Most (82%) of these observed economic costs are associated with damage, with comparatively little invested in management (18%). Reported costs are increasing over time, with damage averaging US$120 million per year and exceeding management expenditure in all decades. Where specified, most reported costs are from terrestrial plants and animals, with damages principally borne by primary industries such as agriculture and forestry. Management costs are more often associated with interventions by authorities and stakeholders. Relative to other countries present in the InvaCost database, New Zealand was found to spend considerably more than expected from its Gross Domestic Product on pre- and post-invasion management costs. However, some known ecologically  impactful invasive species are notably absent from estimated damage costs, and management costs are not reported for a number of game animals and agricultural pathogens. Given these gaps for known and potentially damaging invaders, we urge improved cost reporting at the national scale, including improving public accessibility through increased access and digitisation of records, particularly in overlooked socioeconomic sectors and habitats. This also further highlights the importance of investment in management to curtail future damages across all sectors.Biological invasions are a major component of anthropogenic environmental change, incurring substantial economic costs across all sectors of society and ecosystems. There have been recent syntheses of costs for a number of countries using the newly compiled InvaCost database, but New Zealand—a country renowned for its approach to invasive species management—has so far not been examined. Here we analyse reported economic damage and management costs incurred by biological invasions in New Zealand from 1968 to 2020. In total, US$69 billion (NZ$97 billion) is currently reported over this ∼50-year period, with approximately US$9 billion of this considered highly reliable, observed ( Biological invasions are a major component of anthropogenic global change, causing significant disruption to ecosystems across regions, habitat types and taxonomic groups . Across i.e., the scale at which policy decisions are made, have no centralised, systematic or comprehensive means of reporting, nor collating, economic costs of biological invasions  can impact economies in both conspicuous and inconspicuous ways. Particularly, impacts from invasions can encompass resource damages and losses , proactinvasions . For exanvasions . Previounvasions  and Euronvasions  brought ns e.g.,  given unTe Ika-a-Māui , the South Island Te Waipounamu , and Stewart Island Rakiura —scattered throughout the southwest Pacific Ocean . New Zealand has been heavily affected by IAS and is widely regarded as a country at the forefront of IAS management , and a greater reliance on individual responsibility to declare biological material, is typical. New Zealand has also led the world in pioneering the eradication of IAS—including established mammal species from small islands  has recently been published , providihttps://doi.org/10.6084/m9.figshare.12668570). Full details on the process of the literature search are provided elsewhere (n = 2 pre-date this point [1883 and 1945]). We report results as both cost values and number of reports—the latter reflecting a standardised measure  of cost reporting effort for a given variable. We note that the searches made to compile the database may under-represent groups such as microbes  because, although their spread can be dramatically enhanced by invasive hosts, they are frequently not defined by authors as IAS in their own right , as of June 2021 ; data oplsewhere . Brieflylsewhere . Using twn right . The subn = 12) because they had unclear durations because either the start, end or both years were not specified in the original source. We opted to remove these entries to avoid biases when assessing temporal dynamics of costs . To calculate the total cumulative cost, we first standardised all the cost entries on an annual basis for their defined period of occurrence. Hence, for example, a single cost entry recorded as occurring over a six-year period was transformed to six cost entries, with the total cost divided to get an annual cost that was repeated for each of the six entries. Our dataset therefore initially consisted of 810 annualised cost entries from 124 unique references.Deriving the cumulative cost of invasions over time requires consideration of the duration of each cost occurrence. We calculated this cost duration as the number of years between the database columns “Probable_starting_year_adjusted” and “Probable_ending_year_adjusted”. In a few instances, costs were omitted (https://doi.org/10.6084/m9.figshare.12668570 for complete details on these descriptors):Invasion costs were then considered and estimated according to six descriptive columns present in the database (see (i)vs “Low”). This is based on the type of publication and method of estimation i.e., peer-reviewed or other official documents from the grey literature are likely validated prior to publication so are classified as “High” reliability. Other materials were only classified as “High” if the original sources, assumptions, and methods were accessible and fully described.“Method_reliability”: a conservative but objective evaluation of the traceability of the cost estimation (“High”  (ii)“Implementation”: referring to whether the cost estimate was actually realised in the invaded habitat (“Observed”) or whether it was extrapolated . (iii)i.e., either unspecified or a combination of species and hence biomes).“Environment_IAS”: whether the cost was incurred from biota that are either “Aquatic”, “Terrestrial” or “Diverse/Unspecified” ( (iv)e.g., costs for damage repair, resource losses); (b) “Management”, comprising management-related expenditure ; and (c) “Mixed” costs, including a mixture of damage and management costs. We also used the “Management_type” column to compare management expenditure between pre- and post-invasion actions. Here, pre-invasion management comprised monetary investments for preventing successful invasions in an area including quarantine or border inspection, risk analyses, biosecurity management etc.; and post-invasion management includes money spent on managing invaded areas, and so includes control, eradication, or containment. Additional categories comprised: (a) “knowledge/funding”—money allocated to any action or operation that could be relevant to management at pre- or post-invasion stages, but is not specifically attributed within the source e.g., administration, communication, education or research costs;  (b) “mixed”—costs that included at least (and without possibility to disentangle the specific proportion of) two of the previous categories; and (c) “unspecified”—costs where the exact nature was not clearly defined. “Type_of_cost_merged”: collation of costs according to principal categories: (a) “Damage”, referring to damages or losses incurred by invasion ( (v)e.g., “Agriculture”, “Health” or “Authorities and Stakeholders”—this latter category representing official structures and organisations allocating efforts to manage IAS). Individual cost entries not allocated to a single sector were classified as “Mixed”, and records without an identifiable sector, or those that were unreported, were classified as ”Unspecified”. These are relatively broad groupings as the level of granularity provided within references varied substantially. Importantly, there was very rarely sufficient detail to attribute costs to, for example, specific stakeholders, communities or specific health impacts, even if these were mentioned, rather than to the collective sector groupings detailed above.“Impacted_sector”: the activity, societal or market sector that was impacted by the cost ( (vi)“Species”: the taxonomic nomenclature of the species causing the cost.i.e., had actually occurred) and having ‘high’ reliability , hereafter called ‘robust’ costs.We focussed the majority of our analyses on a conservative approach to the reported costs, retaining only those which were graded as both observed , but carefully checked the data for duplicates across scales, e.g., the same species-location pair reported twice. Identified duplicates were eliminated. We did not extrapolate cost estimates from smaller to larger scales, and such extrapolations are only included in InvaCost if the underlying cost documents did so and explicitly described their estimation methods (see “Implementation” above). In many cases, a lack of extrapolation could render our results underestimates, but we stress that InvaCost is a compilation of reported costs from underlying studies, that extrapolations can come from either “High” or “Low” reliability sources (see above), and that such extrapolations from smaller scales have been previously recognised as potentially problematic  and total (a) pre-invasion management (b) post-invasion management and (c) damage costs for New Zealand, as well as for all other countries represented in the InvaCost database . Accordingly, three linear models were fit with each of these log-transformed cost types as a response variable, and with log-transformed GDP as an explanatory variable per country. From these we extracted the ratio of [observed over expected] expenditures within a country for pre-invasion and post-invasion management, n = 453) were directly observed from actual costs, with the remainder being predicted, potential costs. Of the directly observed cost entries, over four fifths (n = 368) were classed as highly reliable, with a total figure for these highly reliable, observed costs of US$8.83 billion  , and most annualised entries (62%), occurred within the terrestrial environment . Cost envs. 82%) . The grevs. 82%) . The majEstimates of annual management costs since the 1960s averaged at US$26.16 million while damage costs were an order of magnitude higher at US$120.41 million; management costs were first reported a decade later than damages . Despitee.g., creeping thistle (Cirsium arvense), yellow foxtail (Setaria pumila), giant buttercup (Ranunculus acris)) and pest arthropods ). Introduced species reported as incurring the greatest management costs (>US$50 million) were also associated with primary industries but were a different group of species that included the established pest animal brushtail possum (Trichosurus vulpecula) and the Varroa mite (Varroa destructor). Noteworthy management costs included those to thwart the establishment of invading pest arthropods , red imported fire ant (Solenopsis invicta) and gypsy moth (Lymantria dispar)) and eradications of established pest mammals on isolated islands  and European rabbits (Oryctolagus cuniculus))  being agricultural weeds  and post-invasion management , but not pre-invasion management , at the global scale. In terms of these global economic cost patterns, reported costs across both management types are relatively high in New Zealand, and higher than would be predicted based on GDP  over the last 50 years based on only the most robust estimates. Most costs have resulted from damage caused by terrestrial IAS, and costs are rapidly increasing over time. Despite New Zealand’s well-deserved reputation as a world leader in addressing IAS from a variety of pre- and post-invasion perspectives, this situation where damage costs are growing and substantially exceeding management investments is similar to many other countries and regions e.g.,  and confe.g., in terms of biosecurity staff salaries, facility costs, research grant breakdowns etc.—are rarely within the public domain (for example as a result of privacy legislation) and thus are unlikely to be captured within this database. This makes a completely accurate comparison between proactive biosecurity management  and post-invasion control/eradication challenging due to data deficiencies. However, New Zealand now invests a relatively high amount in IAS prevention activities such as pathway management border controls, and is a world leader in pre-border pest risk management (vs. post-invasion at a minimum) to the same invader and ecosystem across comparable time periods. Nevertheless, due to a long history of, and ongoing dependency on, primary production, and more recently tourism, New Zealand continues to emphasise a proactive approach to biosecurity, which has benefits for multiple industries ); ); , often eledge)); , with thledge)); , althouge.g., chamois (Rupicapra rupicapra) and common pheasant (Phasisanus colchicus) and freshwater fish such as trout , which engender conflict through opposing values on whether they are pests (thereby incurring a damage cost) or resources  (Bonamia ostreae which can adversely affect oysters (Ostrea chilensis) (Sitona lepidus), pea weevil (Bruchus pisorum) and termites (Order Isoptera) have had large amounts spent on them by the Ministry of Primary Industries and Biosecurity New Zealand, but this information remains in internal reports and spreadsheets that have yet to leave the organisation . This level of difficulty in accessing relevant data, and the inability of searches to locate such information without the need to specifically target key, potentially unknown, individuals or researchers, represents a major barrier to obtaining accurate pictures of the economic impacts of IAS. Therefore, parties that collect these data, including central government, regional agencies and scientists, should also prioritize their publication such that their availability and impact are maximized, facilitating, for example, independent advice to governments or the linking of environmental management to broader concepts of wellbeing.In addition to differential reporting of ecological costs, there are other costs of biological invasions missing from the database for a range of reasons. There are undoubtedly true gaps in reported damage costs, and consequent lack of, or limited, investment in management. These include introduced game animals enefits) . This coenefits) . Economienefits) . This isilensis) . Reporteilensis) ; althougEmerging threats in biological invasions, and a wider acknowledgement that costs are simultaneously incurred, and borne by, multiple sectors, has led to greater cross-agency partnership in the management of biological invasions in New Zealand . HoweverThe information contained in the unique source of cost data analysed here provides a basis for helping to guide and encourage open reporting practice to facilitate research, management, and active steps to address the socio-economic impacts of IAS in New Zealand and beyond. We again stress that knowledge gaps and inaccessibility of potentially incurred costs make the costs reported here substantial underestimates. Nonetheless, as a centralised platform for standardised cost reporting, we encourage researchers and organisations to submit their data to the InvaCost database to enhance the comprehensiveness of future cost appraisals as biological invasions, their impacts and mitigation measures ensue. This will ensure a more complete understanding of economic costs and benefits under different strategies, a critical requirement for successful prevention and management of biological invasions.10.7717/peerj.13580/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13580/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.13580/supp-3Supplemental Information 3Click here for additional data file.


Citrus australasica) is tolerant to Huanglongbing . This species can be utilized to develop HLB tolerant citrus cultivars through conventional breeding and biotechnological approaches. In this report, we conducted a comprehensive analysis of transcriptomic data following a non-choice infection assay to understand the CaLas tolerance mechanisms in the finger lime. After filtering 3,768 differentially expressed genes (DEGs), 2,396 were downregulated and 1,372 were upregulated in CaLas-infected finger lime compared to CaLas-infected HLB-susceptible ‘Valencia’ sweet orange. Comparative analyses revealed several DEGs belonging to cell wall, β-glucanase, proteolysis, R genes, signaling, redox state, peroxidases, glutathione-S-transferase, secondary metabolites, and pathogenesis-related (PR) proteins categories. Our results indicate that the finger lime has evolved specific redox control systems to mitigate the reactive oxygen species and modulate the plant defense response. We also identified candidate genes responsible for the production of Cys-rich secretory proteins and Pathogenesis-related 1 (PR1-like) proteins that are highly upregulated in infected finger lime relative to noninfected and infected ‘Valencia’ sweet orange. Additionally, the anatomical analysis of phloem and stem tissues in finger lime and ‘Valencia’ suggested better regeneration of phloem tissues in finger lime in response to HLB infection. Analysis of callose formation following infection revealed a significant difference in the production of callose plugs between the stem phloem of CaLas+ ‘Valencia’ sweet orange and finger lime. Understanding the mechanism of resistance will help the scientific community design strategies to protect trees from CaLas infection and assist citrus breeders in developing durable HLB tolerant citrus varieties.The Australian finger lime ( The genus Citrus originated in tropical and subtropical southeastern Asia . When coCandidatus Liberibacter asiaticus (CaLas), being the most destructive , a phloem-limiting bacterial disease caused by the bacterium tructive . In the tructive , when ittructive . Widesprtructive . Most ofCitrus ichangensis ‘2586’  have been reported to be HLB resistant , and effector-triggered immunity (ETI), which is induced by pathogen effector proteins via intracellular receptors that detect intercellular pathogen-derived molecules and intracellular receptors that activate plant defense response upon detection of pathogen-secreted effector proteins that function inside the plant cell , many of which are conserved across CaLas isolates. Sec-delivered effector 1 (SDE1), a secreted protein biomarker used for the detection of HLB, is highly expressed in infected citrus tissue at a relatively early infection stage  proteases have been demonstrated to participate in different pathosystems . Citrus esponses . Additioesponses . Plasma esponses . RLKs aresponses .Citrus sinensis) sweet orange trees with CaLas and evaluated their transcriptome to provide insights into the mechanism of tolerance to HLB. The transcriptome data was also validated in five-year-mature trees growing in the field.In this study, we shed light on the potential mechanism of HLB tolerance in the finger lime. To this end, we graft-inoculated one-year-mature finger lime and ‘Valencia’  and ‘Valencia’ sweet orange clone SPB-1-14-19 were obtained from Florida’s Division of Plant Industry budwood repository and budded onto 6-month-old Swingle citrumelo rootstock. One-year-old budded trees were subsequently side grafted . The trees were periodically evaluated for infection, and 1 to 2 year-old infected trees were utilized for subsequent experiments . Leaves were also collected in the late fall (November) and early spring (March) from 5-year-old finger lime DPI 50-36 and ‘Valencia’ SPB-1-14-19 trees growing in the field (Swingle rootstock) to estimate the CaLas titer in the sampled tissues for three years. The DNA concentration was normalized to 25 ng/μL before performing qPCR using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Detection of CaLas genomic DNA was determined by qPCR using TaqMan™ Gene Expression Master Mix and CQUL primers . High-quality RNA samples with an RNA integrity number (RIN) > 6.5 were used for cDNA synthesis and RNA sequencing (RNAseq). Single-stranded cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit . The cDNA concentration was determined using a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific).Two years following infection, RNA was extracted using TRIzolThe cDNA libraries were sequenced using an Illumina HiSeq platform configured for a 2x150 read length. The generated base callings of the cDNA reads were presented in a paired-ended format. The reads were cleaned, and their adapters were removed using AdapterRemoval v2.2.2 , with thC. sinensis genome using STAR v2.6.0C (C. sinensis genome and annotations used in STAR were obtained from Phytozome (https://phytozome-next.jgi.doe.gov/) The BAM files were indexed using SAMtools v1.7  analysis via DESeq2 v3.10, an R Bioconductor package (2(fold-change)|< 2 and an adjusted P value ) ≥ 0.05.The cleaned reads were mapped to the  v2.6.0C  with theols v1.7 . The BAMols v1.7  with def package . The couCitrus sinensis, statistical test - Fisher’s exact test, adjusted according to the Benjamini–Yekutieli method (C. sinensis pathways file (m02) in MapMan , and selected gene-specific primers  in a final mixture of 20 μL, according to the manufacturers’ instructions. qPCR was performed in a StepOnePlus™ Real-Time PCR System . The citrus β-actin housekeeping gene was used as a reference gene  reaction mix consisted of 1 µL of DNA (25 ng/µL), SYBRnce gene ; each saeviously . The relTIN gene  and calcT method . To confAssessment of Cys protease activity was performed by recording the liberation of fluorogenic peptide substrate VIII , given that protease activity correlates with an increase in detectable relative light units (RFUs) over time . The leaInhibition % = [Absorbance (blank)-Absorbance (test)]/Absorbance (blank) x 100CaLas-infected finger lime and ‘Valencia’ sweet orange leaves  were collected from the greenhouse. The petioles were cut and fixed in 4% paraformaldehyde in 1x PBS. The samples were rinsed three times in 1x PBS and then dehydrated in an ethanol (EtOH) series for 1 hour each. The samples were transitioned from 100% EtOH to 100% tert-butanol  at room temperature (RT) for 8-16 hours each and then cleared in 100% tert-butanol for one hour prior to paraffin infiltration. The samples were infiltrated using increasing concentrations  of Paraplast Plus paraffin  for 24 hours each and then incubated for 48-36 hours in 100% paraffin, which was changed three times. The samples were embedded in paraffin and allowed to harden for 24 hours at 4°C. Afterward, ten micrometer sections were cut using a Leica 2155 microtome , and the sections were floated on a drop of water on a slide. The slides were subsequently incubated overnight on a slide warmer at 37°C to allow the sections to adhere to the slide. The slides were dewaxed in 100% Histoclear II  for an hour each, and the solution was changed twice. The sections were stained with 0.05% toluidine blue O for 30 seconds and then rinsed in dH2O. The slides were dehydrated in an EtOH series for 5-10 minutes each. Coverslips were mounted using Fisher Scientific’s mounting media with toluene . The slides were observed under an Olympus BX61 epifluorescence microscope , and images were captured using a 14 MP OMAX digital camera . The phloem and xylem ring distances were measured using FIJI  test or t tests to compare the means of the different treatments. Statistical significance was established at P < 0.05. Pearson Correlation Coefficient (r) was calculated to validate the modulation in gene expression for RNAseq data and quantitative PCR using JMP Pro v16 software. As for statistical Testing of Phloem Callose Deposits, ANOVA was performed using R statistical software to assess the significance of the model and interactions, any non-zero counts of callose plugs were analyzed with a negative binomial regression in R  or had high Ct values (37.88 ± 0.28)), whereas ‘Valencia’ sweet orange trees had low Ct values of 25.14 ± 0.82  was sequenced using the Illumina HiSeq next-generation sequencing platform. The average total number of raw reads produced was 37,550,067 and 31,506,847 for the CaLas-infected finger lime replicates and the CaLas-infected ‘Valencia’ sweet orange replicates, respectively  and 22,595,692 (86.14%) clean reads were mapped. The mapped reads were analyzed by differential expression analysis. After filtering the differentially expressed genes (DEGs) according to |log2(fold-change)| < 2 and an adjusted P value (FDR) ≥ 0.05, 3,768 remained. Of the 3,768 DEGs, 2,396 were downregulated in HLB-infected finger lime compared to HLB-infected ‘Valencia’ sweet orange, while 1,372 were upregulated  and “ADP binding” (GO: 0043531). “Amine metabolic process” (GO:0009308), “polysaccharide catabolic process” (GO:0000272), and “response to chemical” (GO:0042221) were the top downregulated GO terms in the biological process category. Unlike the upregulated GO terms, the downregulated GO terms were assigned to two cellular component categories: “cell wall” (GO: 0005618) and “external encapsulating structure” (GO: 0030312). A graphical view of these data can be found in via PageMan to investigate the functional categories. Out of the 3,768 DEGs, 1,162 were assigned to disease response categories. Nearly all the DEGs belonged to one of the following categories: cell wall, β-glucanase, proteolysis, R genes, signaling, respiratory burst, abiotic stress, redox state, peroxidases, glutathione-S-transferase, secondary metabolites, and pathogenesis-related (PR) proteins DEG functional analysis is important to understand the biochemical responses elicited by these genes and the roles they play in the overall function of the plant. The significant DEGs were analyzed 2(fold-change)) were recorded for the mitogen-activated protein kinase-encoding DEG (MAPK) in finger lime and ‘Valencia’ sweet orange when the uninfected tissues were compared with the infected tissues.It has been widely reported that microbe infection induces plant defense through two mechanisms, namely, PTI and ETI, play a substantial role in plant disease resistance . Seven cTo validate the reliability of the RNA-seq data in terms of the overexpression of CRKs, we randomly selected 9 DEGs encoding Cys-rich RLKs for confirmation by qPCR Figure 3CaLas-infected finger lime. Indeed, 114 pathogenesis-associated DEGs constituted 3% of all the DEGs mapped, 48 of which were upregulated. Twenty-nine of these upregulated genes were PR protein-encoding DEGs of the Tir-NBS-LRR class, 4 were of the NBS-LRR class, and 7 were of the CC-NBS-LRR class. In addition, there were 33 Tir-NBS-LRR class genes, 7 CC-NBS-LRR gene class genes, and 14 NBS-LRR class genes that were downregulated. Transcript levels of orange1.1g035344m, similar to the stable antimicrobial peptide (SAMP)  and/or LRR domain were differentially expressed in e (SAMP)  was not CaLas-uninfected and infected ‘Valencia’ sweet orange , and this expression was negatively associated with CaLas presence (Fortunella margarita) and Meiwa (Fortunella crassifolia)), Poncirus trifoliata (50-7 and Flying Dragon), Mandarin (Ponkan and Cleopatra (Citrus reticulata), lime (key lime (Citrus aurantifolia) and Rangpur lime (Citrus limonia)), pummelo (Hirado Buntan pummelo and Siamese Sweet pummelo (Citrus maxima)), grapefruit (Ruby Red and Duncan (Citrus × paradisi)), sweet orange ), Volkamer lemon (Citrus volkameriana), Sydney hybrid (Citrus x virgata), Citrus papuana, and Citrus inodora, showed lower expression of this gene (orange1.1g043403m) than did finger lime . Eleven of the DEGs identified belonged to the WRKY transcription factor family, the members of which play important roles in plant development and stress responses. Nine of the WRKY transcription factors were upregulated . Additionally, a Dof-Type zinc finger DNA-binding family protein (DAG1) and two BZIP transcription factor family proteins (TGA9 and BZIP42) were found to be upregulated in finger lime, while bZIP58, BZIP61, and PAN were downregulated.Hormone signaling is important in plant physiology and regulates many aspects from the transduction of messages through plants to adaptation to the environment to the timing of fruit development and ripening. Out of the mapped genes, 2.6% (99 DEGs) were associated with hormone synthesis. Nine abscisic acid-induced genes were identified. Three of these were Hva22-like protein-encoding DEGs  and were upregulated. Two Gram Domain-Containing Protein 2-encoded DEGs were downregulated. Abscisic acid degradation-related DEGs were downregulated only in finger lime; these genes included epoxycarotenoid dioxygenase, aldehyde oxidase, and zeaxanthin oxidase. Auxin induced-regulated related DEGs were expressed to a higher degree in ‘Valencia’ sweet orange than in finger lime, with 25 out of the 34 DEGs upregulated in ‘Valencia’ sweet orange. The upregulated genes in finger lime included those encoding ILR1, TIR1, DFL1, and GH3.1 proteins. Of the six brassinosteroid-related DEGs, cytochrome P450 (orange1.1g037705m) was upregulated, while the STE1-, HYD1-, and SMT1-encoding genes were downregulated.There were 33 ethylene-related DEGs mapped, 17 of which were upregulated, including Gibberellin 2-Oxidase, 2-Oxoglutarate, Kar-Up Oxidoreductase 1, Integrase-Type DNA-Binding, GASA1 and SRG1. Seven jasmonate-related genes were also identified, which were upregulated and included three of LOX2 and JAZ1 gene. Finally, we identified ten salicylic acid (SA)-related genes, and three genes encoding methyltransferase were found to be upregulated, namely, orange1.1g017363m, orange1.1g044676m, and orange1.1g043411m in the infected finger lime.Among the DEGs mapped, 139 were associated with biotic proteolysis, accounting for 3.6% of all the mapped genes. Fifty-one of these genes, including orange1.1g044297m, which encodes a P-Loop containing Nucleoside Triphosphate Hydrolases Superfamily Protein, were upregulated in finger lime. However, the Aaa-type gene Aaa-Atpase 1, several Cytochrome Bc1 synthesis-related genes, aspartate protease, autophagy-related DEGs, and a metalloprotease-related DEG were downregulated. Of the 10 Cys protease-related DEGs that were mapped, only one of the Cys protease inhibitor-encoding genes (orange1.1g018968m) was upregulated in finger lime. There were 11 serine protease (SP)-encoding, 2 Kunitz Family Trypsin- and protease inhibitor protein-encoding, and 15 Subtilase-related DEGS downregulated in finger lime. In contrast, 20 Ubiquitin E3 Scf F-box and one Ubiquitin E3 Scf Skp (orange1.1g030652m) genes were overexpressed in finger lime, while 16 Ubiquitin E3 Ring, 1 Ubiquitin E2, 2 Ubiquitin Proteasome, 1 Ubiquitin 4, and 1 Polyubiquitin 10 genes were downregulated.CaLas infection in ‘Valencia’ sweet orange. In contrast, one Xylem Serine Peptidase 1 gene (orange1.1g004503m) was highly upregulated in the infected finger lime trees  and one of xylem Cys protease 2 encoding genes in the finger lime. Comparatively, these genes were upregulated in response to me trees Table 5.CaLas-free trees  and A20 (orange1.1g025617m) were also upregulated in finger lime. Of the 17 PME-related genes identified by mapping, the PME inhibitor (orange1.1g010441m) was the only DEG upregulated in finger lime. Additionally, six arabinogalactan protein (AGP)-related DEGs were identified, five of which were downregulated, while the gene encoding FLA3 protein (orange1.1g042255m) was upregulated.To evaluate the accuracy of the RNA-seq data and the presence of technical artifacts or errors introduced during the RNA-seq library preparations, the expression of several highly conserved genes and genes encoding transcription factors were assayed through qPCR. Eight of the selected genes  were upregulated in finger lime Figure 6CaLas– and CaLas+ petioles of finger lime and ‘Valencia’ sweet orange  and between healthy and HLB-infected stems    and stems   and no significant difference in the stems  and finger lime (26%); We found increased accumulation of callose in the infected ‘Valencia’ sweet orange compared with the infected finger lime. Analysis of the callose formation counts revealed a significant difference in the percentage of images with few callose plugs in the stem phloem of via qPCR. CsPP2-B1 transcripts were detected in both finger lime and ‘Valencia’ but were not statistically significant when the uninfected and infected trees were compared. CsPP2-B13 transcripts were almost undetectable in finger lime samples, while they were upregulated in ‘Valencia’. The relative transcript levels of CsPP2-B15 were highly upregulated in the infected ‘Valencia’ sweet orange leaves compared with the finger lime leaves  disorder of carbohydrate metabolism affecting the flow of nutrients and source–sink disruption due to starch accumulation in leaves; (II) phytohormones alteration in response to stress; and (III) activation of detoxification proteins, particularly glutathione-S-transferases (GSTs) and modulation of antioxidant pathways.The possible genetic mechanisms of the symptoms of l groups . Nehela CaLas against the host is an important step to identify an approach to increase plant defense. Insect-transmitted bacteria such as CaLas utilize the general Sec secretion system to release effectors  were found to be upregulated in HLB infected finger lime. Our findings are similar to those reported by Poncirus trifoliata was reported. CRKs are characterized by the presence of one to four copies of Domain of Unknown Function 26 (DUF26) and a C–X8–C–X2–C motif in the extracellular receptor region at the N-terminus  serves as an early warning system for detecting the presence of potential pathogens and activating protective immune-related signaling in plants . Interesterminus . These cterminus  and can terminus . Severalnfection . Curtolovia induction of antioxidant pathways and plant growth hormones (particularly GA). Additionally, the involvement of GA signaling in HLB resistance has also been reported by CaLas induced ROS detoxification pathways and activated glutathione-S-transferases (GSTs), catalase and thioredoxin in finger lime. Glutathione-S-transferases was suggested to be as an important modulator of citrus tolerance to HLB disease  and other Cys-rich peptides are major components of innate immunity in various groups of organisms, including insects, mammals and plants . Some euCaLas infection resulted in a decreased ability to synthesize new cellular components, whereas the finger lime was not affected in the same manner and thus could continue with cell metabolism and growth. These findings are agreed with CaLas infection. They reported that cell wall-related pathways were upregulated in rough lemon during the late stage of infection, while there was downregulation in the susceptible sweet orange. Thus, the rough lemon trees generated healthy new growth following infection despite older leaves having some blotchy mottle, while the growth was inhibited in sweet orange trees.Cell wall integrity sensing is a mechanism by which plants can be induced to mitigate biotic stress. Various strategies involving resistance of the cell wall against plant pathogens have evolved, such as the remodeling of the cell wall and alterations to cell wall-associated protein distribution and accumulation . Both ceCaLas-infected finger lime. CaLas infection in ‘Valencia’ sweet orange resulted in the repression of genes encoding cellulose synthase-like D4 (CSLD4) and CSLC7 that mediate synthesis of β-1,4 linkages in the hemicellulose backbones  and downregulated in the HLB tolerant sour pummelo (C. grandis Osbeck). Similarly, CsPP2-15 was also found to be downregulated in the tolerant C. ichangensis  assays as interacting with CaLas effectors (SDE1). Papain-like Cys proteases are important regulators involved in numerous plant biological processes, including programmed cell death (PCD) and leaf senescence. It has been demonstrated that activation of cysteine proteases induced programmed cell death (PCD) pathway of plant cells (AtXCP1 and AtXCP2) were found to be expressed at a high level in xylem during the PCD process , catalase, thioredoxin and growth hormones to mitigate HLB symptoms. Primary recognition of ROS leads to stronger activation of defense responses against CaLas infection. The over-accumulation of Cys proteases and their inhibitors in ‘Valencia’ linked with the degradation of citrus defense proteins such as the cysteine rich proteins. Finger lime did not exhibit any imbalance in the expression of the Cys proteases and their inhibitors. Several defense-related-factors were upregulated in the finger lime such as R-proteins, Cys-rich secretory proteins or PR proteins and hormone signaling-related genes. Additionally, we observed that ‘Valencia’ sweet orange phloem constitutively produced more callose and expressed more phloem proteins than finger lime following infection. Taken together, this study provides evidence that HLB can be managed by mitigating ROS and control of the over-accumulation of cysteine protease related genes that induce cell death . Understanding the mechanisms against ll death Figure 9https://www.ncbi.nlm.nih.gov/, PRJNA755969.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: KW, LM, DS and MD - wrote the manuscript. LM - gene expression analysis. KW - transcriptome data analysis. DS – Microscopy. SW – callose density estimation. LM and WQ – RNA extraction. LM - statistical analysis. AL and JG – resources. MD – designed the study, obtained funding, and supervised the project. All authors contributed to the article and approved the submitted version.This work was supported by the Florida State legislative funding for the UF/IFAS Citrus Initiative.We thank E. Nielsen, C. Hardy, H Rubio, J. Thomson and K. Plant for aiding in various aspects of the work. Microscopy work was carried out at the Microscopy Core Facility of the Citrus Research and Education Center. We thank Novogene Corporation Inc. for library construction and RNA sequencing.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.


Cell microencapsulation in gel beads contributes to many biomedical processes and pharmaceutical applications. Small beads (<300 µm) offer distinct advantages, mainly due to improved mass transfer and mechanical strength. Here, we describe, for the first time, the encapsulation of human-bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in small-sized microspheres, using one-step emulsification by internal gelation. Small (127–257 µm) high-mannuronic-alginate microspheres were prepared at high agitation rates (800–1000 rpm), enabling control over the bead size and shape. The average viability of encapsulated hBM-MSCs after 2 weeks was 81 ± 4.3% for the higher agitation rates. hBM-MSC-loaded microspheres seeded within a glycosaminoglycan (GAG) analogue, which was previously proposed as a mechanically equivalent implant for degenerate discs, kept their viability, sphericity, and integrity for at least 6 weeks. A preliminary in vivo study of hBM-MSC-loaded microspheres implanted  in a rat injured intervertebral disc model demonstrated long-lasting viability and biocompatibility for at least 8 weeks post-implantation. The proposed method offers an effective and reproducible way to maintain long-lasting viability in vitro and in vivo. This approach not only utilizes the benefits of a simple, mild, and scalable method, but also allows for the easy control of the bead size and shape by the agitation rate, which, overall, makes it a very attractive platform for regenerative-medicine applications. Cell encapsulation in microparticulate systems has been applied in a variety of biomedical processes and pharmaceutical applications, including the transplantation of pancreatic islet cells to reverse diabetes ,2,3, thed-mannuronate (M-residues) and α-l-guluronate (G-residues), and alternating M and G residues that gel in the presence of divalent cations, such as calcium; in the case of G residues, a so-called “egg-box” structure is formed [Alginate is a biocompatible biodegradable naturally occurring polysaccharide. It is a linear block copolymer that is composed of 1,4-linked-β-s formed . Commercs formed . Due to s formed ,32, and s formed , as theyAlginate beads can be formulated through a wide range of techniques, including extrusion, coaxial air or liquid flow, vibrating jet cutters, and electrostatic potential , by usina of the uronic acid residues, Ca2+ ions are released from the calcium complex, crosslinking the alginate to form a homogeneous hydrogel [Internal gelation, which was first developed by Poncelet et al.  to immobhydrogel , which ihydrogel . This mehydrogel . Moreovehydrogel , and theGenerally, a bead size in the range of 200–1000 µm is used for biomedical applications. However, encapsulation methods require further optimization to reduce the size while maintaining stable structures. Small beads (<300 µm) offer many advantages for transplanted cells, including more rapid mass transfer (due to a large surface-to-area ratio) , less inVarious groups have produced small microspheres using different methods. Landazuri et al.  and KhatThe primary objective of this study is to produce small (<300 µm) alginate beads that are capable of encapsulating mammalian cells and maintaining their long-lasting viability by using one-step emulsification by internal gelation. This study presents data for a range of hBM-MSC-encapsulating alginate beads that vary in size, as controlled by the agitation rate. Three formulations were prepared and characterized for their viability in vitro and in vivo using a rat-caudal-intervertebral-disc model.l-glutamine, penicillin/streptomycin solution, trypsin-EDTA solution B, and sodium pyruvate solution were purchased from Biological Industries (BI), Beit HaEmek, Israel. Fetal bovine serum (FBS) was from Gibco, Life Technology, Grand Island, NY, USA. Alginate  was purchased from DAEJUNG, Siheung-si, Gyeonggi-Do, Korea. Dil  Cell-labeling solution and Live/Dead™ Cell Imaging kit were purchased from Invitrogen™, Waltham, MA, USA. Live Cell Imaging Solution was from Molecular Probes™, Eugene, OR, USA.io: 1.56 ), propidv/v) FBS and 1% (v/v) penicillin/streptomycin.Human-bone-marrow-derived mesenchymal stem cells (hBM-MSCs)  were cultured in a dedicated complete growth medium: MEM-alpha , supplemented with 10% (w/v) were prepared, given that high cell viability has been reported in the range between 1.2% and 2% (w/v) alginate [7 cells) was mixed with alginate solution  in HEPES buffer  and CaCO3 . The alginate mixture was then added to mineral oil (10 mL) to create an emulsion, and agitated using one of three rates  for 12 min. Internal gelation was achieved by an acidification step using mineral oil (10 mL) with acetic acid , and the mixture was allowed to agitate for an additional 8 min. Beads were recovered by phase inversion, first by adding HEPES buffer (40 mL) with complete medium , and agitated at a low rate (~500 rpm) for 1 min, followed by centrifugation (630× g for 5 min). Beads were collected following aspiration of the oil phase and washed with the complete medium through a 70 µm mesh strainer. Recovered microemulsions were ready to be cultured.Cells were immobilized in alginate beads in a stirred flat-bottomed vessel by a simple one-step method, as previously reported . Beads calginate ,41,62. BThe yield of the overall encapsulation process was determined as:The encapsulation efficiency (EE) was calculated as follows:The total volume of the beads formed was assessed by measuring the excluded volume after transferring the beads to a known volume. The number of encapsulated cells was assessed directly by the Trypan blue exclusion method, following recovery of the cells by degelling .2. For beads prepared at different agitation rates, cell viability was assessed at predetermined time intervals , using the dual-staining assay. Briefly, cells were stained using dual-staining solution containing 16 µM FDA and 30 µM PI in Live Cell Imaging Solution. A sample of cell-containing beads was mixed with HEPES buffer, which contained 10% (v/v) complete medium. The staining solution was then added to the sample  and incubated for 40 min on ice. Images were captured by the Apotome 2 microscope , using Filter Set 38 HE (Ex/Em: 470/525) for FDA and Filter Set 63 HE (Ex/Em: 572/629) for PI. For beads embedded within GAG analogues, cell viability was assayed by the Live/Dead assay kit.Cell-loaded beads were cultured in a complete medium at 37 °C with 5% COCells were recovered from the beads, after a wash step through a 70 μm mesh strainer, using a degelling solution  diluted with 9 volumes of MEM medium. Six volumes of the diluted de-gelling solution were further diluted with one volume of beads for 2 min at room temperature to allow recovery of cells from the beads before quantification with the Trypan blue exclusion method.Bright-field images were used to determine bead morphology and distribution of bead diameters, using the NIS-Elements module .GAG analogues were prepared using sulphonate-containing monomers polymerized in the presence of a crosslinking agent, utilizing redox polymerization. Hydrogels were formed using a typical two-part pre-gel component, in which Part (a) contained water, oxone (redox initiator), and KSPA monomer, and Part (b) contained water, AA (redox initiator), NaAMPS monomer, and PEGDA (crosslinker), as previously detailed . A crossThe animal study protocol was approved by the Ethics Committee of the Hebrew University (MD-17-15342-4). Cell-loaded alginate beads were implanted in a damaged disc model using a biomimetic glycosaminoglycan (GAG) hydrogel  as a car6 cells/1 mL serum-free medium were incubated with 5 μL/mL CM-DiI at 37 °C for 5 min, followed by 15 min incubation at 4 °C, and finally washed with PBS. Cells were encapsulated in alginate beads and cultured in a differentiating medium [6/mL beads) were further immobilized in a GAG-analogue hydrogel [v/v) of cell-loaded beads were mixed with hydrogel solutions (containing 1.5% PEGDA) and allowed to settle in vitro using a silicone tube with an inner diameter of 0.7 mm. The cured ‘spaghetti’-like gel was cut to cylinders of 1 mm height, which were allowed to swell for 4 days in PBS. The swollen cylinders (1.5 mm in diameter and 1.5 mm in height) were used for transplantation.Cell-loaded alginate beads were prepared as previously described in g medium . Cell-lohydrogel . BrieflyNine female Wistar rats (~200 g) were used, of which six animals were operated on. Before surgery, each animal was anesthetized by inhaling isoflurane; the use of anesthetic gas allows for faster recovery of the animals. After having the animal anesthetized, tails were cleaned with a surgical scrub, and a longitudinal incision of up to 3 cm long was made to expose the three most cranial tail discs. In each disc, using a blade, the nucleus pulposus was removed using a curette, and one GAG-analogue-hydrogel cylinder containing cell-loaded alginate beads was implanted into the nucleus pulposus cavity. The annulus was then closed with two 5.0 nonresorbable nylon sutures, and, finally, the tail skin was closed using resorbable sutures. Following the procedure, rats were allowed unrestricted activity in separate cages and were monitored for signs of pain or infection. Buprenorphine (0.01–0.02 mg/kg) was administered as needed for pain. At 8 weeks post-surgery, animals were euthanized. Tails were harvested and fixed in 4% formalin and processed for histology using hematoxylin and eosin (H&E) and 4′,6-diamidino-2-phenylindol (DAPI).High-M alginate was chosen (M/G of 1.56), as it exhibited lower permeability compared to high-G alginate . It is aThe effect of the agitation rate on the bead diameter is summarized in p < 0.0001), as is also shown in The average diameter and respective size distribution (represented by the standard deviation) decreased significantly with an increase in the agitation rate  following bead degelling. The average viability for all agitation rates, throughout the period tested, was estimated at 75 ± 7% and 81 ± 4% for beads obtained at the higher agitation rates . SubsequDespite the polydispersity obtained in the bead size, which is typical of emulsion techniques, the average viability at each agitation rate throughout the period tested was reproducible. The total number of cells (live and dead) counted throughout the period tested remained unchanged, suggesting that no proliferation occurred. However, further studies are required to test the long-term effect on the fate of encapsulated hBM-MSCs.As seen in Previous studies using methods based on external gelation produced small beads with a short-term high-viability rate in vivo and in vitro ,56,62. WThe ability of beads to protect cells from an acidic environment and maintain their viability was tested by using GAG-analogue hydrogels, which were previously proposed as mechanically equivalent implants for degenerate intervertebral discs . Due to The capability of the construct  to maintain cell viability in vivo was further tested. To this aim, constructs were implanted in an intervertebral-caudal-disc model with a damaged nucleus pulposus as a preliminary proof of concept for their long-term biocompatibility and viability in vivo. As is seen in This study sought to utilize the benefits of emulsification by internal gelation to form small alginate beads that are capable of maintaining the long-lasting viability of hBM-MSCs. Here, we demonstrate that small-sized (<300 µm) medium-viscosity high-M alginate beads, formed by one-step emulsification using internal gelation, can be used as a microencapsulating system for hBM-MSCs in vitro and in vivo. The present method has been shown to have minimal effect on the viability of encapsulated cells during and post-processing, both in vitro and in vivo. The encapsulated cells maintained their viability in vivo in a rat caudal model, and, thus, they can be potentially used for the treatment of degenerate intervertebral discs, as well as for other in vivo applications. The reproducible viability obtained, despite the polydispersity in size, makes it a relevant tool for clinical translation in biotechnology and regenerative medicine. For the food industry, it may offer an effective way to protect microbes in adverse in vitro and in vivo environments, and it is promising for the high-throughput production of probiotics microencapsulation, as well as for the coadministration of probiotics and antibiotics.


The antigenicity of SARS‐CoV‐2 is a critical issue for the effectiveness of the vaccine, and thus, it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination inhibition (HA/HI) tests are well known as a representative method for antigenic analysis of influenza viruses, but SARS‐CoV‐2 does not agglutinate human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated cell‐based assays using special equipment such as plate reader or ELISPOT analyzer.Based on the HA/HI tests for influenza viruses, we developed the particle agglutination/particle agglutination inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin‐converting enzyme 2 (hACE2)‐bound latex beads. The virus titers were determined by mixing the beads and the virus from culture supernatant, settling it overnight, and then observing the sedimentation/agglutination pattern (PA test). The neutralization antibody titers were determined by mixing virus‐infected hamster antisera in addition to the beads and virus (PAI test).The PA titer was positively correlated with the plaque‐forming units. The PAI titer using the hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The antigenic differences were supported by the results shown in other methods.The PAI test is an easy and rapid method to analyze the antigenicity of SARS‐CoV‐2. The pandemic of SARS‐CoV‐2, with its continuously evolving viral properties, has become a major public health concern that adds to the existing concern regarding influenza virus pandemics/epidemics. In particular, the omicron variants that emerged in November 2021 have a higher number of mutations than the previous variants and are considered to have relatively mild symptoms, but to be highly infectious. Furthermore, the omicron variants are resistant to immunity raised by previous variantsSARS‐CoV‐2, which causes COVID‐19, has infected over 500 million people and killed over 6 million, despite the production of over 11 billion doses of vaccine as of April 2022 .To predict the antigenicity and other viral properties of each isolate, a large number of the viral genomes have been sequenced using next‐generation sequencers and registered /hemagglutination inhibition (HI) tests.In this study, we established particle agglutination (PA)/particle agglutination inhibition (PAI) methods using hACE2‐bound latex beads as a surrogate for blood cells, utilizing the phenomenon that SARS‐CoV‐2 interacts with hACE2 via its spike protein.22.12. The following isolates, which were mainly prevalent in Japan, were used (information on mutations is summarized in Table Eighty to ninety‐five percent confluent Vero E6 cells expressing transmembrane protease serine 2 (TMPRSS2)2.2280 of the hACE2 solution was measured before and after mixing of hACE2 solution and the beads. The beads were blocked with 0.75 ml of PBS with 3% bovine serum albumin  for 30 min at room temperature. The blocked beads were stored in 0.5 ml of PBS with 1% BSA  beads concentration) at 4°C until use.Deep blue‐dyed latex beads, 0.8 μm in average diameter, were purchased from Sigma‐Aldrich . 0.5 ml of 2.5% (w/v) beads was centrifuged at 2400 g for 10 min at room temperature, and the beads were recovered as precipitates. The beads were washed twice with 1 ml PBS  using centrifugation. The washed beads were centrifuged and resuspended in 0.25 ml of 25 mM MES‐NaOH, pH 6.0. The resuspended beads were centrifuged and mixed with 2.5 ml of 25 mM MES‐NaOH, pH 6.0 containing 100 μg hACE2  for 24 h at 4°C using a rotator. The hACE2‐bound beads were centrifuged at 2400 g for 10 min at 4°C and washed twice with 1 ml PBS. The OD2.32.5% (w/v) hACE2‐beads were diluted to 0.003%–0.8% (w/v) with PBS supplemented with 1% BSA. Based on sedimentation/agglutination patterns and detection sensitivity of the virus, the optimum concentration was set at 0.01%. 50 μl aliquots of a twofold dilution series of SARS‐CoV‐2 variants were prepared in a 96‐well plate with PBS. Fifty microliters of 0.01% (w/v) hACE2‐beads was added to each well. After overnight settling at room temperature, sedimentation/agglutination patterns were observed. A mixture of 50 μl of PBS and 50 μl of the beads was used as the sedimentation (no‐agglutination) control.2.450). For the plaque assay, monolayers of VeroE6/TMPRSS2 cells grown in a 96‐well plate were infected with serially diluted culture supernatants of SARS‐CoV‐2, cultured in high‐glucose Dulbecco's modified Eagle's medium  containing 2.5% carboxymethyl cellulose at 37°C under 5% CO2 for 3 days and then fixed with 4% paraformaldehyde and stained with crystal violet. Emergent plaques were counted using an optical microscope. For TCID50, 10‐fold serially diluted viruses were mixed with VeroE6/TMPRSS2 cells (2–3 × 104 cells/well) in a 96‐well plate and incubated at 37°C under 5% CO2 for 5 days. Five days later, the cytopathic effect in each well was checked, and the TCID50 was determined by the Kärber method.The titers of SARS‐CoV‐2 used in this study were determined either by plaque assay or by 50% tissue culture infectious dose (TCID2.53 TCID50 of each SARS‐CoV‐2 strain: WK‐521 (Pango lineage: A), QH‐329‐037 (B.1), QHN002 , TY11‐927 (B.1.617.2), TY11‐908 , TY38‐873 (BA.1), and TY40‐385 (BA.2). The whole blood was collected by cardiac puncture under deep terminal anesthesia 14–16 days after infection, and sera were prepared by centrifugation. The sera were inactivated at 56°C for 30 min to use for the microneutralization and the PAI test.The 5‐week‐old female Syrian hamsters were intranasally inoculated with 50 μl of 102 TCID50 of the SARS‐CoV‐2 strain and preincubated in 96‐well plates at 37°C for 60 min. After preincubation, VeroE6/TMPRSS2 cells (2–3 × 104 cells/well) were added to the virus–serum mixture and incubated at 37°C under 5% CO2 for 5 days. Five days later, the cytopathic effect in each well was checked, and the microneutralization titers of sera were determined as the reciprocal of the highest dilution that did not display the cytopathic effect.Twofold serial dilutions of sera were mixed with 102.6Prior to performing the PAI test, the nonspecific agglutination factor(s) in antisera was removed if antiserum samples showed nonspecific agglutination as follows. Five hundred microliters of 2.5% hACE2‐beads was centrifuged, and the supernatant was removed. One milliliter of 10‐fold diluted antiserum with PBS was added to the precipitated beads and then mixed by a rotator for 60 min at room temperature. The treated antiserum was centrifuged at 2400 g for 10 min at 4°C, and the collected supernatant was used for the following PAI test.Twenty‐five‐microliter aliquots of 2‐fold dilution series (from 10‐ to 1280‐fold) of each antiserum were prepared in 96‐well plates with PBS. Four PA units/25 μl of SARS‐CoV‐2 isolates were added to the diluted antisera and incubated for 60 min at room temperature. The accuracy of the PA titer of the added viruses was confirmed by another PA test (back titration). Fifty microliters of 0.01% (w/v) hACE2‐beads were added to each well. After overnight settling at room temperature, the agglutination/agglutination inhibition patterns were observed. For the sedimentation or agglutination control wells, 25 μl PBS was used in place of antisera or virus, respectively.33.1First, we aimed to establish a PA test as a virus titration method using hACE2‐bound latex beads (hACE2‐beads) as a surrogate for the blood cells used in the HA test of influenza viruses. The hACE2‐beads were prepared based on the method used for SARS‐CoV‐2 antigen‐coated latex beads.To confirm the specific binding of hACE2‐beads to SARS‐CoV‐2, 50 μl of B.1 isolate was mixed with 50 μl of 0.06% hACE2‐beads  and settled at room temperature. As a control, hACE2‐unbound beads (BSA‐beads) were prepared and tested in the same way as the hACE2‐beads Figure . A mixtuThe PA test was performed on representative SARS‐CoV‐2 isolates under the tentative condition. Agglutination patterns were observed in all tested isolates, and the PA titer of the isolate was defined as the highest dilution factor at which complete agglutination was observed Figure . However3.2Next, the inhibitory effect of antibodies against SARS‐CoV‐2 on viral binding to hACE2‐beads was evaluated based on the HI test for influenza virus.https://www.genetex.com/MarketingMaterial/Index/recombinant_antibodies_for_sars‐cov‐2_research; the web page does not list the reactivity for BA.1, but the same region as for the spike protein B.1.1.529 is used for the reactivity check). A normal (noninfected) Syrian hamster serum  was also used a non‐inhibitory control  and the omicron variants have been reported in humanComparing the results of the PAI Table  and the 4In this study, we established the PA/PAI test using hACE2‐coated beads. The PA titers were correlated with the amount of SARS‐CoV‐2. The PAI titers clearly showed the antigenic differences between the omicron and the previous variants. Furthermore, the titers also suggested antigenic differences of the beta variant from the original Wuhan strain (lineage A) and the delta variant (B.1.617.2). These antigenic differences were supported by the results shown in previous reports.Previous reportAs shown above, the PA/PAI assay is an easier method, but its cost might be a concern. This assay can be performed for ~US$30 per 96‐well plate. This study used commercially available hACE2 to save the time to prepare the protein, which was the most expensive (~US$800/100 μg). If hACE2 can be prepared in‐house, the cost can be further reduced to ~US$1 per 96‐well plate. Finally, we note that a PA/PAI test using inactivated SARS‐CoV‐2 was not performed in this study. If the PA/PAI test were to be validated with inactivated SARS‐CoV‐2, the assay could be used without any restriction due to the biosafety level.Jun Kobayashi: Conceptualization; data curation; investigation; methodology; writing‐original draft. Shutoku Matsuyama: Data curation; funding acquisition; investigation; methodology; writing‐original draft. Masayuki Shirakura: Investigation; resources. Tomoko Arita: Investigation; resources. Yasushi Suzuki: Investigation; resources. Hideki Asanuma: Investigation; resources. Shinji Watanabe: Conceptualization; data curation; investigation; resources; writing‐review and editing. Hideki Hasegawa: Conceptualization; funding acquisition; resources; writing‐review and editing. Kazuya Nakamura: Conceptualization; data curation; methodology; supervision; writing‐original draft; writing‐review and editing.Jun Kobayashi, Shutoku Matsuyama, and Kazuya Nakamura are listed as inventors on Japanese patent application no. 2022‐153084 covering the use of the PA/PAI test submitted by the National Institute of Infectious Diseases.All animal experiments were carried out in accordance with the Guide for Animal Experiments Performed at the National Institute of Infectious Diseases (NIID), and were approved by the Animal Care and Use Committee of NIID .https://publons.com/publon/10.1111/irv.13093.The peer review history for this article is available at Table S1. SARS‐CoV‐2 isolates used in this study.Figure S1. Optimization of the PA test.a, The result using 0.03% hACE2‐beads. A 2‐fold dilution series of the viruses (50 μl) was mixed with 0.06% hACE2‐beads (50 μl), and then settled overnight at room temperature. b, Optimization of the hACE2‐beads concentration. The final beads concentration was varied from 0.03 ~ 0.0025%. The PA titers are shown with red lines.Figure S2. The correlation between the PA titer and the plaque‐forming units. The regression line is shown by the blue dot line.Click here for additional data file.


Furthermore, the purified viral therapeutic material was tested in vivo for its efficacy and safety. All these characterization methods demonstrated a therapeutic virus preparation of high purity and yield, which can be readily used for various studies.Purification of viruses, especially for therapeutic purposes, is a tedious and challenging task. The challenges arise due to the size and surface complexity of the virus particles. VSV-GP is a promising oncolytic virus, which has been approved for phase I clinical trials by the Food and Drug Administration (FDA) of United States and Paul Ehrlich Institute (PEI) of Germany. The virus particles of VSV-GP are larger in size than vectors commonly used for gene therapy . The current established proprietary clinical-grade manufacturing process for the purification of VSV-GP encompasses several chromatographic and non-chromatographic steps. In this study, we describe a new single-step purification process for the purification of VSV-GP virus, using cation exchange convective flow column with relatively higher yields. The purified virus was characterized for its quality attributes using TCID Advanced therapeutic and medicinal products (ATMPs), often touted as the “golden bullet,” are a class of new and innovative therapeutics developed as breakthrough therapies for the treatment of “un-treatable or extraordinary diseases” such as against various types of cancer . OncolytVesicular stomatitis virus (VSV) is a negative-strand RNA virus that belongs to the family of rhabdoviridae . It can Recently, a recombinant version (VSV-GP) has been developed as an immune-oncology platform that can induce both direct tumor cell killing as well as therapeutic cancer vaccination . Herein,One of the major challenges when developing a complex therapy, such as oncolytic viral therapy, lies in developing an efficient manufacturing process . This chThere are various types of chromatography columns commercially available for downstream processing and purification of various therapeutic molecules, especially with ion exchangers . HoweverThe challenges due to such complexities include but not limited to, the fact that virus size can be greater than the pore size of circular chromatography beads in the column . Also, tin vitro and in vivo assays to substantiate the critical quality attributes.In this study, we describe a novel, quick and cost-effective bioprocess for the purification of the oncolytic virus, VSV-GP using membrane adsorber based cation exchange chromatography. This process produces, not just a purer form of VSV-GP, but also a more concentrated drug substance. This is achieved without the need for an extensive concentration step, usually involving ultrafiltration resulting in the high concentration of oncolytic viruses, often being a pre-requisite enabling intra-tumor application of the virus. The latter is known to damage the virus particles due to shear stress . Further50) were obtained from ATCC (−/− cells were generated as described previously (BHK-21 (C13) cells used for the infectivity assay  pseudotyped with glycoprotein from LCMV (LCMV WE HPI-GP) and has been described previously . A VSV-G® membrane adsorbers were used for virus capture in CEX. These membranes are cellulose-based cation exchange membrane adsorber capsules with 3 ml membrane volume (MV) and a bed height of 8 mm . The nominal pore size for these membrane adsorbers is > 3 µm . For all CEX experiments, an Äkta™ Pure 150 liquid chromatography system (Cytiva) with a 280 nm UV, pH and conductivity monitoring was operated at room temperature. Prior to virus application, membranes were equilibrated with 50 mM Tris-HCl, 50 mM Arginine, pH 7.5 at a flow rate of 15 ml/min. Sample application was conducted with the same flow rate. The elution of the virus particles was carried out with a 300 mM NaCl, 50 mM Tris-HCl, 50 mM Arginine, pH 7.5 buffer at a flow rate of 3 ml/min. Pooled elution fractions from CEX were diluted 6x in order to reach the final buffer composition with 50 mM NaCl instead of 300 mM NaCl in the elution fraction. Afterwards, virus is sterile-filtered using a 0.22 µm syringe-filter . Virus aliquots were stored at −80°C before use.Sartobind S Nano50 assay was performed as described previously (50/ml) were obtained by the formula of Spearman Kärber .The quantification of host-cell protein (HCP) impurities was determined by performing the Human Embryonic Kidney 293 Host Cell Proteins ELISA kit from Cygnus Technologies (Cat. Reference: F650S) according to the manufacturer´s instructions. The amount of hydrolyzed substrate, which gives a colorimetric signal directly proportional to the HCPs concentration, was measured with a Spark 20 M Tecan Plate ReaderIn order to test purity and identify the viral and host-cell proteins, SDS-PAGE followed by silver staining were carried out . PrecastFor revealing the gel protein bands, the commercial kit “Pierce™ Silver Stain”  was employed. Silver staining is an ultrasensitive method which can effectively reveal protein bands that are in sub-nanogram ranges. Silver staining was performed as detailed by the manufacturer´s instructions. Gel images were acquired using VisionWorks LS software (Version 8) with ChemStudio gel imaging instrument .−/Å2.Cryo-TEM imaging was performed by NanoImaging Services, Inc . Each sample was prepared by applying a 3 μl drop of undiluted sample suspension to a cleaned grid (holey carbon films on 400-mesh copper grid) and immediately proceeding with vitrification in liquid ethane. Electron microscopy was performed using a Thermo Fisher Scientific Glacios Cryo-TEM operated at 200 kV and equipped with a Falcon three direct electron detector. Automated data-collection is carried out using Leginon software . High maXL SEC column  operated at room temperature with an isocratic flow at 0.5 ml/min flow rate using a mobile phase composed of phosphate buffered saline (PBS).In SEC analysis, 40 µl samples were injected through a 1,290 Infinity II HPLC system  with UV detection at 280 nm wavelength. Contaminant analyses were performed using TSKgel 3000 PW−/− tumors were implanted by subcutaneous injection of 100 μl containing 1 × 106 cells in the right flank (2 × 0.5. Treatment commenced when the mean tumor volume reached a size of 80–150 mm3. Virus solutions with 109 TCID50 of virus were used for intravenous (100 μl) injection. Mice were sacrificed when their tumor volume reached 1,500 mm3 or tumors showed signs of ulcerations  mice, were obtained from Charles River Laboratories . CT26. Cl25-IFNAR1ht flank . Tumor serations . Animalsin vivo imaging solution at 15 mg/ml) in DPBS, and imaged 15 min after injection upon transient narcosis. Exposure times were adjusted to bioluminescence intensity to optimize signal levels and avoid saturation of the light sensors. For quantification of bioluminescent signal, regions of interest (ROI) were manually defined and the average intensity within the ROI was measured as photons/sec/cm2/sr. The obtained images were analyzed using M3Vision image analysis software .For Bioluminescence imaging, a charge–coupled device camera  was used. Mice were injected intraperitoneally with 50 mg/kg D-Luciferin . Moreover, it is important to mention that the treatment of feed with nuclease resulted in a host cell DNA concentration of only 4.16 ng/ml, which is already below any regulatory requirements . This ca50 (The current Sartobind S based chromatography method was developed to facilitate the binding of VSV-GP to the membrane adsorber column in the presence of low concentration of sodium chloride (0.1 M NaCl) thereby eliminating non-specific binding of molecules such proteins present in the crude feed . A wash 50 .Furthermore, we wanted to analyze the dynamic binding capacity of the column with a load of 5,800 ml of the feed material on a 3 ml Sartobind S column . DespiteApart from factors such as a higher yield, speed, and cost-effectiveness, it is also imperative to consider the purification efficiency of the process. Therefore, a consolidation run with a feed of 2,300 ml (766.66 membrane volumes) and a fixed step elution with 300 mM NaCl was carried out . This coTo further substantiate the virus quality, the purified virus was analyzed using cryo-TEM . Bullet in vivo. Therefore, we tested and compared it with the virus material purified using a comprehensive downstream process  currently used for manufacturing at large scale for clinical trials. To address whether the different downstream processes have an impact on the efficacy, tolerability, or activity of the virus, we compared relevant read-outs in BALB/c mice bearing CT26. cl25-IFNAR1−/− tumors (9 TCID50 induced a delay in tumor growth with no difference between our new and clinical-grade process (via luciferase signal. The respective treatments resulted in comparable tumor-selective bioluminescence signals. These signals increased over several days, peaking at day 4, indicating similar active viral replication (Finally, we wanted to ascertain that the therapeutic virus was effective in animal models − tumors . A singl process . This re process . Both te process . After tlication .Cation exchange chromatography is a purification platform used frequently for purification of not only proteins and antibodies , but canThere are other chromatographic methods for the purification of certain viruses, such as affinity chromatography which can be highly specific for the target particles with very good removal of contaminants without additional polishing steps . HoweverIn this study we utilized the capabilities of cation exchange chromatography coupled with convective flow media, i.e., membrane adsorber. This is a very important single use technology along with large pore sizes/channels , allowin


Viruses are part of the microbiome and have essential roles in immunology, evolution, biogeochemical cycles, health, and disease progression. Viruses influence a wide variety of systems and processes, and the continued discovery of novel viruses is anticipated to reveal new mechanisms influencing the biology of diverse environments. While the identity and roles of viruses continue to be discovered and understood through viral metagenomics, most of the sequences in virome datasets cannot be attributed to known viruses or may be only distantly related to species already described in public sequence databases, at best. Such viruses are known as the viral dark matter. Ongoing discoveries from the viral dark matter have provided insights into novel viruses from a variety of environments, as well as their potential in immunological processes, virus evolution, health, disease, therapeutics, and surveillance. Increased understanding of the viral dark matter will continue with a combination of cultivation, microscopy, sequencing, and bioinformatic efforts, which are discussed in the present review. Sequencing techniques, described later in this review, are also presented in Virus discovery has been essential to understanding the emergence and re-emergence of viral pathogens, discovery, and characterization of non-pathogenic viruses, establishing model systems to study replication and infection mechanisms , and defining mechanisms that underpin immunological, evolutionary, and environmental processes. Prior the metagenomics era, viruses were typically discovered and understood using a variety of techniques aimed at deciphering replication and infection mechanisms, morphology, and genetic composition. In the following section, culture techniques, electron microscopy (EM), and molecular methods used for virus discovery are described and summarized Table 1in vitro and in vivo culture conditions have been used to identify and isolate a wide variety of viruses, including those that are in mixed culture (in vivo culture of the virus (which was unknown to be a virus at the time) in several different animals, including monkeys. It was then noticed that the virus caused the same effects to the monkey as those originally observed in humans (Culture methods involving cell lines (eukaryotic viruses) and bacterial strains (phage) have long been the gold standard for the discovery and characterization of viruses. Both  culture Table 1. culture . Interes culture . Similarn humans .in vitro or in vivo culture of viruses are the time and specific conditions required  in the 1930s. EM has also enabled the confirmation of certain Monkeypox and other poxviruses  of information , Blautia, Clostridium, Collinsella, Enterococcus, and Faecalibacterium spp., as potential putative hosts for crAssphage and crAss-like phage; however, B. intestinalis was eventually confirmed to be crAssphage bacterial host based on sequencing gut filtrates and each bacterial host  typical of the e family . Additioial host . Discove”, “Flandersviridae” and “Gratiaviridae”     as an antiviral protection in insects, such as ila spp. . Virusesus (DCV) , and Drous (DCV) . Furtherra virus . However novelty . Similaramilies) .As described in the previous sections, virus discovery from the dark matter often relies upon using sequence alignment methods against reference viral genes and genomes, as well as k-mer-based methods, which are used to predict a putative virus based on genomic and sequence signatures. Examples of several bioinformatic tools, as well as advantages and disadvantages for several of these sequence- and k-mer-based methods have previously been described . Other aViruses can be directly or indirectly associated with health and disease; yet, there is much to learn still about the contribution of the viral dark matter in health, disease, therapeutics, and surveillance efforts Figure 3Caudovirales being the predominant viral order  and ulcerative colitis (UC), has an unknown etiology; yet, IBD appears to be multifactorial in nature and has been repeatedly associated with alterations of the human gut microbiome and more recently, the virome –96. Pional order . Subsequfestyles . This stfestyles . NotablyStreptococcus spp., Lactobacillus spp. and Veillonella spp.  virus from a family named Redondoviridae . The application of phage in this way, also known as phage therapy, has shown renewed interest in recent years as a way to treat ARB infections . Initialruginosa , 103, Clifficile , and Entfaecalis . Single Phage therapy can also become personalized when available phage and phage cocktails do not efficiently lyse target ARB. Screening for ideal phage candidates to be used in phage therapy can be time and labor intensive. The search for phage that could efficiently lyse target ARB can involve screening samples, such as sewage  . Once a Microhyla fissipes, and Pacific salmon nidovirus (PsNV), identifying samples containing corona-like virus-aligned reads and/or k-mers and performing de novo assembly using coronaSPAdes (15)-scale genomics, which can aid in reducing the viral dark matter and improve surveillance efforts of potential emergent viruses from unexpected sources.Understanding the source and evolution of potential emerging and re-emerging pathogens is essential for surveillance efforts. This is when viral metagenomics becomes an important surveillance tool as it allows numerous viruses to be identified simultaneously from various sources and facilitates using one or several of the above-mentioned tools. Virus discovery from viral dark matter is increasingly recognized as an important aspect of surveillance efforts. For instance, while bats are recognized as a resource of novel coronaviruses and an important host for coronavirus evolution , 109, vinaSPAdes . This renaSPAdes Table 2.For the last 20 years, viral metagenomics has shown to be a powerful tool for the discovery of viruses. Viral metagenomics continues to be essential in the characterization of known viruses in diverse sample types, and in association with health, disease, immune system, biogeochemical cycles, therapeutics, and surveillance. Moreover, identification of novel viruses from viral dark matter continues to be possible with viral metagenomics. Virus discovery through viral metagenomics, however, does not come without challenges. Many such challenges that are intrinsic to the technique including viral purification, nucleic acid extraction and amplification, library preparation, sequencing, and bioinformatics. Examples of the bioinformatic frameworks that could be applied for virus discovery from viral dark matter are highlighted in the present review and demonstrate that no universal framework applies to the discovery of all viruses, but are rather diverse and should be suited for the virus(es) of interest. Virus discovery from viral metagenomics data may also require the application of culture, molecular and/or EM techniques.TS-R original draft preparation. TS-R and EH: review and editing of manuscript draft. All authors contributed to the article and approved the submitted version.TS-R and EH are current employees of Diversigen, a subsidiary of OraSure Technologies and a microbiome services company.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.