread iscurrently on the rise. The increased spread is attributable to human-influenced social, economic, and environmental factors. Within this risk group, one notablefamily is the Filoviridae, which encompasses various strains of the Ebola virus, namely Sudan, Zaire, Bundibugyo, Tai Forest (previously referred to as Coted'Ivoire), Reston, and Bombali, as well as the closely related Marburg and Ravn virus strains. The Ebola virus is notorious for igniting aggressive epidemics,with prominent acute hemorrhagic illness and also a post-Ebola syndrome. Case fatality rates (CFR) associated with Ebola infections is strikingly high, varyingbetween 25-90%, underscoring the urgent need for comprehensive measures to curb its spread ,2,3,4. Ebola Virus Disease (EVD), first identified in 1976 in the Democratic Republic of the Congo (formerly Zaire), has since seen sporadic outbreaks throughoutWest and Central Africa, notably in countries such as Gabon, DRC, Sudan, Cote d'Ivoire, and Uganda. To predict and mitigate the truly existential threat posedby Ebola and its cognate Marburg virus infections in Central Africa, Ecologic Niche Modeling (ENM) was employed. ENM is a technique modeling insightfulbiogeographical as well as ecological predictions. This work incorporated data from 19 peer-reviewed studies spanning eight countries, examining variablesencompassing natural reservoirs of Filoviruses. The spectrum of reservoirs includes humans, non-human primates such as gorillas, chimpanzees, monkeys, bats,rodents, and arthropods. Unanticipated, some plant virus species form part of this diverse list cf. .[9]Risk factors contributing to the spread of these agents were also considered. These include occupational hazards such as mining, common practices like travel,attending social gatherings, personal contacts, and rarely sexual exposure. Moreover, certain cultural practices can facilitate virus transmission to humansfrom other species. This cross-species transmission, also known as a 'spillover' or 'jump', could arise from activities including keeping pet primates andmonkeys, hunting and consuming bushmeat (and carrion), attending social events, residing in communal dwellings, and camping. Finally, studies also examinedadditional ecological and socioeconomic parameters. These included contrasting ecological environments such as forests versus savannahs, urban versus ruralsettings, and modes of transportation - local residential versus highway travel. Factors such as these could significantly influence the transmission dynamicsand geographical spread of the Ebola and Marburg viruses ,9,10. FMarburg and Ravn viruses are related to Ebola and also are spreading. During 2023, two outbreaks occurred in Equatorial Guinea and Tanzania due to Marburgvirus. It's reservoir is the Egyptian fruit bat. Work is being done to ascertain if the two outbreaks were separate virus jumps or epidemiologically linked.12, Genome s-4 to 8.21 x 10-4for Sudan and Reston ebolaviruses, respectively. In greater detail, about 10,000 years ago, the Filoviridae family had a common ancestor. More recently, theMarburg virus group shared a common ancestor about 700 years ago and the Ebola virus group shared a common ancestor and the Ebola virus groups shared commonancestors 850 years ago. [As mentioned, Ebolavirus and Marburgvirus genera are members of the Filoviridae family. Carrol et al performed Bayesian coalescent phylogenetic analysisof 97 complete virus genomes. Virus molecular evolution rates (nucleotide substitutions/site/year) vary from 0.46 x 10ars ago.  The Marbars ago. ,16 Figurars ago. In addition to Filovirus infections expansion in Africa, including Angola, South Africa, Cote d'Ivoire, Guinea, Sierra Leone, the Democratic Republic ofthe Congo (formerly Zaïre), Uganda, Kenya, and Sudan, Filovirus infections spread out of Africa.  The wideStandard virus virulence theory states that virulent viruses become less virulent over time. However, the history to date of EVD does not conform to thattheory. This is a challenging current central question that is being addressed. ,33Filoviruses are among the planet's most virulent viruses and are spreading. In actuality, five genera are distinguished among the family Filoviridae: Ebolavirus, Marburg virus, Cuevavirus, Thamnovirus, and Striavirus. Continued research and clinical studies are needed. However, major restrictive bottlenecks toaccomplish these goals include the extreme dangers posed by the Filoviruses themselves and the social conflicts, terrorism, wars, suspicions, and violence tostrangers on the part of some risk/susceptible populations. Clinical diagnostic methods, research, and vaccine development require advances in the pharmaceuticalpipeline as well. , 34,35


This Special Issue highlights multiple facets of virus engineering, ranging from the dissection of the biological properties of individual viral functions in the context of safe genomic backbones, virus genetic modification for applications in gene therapy, oncolytic virotherapy and vaccine production, to the hurdles presented by quality control and the delivery of viruses for their final applications and finally to the simulation, prediction and validation of virus evolution.SARS-CoV-2 handling and modification is a hot topic in virus engineering. This previously presented a matter of debate due to the risks posed by the manipulation of a pandemic virus and by the misuse of a technology described in too much detail . In thisVirus emergence and evolution is a relevant theme, and for RNA viruses, it is entangled with their ability to exist and behave as a quasispecies. During the pandemic, SARS-CoV-2 demonstrated quasispecies behavior as well [Li et al. (contribution 3) present an extensive review on viral-vector-based gene therapy, providing the historical background and an overview of the major virus platforms used and the challenges they face according to their specific properties. They also highlight the advantages of virus-based interventions when combined with editing technologies like CRISPR/Cas, even if the latter are not yet fully developed or safe for routine clinical use e [In the framework of virus-based gene therapy, two research papers in this SI address the issue of AAV small cargo capacity and vector purity assessment. One approach to overcoming the small size of AAV is to engineer AAV with genomes optimized for size and that incorporate adenine base editors (ABEs) . FurtherIn the field of virus engineering for vaccine development, the H9N2 threat  has beenFinally, the work by Reale et al. (contribution 8) reports on an approach for the systemic delivery of herpes simplex viruses engineered for oncolytic virotherapy for the treatment of metastases and deeply rooted tumors. They show that human monocytes can be loaded with oHSV and deliver via intravenous injection the oncolytic virus to tumors xenografted on a chicken egg chorioallantoic membrane. Since an off-target delivery by monocytes to non-tumor tissue cannot be excluded, further safety measures to be combined with the delivery strategy include specific oHSV engineering for full retargeting and tumor-specific replication and virolysis ,8.


Based on data from the World Health Organization and World Bank in 2018, mortality rates caused by ischaemic heart disease in the Netherlands are amongst the lowest in Europe, at a relatively high health expenditure [The increase in cardiovascular morbidity represents an enormous challenge for our society and healthcare system. In 2019, healthcare costs amounted to 6.8 milliard euros, with the highest costs for men and a peak between 70 and 74 years of age . ParalleNetherlands Heart Journal, Wiethoff and colleagues offer an introduction to health technology assessment (HTA) and different types of economic evaluations [In this special issue of the luations . We inviWhile global healthcare costs for CVD are known , exact kEarly HTA can give insight into the potential value of important decision problems, i.e. which screening strategies, diagnostic strategies and treatment strategies to study, relating to the management of subpopulations of individuals with inherited cardiomyopathy. A systematic literature search indeed illustrated that there is limited current knowledge of the societal and economic burden of the most common forms of inherited cardiac disease: hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy . To incrWhile HTA in inherited cardiac disease is at an early stage, studies of HTA in the Netherlands on complications and health care costs of implantable cardioverter defibrillators (ICDs) and on management of acute ischaemic stroke are herein presented. ICD implantation is standard of care for the primary prevention of sudden cardiac arrest, but Van Barreveld and colleagues show that complication-related costs should be taken into account when considering the overall cost-effectiveness of device therapy . Van denThis issue thus shows the potential of HTA to quantify the cost-effectiveness of current diagnostics and clinical care from both a patient and societal perspective and the impact of novel therapies. Future research needs to include quality-adjusted life years and a broader range of costs to provide an information base for optimising care for affected patients.


Chikungunya virus (CHIKV) is an Alpha virus which is transmitted by Aedes genus of mosquitoes. It causes febrile illness with severe arthralgia, myalgia and rashes. Since 2000, the re-emergence of this pathogen have made it a global health security threat as it has caused severe form of diseases with epidemics of large magnitude and also spread to European and American continent. Studies have identified different point mutations in E1 and E2 region associated with virulence and infectivity, which is behind these severe epidemics. Pakistan reported first CHIKV outbreak with more than 30,000 infections from its largest city in 2016 which is already endemic for Dengue virus (DENV).32 CHIKV PCR-positive serum samples were collected from molecular pathology section of DUHS followed by RNA extraction and amplifcation of E1 region by PCR. The amplified products were prurified and sanger sequenced by BigDye termination method. Mutation analyses & Bayesian phylogeny was done by bioinformatic tools. CHIKV E1:M407I mutation was cloned in to CHIKV wild type infectious cDNA clone plasmid (SL-CK-1) by site directed mutagenesis (SDS). In vitro viral RNA was synthesized after plasmid linearization, which was electroporated into VERO-CCL-81 cells. M407I mutant virus was recovered from the culture, titrated by plaque assay and sanger sequenced to confirmed the M407I mutation. Viral competition assay was performed in mammalian and mosquitoes cell lines to determine the relative replicative fitness of mutant CHIKV virus over wild type.Sequence analysis revealed 7 unique mutations in E1 including one mutation (M407I) in more than half of the sequenced samples. Phylogenetic analyses revealed this outbreak was caused by CHIKV-IOL genotype with possible inception from India. Virus competition assay showed relative replicative fitness for CHIKV E1:M407I mutant in Vero-CLL-81 and AAG2 cells have greater advantage while in C6/36, it has slight advantage over wild type virus.This study reports a novel mutations in E1 protein of CHIKV with fitness advantage over wild type strain. Phylogeny revealed the outbreak was caused by A226-IOL strain with possible origin from ancestral strain of Indian outbreak.All Authors: No reported disclosures


We propose caution use and supervision when using Molnupiravir, especially when patients are still at risk of spreading virus.N4-hydroxycytidine (NHC), the active compound of the drug Molnupiravir, is incorporated into SARS-CoV-2 RNA, causing false base pairing. The desired result is an “error catastrophe,” but this bears the risk of mutated virus progeny. To address this experimentally, we propagated the initial SARS-CoV-2 strain in the presence of NHC. Deep sequencing revealed numerous NHC-induced mutations and host-cell-adapted virus variants. The presence of the neutralizing nanobody Re5D06 selected for immune escape mutations, in particular p.E484K and p.F490S, which are key mutations of the Beta/Gamma and Omicron-XBB strains, respectively. With NHC treatment, nanobody resistance occurred two passages earlier than without. Thus, within the limitations of this purely   •Molnupiravir might give rise to new SARS-CoV-2 variants when used without precautions•In vitro, the active compound of Molnupiravir gives rise to nanobody-resistant variants•We provide an integrated view on the selection of nanobody-escape mutants Pharmaceutical science; Immunology; Virology; Structural biology Besides an unprecedented vaccination effort, this also raises the urgent need for effective therapies.d-N4-hydroxycytidine (NHC).,,,,,Molnupiravir is orally applicable. Upon resorption and cleavage of an ester bond, the active compound is released, i.e., β-,,,Despite these impressive developments, a drug that causes virus mutations cannot be without concern, and the approval of Molnupiravir for clinical use was heavily debated. One reservation consisted of the possibility that Molnupiravir might also induce mutations in the cellular genome, which would then cause damage to embryos (in case of pregnancy) or perhaps contribute to malignant transformation.,,,,,Another concern, however, was raised more recently: Could Molnupiravir treatment facilitate the occurrence of mutant virus with increased virulencein vitro, recapitulating the treatment of SARS-CoV-2 infection by incubating infected cultured cells with the active compound of Molnupiravir, NHC. The virus population released from such cells indeed contained multiple mutations, as revealed by deep sequencing. Strikingly, two to four passages of this virus pool gave rise to selection of distinct mutants with apparent gains of fitness. The Furin cleavage site within the spike protein was mutated, plausibly contributing to virus replication in Vero cells. Most impressively, however, the rise of immune-escape mutants was recapitulated in this system. When incubating the pool with a potently neutralizing nanobody, resistant virus mutants were rapidly selected that had reproducibly mutated specific residues in the spike protein, most notably p.F490S, p.E484K, or p.G446D. These residues, according to structure analyses and AlphaFold predictions, were major mediators of the spike-nanobody interaction. Hence, at least in an in vitro system, NHC can indeed give rise to virus pools containing readily selectable mutants, with efficient adaptation to the cellular environment and complete resistance to a neutralizing nanobody.In the present study, we tried to model Molnupiravir-induced virus mutagenesis ,Vero E6 cells replicate SARS-CoV-2 with high efficiencyDuring the passage of virus pools in the presence of nanobody, we noticed that the tolerance of the virus toward the nanobody increased. Whereas the initial virus pool was largely neutralized by the nanobody, even 10 nM of the nanobody no longer prevented infection with the virus pool obtained at the end of the selection procedure. We observed this by quantifying both the virus RNA B and the,Before and after the incubation with NHC, we subjected the pool of virus RNA to paired-end RNA sequencing analysis  and S3. To gain further insight into the composition of virus mutations introduced by NHC treatment and/or passaging, we determined stop-gain mutations, i.e., nonsense mutations that prevent the full-length synthesis of the protein corresponding to a particular open reading frame (ORF). Immediately after treatment with NHC, stop-gain mutations were distributed quite evenly across the virus genome. With further passaging, however, the number of such mutations decreased, whereas the few “surviving” stop-gain mutations increased in frequency . When anWe next asked whether different conditions, i.e., presence or absence of a neutralizing nanobody, lead to the accumulation of distinct sets of virus mutants. We compared lower and higher passages in the presence of nanobody with two experiments carried out in parallel for each condition. Comparing the four virus pools that were grown in the presence of the nanobody, we found a high degree of overlap between the identified mutations. On the other hand, when comparing the virus populations obtained with and without nanobody, we found far less overlap of mutations. This was true when restricting our analysis to missense mutations with a high frequency of >20% A–4C but Next, we compared the predominant mutations obtained in the presence or absence of the nanobody against the spike protein. Here, we focused our analysis on mutations that were selected with high frequency under one or several conditions. Mutations were often silent or without obvious functional relevance for the protein, e.g., displaying conserved charge or hydrophobicity . We filt,,Analyzing the accumulated mutations within the spike protein strongly suggested increased virus fitness, in particular concerning its replication despite the presence of an initially neutralizing nanobody. Firstly, however, passaging NHC-treated viruses under any condition led to the accumulation of the mutation p.R682P/Q A. This mMost strikingly, however, selecting the virus pool with the nanobody led to the accumulation of the mutation p.F490S at a frequency of about 80% after prolonged passaging. Of note, p.E484K was also found under such conditions. F490 as well as E484 are both located within the receptor binding domain (RBD) of the spike protein, and they are also at the center of the interaction between the spike and nanobody Re5D06. We thus analyzed the structural consequences of these mutations in more detail, as described in the following.Other accumulating mutations of the spike protein have less obvious explanations B. For inThe mutation p.D198G was also found with increased frequency in the nanobody-selected virus pools. The residue D198 within the N-terminal domain (NTD) of the spike was reported to form a salt bridge contact with residue K462 of the RBD in the “down” conformation.Moreover, growing the virus in the presence of nanobody led to the accumulation of the mutation p.N1187D within the Heptad Region 2 of the S2 domain. The heptad regions are essential for fusion of viral and cellular membranes. HR1 and HR2 undergo dramatic changes in conformation during this process, interacting with the membranes and forming a bundle of six alphahelices.in vitro evolution assays can lead to different solutions to achieve nanobody resistance. When the virus had not been exposed to NHC, selection with nanobody eventually led to resistance, too, albeit at later passages. These resistant virus pools displayed the mutations p.F490S/V and p.E484G, similar to the NHC-treated samples, but they also led to the accumulation of the mutation p.L542R. As outlined in the following, all these mutated residues are located at the interface of the spike protein and the nanobody.Finally, we compared NHC-treated and non-treated virus pools, side by side, as to the selection of nanobody-resistant mutants. The same virus preparation was passaged thrice in parallel, in the presence of NHC or the DMSO control. This was followed by incubations with nanobody Re5D06 A. The viThe indicated mutations were present with high frequencies at passage 7 C. We theIn the presence of nanobody Re5D06, mutations of SARS-CoV-2 were strongly selected that replaced the residues F490, E484, L452, and G446 of the spike protein. Indeed, the selected virus population was almost completely resistant to Re5D06, with no detectable reduction of virus propagation in the presence of this nanobody B and 1C.24. As shown in We have previously reported the structure of the RBD in a complex with nanobody Re5D06The indicated mutations were selected during virus propagation in the presence of nanobody Re5D06. This not only raised the hypothesis that the mutations will disrupt the binding of the nanobody. It also suggested that the mutations should not counteract the binding of the virus to its receptor ACE2 through the spike RBD. The complex of the wild-type RBD with the ACE2 was previously found by cryo-electron microscopy.Next, we sought to interrogate the role of these mutations in the interaction between the RBD and the nanobody in more detail. We used the program AlphaFold, which was established to predict the folding of proteins into tertiary structures with high accuracy.We then subjected the same proteins for modeling, but with the mutations p.G446D, p.L452R, p.E484K, and p.F490S within the RBD. p.G446D did not grossly affect the predicted structure B. HoweveTo further investigate the impact of the most commonly found mutation p.F490S on the interaction between the RBD and ACE2, we modeled the complex of both proteins by AlphaFold, with or without the mutation F490S. The predicted structures of the complex did not grossly deviate from each other regardless of the mutation, and they were in agreement with the experimental structure Figures . AlphaFoThese results should be taken within the limits of a structure prediction algorithm. The use of AlphaFold to model the effects of a single mutation on structures still awaits further evaluation. Limitations when analyzing single proteins in this way were reported,Taken together, direct inspection of the RBD-nanobody structure as well as AlphaFold modeling are each compatible with the following concept: The substitutions p.G446D, p.L452R, p.E484K, and p.F490S allow the RBD to maintain its overall structure, preclude nanobody binding, and preserve ACE2 binding. In agreement, the mutations are also found in naturally occurring variants of SARS-CoV-2, i.e., p.F490S in variant Lambdain vitro system. Even more remarkably, the virus pool obtained in a single flask of NHC-treated cells proved sufficient for the rapid development of cell-adapted and antibody-resistant virus mutants, and NHC considerably accelerated the development of resistance.Our findings not only provide direct evidence that NHC, the active compound of Molnupiravir, is capable of inducing mutations in SARS-CoV-2 in an ,,,Overall, these findings strongly suggest that NHC treatment can give rise to virus mutants with increased fitness. However, our cell culture system is experimental in nature, and it is an open question whether this can be transferred to the situation of a Molnupiravir-treated COVID-19 patient. There can be little doubt that Molnupiravir will also give rise to virus mutants when used in the clinics. Indeed, recent reports provide evidence that such mutations are actually seen in patients.One concern would be that such virus mutants might quickly escape not only the immune response within an individual patient but also the antibody repertoire raised in a vaccinated population. In this context, it seems particularly worrisome that a small NHC-treated culture sample was capable of rapidly evolving resistance to one of the most potently neutralizing antibody structures reported so far, i.e., nanobody Re5D06.Besides immune escape, virus variants may also be selected to accelerate transmission between individuals. This can be achieved by increased stability of the infectious particles, by shifted cell tropism but perhaps most conceivably by enhanced efficiency of the pre-established viral entry mechanism. At least in the experimental system presented here, the most impressive mutagenesis was found within the spike protein. Not only the RBD as such was subject to nanobody-escape mutations. Mutations at the cleavage site of the spike were also found particularly often. Hence, mutations were selected to affect the virus-receptor interactions and the fusion of membranes, and this represents an important opportunity for coronaviruses to vary infectivity and cell tropism. Such mechanisms appear to gain momentum by a drug that mutates the virus genome. Thus, there is a concern that transmissible mutants could occur if Molnupiravir is used inappropriately, especially when treating patients with suboptimal doses, or for insufficient durations, and without isolating them. However, if used with appropriate care, the continued, controlled use of Molnupiravir might still be beneficial.A third concern is that Molnupiravir may promote the resistance of viruses against the drug itself or against additional therapeutics that were applied together with it. Since Molnupiravir does not work through direct inhibition of viral enzymes, Molnupiravir-resistant virus mutants may be unlikely to occur. And indeed, even prolonged NHC treatment did not lead to resistance formation in other coronaviruses.HPRT1-deficient mutants,Beside the mutagenesis in virus genomes, another concern of using Molnupiravir consists in the possible risk of mutagenesis in the DNA of patient cells. Such mutations, if they occur, might give rise to cancer. In pregnant women, they might also cause damage to the embryo. Currently, there is limited data to support such a scenario. Prolonged treatment of cultured cells with NHC for several weeks gave rise to low numbers of Why did we observe the accumulation of different mutations in parallel experiments with seemingly identical conditions? As in the real world, identical conditions can lead to the evolution of different variants of an organism, since stochastic processes play an important role and since several genetic solutions can often be found to meet a phenotypic challenge.,Molnupiravir is not the only drug that develops its antiviral activity through mutations in the virus genome.Further studies are required to assess the occurrence of mutant SARS-CoV-2 in Molnupiravir-treated animals and patients. However, even the available data are raising concerns regarding the use of Molnupiravir outside a controlled clinical setting. We propose that treatment with Molnupiravir should be limited to patients at high risk of developing severe COVID-19. Treatment doses and duration should be chosen to eliminate the virus below detectability. And the patients, while still being infectious, should avoid social contacts to limit the possible spread of virus variants that were formed due to Molnupiravir-induced mutagenesis.in vitro, i.e., in a cell culture system and using a single species of a spike-binding nanobody. This is different from the in vivo situation by several aspects: An infected body replicates the virus with different kinetics and in multiple cell types. These cells are likely to take up Molnupiravir and its metabolites with different efficacies. The immune response comprises a multitude of antibodies, flanked by T cells as well as the natural immune response. Finally, the virus spreads to other individuals through aerosols, not through incubation with tissue culture supernatant. In addition to these considerations, it should also be noted that we deliberately used a suboptimal dose of NHC in our study. Obviously, an NHC dose that eliminates all virus would not give rise to any mutants. Conceivably, keeping drug concentrations well above full efficiency levels would likely reduce the likelihood of raising escape mutants in vivo, too. Thus, the results of our study should be taken as what they are: a strongly simplified model system that reflects only single aspects of the infection of an organism. We show that Molnupiravir has the potential of selecting antibody-resistant virus. It remains to be seen whether this actually happens in a real-world setting, especially when patients are treated with Molnupiravir without fully suppressing virus propagation.The experiments shown here were all performed mdobbel@uni-goettingen.de).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Matthias Dobbelstein (This study did not generate new unique reagents.2. Vero E6 cells were authenticated in 2021 by means of Cytochrome C Subunit I (COI) DNA Barcoding by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Furthermore, the cells were routinely tested to ensure they were negative for mycoplasma contamination, using the MycoAlert Assay Control Set (Lonza).Vero E6 cells (Vero C1008) were obtained from the German Primate Research Center Göttingen. Cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 50 units/mL penicillin, 50 μg/mL streptomycin (Gibco), 2 μg/mL tetracycline (Sigma) and 10 μg/mL ciprofloxacin (Bayer) at 37°C in a humidified atmosphere with 5% CO4-hydroxycytidine  at the concentrations indicated in the legends to 250,000/750,000 cells were seeded into T25/T75 flasks, respectively, using medium containing 2% FBS, and incubated for 8 h at 37°C. Cells were treated with β-D-NFor RNA isolation, 100 μL of the SARS-CoV-2-containing cell culture supernatant was mixed (1:1 ratio) with the Lysis Binding Buffer from the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) to inactivate the virus. The viral RNA was isolated using Trizol LS, chloroform, and isopropanol. After washing the RNA pellet with ethanol, the isolated RNA was re-suspended in nuclease-free water. Quantitative reverse transcription and polymerase chain reaction (RT-PCR) was performed involving a TaqMan probe,50), the virus-containing supernatant was titrated (endpoint dilution assay). Vero E6 cells were seeded in 96-well plates and incubated with 10-fold dilutions  of virus for 48 h and then fixed with 4% formaldehyde in PBS for 1 h at room temperature. After permeabilization with 0.5% Triton X-100 in PBS for 30 min and blocking in 10% FBS/PBS for 10 min, a primary antibody was used to stain the SARS-CoV-2 Nucleoprotein . The secondary Alexa Fluor 546-coupled donkey anti-rabbit IgG antibody  was added together with 4′,6-diamidino-2-phenylindole (DAPI) for 1.5 h at room temperature. Fluorescence signals were detected by automated microscopy using a Celígo® Imaging cytometer. The titer was determined according to Spearman and Kärber.To determine the Median Tissue Culture Infectious Dose  was used to trim the adapters, followed by removing the contaminating human host reads by kraken2 (version 2.1.2).FastQC (version v0.11.9)https://www.ncbi.nlm.nih.gov/sars-cov-2/. The filtered alteration allele frequency along genome was plotted against the nucleotide positions using R 4.2.2. Mutation lolliplot was made using trackViewer R package.SARS-CoV-2 genome annotation GCF_009858895.2_ASM985889v3_genomic.gff was obtained from Predictions were carried out using AlphaFold-Multimer version 2.1.1 and 2.2.0. The Multiple Sequence Alignments (MSAs) used for the structure inference were built with the standard AlphaFold pipeline. Template modeling was enabled, but only templates that were released before 12–2019 were used. To model the RBD-ACE2 dimer, we also included more recent templates. As AlphaFold only uses single-chain templates and excludes templates with identical sequence, a simple replication of published structures is impossible.Structures were inferred with eight MSA recycling iterations and all five different model parameter sets. After prediction, models were ranked by the pLDDT score for monomers and pTM score for dimers.modelarchive.org) with the accession codes ma-qp22j (RBDwt-Re5D06), ma-0pnx1 (RBD p.G446D-Re5D06), ma-v4odj (RBD p.L452R-Re5D06), ma-jgqs4 (RBD p.E484K-Re5D06), ma-r62mw (RBD p.F490S-Re5D06), ma-lxnac (RBDwt-ACE2), and ma-eh742 (RBD p.F490S-ACE2). Model confidence predictions per residuum (pLDDT-score) are stored in the b-factors column of the .pdb file. PAE-value-plots for each structure are included in the ModelArchive entries.Predictions are available in ModelArchive . The significance of virus load difference between "Ctrl., +Nb" and "+NHC, +Nb" groups within each passage were estimated using Wilcoxon rank-sum test. Statistical details of each experiment can be found in the corresponding figure legend.


An in-house tomato inbred line, YNAU335, was planted in a greenhouse in spring from 2014 to 2017, and showed immunity to tomato spotted wilt virus (TSWV). YNAU335 was infected with TSWV in the spring from 2018 to 2020, and disease was observed on the leaves, sepals, and fruits. In 2021 and 2022, YNAU335 was planted in spring in the same greenhouse, which was suspected of being infected with TSWV, and visible disease symptoms were observed on the fruits. Transmission electron microscopy, deep sequencing of small RNAs, and molecular mutation diagnosis were used to analyze the pathological features and genetic polymorphism of TSWV infecting tomato fruit. Typical TSWV virions were observed in the infected fruits, but not leaves from YNAU335 grown between 2021 and 2022, and cross-infection was very rarely observed. The number of mitochondria and chloroplasts increased, but the damage to the mitochondria was greater than that seen in the chloroplasts. Small RNA deep sequencing revealed the presence of multiple viral species in TSWV-infected and non-infected tomato samples grown between 2014–2022. Many virus species, including TSWV, which accounted for the largest proportion, were detected in the TSWV-infected tomato leaves and fruit. However, a variety of viruses other than TSWV were also detected in the non-infected tissues. The amino acids of TSWV nucleocapsid proteins (NPs) and movement proteins (MPs) from diseased fruits of YNAU335 picked in 2021–2022 were found to be very diverse. Compared with previously identified NPs and MPs from TSWV isolates, those found in this study could be divided into three types: non-resistance-breaking, resistance-breaking, and other isolates. The number of positive clones and a comparison with previously identified amino acid mutations suggested that mutation F at AA118 of the MP (GenBank OL310707) is likely the key to breaking the resistance to TSWV, and this mutation developed only in the infected fruit of YNAU335 grown in 2021 and 2022. Orthotospovirus, family Tospoviridae, and order Bunyavirales. Listed as one of the ten most harmful plant viruses in the world, it is the only genus with plant-infecting members within the family Bunyaviridae [Solanaceae [Tomato spotted wilt virus (TSWV) is an important virus in the genus aviridae ,2. TSWV lanaceae ,4. Chinalanaceae .Sw-1a, Sw-1b, Sw-2, Sw-3, Sw-4, Sw-5a, Sw-5b, Sw-6, and Sw-7, were derived from common tomato (Solanum lycopersicum L.) or wild tomato plants [Sw-5 [Sw-5 [Sw5-a and Sw5-b, which were located in the Sw-5 locus, were cloned, but only Sw5-b increased resistance to TSWV [TSWV disease-resistant tomato resources were distributed mainly in wild tomatoes. In particular, Peruvian and Chilean tomatoes represented the most comprehensively resistant resources in which a number of excellent disease-resistant tomato materials and genes were identified ,7. Multio plants ,9,10,11.ts [Sw-5 . Multipl-5 [Sw-5 ,14,15. T to TSWV .Tospovirus in the endoplasmic reticulum pool in the host cytoplasm was the most important cytopathological feature of this virus genus [Tospovirus, was observed to have this feature in infected tomato fruits [Under an electron microscope, typical quasi-spherical and dumbbell-shaped particles of 80–100 nm in diameter were found in cells infected with TSWV ,18. The us genus . TSWV, aReplicating viral RNA genomes into small interfering RNAs (siRNAs) of discrete sizes were processed to guide virus clearance by RNA interference . It was Sw-5 gene, with the results indicating that a small number of tomatoes not only had local allergic reactions but also showed susceptibility in the whole plant, suggesting that some TSWV isolates had overcome the Sw-5 gene-based resistance. Subsequently, other breaks in Sw-5 resistance were observed [Sw-5 resistance [The genome of TSWV comprised a negative single-stranded RNA molecule consisting of three parts: L RNA, M RNA, and S RNA, and containing five open-reading frame coding for four structural and two non-structural proteins . Latham observed ,27,28,29observed . It was Sw-5 gene [In this study, the tomato inbred line YNAU335, which previously showed immunity to TSWV, was investigated and demonstrated not to have the w-5 gene . YNAU335w-5 gene . In 2021Solanum lycopersicum L.) that shows the characteristics of typical cultivated species, collected from Yuanmou County, Yunnan Province, China, and was used in this study. Leaves and fruits of YNAU335 were sampled annually from 2014 to 2022.The inbred line, YNAU335, is the cultivated species  was added to the dried films for 3 min and was dried to dye the virus material, which was then observed under a JEM100CX-II transmission electron microscope  .4 solution at 20 °C for 2 h. After ethanol gradient dehydration, epoxy resin Ep812 embedding, AO ultrathin microtome sectioning, and staining with 5% lead citrate and 1% uranyl acetate, the samples were observed and photographed under a JEM100CX-II transmission electron microscope  [The parts ~0.2 cm below the tomato fruit surface were cut into small pieces of 1 mm × 1 mm × 3 mm and fixed in 2.5% glutaraldehyde solution for 12 h at 4 °C. A phosphate-buffered solution  was used to rinse the sample, which was then fixed in 1% OsO, Japan) .TSWV, tomato zonate spot virus (TZSV) and tomato brown rugose fruit virus (ToBRFV) display similar symptoms in tomato fruits, and the virions of TZSV and ToBRFV are similar in shape. RT-PCR was used to further verify the virus species. The total RNA of fruits and leaves of YNAU335 of 2021–2022 was extracted using the Quick RNA Isolation Kit , and then treated with RNase-free Dnase I  to remove the genomic DNA. Total RNA (2 μg) was reverse transcribed into first-strand cDNA using the M-MLV Reverse Transcriptase Kit , according to the manufacturer’s protocol, and oligo (dT) primer and random primers were used in the reverse transcription reactions. The cDNA samples were used as templates for RT-PCR. Primers pMP, pNP, pTZSV, and pToBRFV for the TSWV-MP, TSWV-NP, TZSV, and ToBRFV, respectively, are shown in 2O were combined to yield a final volume of 50 μL. PCR conditions: pre-denaturation at 98 °C for 2 min, followed by denaturation at 98 °C for 10 s and annealing for 5 s, with the annealing temperature set according to the base composition of each primer. Extension was performed at 72 °C for 1 min for 40 cycles. The final extension lasted 5 min.PCR reaction system: 25 μL of PCR high-fidelity enzyme , 1 μL each of the upstream and downstream primers, 1 μL of cDNA template, and sterile ddHTM Small RNA Sample Prep Kit. First-strand cDNA was obtained via reverse transcription with random primers using the TruseqTM Small RNA sample prep Kit. The library was enriched using PCR for 11 to 12 cycles. The product was recovered using PAGE electrophoresis and quantitated on a TBS380 . The clusters were generated using the cBot system and sequenced on an Illumina HiSeq 2000 platform [Three grams of fresh material was sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd.  for deep sequencing. Total RNA was spliced at the 5′ and 3′ ends using a Truseqplatform . The rawhttp://hannonlab.cshl.edu/fastx_toolkit/, (accessed on 11 November 2022)). After quality control, the high-quality sequencing reads were assembled de novo to generate contigs and singletons, and the small RNAs that ranged from 18 to 40 nt were also counted. The assembled data were compared with the virus database ) and annotated. According to the results of the assembly and comparison with the plant virus database, the virus with the highest proportion was selected, and the corresponding viral genome sequence was downloaded from NCBI to predict the virus-induced siRNA.The raw sequencing data of the fruits and leaves of YNAU335 planted in 2014–2017, 2018–2020 and 2021–2022 were assessed for quality and subjected to data filtering using the Fastx-Toolkit program . MEGA5.0 was used to analyze the amino acid mutation sites on the NP and MP.The fruits of infected tomatoes in 2021 and 2022 were used as experimental materials. Sample RNA extraction refers to the above-mentioned method. Forty positive colonies were randomly selected to identify the NP and MP genes of TSWV. The colonies were cultured for development and identification, followed by sequencing at Beijing Tsingke Biotech Co., Ltd. . The gene accession number of the sequenced and spliced coding sequence was obtained through the BankIt submission system at Throughout the growth period from 2021 to 2022, the tomato leaves of YNAU335 were normal in appearance and showed no symptoms of TSWV. The green fruit showed chlorotic ring spots, tumor-like protrusions, and blurred ring patterns A. DuringIn order to preliminarily identify the species of virus infecting tomato fruit of YNAU335 planted in 2021 and 2022, a negative staining technique was used to analyze the virus. Typical TSWV virions, with a diameter of ~80 nm and aggregates with a nearly spherical shape, were observed .The infected tomato fruits showed severe subcellular pathological changes with plasmolysis observed. The nucleus, cell membrane, endoplasmic reticulum, Golgi apparatus, and other substructures disappeared, with only the cell wall, mitochondria, chloroplasts, vacuoles, and peroxisomes observed A. The nuTSWV, TZSV, and ToBRFV display similar symptoms in tomato fruits, and it is difficult to distinguish them based on symptoms alone. RT-PCR was used to determine which virus the tomato fruit was infected with. The results showed that the NP and MP genes of TSWV were cloned from the fruits of the infected tomatoes of 2021 and 2022  and five NPs  were selected as controls . The seqIn each spring from 2014 to 2022, three inbred tomato lines, No. 5, CLN2037E, and 96172I in a greenhouse planted with YNAU335, were infected with TSWV every year, or even failed to harvest. Therefore, YNAU335 was guaranteed to be in a TSWV-susceptible environment every year. After small RNA deep sequencing, abundant virus species were detected in infected and non-infected YNAU335 plants. TSWV was not detected in YNAU335 plants showing no symptoms of TSWV infection. Most of the detected viruses were bacteriophage viruses and a small amount were animal viruses. The number of TSWV sequences in TSWV-infected tomato tissues accounted for more than 50% of all virus sequences. However, other kinds of viruses were present in TSWV-infected tissues that were roughly the same as those in non-infected plants. The exclusion of these same viruses indicated that TSWV played a dominant role in YNAU335 infection.Tospovirus in the endoplasmic reticulum pool in the host cytoplasm is the most important cytopathological feature of the genus virus [Tospovirus, was also observed with this feature in the infected tomato fruits in this study. Under natural conditions, the mixed infection of two or more viruses on the same host plant is quite common. Mixed infection caused by different viruses were antagonism or synergism [Datura stramonium [Cymbidium hybridum [The accumulation of us virus . TSWV, aynergism . TSWV anramonium ,37. Therramonium . Zhang ehybridum . This stCapsicum annuum) plants harboring the Tsw resistance gene was found after simultaneous inoculation of resistance-breaking (RB) and non-resistance-breaking (NRB) isolates [citrus tristeza virus was found to be overwhelmingly dominated by the severe strain, which shows that CTV-B2 does not provide a useful level of cross-protection to citrus against CTV-B6 [Disease synergism in pepper  and NPs  from resistance-breaking isolates that were found in previous studies. This result revealed rich genetic diversity in the MP and, in particular, the NP of the tomato fruit. Based on a combination of their frequencies, the amino acid mutations, especially that at F118 in the MP, were suggested to play a major role in YNAU335 resistance-breaking by TSWV. A previous study showed that the mutation at F118 from C to Y and mutation T120 to N were related to breaking . This re to TSWV . HoweverThe immune characteristic of tomato inbred line YNAU335 to TSWV was broken, and the disease symptoms only occurred on fruit. In addition to other plant viruses, TSWV accounted for the highest proportion of infected fruits. Mutation F at AA118 of the MP, developing only in the infected fruit, was likely to be the key to breaking the resistance to TSWV.


Rheumatoid arthritis (RA) is a common chronic condition with substantial morbidity that can now be treated with disease-modifying biologic agents that target tumor necrosis factor (TNF) or related mechanisms. The anti-TNF biologic agents are available in either intravenous (IV) or subcutaneous dose forms. The biologic agents with an indication for rheumatoid arthritis and administered only by IV infusion in medical offices include abatacept, infliximab, and rituximab. Although the literature on RA treatments, their outcomes, and aspects of their costs is substantial, the costs of administration by the IV route have not been directly studied.To assess the detailed costs of administering IV biologic agents for the treatment of RA in relation to the total cost of the medication itself in the United States.The sample included all patients with at least 1 medical claim with an ICD-9-CM diagnosis code for RA (codes 714.XX) in any claim field and at least 1 claim for infliximab, abatacept, or rituximab  at any time from January 1, 2006, through December 31, 2008, in a database associated with billing and claims administration for 72 U.S. medical clinics. Costs were determined using the payer allowed payment, which is the total contractual amount that the provider should receive, including the patient cost share. Costs were measured as the average cost per IV administration visit and in relation to the dose of medication billed. The authors verified that an RA diagnosis was present on 100% of infusion claims for the study drugs.Over the study period for claims with dates of service from January 1, 2006, through December 31, 2008, 72 medical clinics had claims for a total of 4,248 unique patients with RA and a total of 33,354 clinic visits in which these patients received at least 1 infusion of 1 of 3 biologic agents . Mean (SD) total payment for all drugs and other cost components was $2,874  per visit, of which IV administration costs were $226 (7.9%); the mean cost of the biologic agent itself was $2,616 (91.0%), and other visit-related services were $33 (1.1%). For individual agents, the total costs of visits were $2,828, $1,827, and $6,076; and the costs of IV administration were $224, $171, and $390, respectively, for infliximab, abatacept, and rituximab.For patients who received an IV biologic agent to treat RA, IV administration costs accounted for 7.9% of the total cost of the visit.


Viruses infect all cellular life forms and cause various diseases and significant economic losses worldwide. The majority of viruses are positive-sense RNA viruses. A common feature of infection by diverse RNA viruses is to induce the formation of altered membrane structures in infected host cells. Indeed, upon entry into host cells, plant-infecting RNA viruses target preferred organelles of the cellular endomembrane system and remodel organellar membranes to form organelle-like structures for virus genome replication, termed as the viral replication organelle (VRO) or the viral replication complex (VRC). Different viruses may recruit different host factors for membrane modifications. These membrane-enclosed virus-induced replication factories provide an optimum, protective microenvironment to concentrate viral and host components for robust viral replication. Although different viruses prefer specific organelles to build VROs, at least some of them have the ability to exploit alternative organellar membranes for replication. Besides being responsible for viral replication, VROs of some viruses can be mobile to reach plasmodesmata (PD) via the endomembrane system, as well as the cytoskeleton machinery. Viral movement protein (MP) and/or MP-associated viral movement complexes also exploit the endomembrane-cytoskeleton network for trafficking to PD where progeny viruses pass through the cell-wall barrier to enter neighboring cells. Viruses are intracellular biotrophic parasites that infect all living organisms and exclusively replicate themselves in the infected cells. The majority of viruses that infect animals and plants are positive-sense single-stranded (+ss) RNA viruses . Plant-iThe plant endomembrane system consists of the endoplasmic reticulum (ER), Golgi apparatus, vacuole, nuclear envelope, plasma membrane (PM), trans-Golgi network/early endosome (TGN/EE), and prevacuolar compartment/multivesicular body/late endosome (PVC/MVB/LE) and transport vesicles . As the The plant endomembrane system controls the secretion of biomolecules and mediates the uptake of substances from the exterior of the cell and the delivery to specific intracellular locations . The secIn plant cells, the endomembrane compartments interact extensively with the cytoskeleton system that is composed of actin filaments (AFs), microtubules (MTs), and associated proteins ,32. The Upon infection, plant viruses preferentially target and remodel specific membranous organelles, such as the ER, peroxisomes, mitochondria, chloroplasts, and/or tonoplasts for the formation of VROs. In addition to the origin of membranes, the shape and size of VROs may also vary from virus to virus. Some viruses may not have strict requirements for a particular type of membrane source, as they can change to alternative organelles for replication ,43,44. TAs mentioned above, different viruses target different organelles to form VROs. Based on their morphology, VROs may be divided into two groups: the invaginated/spherule type, and the protrusion type ,49,50. TBrome mosaic virus , Tobacco mosaic virus , Red clover necrotic mosaic virus , and Cucumber leaf spot virus  induce membrane invaginations towards the ER lumen to form spherules or vesicles in the perinuclear region, or randomly dispersed in the cytoplasm [Beet black scorch virus , three-dimensional electron tomographic analysis reveals the formation of multiple ER-originated vesicle packets (VPs), which enclose anywhere between a few and hundreds of 50–70-nm independent invaginated spherules [Turnip mosaic virus (TuMV), convoluted membrane (CM) structures appear in close proximity to the rough ER, followed by the production of both SMV- and DMV-like structures [Peanut clump virus  remodels the ER and induces the formation of MVBs in tobacco protoplasts [Wheat yellow mosaic virus  remodels the ER to form membrane inclusion bodies (MIBs) [The ER is the most frequently targeted organelle to form VROs for viral genome replication. Viruses, such as ytoplasm ,53,54,55pherules . In planructures ,56. Thesructures . Peanut toplasts . Remarkas (MIBs) , lamellas (MIBs) . Tomato bushy stunt virus (TBSV) and Cucumber necrosis virus (CNV) to form VROs [Melon necrotic spot virus  and Carnation Italian ringspot virus , target and remodel mitochondria for VRO formation [Cymbidium ringspot virus (CymRSV) that carry mitochondrial or peroxisomal targeting signals are able to form VROs in the newly targeted organelles [Peroxisomes are preferred by some tombusviruses such as orm VROs ,60,61. Torm VROs . These sormation ,64,65. Iormation ,46. TBSVormation . Consistganelles . Turnip yellow mosaic virus , Barley stripe mosaic virus , and Tobacco rattle virus , build their VROs on chloroplasts [Chloroplasts are an organelle unique to the plant cell and some protists. Many viruses, such as roplasts . In TYMVroplasts . During roplasts . Thus, Broplasts ,72.Cucumber mosaic virus  or Tobacco necrosis virus A Chinese isolate  [The tonoplast, the semipermeable membrane surrounding the vacuole, is also targeted for viral replication by several plant viruses, such as rovirus) ,74. InfeTaken together, different plant RNA viruses have their preferred organelles for VRO formation, but at least some of them seem to have the ability to utilize alternative cytoplasmic membranes for viral genome replication. A few examples of modified membranous structures are presented in VRO biogenesis is initiated when newly translated viral proteins target the preferred organelle(s) and remodel the organellar membrane. These viral proteins are usually membrane-associated proteins ,75. The Many of these viral membrane-associated proteins have one or several transmembrane domains (TMDs) which are typically 12–35 residues (mostly hydrophobic amino acids). The TMD(s) are required for membrane insertion ,77,78. STomato ringspot virus , BMV 1a, and the p33 and p36 proteins from tombusviruses, have been studied. The potyviral 6K2 protein is an integral membrane protein and expression of 6K2 alone induces ER proliferations and the formation of numerous cytoplasmic vesicles [Since the late 1990s, membrane proteins encoded by many plant RNA viruses, such as 6K2 from potyviruses, the nucleotide triphosphate-binding protein (NTB) of vesicles ,82. The vesicles .In the case of ToRSV, the NTB-VPg polyprotein, which is the precursor of NTB and VPg  is associated with ER-derived membranes active in virus replication ,83. The Different from 6K2 and NTB, the BMV replication protein 1a does not contain any TMD and instead has two AHs  that are adjacent to each other. BMV 1a is a multifunctional protein and consists of a capping domain at the N-terminus and a helicase-like domain with NTPase activity at the C-terminus. AH A and AH B are located in the region E (114 amino acids in length) between the capping and helicase-like domains. Previous studies have suggested that the region E is necessary for nER membrane association, and AH A plays a role in nER targeting and regulates the size and frequency of VROs ,85. A moFor tombusviruses, such as TBSV and CIRV, viral proteins p33 and p36 that contain an N-terminal hydrophilic region and two TMDs are required for the membrane association of VROs. TBSV and CIRV target peroxisomes and mitochondria, respectively, to induce the formation of multivesicular bodies (MVBs). The N-terminal hydrophilic region and the TMDs specify which organelle is involved in VRO biogenesis ,67. In tWhile viral membrane-association proteins are critical for membrane modification, VRO biogenesis requires various host factors such as lipids, cytoskeleton, membrane-shaping proteins, and components of the membrane trafficking pathway.Soybean mosaic virus  and contributes to viral replication. However, it remains unclear how RHPs are involved in the formation of potyviral VROs [Dengue virus , depletion of ATL2 alters the formation of ER-derived VROs by distorting vesicle size and shape and condensing the vesicles to a small perinuclear region [In high eukaryotes, at least four curvature-generating and -stabilizing protein families, including the Bib-Amphiphsin-Rvs (Bar) domain proteins (Bars), the reticulons (RTNs), the membrane-fusing GTPase atlastin (ATL), and the lunapark protein (Lnp), are known to affect the shape of membranes. Among them, BARs, RTNs and ATLs have been shown to be implicated in VRO biosynthesis ,7. BARs ral VROs . ATLs arr region . In planr region ,93,94. Tr region .The endosomal sorting complexes required for transport (ESCRT) machinery, made up of cytosolic protein complexes, known as ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, play a vital role in a number of cellular processes, such as MVB biogenesis and autophagy, through a unique mode of membrane remodeling that results in membrane bending/budding away from the cytoplasm ,97,98. DThe early secretory pathway is composed of the ER, Golgi bodies, and vesicles that travel between them . The traThe early secretory pathway has been shown to make essential contributions to VRO biosynthesis for diverse plant +ssRNA viruses. RCNMV p27 interacts with Arf1 in the virus-induced structures originating from the ER membrane. Inhibition of Arf1 using the inhibitor brefeldin A (BFA) disrupts p27-mediated ER remodeling and VRO assembly, and expression of a dominant-negative mutant of Arf1 or Sar1 compromises RCNMV RNA replication . These fCowpea mosaic virus , the SNARE-like ER protein Vap27-1 interacts with the CPMV 60K membrane protein and colocalizes with the 60K-induced membranous vesicles [In the case of TBSV, a screening of Legionella effectors for antiviral effects led to the discovery that TBSV hijacks Rab1  and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within VROs . Rab1 isvesicles . Recentlvesicles . The TMVvesicles . An increasing body of evidence supports the involvement of endocytic pathway regulators in viral replication. Dynamins are important regulators of clathrin-mediated endocytosis (CME), which is a predominant endocytosis pathway in plant cells . RecentlRetromer plays a key role in the recruitment of cargo for endosomal trafficking and recycling . It contEarly and late endosomes participate in VRO biosynthesis too. For example, endosomal Rab5 small GTPase  is a key regulator of early endosomal biogenesis, maturation, trafficking, and membrane fusions. TBSV recruits Rab5 into VROs via the interaction with TBSV p33 . Phosphapah1 mutant, TBSV switches from the peroxisome to the expanded ER membrane for VRO formation [Tombusviridae family induces VROs that contain PA-producing enzymes phospholipase Dα (PLDα) and Dβ (PLDβ) [Lipids are essential components of cellular membranes and directly affect membrane asymmetry, curvature, and proliferation ,125. Plaormation , whereasormation . Regardlβ (PLDβ) . RCNMV pβ (PLDβ) .Sterols are unique among lipids as they have a multiple-ring structure. Similar to common lipids, sterols also play a role in viral replication ,133. TBSAccumulated evidence suggests that plant RNA viruses hijack the cytoskeleton for VRO biosynthesis. One such example is that TMV uses MTs for the anchorage of VROs and further development of these VROs into large virus factories ,41,139. To establish systemic infection, plant viruses must cross the cell wall barrier via PD to enter and infect the adjacent cells. Viral cell-to-cell movement requires the co-ordinated action of viral MPs and the endomembrane system and associated cytoskeleton. For some plant RNA viruses, viral replication and intracellular movement are structurally and functionally linked processes . This inPotato virus X  and Poa semilatent virus  encoding triple gene block (TGB), seem dependent on the ER network, but not the secretory pathway for movement as well. TGB2 and TGB3 are small membrane-integrated proteins that are involved in the delivery of TGB1 and the viral genomic RNA bound by TGB1 to PD-associated sites for intercellular movement [Among eukaryotic living organisms, the plant ER is a unique structure that is continuous between adjacent cells via the PD desmotubules to form an ER network throughout the entire plant, and thus provides a direct pathway for the movement of ER-associated proteins or macromolecular complexes, including the viral MP-associated movement complex to PD and subsequently into neighboring cells. Indeed, TMV intercellular movement requires the ER membrane, but is independent of the secretory pathway. TMV MP is associated with the cytoplasmic face of the ER . The intmovement ,149. TGBmovement . Sar1 inmovement . Tomato spotted wilt virus  and its MP NSm [The ER membrane transport system is also critical for the intercellular movement of the negative strand RNA s MP NSm . TSWV MPs MP NSm . The ER-s MP NSm .Grapevine fanleaf virus  MP assembles tubules at PD with the assistance of PD-located proteins (PDLPs) for viral intercellular movement. Both GFLV MP and PDLPs localize to PD via the ER-Golgi secretory pathway, as disruption of the early secretory pathway by expression of the Sar1 dominant-negative mutant or genetic mutation of PDLP genes prevents the PD targeting of the GFLV tubule-forming MP [Arabidopsis thaliana sec24a mutant defective in the secretory pathway, TuMV systemic movement is delayed [Unlike TMV, TSWV and viruses with TGB MPs discussed above, many other plant viruses require the ER-Golgi secretory pathway for cell-to-cell movement. rming MP . TuMV inrming MP . Endogenrming MP . In the  delayed . In addi delayed ,160,161.Bamboo mosaic virus  moves from the ER membrane to the PM via the endosomal system by vesicle trafficking with the activation of NbRabF1, a plant-specific Rab5 small GTPase from Nicotiana benthamiana [Potato mop-top virus  traffic in the endocytic recycling pathway [Cabbage leaf curl virus  [Turnip vein clearing virus  [The endocytic pathway appears to be involved in viral intercellular movement for several plant viruses from viruses encoding TGB MPs, tobamoviruses, and potyviruses. In the case of viruses with TGB MPs, the viral movement complex of thamiana . The two pathway . Synaptomovirus) . Moreovemovirus) ,165. Recmovirus) . Treatmemovirus) . These vmovirus) . CI intemovirus) .Barley yellow striate mosaic virus  [Rice black-streaked dwarf virus  [Rice grassy stunt virus (RGSV) and Rice stripe virus (RSV), respectively [Trafficking of organelles and virus-induced complexes in plant cells is largely dependent on the dynamics of the cytoskeleton ,36,37. Tdovirus) ,168. Disdovirus) . Specifidovirus) . These tdovirus) ,171. Thejivirus) , and pC6ectively ,161. Difectively . MyosinsThe involvement of MTs in plant viral movement has also been studied in TMV ,173. MTsIn this review article, we have briefly summarized the current knowledge about how plant viruses deploy their membrane protein to target preferred cellular organelles and further recruit various host factors to modify membranes for VRO biogenesis. We have discussed that some viruses have the ability to target alternative organelles for viral replication. The virus-induced cellular membranous structures are in different forms, such as spherules, vesicles, and MVBs. We have highlighted progress in the recruitment of the endomembrane system and cytoskeleton by plant RNA viruses to move intracellularly to PD for their intercellular movement . R gene Rsv4 can target SMV VROs to inhibit viral multiplication [Despite the significant advances in the role of the endomembrane-cytoskeleton network in VROs biogenesis, many intriguing questions remain to be answered. For example, it has been shown that some ER-derived small virus replication structures are mobile at the early stage and form large static aggregated structures (VROs). The mechanical details underlying this transition are largely unknown. VRO intercellular movement is still an understudied area. It is not clear if VRO mobility is a conserved feature for all plant RNA viruses. What is the minimum requirement of the host factors for VRO biogenesis? Are any host factors conserved among different hosts and viruses? How do VROs concentrate essential materials, including energy supplies, for viral replication? Moreover, as always, novel discoveries lead to more questions. For example, a recent study has shown that a dsRNase with dsRNA-degrading activity encoded by the broad-spectrum SMV lication . This woThe molecular identification and functional characterization of host factors essential for viral replication and movement are of great interest, as host factors may be targeted for the development of genetic resistance in plants through advanced technologies such as the precise genome editing technology. Effective methods for the discovery of host factors, such as proximity labeling , lipidom


Pulses have gained popularity over the past few decades due to their use as a source of protein in food and their favorable impact on soil fertility. Despite being essential to modern agriculture, these species face a number of challenges, such as agronomic crop management and threats from plant seed pathogens. This review’s goal is to gather information on the distribution, symptomatology, biology, and host range of seedborne pathogens. Important diagnostic techniques are also discussed as a part of a successful process of seed health certification. Additionally, strategies for sustainable control are provided. Altogether, the data collected are suggested as basic criteria to set up a conscious laboratory approach. Brazil The Rural Development Policy 2014–2020 encourages the use of legumes in new agricultural systems, because they boost soil fertility by contributing to the recovery of natural organic matter. It is well known that legumes secrete organic acids from the roots, which enables them to solubilize phosphate and, when grown in rotation with other crops, to induce pathogen suppression. Legumes also fix nitrogen (fixation), which allows the enrichment of nitrogen content in the soil . At a glIn this scenario, legumes assume a major role in food production. In order to obtain high production, challenges in crop management have to be faced, and research needs to be conducted to increase yields by means of preventing pest spread and devastating diseases. In fact, even though legumes are promising for future food production, current trends have been more focused on the optimization of cereal and oilseed cultivation, leading to the marginalization of Leguminosae crops . BecauseFusarium spp. are not included in this review, since it is a complex genus that is extensively described in several commendable reviews . In. InAscocng vigor ,61; sympng vigor . Similarn leaves . Symptomare huge ,65. Regater & Aa . In thister & Aa . The seeter & Aa . Additioin seeds . In 2007chickpea . Then, iRhizoctonia solani J.G. Kühn is one of the most widespread soilborne pathogens that inflict massive damage to economically important crops worldwide . Ho. HoDiapomycelium . ExtensiPhillips . Similar soybean . FurtherBotrytis is an Ascomycota, taxonomically classified in the kingdom Fungi, phylum Ascomycota, subphylum Pezizomycotina, class Leotiomycetes, order Helotiales, family Sclerotiniaceae, and genus Botryotinia . T. T121]. tic spot . The seePhaseolus acutifolius, P. coccineus, P. lunatus, Cajanus cajan, Centrosema sp., Desmodium spp., Glycine maxima, Lablab purpureus, Lens culinaris, Macroptilium atropurpureum, Neonotonia wightii, Pachyrhizus erosus, Pisum sativum, Pueraria lobata, P. thunbergiana, Vigna angularis, V. radiata, and V. unguiculata [Psp is one of the most studied bacterial pathogens, and it was the first plant pathogenic bacterium in which the T3SS was described. It is responsible for the halo blight of beans, and it is spread wherever the host is cultivated (more than 50 countries on five continents) . The Pspuiculata .Pseudomonas viridiflava (Pv) is a Gram-negative, fluorescent bacterium. Taxonomically, it is classified as Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; Pseudomonas viridiflava. It exhibits endophytic, epiphytic, saprophytic, and phytopathogenic behavior and is phylogenetically included in the P. syringae complex in genomospecies 6, and it is diffused in PG7 and PG8. Its ability to cause soft rot on potato tubers, its phenotypic phase variation, and its noncanonical T3SS are typical features of PG7, while strains belonging to PG8 produce a typical toxin when tested on Geotricum candidum. Since 2000, Pv has been found to be responsible for 13 outbreaks and has affected about 50 hosts, classifying itself as a generalist plant pathogen. In legumes, the source of the inoculum can be represented by contaminated environments, such as biofilms, irrigation water, and rain, and can be transferred to plants by droplets, aerosols, and wind. The bacteria enter the plant tissues through stomata, hydathodes, and mechanical wounds. The infection process is favored in stressed plants by high humidity, intensive rainfall, and low temperatures [eratures . Pv  is a Gram-positive bacterium that is responsible for bacterial wilt or tan spot in common bean, cowpea, mung bean, and soybean. The EPPO database taxonomically reports Cff as Bacteria; Actinobacteria; Micrococcales; Microbacteriaceae; Curtobacterium; Curtobacterium flaccumfaciens pv. flaccumfaciens. According to the EPPO Global Database, Cff was recorded in 43 countries on five continents , but other non-official reports are documented in European countries. In Asia, the only affected country is Iran. In general, the most important outbreaks have occurred since the beginning of the 21st century.Xanthomonas or Pseudomonas infections. The bacteria can overwinter in plant debris, soil, or weeds or can survive for long periods (for 24 years) in seeds, under the seed coat; it is transmitted from residues or infected plants through water , reaching wounds on plant tissues either in the aerial parts or in the roots (root-knot nematodes can favor the entry of the pathogen). Additionally, Cff can enter the ovules through the funiculus or the micropyle and reach the seed, but the contamination of the pods is hampered by the vascular system closing during ripening. Seed infection is considered the main pathway for transmission to plants, with an efficacy that has been evaluated to range from 6 to 70%. Colonies of Cff isolated in 1950–1960 from diseased plants have a yellow or orange color, and only in 2007–2008 were pink and red colonies observed. Even though yellow and orange isolates appear to be more virulent on bean and cowpea than red and pink ones, all the isolates were thought to belong to the same species and pathovar following biochemical, physiological, and molecular characterization. Molecular tools can distinguish among C. flaccumfaciens pathovars. Combining the results of AFLP, rep-PCR, and pulsed-field gel electrophoresis (PFGE), it is possible to divide the differently colored isolates into two clades: yellow, orange, and pink strains fall into clade A, while, in clade B, only yellow strains are allocated. With the same techniques, it is possible to distinguish among pathovars: AFLP and rep-PCR recognize almost all the pathovars, but the use of PFGE, followed by digestion with enzymes, perfects the pathovar identification.The main symptoms of the disease on common bean are cotyledon and leaf wilting, which progress during the colonization of plant tissues. As generally occurs in vascular diseases, in the initial phase of the infection, the basal leaves wilt, but, after watering, they recover turgidity. As the disease progresses, watering does not allow the leaves to recover normal turgidity, and the leaves turn yellow and irreversibly wilt. With the worsening of the disease, the leaves necrotize, with lesions surrounded by chlorotic halos (more evident in cowpea), and the whole plant wilts until it dies off. The seeds can also be symptomatic, showing external yellow, orange, or red discoloration due to the presence of bacterial biofilms; on the contrary, symptoms are not visible on the external surfaces of pods . GerminaSeed contamination: The virus remains on the coat of the seed, and infection occurs mainly through wounds of plantlets during emergence. Viruses harbored externally can be readily inactivated by a number of treatments to totally or partially eliminate seedborne infections. This type of seed transmission is specifically associated with tobamoviruses  and also with some potexviruses, such as Pepino mosaic virus (PepMV) ,149, usuTrue seed transmission: The virus resides within the tissues of the embryo. The developing embryo can be infected either prior to fertilization (by gametic transmission) or after fertilization (by direct invasion). The latter type of transmission is more common.Viruses are obligate, acellular agents that reproduce inside living cells, spread between tissues, and frequently cause diseases in plants. Among all plant viruses, about one-quarter are transmitted through seeds . Since vFabaceae family, in this review, attention is focused on seed-transmitted viruses affecting both annual cool- and warm-season legumes: chickpea (Cicer arietinum L.), naturally infected by 36 pathogenic viruses belonging to 24 genera of 11 families [Vicia faba L.), including 40 species, 24 genera, and 11 families [Lens culinaris Medik.), including 24 species, 16 genera, and 9 families [Pisum sativum L.), including 42 species, 18 genera, and 11 families [Phaseolus vulgaris L.), including 83 species, 24 genera, and 12 families [Glycine max), including 35 species, 16 genera, and 12 families [Vigna unguiculata (L.) Walp.), including 40 species, 16 genera, and 11 families [Arachis hypogaea L.), including 27 species, 11 genera, and 10 families [Medicago sativa L.), including 36 species, 24 genera, and 11 families  (at least 4 seed-transmitted species), and clovers (Trifolium spp.), including 32 species, 15 genera, and 10 families [The percentage of contaminated seed and seed transmission rates can be impacted by a variety of parameters: (i) virus epidemiology and genomic features; (ii) host–pathogen interaction; (iii) the location of the seed in the plant; (iv) the age of the seed; (v) environmental conditions; (vi) host resistance ,151. Virfamilies ,160,161 families ,166,167 families ,160  was described in alfalfa (Medicago sativa L.) in 1931 and later in pea (Pisum sativum L.) in 1933 [Alfalfa mosaic virus (AMV)  was discovered in common bean (Phaseolus vulgaris L.) in 1925, and it was the first pathogenic virus reported in chickpea (1956) [Trifolium spp. and Melilotus albus), vetch (Lathyrus spp.), and alfalfa (Medicago sativa) are common field sources of the virus, while, among cultivated legumes, faba bean and pea represent the favored hosts of aphids that act as foci for the virus. BYMV is also seed-transmitted in most temperate pulses, including bean (7%), faba bean (up to 9%), field pea (10–30%), lentil, and lupin [Bean yellow mosaic virus (BYMV) (taxonomic position: family: a (1956) ,189. BYMa (1956) . BYMV isa (1956) . BYMV isIn severe infections in faba bean, stem and tip necrosis and early death may occur. Crinkling, downward cupping, yellow mottling, and dead areas along the veins of infected leaves can be observed in bean. Measures to control BYMV include the management of the sowing date, spraying with mineral oils, soil mulching with reflective polyethylene sheets, and crop isolation from known overwintering disease sources .Bromoviridae; genus: Cucumovirus; species: Cucumber mosaic virus) was first described in 1916 (Doolittle and Jagger), and it is distributed worldwide in both temperate and tropical climates, affecting agricultural and horticultural crops of 1000 plant species in 85 families [Cucumovirus species Peanut stunt virus and Tomato aspermy virus. In many weed species, as well as in green vegetation and crops in temperate zones, the virus can survive from year to year. Even if they show no symptoms, perennial legumes such as clover and alfalfa can serve as easy sources of inocula. Over 80 distinct aphid species can spread the virus non-persistently [M. persicae and A. gossypii are two of the most prevalent aphid vectors among them. Mechanical inoculation and transmission through seeds are further known routes of virus spread. The rate of seed transmission is reported to be from 10% to 100% in bean, from 50 to 90% in soybean [Cucumber mosaic virus (CMV)  was first identified in Europe in 1966 and then in Japan in 1967. It was subsequently discovered in the US in 1969 and in New Zealand in 1980 [bean yellow mosaic virus. Pathotypes designated P-1, P-2 , P-3, and P-4 have been distinguished by the susceptibilities of different lines of pea and based on sequence variability in two coding regions of the RNA [Pea seedborne mosaic virus (PSbMV) .Potyviridae; genus: Potyvirus; species: Bean common mosaic virus) and bean common mosaic necrosis virus  are two potyviruses considered to be the most important viruses causing diseases in common bean [A. pisum, A. fabae, and M. persicae) act as vectors in a non-persistent manner. The rate of seed transmission for both viruses has been shown to be 35% [Bean common mosaic virus  [Glycine max), its wild relative (G. soja), and a limited number of natural hosts [Aphis glycines, whose flight is correlated with the incidence of the disease [Soybean mosaic virus (SMV) (taxonomic position: family: c virus)  was firsc virus)  since itc virus) . In contal hosts . The viral hosts ,209. SMVal hosts . SMV is  disease . The inf disease ,212. Pla disease . Infecti disease ,214. Fol disease .Lasiodiplodia theobromae, which is a non-specific pathogen with a wide host range and is responsible for fruit rot, root rot, dieback, and cankers [L. theobromae caused dieback, decay, cankers on stems, and plant decline [Corynespora cassiicola, which is usually associated with the target spot of soybean and cotton, was also reported in common bean seeds. In this case, dark or brown-dark lesions on stems, softer and thinner stems, root rot, and blight were reported as experimental symptoms. The authors also found Colletotrichum gleosporioides, known as the causal agent of anthracnose of the olive tree, and Colletotrichum capsici, which is linked to anthracnose of pepper [C. gleosporioides is able to induce necrosis, brown lesions, spots, cankers on stems, seed rot, soft stem and leaf blight, dieback, and small dark spots on the stem, while C. capsici is responsible for the discoloration of roots and dark spots on the cotyledon [Botryosphaeriaceae family were also found on the seeds of Italian common bean landraces, namely, Botryosphaeria dothidea and Diplodia mutila [B. dothidea and D. mutila were shown to inhibit germination by up to 50%, and the seedlings that developed were stunted, mainly due to root rot. When inoculated on grown plants, they were able to produce necrotic lesions that worsened over several days [Apiospora arundinis was isolated from the seeds of common bean, and, when inoculated into plants, typical necrotic spots on the leaves confirmed its pathogenicity in the new host [Sclerotinia sclerotiorum was isolated for the first time from the seeds of faba bean and was shown to be able to induce leaf chlorosis and severe stem rot when artificially inoculated [Curvularia verruculosa was reported in China, and the symptoms were described as leaf spots [In the last few decades, the climate emergency and the improvement in global trade have fostered the spread of novel seedborne pathogens ,216. A s cankers , was fou decline . In the f pepper . Pathogeotyledon . Interesa mutila . The pata mutila ,221. In ral days . Similarnew host . Moreoveoculated . In 2022af spots . The devincubation methods, to remove external contaminants, a stage of surface sterilization is frequently included. External disinfection is the first step that must be assessed on a case-by-case basis since it allows for the eradication of external saprophytic microflora that may compromise the accuracy of the diagnosis. Surface sterilization, however, may help prevent phytopathogenic microorganisms that dwell in the seed’s outermost layers from being missed during the diagnostic process. In this situation, it is important to keep in mind that internal pathogenic bacteria in the seeds pose the greatest challenge to seed health, whereas external pathogens can be easily combated by seed coating or other seed treatments.Seed health is a key factor in preventing the spread of seedborne pathogens in new environments, shedding light on the crucial role of early diagnosis. This field is tricky since most of the seeds affected by pathogens are symptomless, and concomitantly, only a low percentage of these infections will be translated into symptomatic plants . For thiIncubation is also used to detect bacteria. In this case, the protocols are based on the soaking method: seeds are soaked in an appropriate amount of buffer and macerated for 2–18 h at predefined temperatures. During this process, the bacteria are released from the seeds into the buffer, which is then plated on semi-selective agar media. This approach also enables the isolation of bacterial pathogens from the endosperm and embryos. After 5–15 days of incubation (depending on the species), colonies develop, and then bacteria can be recognized by visual observation. This stage is critical since it is influenced by the analysts’ experience, and the use of reference strains to compare with suspected colonies appears to be particularly useful. After detecting putative pathogenic isolates, confirmation assays are used to establish the identity and to verify the pathogenicity. Pathogenicity tests are performed while trying to balance between the reproduction of the natural infection process and laboratory feasibility. For details about the most used inoculation protocols, refer to the “International rules for seed testing”  and, for Cff, to the diagnostic protocol available on the EPPO website . Once purified, colonies with the typical morphology can be identified by using molecular methods: PCR assays were developed by Tegli et al. (2002) and Guimaraes et al. (2001) to confirm the identity of Cff [Fungal pathogens contained in the interior layers of seeds  are those that may develop into colonies when surface sterilization is performed. In these techniques, non-selective media, such as Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA), are used, but because they do not always allow the formation of reproductive structures, less rich media, i.e., Water Agar (WA) and Cornmeal Agar (CMA), are preferable. Moreover, to facilitate the process of seedborne pathogen isolation, several selective media were developed by adding antibiotics or chemicals, such as streptomycin or bromophenol blue. The final step of these procedures involves the microscopic observation of the colonies and the detection of species-specific structures that can allow the final identification. y of Cff ,226; Audy of Cff , and Arny of Cff .Serological methods, which use polyclonal or monoclonal antibodies to detect seedborne infections, can be a viable alternative. The most commonly used technique in this field is the double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA). The ELISA has the advantage of being applicable to biotrophic and necrotrophic pathogens and is largely employed in the detection of seedborne viruses. Finally, molecular methods have gained popularity in seedborne pathogen diagnosis due to their rapidity, sensitivity, specificity, and ease of interpretation. These methods need a preliminary step in which fungal DNA has to be extracted and purified [purified . Many lapurified ,230,231.purified . In factpurified . In addipurified .In the last few decades, rapid and specific serological  and molecular techniques (molecular hybridization and DNA amplification) have also been developed for the detection of plant viruses. For all the viruses included in this review, it is possible to apply serological and/or molecular detection methods. In pathogenic virus diagnosis, serological approaches are usually enough for regular testing, while a molecular-based detection approach can be effective for strain differentiation when high sensitivity is required. In particular, AMV can be detected by serological methods   using poIn recent years, global seed trade has fostered the spread of seedborne diseases. Several techniques can be implemented to mitigate their effects on crops, such as reducing long-distance spread, seed health testing, breeding, and pest management in the field.specificity, defined as the ability to distinguish the target microorganism from others found in the seeds tested or in the field analyzed and to avoid false-positive results; (ii) sensitivity, defined as the ability to detect the target pathogen even at low concentrations in seed lots; (iii) speed; (iv) simplicity, outlined as the minimization of steps in a procedure and the feasibility for non-expert operators; (v) cost-effectiveness, suitable to be part of routine processes; and (vi) reliability, indicating that the test must be robust enough to ensure repeatability on the sample, between samples, and regardless of who carries out the method [C. lindemuthianum have been identified based on 12 differential cultivars [Ascochyta spp. in legumes highlighted heterogeneity in resistance genetics, and the use of wild cultivars as a source of genetic diversity is a promising strategy to develop resistant materials [R. solani was scarce: it was defined as quantitative in nature and it was influenced by environmental conditions. In recent studies (2019), the knowledge of resistance to R. solani in common bean was improved: Oladzaz and coworkers analyzed a pool of varieties derived from two different geographic areas, namely, Andean and Middle American areas, and they found that 2 varieties out of 552 showed resistance to the fungus [The European and Mediterranean Plant Protection Organization (EPPO) is an international organization involved in plant protection cooperation, and among its objectives is the development of an international strategy against the introduction and spread of pests in agricultural and natural ecosystems. The EPPO Global Database provides information about pathogen distribution in worldwide territories and archives pest categorization and restriction requirements. Of course, field inspection and diagnostic analyses continuously implement the information provided by EPPO GD. The categorization established in different countries for the pathogens dealt with in this review is reported in e method . In thisultivars . Studiesaterials . In the e fungus .Avr genes were molecularly characterized in Psp, allowing the development of bean varieties resistant to halo blight; however, no complete resistance was found to race 6, which is the most widespread [P. coccineus, P. lunatus, and P. acutifolius, suitable to be used in intraspecific breeding research [Xanthomonas, pathogenic Pseudomonas species have plastic populations, and chemical control is not currently considered a solution.Disease resistance is also studied to counteract bacterial diseases. The breeding of resistant soybean varieties is considered to be the most reliable approach, even if the development of plant resistance is hindered by the evolution of the pathogen virulence . Avr gendespread . The bredespread . Extensiresearch . In addiC. lindemuthianum [Trichoderma spp. or Plant-Growth-Promoting Rhizobacteria (PGPR) increases the germination rate and reduces the incidence of seedborne diseases. In fact, El-Koly et al. demonstrated that coating seeds with Bacillus megaterium, B. subtilis, Pseudomonas fluorescens, Serratia marcescens, Trichoderma album, T. harzianum, T. lignorum, and T. viride strongly reduced the pre- and post-emergence damping off caused by R. solani and did not affect the nodulation of rhizobia [M. phaseolina by using strains of Bacillus spp. and Trichoderma spp. and both were shown to be able to reduce the incidence of charcoal rot in common bean [T. viridae and B. subtilis were tested. The results highlighted the ability of the microorganisms to counteract charcoal rot, although chemical agents were shown to be more adaptable to climatic conditions [B. amyloliquefaciens showed a reduced incidence of R. solani root rot, along with the increased tolerance of the seedlings to drought [Ascochyta spp., effective alternative strategies involving the combination of Curtobacterium rosae, thyme oil, and high temperature were shown to significantly increase seed germination and reduce Ascochyta presence in pea seeds [A. pinodes: in this study, PGPR, identified as Pantoea agglomerans, B. subtilis, and B. amyloliquefaciens, showed strong biocontrol activity, reducing the incidence of the disease both in the greenhouse and in the field [Bacillus have been tested to control S. sclerotiorum infection in common bean. In 2018, Sabatè and coworkers demonstrated that when incubating common bean seeds with Bacillus strain B14, the growth of bacterial contaminants and the presence of pathogenic fungi were completely inhibited. Moreover, they proved that Bacillus strains are also able to mitigate S. sclerotiorum infection in common bean seedlings [Botrytis spp., traditionally controlled by crop rotations or by using fungicides, have been developed and are already present on the market. Indeed, Gliocladium catenulatum strain JI446 and a B. subtilis strain have been used to formulate two products, Prestop and Serenade, respectively, to mitigate the effects of B. cinerea and B. fabae on faba bean (Vicia faba L.) [Nowadays, a consolidated and practical approach is to set up plans to control plant pathogens based on the use of chemical fungicides, biological antagonists, or a combination of both. The choice of treatment to be used in counteracting seedborne diseases is based on the pathogen’s localization in the seeds. For instance, contact fungicides can control only surface contaminants and are useless against those microorganisms that are stored in the internal tissues. On the contrary, fungicides defined as translaminar or cytotropic are also effective against slight infections in the external layers of the seeds. However, when the infection involves the inner layers of the seeds (embryos), only systemic compounds can be effective in preventing the disease . Compounuthianum . On the rhizobia . Biologimon bean ,254. Simnditions . In 2018 drought . Regardiea seeds . Similarhe field . Strainseedlings . On the faba L.) .Bacillus spp. and Pseudomonas spp. strains against Psp, obtaining a 50% reduction in infection [Rahnella aquatilis was shown to be able to inhibit the growth of Xcf in vitro and to induce host defense by enhancing phenolic compound synthesis and peroxidase activity in common bean plants [Bacillus and Pseudomonas spp., alone or in combination, against Xcf, and the results demonstrated the effectiveness of the treatments, depending on the timing of the incubation [Integrated control strategies are also implemented for the management of bacterial diseases. Psg bacterial blight can be successfully controlled using integrated disease management. In this context, crop rotation, the use of pathogen-free seeds, the avoidance of planting in areas prone to frequent frosts or extreme wet weather, crop hygiene, and the management of sowing time can be adopted. Donmez and Alyieva tested nfection . A similn plants . Moreovecubation .Ghunavirus, subfamily Studierviriae, were studied against Psg, and their activity in devitalizing P. savastanoi pv. phaseoli and P. s. pv. pisi was also demonstrated. The authors also proved that these phages were not able to infect beneficial epiphytic Pseudomonas fluorescens, Rhizobium, and Bradyrizobium [B. alcalophilus, B. atrophaeus, B. megaterium, B. mycoides, B. subtilis, Lysinibacillus sphaericus, P. fluorescens, and P. putida were recently studied with promising results in an in vivo biocontrol assay on halo blight [B. cereus, P. fluorescens, and Pantoea agglomerans were successfully used as seed treatments to control bacterial wilt caused by Cff; moreover, it was demonstrated that P. agglomenrans is able to colonize seedling tissues and protect the plant for 7 days post-emergence [Bacteriophages are viruses that affect several plant pathogenic bacteria. They can be used as a biological control means against bacterial diseases because they are able to selectively affect pathogenic bacteria by lysing their cells. Commercial products based on phages are already available, and several research trials were conducted to counteract BBS. In 2022, phages belonging to the genus rizobium . Strainso blight . B. ceremergence .I gene, a dominant gene conferring resistance to most BCMV strains. In the absence of resistant cultivars, the use of virus-free common bean seeds minimizes the risk of outbreaks.A separate discussion is presented for viruses because the control methods are strictly dependent on the way they are transmitted. Namely, the control of vectors (insects and weeds), the employment of virus-free seeds, and the use of resistant cultivars are the main strategies in virus control. AMV is both seed- and aphid-transmitted, and thus, a range of general control measures can be applied: (i) the use of virus-free seeds, (ii) the control of virus spread from overwintering hosts by spatial separation, and (iii) aphid vector control. However, insecticides have limited use, as the virus is non-persistently transmitted by aphids. Finally, no source of resistance to the virus has been reported in bean . RegardiSMV management could include the use of virus-free seeds; as the virus is non-persistently transmitted by aphids, vector controls using insecticides have limited use. The use of resistant varieties represents a feasible aim and depends on the type of resistance the plant has and on the strain of the virus . ResistaAltogether, these findings open interesting perspectives on the biological and eco-friendly management of legume crops.In conclusion, climate change and the world’s population growth have created new concerns, such as food scarcity. As a result, legumes have the potential to increase agricultural diversity and production while also contributing to the protein requirements of the human diet. In this context, successful legume management is critical in order to enhance productivity and market value. This procedure necessitates the advancement of information about these crops, including the optimal agronomic strategies for generating profits, as well as phytopathological evidence to combat plant infections. The target microorganisms in seed health assessments are chosen based on the plant species, but also on information about the presence or absence of a specific disease in the country where the samples are produced. In recent years, in addition to species-specific seedborne diseases, novel microbes, particularly fungi, have been described on a daily basis. Indeed, the use of non-selective media in seed analysis allows for the recovery of unanticipated infections, and their identification and disease description aid in preventing their spread to new areas. On the contrary, despite the recovery of novel putatively hazardous bacteria, the selective procedures utilized in bacterial analyses may result in the selection of well-known pathogens. This review compiles the most important information on seedborne infections on legumes in order to aid in their identification. Furthermore, the report of novel infections sheds light on the critical importance of an open-minded approach to seed health and provides a broad perspective on crop management based on innovative and environmentally friendly solutions.


Here, we demonstrate how Color-flu, four fluorescent IAV strains originally developed for mice, can be used to study host-virus interactions by simultaneously monitoring virus particles, neutrophils, and macrophages in vivo. Using this model, we show how the angiotensin-converting enzyme inhibitor, ramipril, and mitophagy inhibitor, MDIVI-1, improved survival, decreased viral burden, and improved the respiratory burst response to IAV infection. The Color-flu zebrafish model of IAV infection is complementary to other models as it is the only model where interactions between virus particles and host cells in an intact vertebrate can be visualized in vivo.Influenza virus infection can cause severe respiratory disease and is estimated to cause millions of illnesses annually. Studies of the contribution of the innate immune response to influenza A virus (IAV) to viral pathogenesis may yield new antiviral strategies. Zebrafish larvae are useful models to study the innate immune response to pathogens, including IAV,  An estimated 10-37 million influenza infections occur each year in the US, with 114,000-624,000 needing hospital care for associated respiratory and heart symptoms. Antivirin vivo. Mammalian influenza virus infections originate in respiratory epithelial cells and alveolar macrophages. While ACE overexpression in myeloid cells benefit immune responses, ACE deficiency can also be beneficial. A study of mouse models of acute respiratory distress syndrome showed that ACE deficient mice have less lung damage[Ramipril is an ACE inhibitor that is frequently prescribed for hypertension as ACE produces angiotensin II which, in turn, elevates blood pressure. ACE hasneumoniae. ACE oveng damage. Interesng damage. Our stuMitochondria have important roles in immune function, including regulating inflammation. MitophaZebrafish embryos and larvae have been used screen small molecule drugs where the response of separate larvae to different treatments can be monitored in multi-well plates. Large-sin vivo. The roles of neutrophils and macrophages during IAV infection can also be readily characterized using this model. Using Color-flu, we characterize how ACE inhibition by ramipril and mitophagy inhibition by MDIVI-1 both improve the response to IAV infection by limiting inflammation through distinct mechanisms. These studies demonstrate how larger small molecule screens are possible using this zebrafish Color-flu model of IAV infection. Such screening studies are needed to find small molecules that could be developed into novel antiviral therapies for influenza virus.In summary, we demonstrate that Color-flu can be used to study the innate immune response to IAV infection in zebrafish larvae. This model is complementary to other models of IAV infection. The model is the only IAV model that allows for the visualization of interactions between virus particles and host cells in an intact vertebrate Supplement 1