Didymella glomerata is a new fungal disease of maize (Zea mays), first detected in 2021 in Panjin, Liaoning province of China. Here we report the reference genome assembly of D. glomerata to unravel how the fungal pathogen controls its virulence on maize at the molecular level. A maize-infecting strain Pj-2 of the pathogen was sequenced on the Illumina NovaSeq 6000 and PacBio Sequel II platforms at a 575-fold genomic coverage. The 33.17 Mb gapless genome assembly comprises 32 scaffolds with L/N50 of 11/1.36 Mb, four of which represent full-length chromosomes. The Pj-2 genome is predicted to contain 10,334 protein-coding genes, of which 211, 12 and 134 encode effector candidates, secondary metabolite backbone-forming enzymes and CAZymes, respectively. Some of these genes are potentially implicated in niche adaptation and expansion, such as colonizing new hosts like maize. Phylogenomic analysis of eight strains of six Didymella spp., including three sequenced strains of D. glomerata, reveals that the maize (Pj-2)- and Chrysanthemum (CBS 528.66)-infecting strains of D. glomerata are genetically similar (sharing 92.37% genome with 98.89% identity), whereas Pj-2 shows truncated collinearity with extensive chromosomal rearrangements with the Malus-infecting strain M27-16 of D. glomerata (sharing only 55.01% genome with 88.20% identity). Pj-2 and CBS 528.66 carry four major reciprocal translocations in their genomes, which may enable them to colonize the different hosts. Furthermore, germplasm screening against Pj-2 led to the identification of three sources of DLB resistance in maize, including a tropical inbred line CML496. DLB resistance in the line is attributed to the accumulation of ROS H2O2 in the apoplastic space of the infected cells, which likely restricts the fungal growth and proliferation.Didymella leaf blight (DLB) caused by  Didymella (syn. Peyronellaea or Phoma) glomerata (Corda) Q. Chen & L. Cai is an ascomycete fungal pathogen, which causes Didymella leaf blight (DLB). The pathogen has a broad host range and is known to infect both monocots and dicots, including although not limited to flax (Linum usitatissimum) (Brassica napus) (Triticum aestivum), durum wheat (Triticum turgidum) and triticale  (Pisum sativum) (Helianthus annuus) (Vitis vinifera) (Prunus persica) (Actinidia chinensis) (Pistacia vera)  .issimum) , oilseeda napus) , bread waploide) , pea (Pisativum) , sunflow annuus) , grapeviinifera) , peach (persica) , kiwi (Ainensis) , pistachia vera) , Chinesecinalis)  and applD. zeae-maydis, the only Didymella species infectious on maize and the causal agent of yellow leaf blight. Culture characteristics and molecular phylogeny based on multi-locus DNA barcodes  showed that the single spore culture isolate Pj-2 retrieved from the infected leaf tissues was morphologically and genetically identical with the D. glomerata isolate D/034 from soybean (Glycine max)  . This waon maize .D. glomerata is likely a heterothallic fungal pathogen as it harbors the mating-type gene scaffold1.t522 (MAT1-2), whose protein product contains the characteristic high mobility group box domain. The pathogen likely deploys a subcuticular intramural necrotrophic infection strategy, whereby the pathogen colonizes maize leaves, resulting in DLB. The infection cycle starts when fungal spores (conidia) land on the leaf surface and germinate to form a germ tube. However, unlike hemibiotrophic fungal pathogens, the germ tube does not differentiate into appressoria  but instead directly perforates the host cuticle, presumably via an infection peg. The pathogen can also gain access to host tissues through natural openings, such as stomata. Following successful penetration, the invasive hyphae emanating from the infection pegs rapidly colonize the host parietal layers and eventually kill and macerate the host cells. Black lesions appear on the maize leaves five to seven days after infection. Single-celled ellipsoidal conidia are produced in glabrous pycnidia developing in these lesions and are dispersed by splashing raindrops and wind to infect healthy neighboring plants.D. glomerata produces phytotoxins zinniol , whereas five are pathogenic to plants . More recently, fragmented genome assemblies of two D. glomerata strains CBS 528.66  and M27-16  were deposited to GenBank.Thus far, studies have revealed that  zinniol  and 5-di zinniol , which cD. glomerata strain Pj-2, which infects maize and causes DLB. Therefore, the high-quality gapless assembly reported in the study represents the first sequenced genome of the maize-infecting D. glomerata strain. Furthermore, we also identified three genetic sources of DLB resistance in maize germplasm, including an elite tropical inbred line CML496, for potential introgression of resistance alleles into elite cultivars lacking resistance through intraspecific hybridization.In this study, we sequenced, assembled and annotated the genome of D. glomerata strain Pj-2 genome on the second-generation (Illumina NovaSeq 6000) and third-generation (PacBio Sequel II) sequencing platforms, which yielded a de novo reference genome assembly of 33.17 Mb with a 575-fold genomic coverage  and the apple-infecting strain M27-16 (35.52 Mb) of D. glomerata.We sequenced the coverage Table 1.50 of the D. glomerata Pj-2 genome is 11/1,359,674 bp, which is far superior than the other two genome assemblies of D. glomerata strains CBS 528.66  and M27-16 . In addition, the Pj-2 genome assembly is either comparable or better than that of other sequenced Didymella species: D. keratinophila 9M1 , D. heteroderae 28M1 , D. pinodes WTN-11-157 , D. lethalis GRM-16-623 , D. arachidicola YY187  , D. rabiei ArMe14    ; D. exig812,190)  and D. s254,797) .n or (TTAGGG)n were present in at least one end of the 23 scaffolds of the D. glomerata Pj-2 genome assembly. Of these 23 scaffolds, four  contain the telomeric repeats at both ends and hence represent full-length chromosomes. Transposable elements (TEs) comprise only 1.93% of the D. glomerata genome assembly  and are scattered in the low GC content regions of the genome  out of 6,641 pleosporales (the order within Dothidiomycetes) single-copy genes were mapped on the D. glomerata Pj-2 genome, suggesting a near-complete genome assembly with regard to the genic content thereof. Overall, the assembled PacBio reads corrected by the Illumina reads produced a high-quality (32 scaffolds with 575-fold genome coverage), nearly gapless (single gap of 100 bp) genome assembly of the maize-infecting strain Pj-2 of D. glomerata.Tandem telomeric repeats (CCCTAA)e genome Table S1D. glomerata Pj-2 genome is predicted to contain 10,334 protein-coding genes, whose number is markedly lower than that of the Chrysanthemum-infecting strain CBS 528.66  and the Malus-infecting strain M27-16 . Similarly, the predicted gene content of Pj-2 encodes is markedly lower than that of other Didymella spp., e.g., D. keratinophila 9M1 , D. heteroderae 28M1 , D. exigua CBS 183.55  and D. rabiei ArMe14 , except D. pinodes WTN-11-157 . The clustering of 90,823 proteins coded by the eight strains based on their sequence similarity  showed that the strains carry 1,027 core orthologous proteins. Pj-2 shares 2,734 more orthologous proteins with CBS 528.66  than M27-16 , suggesting that the maize-infecting D. glomerata strains are phylogenetically closely related to the Chrysanthemum-infecting D. glomerata strain. Pj-2 shares 5,444 orthologous proteins with 9M1, 4,771 with 28M1, 3,847 with CBS 183.55 and 1,958 with ArMe14 (The h ArMe14 Table S2Chrysanthemum-infecting strain CBS 528.66 (176) and the Malus-infecting strain M27-16 (196) even though the latter two strains of D. glomerata have a larger gene content than the former , Fusarium spp., Magnaporthe oryzae  and Ustilago maydis , are localized in chromosomal regions enriched with transposable elements, including telomeres, or clustered in AT-rich blocks of the genomes  and secondary metabolism enzymes. Effectors are the pathogen-secreted small proteins and are an example of the extended phenotype as they function in hosts instead of pathogens, whose genomes carry the effector-coding genes. Pathogens deliver effectors into plants to subdue the innate immune system of plants, thereby enabling colonization and proliferation . Only 1.e former S5. Simines 180;  predicte genomes . Such a recently .D. glomerata strains possess a slightly lower number of CAZymes, i.e., CBS 528.66 , and each BGC contains a backbone gene and a few decorating genes, and encodes a specific secondary metabolite . SecondaD. glomerata strain Pj-2 genome (33.17 Mb) is similar in size to the Chrysanthemum-infecting strain CBS 528.66 (33.98 Mb), apple-infecting strain M27-16 (35.52 Mb), as well as other Didymella spp., e.g., D. pinodes WTN-11-157 (33.77 Mb), and D. exigua CBS 183.55 (34.39 Mb). However, the other four sequenced Didymella spp. have slightly larger genomes, e.g., D. rabiei ArMe14 (40.92 Mb), D. keratinophila 9M1 (36.22 Mb) and D. heteroderae 28M1 (37.07 Mb).The Didymella spp., we performed genome similarity and synteny analyses among eight strains, representing six species. An eight-strain whole-genome alignment was contrived using the following parameters: 50-bp seeds and extension thereof with HOXD scoring matrix  of its genome with CBS 528.66, followed by 57.71% with 9M1, 55.01% with M27-16, 45.04% with WTN-11-157, 43.14% with 28M1, 23.31% with CBS 183.55 and 5.19% with ArMe14. The shared genomic regions showed ≥85% ANI; Pj-2 displayed 98.89% nucleotide similarity with CBS 528.66, followed by 88.79% with 9M1, 88.20% with M27-16, 87.01% with 28M1, 86.99% with WTN-11-1, 85.11% with CBS 183.55 and 84.56% with ArMe14. A neighbor-joining tree constructed from the pairwise distance matrices revealed that a clade comprising the three D. glomerata strains Pj-2, CBS 528.66 and M27-16, and D. keratinophila 9M1. These strains share 55.01% to 92.37% genomes (AP), with ANI values ranging from 88.20% to 98.89%. Within this clade, Pj-2 and CBS 528.66 formed a subclade, which is in agreement with high AP (92.37%) and ANI (98.89%) between their genomes  exist between their genomes , Pj-2 scaffold22/CBS 528.66 scaffold6 and Pj-2 scaffold3 (166 kb), Pj-2 scaffold26/CBS 528.66 scaffold17 and Pj-2 scaffold13 (131 kb translocation), and Pj-2 scaffold26/CBS 528.66 scaffold17 and Pj-2 scaffold8 (217 kb translocation)  and CBS 528.66 scaffold12 (size = 763.58 kb) are collinear with a high degree of similarity in the genomic region located between 282 kb and 764 kb, whereas a 274-kb genomic region of CBS 528.66 scaffold12 (8 to 282 kb) shows alignment with a high degree of homology with a similar size genomic region located at the 5’-end of Pj-2 scaffold18 . This is called the reciprocal translocation between Pj-2 scaffold14/CBS 528.66 scaffold12 and Pj-2 scaffold18. The 274 kb translocation region contains 92 genes in Pj-2 and 100 genes in CBS 528.66, 78 of which are homologous. This translocation encodes three apoplastic effector candidates with unknown functions. The Pj-2 effector candidates scaffold18.g41 (141 aa) and scaffold18.g46 (238 aa) are 100% identical to CBS 528.66 scaffold12.g4610 and scaffold12.g4605, respectively. The third effector candidate scaffold18.g5 (269 aa) is aligned with 100% identity from 6 aa to 265 aa of the 526 aa CBS 528.66 protein scaffold12.g4649 (526 aa); likewise, the third CBS 528.66 effector scaffold12.g4639 (186 aa) is matched with 97% identity from 1 aa to 184 aa of the 675 aa Pj-2 protein scaffold18.g16. Therefore, these proteins represent the truncated version of larger proteins and thus are unlikely effector candidates. In addition to effector candidates, the translocation codes for three CAZymes. The Pj-2 cellulase scaffold18.g13 (394 aa) and the CBS 528.66 protein scaffold12.g4641 (439 aa) are homologous proteins, showing 98% identity over the first 327 aligned aa, whereas the C-termini thereof are highly variable and former lacks 45 aa in this region. The Pj-2 hemicellulase scaffold18.g6 (837 aa) is homologous to the CBS 528.66 hemicellulase scaffold12.g4648 (837 aa), exhibiting 99% identity over the entire length of proteins. The Pj-2 pectinase scaffold18.g16 (675 aa) and the CBS 528.66 scaffold12.g4639 (186 aa) are homologous proteins with 99% identity over the first 184 aligned aa; however, scaffold12.g4639 was predicted as an effector candidate due to its length ≤300. Therefore, scaffold12.g4639 is a truncated pectinase rather than an effector candidate. The translocation does not carry any secondary metabolite backbone enzyme.Pj-2 scaffold22 (size = 915.48 kb) and CBS 528.66 scaffold6 (size = 1123.25 kb) are collinear with a high degree of similarity in the genomic region located between the 7–911 kb (Pj-2 scaffold22) and 174–911 Kb (CBS 528.66 scaffold6); however, a 166-kb genomic region of CBS 528.66 scaffold12 (8–174 kb), which Pj-2 scaffold22 lacks, aligns with a high degree of homology with a similar size to the genomic region (25–191 kb) located at the 5’-end of Pj-2 scaffold3 (size = 1813.70 kb). This is the reciprocal translocation between Pj-2 scaffold22/CBS 528.66 scaffold6 and Pj-2 scaffold3. The 166 kb translocation contains 54 genes in Pj-2 and 60 genes in CBS 528.66, 53 of which are homologous. This translocation encodes two apoplastic and/or cytoplasmic effector candidates with unknown functional domains and one CAZyme hemicellulase; however, it does not code for any secondary metabolite backbone enzyme. The Pj-2 effector candidates scaffold3.g27  and scaffold3.g51  are 100% identical to the CBS 528.66 effector candidates sacffold6.g2757 and scaffold6.g2777, respectively. The Pj-2 hemicellulase scaffold3.g49 (338 aa) is homologous to the CBS 528.66 hemicellulase scaffold6.g2776 (1120 aa), displaying 99% over the 334-aligned aa, whereas the former lacks the remaining aa. Therefore, scaffold3.g49 is a truncated protein.Pj-2 scaffold26 (size = 703.34 kb) and CBS 528.66 scaffold17 (size = 703.44 kb) are collinear with a high degree of similarity in the genomic region located between 1–277 kb (Pj-2 scaffold22) and 434–703 Kb (CBS 528.66 scaffold6). The remaining genomic region of Pj-2 scaffold26, spanning from 277 kb to 703 kb, is collinear with CBS 528.66 scaffold29 (size = 481.49 kb). However, a majority of the 430-kb genomic region located at the 5’-end of CBS 528.66 scaffold17 (15–430 kb), which Pj-2 scaffold26 lacks, matches with a high degree of homology with two genomic regions located at distinct Pj-2 scaffolds, i.e., scaffold13  and scaffold8 . Therefore, there are two reciprocal translocations: Pj-2 scaffold26/CBS 528.66 scaffold17 and Pj-2 scaffold13 (131 kb translocation) and Pj-2 scaffold26/CBS 528.66 scaffold17 and Pj-2 scaffold8 (217–250 kb translocation). The 131-kb translocation contains 46 genes in Pj-2 and 55 genes in CBS 528.66, 45 of which are homologous genes. The translocation codes for one apoplastic and/or cytoplasmic effector candidate with an unknown functional domain; however, it does not encodeCAZymes and secondary metabolite backbone enzymes. The Pj-2 effector candidate scaffold13.g401(239 aa) is homologous to CBS 528.66 Scaffold17.g5676, showing 100% identity. The 217–250 kb translocation contains 77 genes in Pj-2 and 89 genes in CBS 528.66 , 77 of which are homologous genes. The translocation codes for two apoplastic and/or cytoplasmic effector candidates with an unknown function; however, it does not encode CAZymes and secondary metabolite backbone enzymes. The 136 aa effector candidates scaffold8.g410 (Pj-2) and scaffold17.g5766 (CBS 528.66) are homologous proteins, exhibiting 95% identity over their entire length; seven aa differ between the two effector candidates. Both are predicted to be localized in the apoplast and/or cytoplasm. However, the second effector candidate  does not show any polymorphism between the two strains and is likely localized in the cytoplasm.A spurious inversion is evident in the collinearity plot between Pj-2 and CBS 528.66. Pj-2 scaffold27 is flipped with respect to the 3’-end of CBS 528.66 scaffold4; the 3’-end of CBS 528.66 scaffold4 is aligned with Pj-2 scaffold 24. Therefore, we conclude that Pj-2 scaffold24 and scaffold27 are part of the same contiguous sequence.D. glomerata are genetically similar, sharing 92.37% genome with 98.89% sequence similarity. However, the Malus-infecting strain of D. glomerata M27-16 is highly diverse from the other two strains, Pj-2 (sharing only 55.01% genome with 88.20% sequence similarity) and CBS 528.66 (sharing 54.28% genome with 88.22% sequence similarity)  and CBS 528 (scaffold17.g5766). It would be interesting to investigate whether the targeted deletion of these two effectors in their respective strains contributes to virulence on their hosts.The structural variations in the genomes are the major drivers of genetic diversity within and between species of the same genus, which enable the species or strains within the same species to colonize distinct ecological niches. For example, the maize- and Chrysanthemum-infecting strains of ilarity) 3B. The D. glomerata isolate Pj-2 in a growth chamber through spray-inoculation following an augmented randomized block design  and susceptible  reactions  and carries a major QTL RppCML496 for resistance to SCR (2: 12.7 ± 19.2 mm2), Zheng58 (16.7 mm2), Chang7-2 (39.7 mm2), CML470 (51.0 mm2), PH4CV (32.6 mm2), PH6WC (6.5 mm2) and B73 (66.6 mm2)  and Puccinia sorghi (common rust pathogen), whereas they are susceptible to Curvularia lunata  and P. polysora. In addition, Zheng58 and Chang7-2 are moderately resistant to Exserohilum turcicum (northern corn leaf blight pathogen), and the latter line also manifests a similar resistance response to Cercospora zeae-maydis (gray leaf spot pathogen)  and a donor parent  that carries alleles conferring genetic resistance to the disease of interest. Identifying such genetic sources of disease resistance is the first key step in cultivar development. With the above objective and given that DLB is a new disease of maize, we conducted germplasm screening to identify the genetic source of DLB resistance in maize. Thirty maize lines representing inbred lines, cultivars and landraces were screened for reactions to the e to SCR . Among t6.6 mm2) . All thrathogen) . CML470,olysora . PH4CV aP. polysora SCR (six QTL for SCR resistance)  while the CML496 leaves remained healthy  in the P178 epidermal cells, facilitating fungal growth and proliferation  and the most resistant inbred line (CML496) for histochemical analysis. P178 is an inbred line, which is resistant to istance)  and Exseurcicum . However in P718 . The CMLD. glomerata strain Pj-2, which comprises 32 scaffolds, including four full-length scaffolds (representing chromosomes) carrying telomeres at both ends. Phylogenomic analysis shows that the maize- and Chrysanthemum-infecting strains of D. glomerata are genetically similar and share 93.37% genome with 98.89% sequence similarity. However, they carry four major reciprocal translocation regions in their genomes, which may allow their adaptation to different hosts. Furthermore, germplasm screening against DLB identified three maize inbred lines  expressing complete resistance to the D. glomerata strain Pj-2. Therefore, these lines can be used as a donor parent to transfer genetic resistance into other elite cultivars lacking DLB resistance through intraspecific hybridization introgression. Further research, however, is required to identify genes in CML496 conferring resistance to DLB. In addition, field research should be undertaken to assess the economic impact of this new maize disease, both in terms of yield loss and down-gradation of grain quality.DLB is a new disease of maize, which we recently identified in Panjin, Liaoning, a northeastern province of China . In thisD. glomerata strain Pj-2 was routinely maintained on PDA plates under a 12 h photoperiod at 25°C. Maize germplasm involving 30 lines representing cultivars, inbred lines and landraces (The 7) were collected by swamping the five-day-old six-cm PDA culture plates of Pj-2 with five ml of distilled deionized H2O. One ml of conidial suspension was incubated in a one-liter flask containing 200 ml of complete medium for four days in an orbital shaker set at 25°C and 120 rpm. Mycelia balls were collected by filtering the liquid culture through a one-layered Miracloth. The ENZA Fungal DNA kit  was used to isolate genomic DNA (gDNA) from the mycelia.Conidia (ca. 10K-mer (17) frequency in the paired-end reads. Finally, Jellyfish v2.0 (K-mer (17) within the high-quality paired-end reads to estimate the D. glomerata Pj-2 genome size.Covaris E220  was used to fragment one µg of high-quality gDNA, and end-repaired fragments were used to construct a short insert (500 bp) paired-end library using TruSeq DNA LT Kit . The library was assessed for the insert size and quality using BioAnalyzer 2100 using Agilent 1000 DNA Chip  and sequenced on NovaSeq 6000 , generating 2 x 250 bp reads. AdapterRemoval v2  was used to clip adapters and low-quality bases from the paired-end reads. SOAPec v2.0  was usedish v2.0  was usedg-TUBE  was used to shear five µg of high-quality gDNA, and BluePippin  was utilized to size-select ~10 kb gDNA fragments. Finally, the end-repaired large fragments were used to construct a long insert (10 kb) library using SMRTbell Express Template Prep Kit 2.0 . The resulting library was assessed for insert size and quality on BioAnalyzer 2100 and sequenced on PacBio Sequel II  using Continuous Long Read mode. The polymerase reads generated on PacBio Sequel II were checked for adapter contamination, quality and size using SMRT analysis v2.3.0; the subreads  were extracted from the adapter-free polymerase reads. Unicycler  was emplDidymella spp.  were aligned using the Whole Genome Alignment tool plugged in CLC Genomics Workbench  with the following parameters: 50-bp seed and minimum alignment block size ≥100 bp. AP and ANI were calculated from the whole-genome alignment using pairwise distance matrices.The whole genomes of eight strains of six D. glomerata strains , D. exigua CBS 183.55, D. pinodes WTN-11-157, D. rabiei ArMe14, D. keratinophila 9M1 and D. heteroderae 28M1. The masked genome of Pj-2 was assessed for its completeness using BUSCO v5.1.3  were mapped onto the D. glomerata genome using Exonerate v2.2.0 (http://www.ebi.ac.uk). The ab initio predictions and the Exonerate alignments were combined using EVidenceModeler v.r2012-06-25  following the method described previously (2) was measured using ImageJ (https://imagej.net/). A linear mixed-effects model using the R lm function was used to calculate least-squares (LS) means of lesion areas on the maize lines (a fixed effect) while considering blocks  as a random effect.Thirty maize lines were individually grown in pots (6 x 10 cm) containing peat moss (soilless medium) and perlite in a 2:1 ratio in a growth chamber following an augmented randomized block design. Plants were spray-inoculated at the V3 growth stage with the Pj-2 conidia  staining of Pj-2 infected leaf tissues of the inbred lines P178 and CML496 were performed by methods described by D. glomerata Pj-2 (BioSample: SAMN23554018) genome (32 scaffolds) has been deposited to GenBank/ENA/DDBJ under the accession number JAJOHN000000000 (BioProject: PRJNA785361).The VB conceived and performed research. Y-LP, VB, JD, WZ, JY and WM analyzed data. JD provided maize germplasm and VB, Y-LP, JD, WZ and JY wrote the paper. All authors contributed to the article and approved the submitted version.The research was supported by grants from the National Natural Science Foundation of China (Grant No. 32172363) and the Chinese Universities Scientific Fund (Grant No. 10092004). Technical support for the incumbent study was provided by Hongjie Yuan at the Communication University of China, Beijing, China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Phylogenetic analysis of monkeypox virus genomes showed statistically significant divergence and nascent subclades during the 2022 mpox outbreak. Frequency of G>A/C>T transitions has increased in recent years, probably resulting from apolipoprotein B mRNA editing enzyme catalytic polypeptide 3G (APOBEC3) deaminase editing. This microevolutionary pattern most likely reflects community spread of the virus and adaptation to humans. Initial epidemiologic studies provided evidence of sustained human-to-human transmission in some non–MPXV-endemic countries in Europe, through close contacts, including in sexual networks (Monkeypox virus (MPXV) is a double-stranded DNA virus mostly associated with rodents and occasionally spilling over to humans, causing outbreaks of mpox (formerly monkeypox) that have been relatively short-lived and self-limiting because of ineffective transmission among humans  , which catalyzes strand-specific C>U deamination, resulting in G>A substitutions in the complementary strand of viral genomes  of all substitutions were G>A/C>T transitions, followed by 6/70 (8.6%) T>C/A>G transitions, and 2/70 (2.8%) were C>A/G>T transversions and 1/70 (1.4%) A>C/T>G transversions. Comparison of these substitution proportions with those observed in clade II up to 2018 and those from the clade I showed a striking pattern  Figure. <0.0001)  Figure. In conclusion, our analyses of MPXV genome sequences indicate that the virus has been circulating silently and undetected for about 2 decades, probably in multiple non–MPXV-endemic countries outside of Africa. Also, a clear genomic signature of a recent change in hosts is evidenced by major changes in its nucleotide substitution pattern. Our observations have major public health implications; the changing epidemiology of MPXV infections and human circulation of the virus in non–MPXV-endemic countries call for increased surveillance (Additional information for study of monkeypox virus evolution before the 2022 outbreak.


In this paper, we report on the utilization of micro-technology based tools to fight viral infections. Inspired by various hemoperfusion and immune-affinity capture systems, a blood virus depletion device has been developed that offers highly efficient capture and removal of the targeted virus from the circulation, thus decreasing virus load. Single-domain antibodies against the Wuhan (VHH-72) virus strain produced by recombinant DNA technology were immobilized on the surface of glass micro-beads, which were then utilized as stationary phase. For feasibility testing, the virus suspension was flown through the prototype immune-affinity device that captured the viruses and the filtered media left the column. The feasibility test of the proposed technology was performed in a Biosafety Level 4 classified laboratory using the Wuhan SARS-CoV-2 strain. The laboratory scale device actually captured 120,000 virus particles from the culture media circulation proving the feasibility of the suggested technology. This performance has an estimated capture ability of 15 million virus particles by using the therapeutic size column design, representing three times over-engineering with the assumption of 5 million genomic virus copies in an average viremic patient. Our results suggested that this new therapeutic virus capture device could significantly lower virus load thus preventing the development of more severe COVID-19 cases and consequently reducing mortality rate.The online version contains supplementary material available at 10.1007/s00604-023-05671-9. Since the first hospitalizations in December 2019 , and appApproximately 17–40% of SARS-CoV-2 positive patients reported no typical symptoms , 20 whil2 removal can be used to address respiratory failures. Furthermore, cytokine removal by continuous renal replacement therapy or direct hemoperfusion with specific filters, HA resin hemoperfusion cartridges, and Toraymixin polymixin-B endotoxin removal are available therapeutic strategies during cytokine storm caused by SARS-CoV-2 infection [In general, separation and purification technologies are frequently studied and utilized for biomedical and clinical applications –45. Extrnfection , 46, 47.nfection , 49, extnfection –52.Next to the hemofilters, hemoperfusion and plasmapheresis procedures, such as heparin-immobilized polyethylene beads, are also used for non-specific pathogen removal from the bloodstream. The attached heparin binds viruses, bacteria, fungi, and toxins similarly to heparane-sulfate interacting with cell surface , 54. HemIn this paper, we report a feasibility study for the development and implementation of a SARS-CoV-2 capture device to specifically remove virions from the circulation, preventing the development of serious clinical outcomes. The approach combines the specificity of affinity chromatography with the high throughput hemoperfusion technique to fight the coronavirus pandemic. A laboratory scale hemoperfusion system was designed and fabricated to study the feasibility of the proposed approach. The setup has been tested in a BSL-4 laboratory environment with live virus suspensions as representative conditions and models to explore any potential risk factors before further development.2PO4 from Merck  and NaCl from VWR . Imidazole for buffer “B” was purchased from Merck.Picoline borane, DMEM cell culture media, and phosphate buffered saline  were from Sigma-Aldrich . Polyethylene-glycol was purchased from Merck . Fetal bovine serum, trypsin-EDTA, and 3-aminopropyltriethoxysilane (APTES) were from Thermo Fisher Scientific . Glutaraldehyde was purchased from Carl Roth Chemicals . Methanol and absolute ethanol were purchased from VWR . Hydrochloric acid, sulfuric acid, and dry toluene were purchased from Molar Chemicals Kft. . Kanamycin was from SERVA Electrophoresis GmbH . Isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Biosynth AG . The SDS PAGE gel was made from 40% acrylamide solution from Bio-Rad  and HPLC grade water in a ratio of 37.5:1. Buffer “A” contained NaHE. coli [E. coli strain, followed by purification using Ni-affinity chromatography as published earlier [The coding sequence of the publicly available single-domain antibody (sdAb) against SARS-CoV-2 spike protein was codon optimized for E. coli  and the  earlier , 58. TheIt should be noted that increased stress level of >150 Pa may cause extensive cell damage. To avoid any damage of the different blood constituents, a hydro-dynamically similar model system to an approved extracorporeal technology, i.e., Cytosorb , was utilized. For this purpose, commercially available monodisperse 450 μm diameter soda-lime glass beads were obtained from Glass Sphere  due to their available immobilization chemistry and biocompatibility . The SARS-CoV-2 spike protein–specific sdAb was immobilized onto the surface of the beads as follows. First, the beads were treated with a 1:1 mixture of cc. hydrochloric acid and methanol for 30 min at room temperature, followed by washing with HPLC grade water. After the washing step, the beads were treated with cc. sulfuric acid for 30 min at room temperature. The beads were washed and treated by boiling HPLC grade water for 30 min  and drie2.SARS-CoV-2  virus isolate was used for the experiments. Propagation of the viruses was carried out in VeroE6 cells  cultured in DMEM cell culture media containing 10% heat-inactivated fetal bovine serum. Cells were incubated at 37 °C in humidified air supplied with 5% COth Edition) [All virus culturing and capture experiments were carried out in the facility of the National Virology Laboratory, University of Pecs, Hungary, which is a Biosafety Level 4 (BSL-4) facility fulfilling all the requirements of the US Centers for Disease Control and Prevention guide for Biosafety in Microbiological and Biomedical Laboratories (6Edition) . ShortlyStandard stainless steel HPLC columns  were modified to obtain laboratory-scale test cartridges. Four hundred micrometer hole size meshes were placed in the Swagelok fittings to retain the beads during the circulation. Four grams of anti-SARS-CoV-2 sdAb immobilized beads was loaded into the cartridges for the virus capture experiment. The viruses, at the average of ~350 viral genome copies/μl concentration, were spiked into 30 ml DMEM medium and the virus suspension was circulated through the cartridges under investigation using a Masterflex peristaltic pump  for 2 h . An open-lid vessel was inserted in the circulation system to equilibrate the fluid level and ensure bubble-free flow. Furthermore, the vessel served as injection and sampling point as well. The schematics and photo of the experimental setup are shown in Fig. Duplicate samples of 100 μl were taken from the vessel  as well as from the supernatants (referred to as eluent) to determine the initial virus concentration and the number of captured virion particles, respectively. The number of captured virus particles was quantitatively determined by ddPCR  as published earlier , 62. ShoCaptured virus particles were visualized using scanning electron microscopy (FEI/ThermoFisher Apreo S LoVac). Observation by STEM was carried out in transmission mode under high-vacuum with 30 kV accelerating voltage. Samples were observed without fixation by following a standard air-drying procedure . UV-C inIn this paper, we report a feasibility study, first approach towards implementing a novel method to fight SARS-CoV-2 pandemic. A high throughput extracorporeal device has been designed to specifically remove virions from the circulation with the goal to avoid serious clinical outcome. A laboratory scale hemoperfusion system has been fabricated to study the feasibility of the proposed approach, which combined the specificity of affinity chromatography with the high throughput of hemoperfusion. The scheme of the proposed virus capture technique is shown in Fig. Capture efficiency was investigated by using an actual strain of SARS-CoV-2 in a BSL-4 classified laboratory. In the presented proof of concept experiments, the virus particles were captured from Dulbecco Modified Eagle Medium containing L-glutamine, sodium pyruvate, sodium bicarbonate, and phenol red indicator as described in the experimental section. Albeit, its complexity lags behind that of blood complexity, it can adequately model the virus capture efficiency from fluidic circulation. Virus concentrations were determined by ddPCR, both in the active and control experiments Table .Table 1Rinitial-residual+eluent. However, it should be emphasized that the surface-to-volume ratio of this laboratory scale experimental setup was significantly higher than that of in an actual hemoperfusion device; thus, non-specific binding was considered to be overrepresented. The measure of non-specific binding was determined by balancing the captured virus copies in the active and control experiments. Non-specific binding of virions or any proteinous macromolecules is governed by physical adsorption (or physisorption), in which van der Waals forces, hydrophobic interactions, electrostatic interactions, and hydrogen bonds play essential roles. Physisorption is resulted due to the interaction of electron configuration of the contacting atoms or molecules. In spite of the numerous technological advancements in the field of protein immobilization [As apparent from Table lization , non-spelization , 68. FroNote, only those virus particles were involved in the evaluation, which remained in the circulation and did not bind non-specifically, i.e., not removed from the circulation. Considering the reported genomic virus concentrations Table , the vol5 PFU/ml concentration virus suspension was dried onto a microscope slide-grid to observe certain morphologies of the virions under the conditions of the actual experimental setup  imaging was utilized to visualize the captured viruses on the surface of the beads. To this end, ~4·10see Fig. A and B. 3 of glass beads, the difference in the number of specifically captured viruses was considered to be the actual performance, i.e., found to be 120,000 virus copies. The captured 120,000 virus particles from  the virus culture media circulation, suggested the partitioning ability of 15 million virus particles with an actual therapeutic size column. In other words, since the actual size of an average hemofiltration cartridge is about 330 cm3, the virus-binding capacity of our design is expected to be around 15 million. There is a reported correlation between human plasma SARS-CoV-2 virus concentration and COVID-19 disease course and mortality [According to the current clinical protocols, similar extracorporeal treatments may last up to 24 h , that, bortality , 72. Virortality , 74. Conortality , 75, 76;ortality .Please note the importance of future biocompatibility testing to shed light on any potential drawbacks of the proposed technology, which was not possible at this very early stage of the development. However, it is suspected that shear stress on blood elements, extracorporeal treatment–induced hypothermia, and blood exposure to non-biological surfaces may initiate perioperative systemic inflammatory response syndrome . AnotherA laboratory scale virus capture device was designed and fabricated to evaluate the feasibility of a novel immuno-affinity capture–based extracorporeal treatment approach. Due to the recent SARS-CoV-2 pandemic, its Wuhan (VHH-72) virus strain was chosen as model system to probe the proposed technology. Single-domain antibodies were produced by recombinant DNA technology and immobilized on the surface of glass micro-beads. A laboratory size, i.e., down-scaled model device, was utilized for the feasibility tests. The experiments conducted in BSL4 laboratory environment using live virus suspensions successfully proved the virus capture ability of the reported method. In the lack of effective antiviral drugs, especially during the outbreak of a rear or unknown virus caused pandemic, the suggested technology may serve as the base of an effective extracorporeal therapy, which helps patients to fight against the infection and significantly decrease the chance of being admitted to intensive care units. Furthermore, it is anticipated that after further development, the reported technology could possibly be an artificial organ platform, which represent an option to address organ failure issues and improve survival in case of viremia caused by several types of viral infections.ESM 1(DOCX 30 kb)


Plant viral diseases are major constraints causing significant yield losses worldwide in agricultural and horticultural crops. The commonly used methods cannot eliminate viral load in infected plants. Many unconventional methods are presently being employed to prevent viral infection; however, every time, these methods are not found promising. As a result, it is critical to identify the most promising and sustainable management strategies for economically important plant viral diseases. The genetic makeup of 90 percent of viral diseases constitutes a single-stranded RNA; the most promising way for management of any RNA viruses is through use ribonucleases. The scope of involving beneficial microbial organisms in the integrated management of viral diseases is of the utmost importance and is highly imperative. This review highlights the importance of prokaryotic plant growth-promoting rhizobacteria/endophytic bacteria, actinomycetes, and fungal organisms, as well as their possible mechanisms for suppressing viral infection in plants via cross-protection, ISR, and the accumulation of defensive enzymes, phenolic compounds, lipopeptides, protease, and RNase activity against plant virus infection. Eco-friendly crop disease management is the one of most important pre-requisites for ecological and sustainable farming in the 21st century, as many plant diseases caused by fungi, viral, and bacterial organisms pose major yield-limiting factors and affect the quality of produce in economically important crops. Among the biotic stresses, plant viruses cause severe epidemics in all major agricultural crops of economic importance, posing a severe threat to global food security. Plant viruses are known to cause nearly half 47%) of the emerging and re-emerging plant disease epidemics worldwide , and app7% of theThere are currently very few options available for managing plant viral diseases in the field, since the application of insecticides and other toxic material inhibit the virus transmission by vectors is a desirable and unavoidable management strategy. As a result of the indiscriminate and excessive use of insecticides, insect vectors have developed resistance mechanisms against them. Furthermore, the probability of virus recombination has increased the chances of evolving highly aggressive viral strains and strains, which are capable of overcoming genetically induced resistance in plants by means of resistance breakdown. Chemical control measures are unsuccessful, as viruses are intracellular pathogens. The prophylactic measures include the removal of affected crop debris and using a greater number of applications of pesticides to reduce the population of insect vectors. The need of an hour to identify bioagents, with their beneficial activity, environmental safety, and a combination of diverse biocidal strains against major pathogens and pest with the activation of immune reactions in plants via by activation of specific signaling cascades, induced systemic resistance (ISR), and secondary metabolites in virus-infected plants, is of great interest. When compared to chemical pesticides and transgenic approaches, beneficial and heterogeneous groups, such as actinomycetes, endophytic microbes, plant growth-promoting bacteria, and fungal agents, can suppress viral activity and inhibit the egg-laying capacity of insect vectors ,10,11,12Streptomyces ahygroscopicus [Streptomyces sp. ZX01 [Streptomyces noursei var. xichangensis [TMV infection in tobacco. Actinomycetes inhibit the virus through a variety of mechanisms, including the activation of plant defense pathways and the production of signaling molecules. Marine organisms differ metabolically and physiologically from terrestrial habitants, and marine actinomycetes have been identified as a possible origin of numerous unique bioactive compounds [Cucumber mosaic virus (CMV).Actinomycetes are possible candidates for the production of secondary metabolite compounds, known as antibiotics, for their role as biocontrol agents and plant probiotics potential, due to their plant colonization and in situ antibiotic production ,17,18. Sscopicus ,20, Stresp. ZX01 , and Strangensis , againstompounds . Latake ompounds  investigStreptomyces olivaceus was found to be impressive in controlling the CMV infections under in-vitro and open fields when applied individually as seed and spray treatment. A decreasing trend was observed in various necrotic lesions of Tobacco mosaic virus (TMV) and Potato virus Y (PVY) on Nicotiana glutinosa and Chenopodium quinoa, respectively, by application of the Streptomyces culture filtrates and virus sap mixture [Nicotiana glutinosa and N. tabacum by mixing the different components, such as fermented actinomycetes broth, along with TMV sap, at different time intervals. The antiviral compound, ε-poly-l-lysine (ε-PL) of 3454–4352 Da, produced from Streptomyces ahygroscopicus has anti-TMV property [In general, bioagents have diverse advantages in the reverence of little mammalian toxicity, biodegradability, superior ecological compatibility, and a distinctive mechanism of action. Metabolite of  mixture . Similar mixture  observedproperty . Pseudomonas aeruginosa, Burkholderia sp. and Bacillus sp., were evaluated against the Cotton leaf curl virus (CLCuV) under artificial conditions by applying individually and in different combinations. The highest inhibition of CLCuV was observed in plants applied with a mixture of bacterial strains and only 0.4%, and the viral load was estimated in challenged plants, as compared to 74% in control plants. The principal component biplot analysis (PCA) revealed that a highly significantly correlation was found among the attributes, the viral load, and the incidence of disease [Monitoring the global biocontrol market revealed that there are no antiviral biopreparations in the biopesticides group that act directly as antiviral agents of biological origin in nature . The dif disease .Pseudomonas sp. consortium of chitosan-based formulation, three demonstrated the increased effect of ISR and resulted in the accumulation of chitosan, which led to the enhancement of biocontrol efficacy against Tomato leaf curl virus (ToLCV) by application of Pseudomonas sp. The minimum viral titre was quantified through a semi-quantitative PCR assay with the application of chitosan and Pseudomonas sp. and scanning electron microscopy revealed a significantly higher number of bacterial cells in the roots, with no morphological or other qualitative differences [P. fluorescens was discovered to have field efficacy for CMV and Tomato mottle virus (ToMoV) [Tospovirus [Solanum lycopersicum, and lesion-inducing Tobacco necrosis virus (TNV) disease in Nicotiana tabacum resulted in a reduction in TNV-induced lesion number in P. fluorescens treated plants [Azotobacter vinelandii and Azotobacter chroococcum [B. subtilis Ch13 [Potato virus X(PVX), PVY, and Potato leaf roll virus (PLRV) in Solanum tuberosum; B. cereus (I-35) and Stenotrophomonas sp. (II-10) reduced the infection of TMV, Chili veinal mottle virus (ChiVMV) in Capsicum annuum [Bacillus subtilis 26D and B. subtilis Ttl2 against PVX and PVY [Bacillus spp. against Tobacco streak virus (TSV) in cotton [B. amyloliquefaciens MBI600 against Tomato spotted wilt virus (TSWV) in S. lycopersicum and Solanum tuberosum [B. amyloliquefaciens FZB24, B. pumilus EN16, B. subtilis SW1, Pseudomonas syringae (heat-killed cells) [P. putida A3 [Rhodopseudomonas palustris GJ-22 against TMV in N. tabacum [Pseudomonas chlororaphis O6N against TMV in N. tabacum cv Xanthi-nc [Bacillus pumilus T4 and B. subtilis GBO3 against Bean common mosaic virus (BCMV) in Vigna unguiculata [B. pumilus SE34, B. amyloliquefaciens 5B6, B. pumilus SE34, Kluyvera cryocrescens IN114, B. amyloliquefaciens IN937a, B. subtilus IN937b, Pseudomonas lachrymans against CMV in Cucumis sativus [S. lycopersicum [Capsicum annuum [Paenibacillus lentimorbus B-30488 against CMV in N.tabacum [Serratia marcescens 90-166 against CMV in A. thaliana [Pseudozyma churashimaensis against CMV, Pepper mottle virus (PepMoV), Pepper mild mottle virus (PMMoV), and Broad bean wilt virus (BBWV) in Capsicum annuum [P. fluorescens CHA0 against Urdbean leaf crinkle virus (ULCV) in Vigna mungo [P. fluorescens CHA0 [P. fluorescens P3 [N. tabacum. In tomato seedlings, when subjected to the ferences . P. fluo (ToMoV) , biocontspovirus  in Soland plants . Zehnderd plants  identifid plants . The effoococcum , and B. lis Ch13  inhibitem annuum ; Bacillu and PVY ; Bacillun cotton ; B. amyluberosum ; B. amyld cells) , P. putiutida A3 , Rhodops tabacum ,43,44; Panthi-nc ; Bacilluuiculata ; B. pumi sativus ; S. lycopersicum ,49, Capsm annuum ; Paeniba.tabacum ; Serratithaliana ; Pseudozm annuum ; P. fluona mungo ; P. fluoens CHA0 , P. fluoscens P3  against B. subtilis GB03 + B. pumilus SE34/B. amyloliquefaciens IN937a/B. subtilis IN937b/B. pumilus INR7/B. pumilus T4 in S. lycopersicum [Serratia marcescens 90-166 + P. putida 89B-61/B. pumilus T4 against CMV in Cucumis sativus [B.licheniformis MML2501 + Bacillus spp. MML2551 + Pseudomonas aeruginosa MML2212 + Streptomyces fradiae MML1042 against Sunflower necrosis disease (SND) caused by the Tobacco streak virus (TSV) in Helianthus annuus [B. amyloliquefaciens IN937a + B. pumilus SE34 + B. pumilus T4 against Papaya ringspot virus (PRSV-W) and Tomato chlorotic spot virus (TCSV) by in Carica papaya/S. lycopersicum [P. fluorescens Pf1. + Bacillus spp. EPB22 against Banana bunchy top virus (BBTV) in Musa spp. [Bacterial microbial consortia are also reported to control many viruses. The reduction of CMV infection was observed by using bacterial consortia viz., persicum ; Serrati sativus ,56; B.lis annuus ; B. amylpersicum ; P. fluousa spp. . P. fluorescens CHAO + chitin reduced the infection of BBTV in Musa spp [Pseudomonas sp. 206(4) + B-15+JK-16+chitosan inhibited the ToLCV infection in S. lycopersicum [B. polymixa + P. fluorescens + chitin were used against Squash mosaic virus (SqMV) control in Cucumis sativus [B. pumulus INR7 + benzothiadiazole against CMV in Capsicum annuum [The combinations of individual bacterial isolate/consortium and chemical immunoregulators, such as Musa spp ; Pseudompersicum ; B. poly sativus ; B. pumum annuum  are alsoThe concurrent infection of a single plant by a variety of pathogen is progressively more familiar as a host resistance modulator and pathogen evolution driver. In agro-ecosystems, plants are the target of a large number of pathogenic microorganisms, and co-infection could be regular, and as a result, it is important to consider. Co-infection was directed to raise bacterial specific symptoms, whereas there was a decrease in viral load, compared to the mono-infected plant. This could be due to gene silencing mechanisms intervening within plant interaction between virus and plant pathogenic bacteria. Therefore, pathogen–pathogen–host relations positively justify more consideration, from a hypothetical and practical point of view. A few of the co-infected bacteria in plant virus disease management with host plants and treatment methods in the reduction of virus infection are listed in Colletotrichum legendarium or TNV protects plants systemically against diseases caused by several pathogens. Muvea et al. [Hypocrea lixii F3ST1) inoculation on onion reduced the thrips vector population, resulting in higher death rate of vector population, and due to antixenotic repellence activity of the applied agents, reduced feeding behavior and oviposition could be observed. Furthermore, the reduced vector feeding activity of endophyte-colonized onions may decrease the virus spread of vectors. The endophytic interaction increases the incompetence of viruliferous thrips to transmit Iris yellow spot virus (IYSV) (Genus-Tospovirus), transmitted by Thrips tabaci, which has negative effects on IYSV replication in the infected plants. This may be due to the possibility of endophytes, such as fungi, eliciting the activation of gene expression in defense cascade pathways, in turn enhancing the accumulation of defensive compounds for development of resistance [Endophytes and fungal bioagents are able to recognize the changes in physiological means in stress-induced plants as defense machinery, thus regulating the plant gene expression ,68. Inoca et al. ,70 discosistance ,72. Rhopalosiphum padi transmitting Barley yellow dwarf virus (BYDY), which showed a reduced aphid population and percentage of BYDV infections in fungal endophyte (Neotyphodium uncinatum) infected in meadow ryegrass (Lolium pratensis), in comparison to endophyte-free plants [The influence of fungal secondary metabolites synthesized by endophytic association could be responsible in fungal-viral interplay mechanism. The alkaloids produced as a result of fungal endophytes application are found to have antiviral properties . Endophye plants , inferrie plants ,76.Arachis by application of bioagents [viz., enhanced plant growth vigor, and disease resistance caused by fungi, bacteria, nematodes, viruses, and viroid’s [A probable reason for the low virus load observed between the plants is most probable and likely to inhibit or down-regulate the coat protein gene expression, resulting in impaired virus replication during the initial phases of the infections. Peanut bud necrosis virus, for example, was also managed by blocking the systemic movement of the virus in wild ioagents . Virus rioagents ,79. Therviroid’s . The funPepino mosaic virus (PepMV) of S. lycopersicum, which depends on the mild variants of PepMV for its induction [The virus load in infected plants can trigger for the release of secondary volatiles and other compounds, which are highly congenial for attracting vectors. When the vector tends to feed on the plant, the virus can produce an anti-feeding substance, causing the vector to flee to a new plant, as well as influence insect vectors to feed on healthy plants ,99. Virunduction . The comCitrus tristeza virus (CTV), belonging to the genus Closterovirus transmitted by aphid vectors, has been successfully controlled by cross-protection in several parts of the world [Microbial biological agents control and protect crops against diseases through diverse mechanisms. The application of biological agents is an alternative option for pathogen control using cross-protection. Cross protection is a type of induced resistance that develops in plants against plant viruses. It is most effective among the closely related strains of the same virus, even though reports of viruses that are phylogenetically distinct protecting against each other exist. This phenomenon was described by the Dutchman Thung and the English man, Salaman, about seventy years ago, the prior immunization of host plants with a mild strain to defend against the challenge inoculation by aggressive or severe strain of the same plant virus, which leads to fits with future-proofing of production systems, known as mild strain cross-protection (MSCP) . Citrus he world . The mechanisms of cross-protection are still unknown; however, several plausible mechanisms are proposed, such as antibody formation, specific adsorption by new cell compounds, exhaustion of essential metabolites, limited replication sites available to the plant virus multiplications, competitiveness between main and severe viruses for host components, and point of replication sites within the cells, interfering with disassembly, secondary virus translation, or replication ,105,106 ZYMV in squash, melon, and watermelon [Cocoa swollen shoot virus in cocoa [Tomato mosaic virus (ToMV) in S. lycopersicum and pepper [Papaya ringspot virus in papaya [Additionally, termelon ,114,115,d pepper , and Papn papaya  are somen papaya . Howevern papaya . For plan papaya ,121.Coriolus Versicolor, Coprinus comatus, Lentinus edodes (Berk.) sing, Pleurotus ostreatus, and Flammulina velutiper, polysaccharides and their peptides with antiviral abilities have been studied [Coriolus versicolor showed 85.4% and 64.8% anti-TMV action lower concentrations [−1 concentration [Polysaccharides, polysaccharide peptides, and proteins are the main antiviral compounds existing in fungi. For the past few years, from fungal organisms such as  studied . A polystrations . The lenntration . The lenAlternaria tenuissima can successfully help the immunization of plants. A protein with higher capacity may induce plant immune responses, plant resistance, and growth metabolism. Other fungi having antiviral-active compounds include Agrocybe aegerita, Flammulina velutipes, Lentinus edodes, Pleurotus citrinopileatus, and P. eryngii. Additionally, Neosartorya fischeri and Penicillium oxalicum fungal methanolic extracts have shown repressive action to TMV infections [Trichoderma pseudokoningii SMF2, are an ISR determining factor that exhibited resistance against TMV [Trichoderma harzianum has condensed TMV symptoms expression on S. lycopersicum through ISR activity [Kulye et al.  reportedfections . The antinst TMV . Alkaloiinst TMV . Similaractivity .The understanding of the effectiveness of bacteria and other microorganisms in improving growth of plants and inhibiting virus infectivity benefit the plant virus control in the field, particularly for those apprehensive about the eco-friendly control of crop diseases. The use of antiviral compounds that activate systemic resistance in plants has been described for many viruses in plants. Microbe-derived antiviral agents afford environmentally friendly, efficient, and degradable backup strategies for traditional chemical agents to control plant viral diseases. Their mechanisms against plant viruses are viable for directly acting on viral nucleic acids or proteins, or ultimately, restrain viruses by regulating host reaction . Ningnanmycin (NNM), an antiviral substance extracted from actinomycetes-Strepcomces noursei var. xichangensis, a new cytosine nucleoside peptide antibiotic, is most successful in inhibiting TMV infections, which is widely used in crop production [The actinomycete group comprising 80 genera produces a variety of secondary metabolites with excessive inference in crop protection. More than 50% of the identified antibiotics are sourced from actinomycetes . Ningnanoduction . It was oduction . The NNModuction .Streptomyces ahygroscopicus, showed competent virucidal action [Streptomyces sp. ZX01, with an 80% anti-TMV rate at 1 mg mL−1 concentration. Peroxidase, is well-thought of as an authoritative defensive enzyme, is evidenced in numerous physiological responses against the biotic stresses of plants [The cytosine peptidemycin, extracted from l action , and it l action  reportedf plants ,129. Streptomyces strains induced the systemic acquired resistance (SAR) to virus infections, whereas P. aeruginosa enhanced the resistance to TMV in tobacco plants [S. Noursei var xichangensis, known to induce ISR [S. pactum Act12 ISR against TYLCV and salicylic and jasmonic acid concentrations have amplified in S. lycopersicum plants. The various bioactive compounds derived from Streptomyces strains were found to be effective in minimizing the TMV local lesions on the leaves of Datura metel weed plant [l-lysine, released from by S. ahygroscopicus, has revealed substantial defense and curative action against TMV [Cucumis sativus with S. Albovinaceus and S. sparsogenes, separately [Cytovirin is a broad-spectrum antibiotic that reduces virus concentration in the host and delays symptom emergence [Actinomycin D) protects soybeans from Bean pod mottle virus (BPMV) [Pea streak virus (PSV) may be controlled by spraying cycloheximide [Sunn-hemp mosaic virus (SHMV) in leguminous plants by using pentaene G 8, an antifungal antibiotic from Streptomyces anandii. Leaf hoppers were unable to pick up viruses when the host plants were applied with tetracyclines [Bromegrass mosaic virus has been controlled by actinomycin D and blasticidin S [S. lycopersicum. The bacterial bioagent Bacillus amyloliquefaciens (VB7) proficiently reduced the expression of GBNV disease symptom up to 84% through the transient expression of MAMP genes, triggered immune reaction, and decreased virus titer [Galal  describeo plants , as wellduce ISR . Li et aduce ISR  reporteded plant . Additioinst TMV . The mosparately . The usemergence ; ACD (Acs (BPMV) . TMV inhs (BPMV) , oxytetrs (BPMV) , puromycs (BPMV) , laurisis (BPMV) , cyclohes (BPMV) , chlorams (BPMV) , etc. Peheximide , actidioheximide , and blaheximide . Phatak heximide  reportedcyclines . Bromegricidin S . Root diicidin S ,150, andus titer .In some of the reports, the treatment of antibiotics to virus-infected plants leads to reduced symptom expression, besides the inhibition of plant viruses ,153. StiActinomycin D and mitomycin C antibiotics are also identified to affect the metabolism of nucleic acid and form complexes with DNA. Excluding in the very early stages of infection with specific virus-host amalgamations, they do not obstruct the plant pathogenic RNA virus synthesis ,158. CMV−1 concentration [−1 concentration. Nagorskaia et al. [Tichocarpus crinitus, released kappa/β-carrageenan that can decrease TMV infection in Xanthi-NC tobacco leaves, and the lectin from the marine algae, Ulva pertusa, have anti-TMV activity [The algae group contains a diversity of plants ranges, from diatoms observed as microscopic and unicellular to seaweeds spreading over 30 m. The first discovered sources of natural compounds are microalgae against HIV . The polntration . From tha et al.  found thactivity .−1 concentration inhibits 50.41% TMV infection. Since then, oligosaccharide has been marketed as a vital anti-plant virus compound in China. Similarly, melittin and its analogue and chondroitin sulfate, a whey protein, and its esterification products were identified as anti-plant virus compounds [Animals have an insufficient number of anti-plant-virus compounds than plants and microbes. Amongst them, oligochitosan is the most successful anti-plant-virus compound produced by enzymatic hydrolysis of chitosan polymer discovered in animals. Chitin and chitosan have been discovered to be nontoxic, biodegradable, and biocompatible substances that stimulate the broad-spectrum defensive response in plants ,168. Theompounds ,172,173.ompounds ,176,177.P. fluorescens CZ against TMV. Thapa et al. [Serratia marcescens culture filtrate has stable antiviral capacity against CMV. Cell-free supernatant consisting of cyclic peptide synthesized from Pseudomonas chlororaphis O6 exhibited strong antiviral activity against TMV [Lipopeptides are antimicrobial peptides synthesized by a wide range of microorganisms via non-ribosomal synthesis, including secondary metabolites, such as peptaibols, cyclopeptides, and pseudo peptides . Many rea et al.  describeinst TMV .Bacillus and Pseudomonas sp. [Lipopeptides are 1000–2000 Da cyclic, lower antimicrobial compounds that are primarily synthesized by bacteria, such as onas sp. . Indeed,onas sp. . Additioonas sp. .Bacillus spp. produces surfactin, iturin, and fengycin of three major families containing hydrophilic amino acids (7–10 amino acids) connected with a hydrophobic fatty acid tail. These compounds perform as effective antagonists by inhibiting plant pathogens by diverse mechanisms by inducing and establishing the plant defense apparatus in defense-related systems recognized as ISR [Bacillus spp were evidenced as elicitors to activate ISR in plants. Lipopeptide surfactants exhibit an exceptional aperture and ion networks establishing possessions and, thus, may interrupt the normal integrity and absorptivity of the lipid layer of the plasma membrane. Lipopeptides, by virtue of their capability to interrupt the physical integrity of the living membrane, create their primary means of anti-microbial activity against diverse microbes, such as bacteria, fungi, viruses, mycoplasma, etc. [The d as ISR . Of lated as ISR , releasema, etc. .B. amyloliquefaciens S499 releases cyclic lipopeptides and affords an efficient resistance in both the leaves and roots of sugar beet, consequential in biocontrol rhizomania disease through the reduction of the virus vector P. betae infection [Beet necrotic yellow vein virus (BNYVV) causes rhizomania disease transmitted by the obligate root-infecting parasite Polymyxa betae [Bacillus Cyclic lipopeptides (CLPs) significantly decrease the infection by P. betae in sugar beet through ISR. McGrann et al. [P. betae in sugar beet with decreased virus concentration. It seems that CLPs offers a new technique for the sustainable management of rhizomania disease.nfection . Beet nexa betae . Bacillun et al.  and Barrn et al.  revealedN. tabacum plants treated with Rhodopseudomonas palustris GJ-22 strain, reduced TMV infection by stimulating the salicylate (NbPR1a and NbPR5) and jasmonate-mediated (NbPR3 and NbPDF1.2) signaling pathways genes [B. amyloliquefaciens MBI600 strain induced host resistance to PVY and TSWV in S. lycopersicum by the expression of SA-induced signaling pathway genes [The peptide compound indolil acetic acid and non-proteinogenic amino acid, such as the 5-aminolevulinic acid produced in ys genes . Howeveray genes .Ribonucleases are a group of hydrolytic enzymes that have the ability to dimerize and catalyze the breakdown of ribonucleic acid (RNA) into smaller units in several critical transcription phases and inhibit virus reproduction. Ribonucleases are the classical components of immune system [Buckwheat burn virus (BBV) by the expression of enhanced ribonuclease activity [Schizosaccharomyces pombe has improved resistance to CMV, ToMV, PVY, and TSWV in chrysanthemum, tobacco, and impatiens plants [Bacillus sp. TS2 and B. subtilis 26D, are used for the development of biocontrol agents. Several viral infections  hasten plant impairment substantially, compared to a sole virus infection, which was discovered by Hameed et al. [B. thuringiensis B-6066, Bacillus sp. TS2, Bacillus sp. STL-7, and B. subtilis 26D, as well as strains with the enormous capacity to colonize internal plant tissues collective with increased RNase activity and decreased viral disease incidence and severity of potato viruses M, S, and Y. They distinguished that Bacillus spp. lessened the Leptinotarsa decemlineata egg clusters and larvae number and revealed their antifeedant activity on treated plants.e system . Variousactivity . The exps plants ,193,194,s plants . Endophyd et al. . Sorokand et al.  found a viz., B. pumilus, B. amyloliquefaciens, and B. licheniformis  have extracellular RNase secretive capacity to utilize organic phosphates in aiding bacterial adaptation mechanisms to changing ecological situations and slice RNA containing viral particles continuously in plant tissues [Pantoea, Cronobacter, Microbacterium, and Staphylococcus isolates, which originated from the Cucurbitaceae family, produce nucleases, which cleave viral nucleic acids [Pseudomonas putida A3 [B. pumilus 7P/3-19 [The bacterial species  tissues ,199. Lesic acids . The virutida A3  and B. p 7P/3-19 .Schizosaccharomyces pombe in soybean crop caused a significantly higher concentration of Soybean mosaic virus (SMV) in SC3 strain-free soybean plants [Nicotiana benthamiana plants holding CRISPR/Cas13 a cassette that included class 2 type VI-A RNase, created by genetic engineering capable of identifying and slicing ssRNA, was extremely resistant to Turnip mosaic virus (TuMV) [B. amyloliquefaciens showed complete resistance to ToLCV infection [Bacillus and other bacteria can protect plants against viral infection by upsetting vectors of viral particles, such as insects and plant pathogens.There was a strong and stable constructive association between the RNase activity in several potato cultivars and their resistance to PVX, PVY, PVM, and PVS . The RNan plants . The Nics (TuMV) . Approxinfection . Thus, tThe PGPRs are endophtic or rhizospheric bacteria which enhance yield sustainability, and growth promotion and defend plants from invading pathogens . As a paEnterobacter asburiae activated the TYLCV resistance and lower disease rates, approaching 42%, even at 45 dpi, under greenhouse conditions. Regulation of disease resistance was detected against ToMoV in S. lycopersicum plants by the different strains of B. amyloliquefaciens [The presence of bioagents/endophytes in plants recognizes the plant virus proteins, particularly viral RNA, as well as Coat protein (CP) of a virus, and the small conserved molecular motifs present in microbes called Pathogen-associated molecular patterns (PAMPs) are recognized by the receptors of plant cell, which results in the development of plant defense reactions  with rapefaciens .2O2 levels. HR reactions against PVYC and PVYO strains of PVY are regulated by potato Nytbr and Nctbr genes, respectively, in Solanum tuberosum crop [Nx-mediated hypersensitivity and Rx-mediated resistance were produced by various coat protein (CP) subunits of PVX [B. subtilis 26D and B. subtilis Ttl2, secreted ribonucleases and phytohormones, inhibited PVX and PVY in S. lycopersicum plants. Both the 26D and Ttl2 strains triggered ISR by activating the transcriptional genes related to salicylate and jasmonate-dependent reaction and increased the content of cytokinins and decreased the level of indolacetic acid in PVX- or PVY-infected plants [B. amyloliquefaciens 5B6 treatment with pepper plants reduced the CMV incidence by induction of transcription-encoding genes, such as PR-4, PR-5, and PR-10 proteins [Some of the rhizobacteria interacting with the plant roots result in a resistance to bacteria, fungi, and viruses, causing plant diseases, and this phenomenon is called ISR. In addition, ISR also involves jasmonate and ethylene signaling, which stimulates the defense responses in the host plant against diverse range plant pathogens . PGPRs psum crop . The avis of PVX . This mes of PVX . The apps of PVX ,221. Thes of PVX . The twod plants . Polyphed plants . The prod plants ,225,226.d plants . The infd plants . B. amylproteins .Streptomyces strains stimulated SAR for virus infections, while P. Aeruginosa was extremely potent at enhancing TMV resistance in tobacco [S. Nourseivarxi changes has been shown to induce SR against TMV [Capsicum annuum pathogenesis-related 4 (CaPR4), Ca chitinase 2 (CaChi2), CaSAR8.2, Ca phenylalanine-I ammonia-lyase (CaPAL), Ca 1-aminocyclopropane-1-carboxylic acid oxidase (CaACC), and Ca proteinase inhibitor 2 (CaPIN2), which was similar to the increase in genes expression in plants received benzothiadiazole [An authoritative PR protein or defense protein, such as the peroxidase gene, is involved in various useful responses against plant biotic stresses, along with studying pollutant degradation and management ,129. Gal tobacco . Furtherinst TMV . The bacadiazole .P. fluorescens Pf1 and endophytic Bacillus spp. EPB22 bacterial mixture treated banana plants stimulated the defensive enzymes, such as peroxidase (PO), polyphenol oxidase (PPO), and PAL, in addition to the phenolic substances, which reduced the BBTV incidence up to 80% [S. lycopersicum plants [Urdbean leaf crinkle virus (ULCV)-infected black gram [The rhizospheric p to 80% . Similarp to 80% , TSWV-inm plants , and Urdack gram .Cladosporium herbarum is peptidogalactomanann (pGM), induces defense-related genes for the expression of SAR and ROS and its accumulation with BY2 tobacco cells responsible for weakening viral infection [N. tabacum displayed strong and durable antiviral action to TMV. Additionally, defense enzymes, such as peroxidase and PAL transcripts, showed strong antiviral activity [The cell wall glycoprotein of fungal phytopathogen, nfection , with ennfection . Sindelanfection  reportedactivity ,206,232.Bacillus spp. and P. fluorescens (Pf1) inhibit the BBTV. Chitosan and PGPR had reduced disease severity and Squash mosaic virus (SqMV) titre, which infects Cucumis sativus plants by deferring the incubation period in the reproductive phase (4-7 weeks after planting) [B.subtilis) and IN937a (B. amyloliquefaciens) application with the chitosan as the carrier was effective against the CMV in S. lycopersicum plants [P. fluorescens-treated tobacco plants controlling the TNV and salicyclic acid production by rhizobacteria are also responsible for ISR against TNV. Pyung-II et al. [P. fluorescens strain EXTN-1 treated tobacco plants treated against the Pepper mild mottle virus (PMMoV).Harish  reportedlanting)  by triggm plants . Maurhofm plants  detectedI et al.  reportedP. fluorescens strains are proficient in inducing significant levels of defensive enzymes in banana that its induced enzyme actions are linked with the biosynthesis of phenolics and other secondary metabolic compounds, which have been projected to be major factors in ISR against the BBTV disease along with higher yield [−1) inoculation. The ALMV produced local infections on bean leaves (were fully controlled with the treatment of the maximum chitosan concentration (0.1%) through spray or addition to the virus inoculum [C. sativus or S. lycopersicum plants with PGPR lead to ISR against the systemic infection of CMV.The er yield . Chirkover yield  defined inoculum . Similarinoculum ,238. Rauinoculum  were the2O2 as a systemic antiviral signaling unit during TMV infection. A higher amount of ROS production is also considered a biochemical marker during SAR induction.The roles of ROS in plant–virus interactions are not well-understood. It is anticipated that ROS can act as a defense substance ,238 and Pepper leaf curl virus (PepLCV) transmitted whiteflies are controlled by T. harzianum, T. Polysporum, T. Atroviridae, and their consortia. They occupy the pepper plants endophytically, with a considerable increase in the phenolic content (183% more), and induce innate host immunity by the activated phenylpropanoid biosynthesis. The competence of Trichoderma bioagents to produce salicylic acid seems to have a prominent role in composing the PepLCV suppression (up to 50%) and ROS accumulation at the infection point resulting in the restricted virus spread [Cotton leaf curl Burewala virus-resistant genotypes than in susceptible ones after the inoculation. Abo-Zaid et al. [Streptomyces cellulosae (isolate Actino 48) at 2 × 107 CFU mL−1 reduced the incidence of TMV in S. lycopersicum through ISR. They applied Actino 48 before TMV inoculation (48 h) and reported significantly increased levels of total phenolic compounds, proteins, peroxidase, and chitinase enzymes in TMV-treated tomato plants + Actino 48, as compared to TMV-treated tomato plants alone. Hence, Actino 48 could be used for the biological management of TMV. Due to the systemic nature of virus infection, effective chemical compounds cannot be applied for the control of plant viral disease in agricultural or horticultural crops. Although, the endophytic plant-growth-promoting bacteria (ePGPB) strains viz., Paraburkholderia fungorum R8, Paenibacillus pasadenensis R16, Pantoea agglomerans 255-7, Pseudomonas syringae 260-02, and chitosan-treated plants exhibited precise biocontrol activity against Cymbidium Ring Spot Virus (CymRSV) and CMV through a considerable decline in severity of virus symptom with increased plant height compared to the control. Furthermore, defense-related genes, such as enhanced disease susceptibility-1 (EDS1) gene up regulation indicated the involvement in salicylic acid (SA) signaling pathway, non-expressor of pathogenesis-related genes-1 (NPR1) involved in mediation of cross talk between SA or jasmonic acid (JA) and ethylene (ET) signaling pathways and induction of SAR in plant system by the activation of PR2B, a PR- protein results in activation of defense against virus infection [The s spread . Siddiqus spread  also repd et al.  found thnfection . The ePGHumans began to consider alternatives as a result of the detrimental effects of synthetic pesticides . PesticiPresently, numerous reports are available on the role of bacteria, fungi, actinomycetes, and other organisms in the involvement of defense mechanisms against plant pathogens, except for viruses. Basically, induced defense mechanisms against viral infection by microbes and their metabolites are impeding virus transmission and replication. It is suggested that the predictions of the exploitation of bacteria and plant RNase for the prevention of virus infection in plants are challenging and unexplored research areas. Thus, the identification of environmentally safe biological agents with antiviral properties for plant protection against virus diseases is a constructive method of plant defense. PGPRs have direct antiviral properties by generating RNases or SR-inducers, which live on surfaces or internal plant tissues, and such microbes indirectly decrease the viral load in the agro-ecological system through vector control by the “RNA biocides” specific for crop pests.


Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-2.  The study of the structural and physicochemical stability of viruses to adverse environments is becoming increasingly relevant to the present or future pandemics. One of the main fronts against the COVID-19 disease are disinfection methods, including hydroalcoholic solutions and detergents. Here we report the mechano-structural stability of individual coronavirus virions using a structurally indistinguishable surrogate, the porcine transmissible gastroenteritis virus (TGEV). We probe virions’ resistance under a range of physicochemical assaults, including mechanical stress, desiccation and treatment with fixative  and biocidal chemical agents (detergent and ethanol). Our experiments reveal that virion stability depends on differences in the packing fraction between virus particles due to their pleomorphic nature. We find that individual viral particles crumble under a detergent concentration three orders of magnitude lower than ethanol. These experiments determined that detergent reduces virus size by 15–30 nm by solubilizing the lipid virus envelope, whereas ethanol dehydrates and shrinks TGEV virions by ~12 nm. Both biocidal agents induced material spreading around virus particles that correspond to externalization of ribonucleoproteins. This work suggests the convenience of using a SARS-CoV2 surrogate for dynamic nanoscale structure studies that can alleviate the use of BSL3 labs that are highly demanded for biomedical and biotechnological research.tour de force, virus particles must be stable enough to protect the genome during transmission but prepared to easily release it at the right time and place [Viruses can be considered to be molecular machines that parasitize the host cellular machinery to perpetuate themselves . The virnd place . A major challenge in the current and future pandemic scenarios is to control virus spread . The proWhen zoonotic animal viruses spread in humans, they can become a serious health threat because the population is immunologically naïve ,11, leadDuring the fight against COVID-19, hygienic and prophylactic practices have been globally recommended to stop virus spread, such as face masks  and disiIn recent years, atomic force microscopy (AFM) has been used to thoroughly characterize the physical properties, structure and stability of many viruses . The corHere, we use AFM to explore in real time the stability of individual TGEV particles as a surrogate model for SARS-CoV-2 in order to elucidate its structural stability under a range of physicochemical assaults, including mechanical stress, desiccation-rehydration cycles and treatment with chemical agents commonly used as biocides, such as detergents and ethanol. Likewise, we aim to show that some structural research can be performed with non-hazardous CoV strains. Swine testis (ST) cells   were grog for 20 min at 4 °C. Virions were then sedimented by ultracentrifugation at 112,000× g for 120 min through a 31% (w/w) sucrose cushion in TEN buffer  with 0.2% Tween 20 . The viruses were subsequently centrifuged through a continuous 15% to 42% (w/w) sucrose gradient in order to separate wild-type virions and defective virions with lower density. Finally, virions were pelleted by ultracentrifugation at 112,000× g for 60 min and resuspended in TNE buffer . Purified virions were disaggregated by sonication (six pulses at medium intensity in a Branson sonifier 450). SDS-PAGE protein analysis of the purified TGEV virions using Coomassie staining showed the presence of the major viral proteins S, N and M. This result confirmed the presence of TGEV virions in the samples used for AFM and cryogenic electron microscopy.Initially, supernatants from TGEV-infected cells were clarified by centrifugation at 4500× Purified TGEV virions were applied to Cu/Rh 300 mesh Quantifoil grids and vitrified by plunge freezing in liquid ethane using a Vitrobot Mark IV  cryo-fixation unit. The vitrified grids were transferred to a Talos Arctica electron microscope  operated at 200 kV under cryogenic conditions. The tomographic tilt-series from −60° to 60° at 3° step were recorded on a Falcon III direct detector using TOMOGRAPHY 4 software  at a nominal magnification of x57K, applying a defocus of ~3 µm and with a sampling ratio of 1.8 Å/pixel. Tilt series were aligned using IMOD software  and CTF 2 was applied to the surface to dry it. Then, a 30 µL droplet with a viral titer of ~2 × 106 virus/mL in TNE buffer was incubated for 40 min and rinsed with a clean buffer 6 times, adding 1× incubation volume and removing it. A final volume of 120 µL was then used for measuring the virus.Muscovite mica treated with poly-L-lysine was used as support substrate. A 30 µL droplet of 0.1% poly-L-lysine was deposited onto freshly cleaved mica for 15 min and rinsed with ultra-pure water 3 times. After every rinse, a highly pure stream of N2 and 60 × 25 µm2 cantilevers, respectively.All the experiments were carried out with NANOSENSORS™ qp-BioAC AFM probes, with a tip radius smaller than 10 nm. The cantilevers were calibrated with Sader’s method  for rectMeasurements were performed with an AFM  in Jumping Plus Mode . In thisFor the nanoindentation assays, individual particles were deformed with the AFM tip by performing single force-distance curves (FDC) at a constant speed (50 nm/s). The first and second FDC were performed with a Z piezo displacement of 150 nm. Thereafter, Z piezo displacement was increased to 200 and 300 nm, consecutively. The elastic modulus of the particles was obtained from the linear regime of the FDC curves. Both breaking force and critical indentation were defined as the point at which a major drop in the normal force was obtained, usually after the end of the linear regime, meaning a failure in the integrity of the shell.All the images and curves were processed and analyzed using the WSxM software .® CA-630  was diluted into a TNE buffer to get 1, 0.2 and 0.08% v/v solutions. This detergent was selected for its non-denaturing properties and well-established protocol for inactivating human CoVs [v/v buffer. Gradients were performed with Genie Syringe Pump .For the time-course experiments, IGEPALman CoVs . EthanolAfter TGEV purification , the integrity of the virions was checked by cryo-electron tomography, revealing the described morphology of the virus with the ribonucleoproteins (RNPs), membrane and spikes . 2 to remove the liquid from the surface  kept their height constant , which consists of pushing one virus at the very top with the AFM tip while registering the cantilever deflection ,56. Thesconstant C, black,constant B and usiconstant  consisticonstant B#7. Thisconstant C, blue. constant , the iniconstant D, a diffconstant , though constant E,F. In tconstant E revealeconstant F. In addconstant .After checking TGEV’s mechanical stability, we investigated the chemical resistance of virions to two biocidal agents that are commonly used for hygiene and disinfection: detergents and ethanol. Ethanol has been proven to be virucidal  and is ov/v) in the AFM liquid cell and monitored the individual virus structure afterwards  to test in situ the effect of mild surfactants on the viral particle. IGEPAL has been extensively used in biochemistry for solubilizing the membrane of CoVs without affecting viral proteins ,59,60. Iterwards A, right.terwards B, black terwards B, blue sterwards C, black.terwards C, blue, terwards A in the terwards . The virterwards , showingv/v) were explored  in a variety of harsh physicochemical environments. Although high resolution tomography A of TGEVFixation of native virus structures with PFA resulted in rugged structures D with anBeyond the intrinsic pleomorphism of enveloped viruses, the broad height distribution of native TGEV G is probWe can correlate our mechanical results with these two assemblies. Deformation of indentation-resistive virions presented a drop in the normal force between 0.1 nN and 0.3 nN . This drIn contrast, indentation-sensitive particles exhibited lower heights than the resistive ones D and shoIt is worth noting the different behavior of FDC after the drop in force presented in Beyond mechanical stress, the effects of chemical treatment on TGEV particles were also deeply related to the virion structure. Our experimental data indicate that virions exposed to 0.2% IGEPAL quickly lost their membranes . Data prIn this work we have elucidated the mechano-structural properties of TGEV and the way in which individual virion structures are altered in situ while varying the presence of virucidal agents gradually. We showed that while larger TGEV particles can withstand consecutive mechanical assaults, smaller structures are prone to physical breakdown under the same conditions. Our experiments in real time revealed that the structure of TGEV virions is disrupted by detergent concentrations three orders of magnitude lower than ethanol. IGEPAL was able to reduce virus height more than ethanol (~15–30 nm vs. ~12 nm). These two facts likely indicate that the infectivity would be more affected when using detergent than ethanol. TGEV’s mechanical properties appear very similar to those of SARS-CoV-2, and the structural similarity between them shows the utility of surrogate virus models as an alternative strategy for investigating highly infectious viruses which require BSL3 laboratory facilities. This work may help optimize sanitizing protocols in both daily life and in industrial processes. Various antiviral elements are currently being used in disinfection at all scales, whether for personal use or on an industrial scale, and they entail a cost overrun that can be alleviated by adjusting the concentration to the one which is strictly necessary.