Phaseolus calcaltus), as an annual summer legume, is always subjected to acid soils in tropical to subtropical regions, limiting its growth and nodulation. However, little is known about its responses to lime and biochar addition, the two in improving soil fertility in acid soils.Rice bean (−1) and three biochar rates . The biochar was produced from aboveground parts of Solanum tuberosum using a home-made device with temperature of pyrolysis about 500 °C.In the current study, a pot experiment was conducted using rice bean on a sandy yellow soil (Orthic Acrisol) with a pH of 5.5. The experiment included three lime rates  and alkali dispelled nitrogen (AN), whereas biochar application increased the concentrations of soil AP, AN and available potassium (AK). However, sole biochar application could not achieve as much yield increase as lime application did. High lime rate (1.5 g lime kg−1) incorporated with low biochar rate (5 g biochar kg−1) could obtain higher shoot biomass, nutrient uptake, and nodule number when compared with high lime rate and high biochar rate.The results indicated that both lime and biochar could reduce soil exchange Al concentration, increase soil pH and the contents of soil microbial biomass carbon and microbial biomass nitrogen, and enhance urease and dehydrogenase activities, benefiting P. calcaltus growing in acid soils when compared with sole lime or biochar addition.Lime incorporated with biochar application could achieve optimum improvement for  Fast-grand rice . The greand rice . Green mand rice .Phaseolus calcaltus Roxb) is an annual legume that can be used as summer green manure , AP, and organic carbon, and improved cabbage growth  with a pH of 5.5. Soils were amended with different lime and biochar rates. We hypothesized that biochar addition might be an alternative in improving rice bean growth in acid soils. The following three aspects were mainly addressed: (1) soil chemical properties; (2) soil biological and biochemical properties ; (3) plant growth and nutrient uptake.In order to evaluate the efficiency of biochar from −1 respectively. The concentrations of alkali dispelled N (AN), available phosphorus , available potassium (AK) and exchangeable aluminum (Al3+) were 100.12, 20.16, 112.5 and 1126.82 mg kg−1 (4.17 cmol kg−1), respectively. The soil cation exchange capacity (CEC) was 8.54 cmol kg−1. The Al saturation was 48.83%. The soil pH was 5.5 (soil: water = 1:2.5).The soil (Orthic Acrisol) used in this study was collected from arable land, located in Mountain Jigong , Chongqing, China. During the last ten years, corn and sweet potato are the main crops in the arable land. The climate in this area is subtropical monsoon humid climate with mean annual precipitation reaching 1133.7 mm and mean annual temperature reaching 18 °C . Soils wSolanum tuberosum, which normally have been discarded after harvesting the tubers, were collected, air dried, pyrolysed at a highest treatment temperature of 550 °C for 4–5 h, and then ground to pass a 2 mm sieve. The pH of the biochar was 9.89 and measured using a biochar to deionized water mixture (1: 5) followed by shaking and an equilibration time of 30 min before measurement with a pH meter  in biochar was determined by oxidation with potassium dichromate in a concentrated sulfuric acid medium and the excess dichromate was measured using Mohr’s salt (NH4)2Fe(SO4)2 ⋅ 6H2O . Aboveground parts of pH meter . The biopH meter . The con2 ⋅ 6H2O . TN was 2 ⋅ 6H2O , TK was 2 ⋅ 6H2O . The conPhaseolus calcaltus in field. They were purified and propagated using YMA medium (Yeast Mannitol Agar). The inoculants were propagated using YMA medium without agar  and 10 g kg−1  and three lime rates, 0 g kg−1, 0.75 g kg−1  and 1.5 g kg−1 , with four replications. The lime and biochar were mixed thoroughly with soils. In a pre-experiment, liming the tested soil with 0.75 g lime kg−1 and 1.5 g lime kg−1 could obtain pH of 6.0 and 6.4, respectively. About 2.0 kg soil was filled into a plastic bag, then put in a pot (15 cm × 20 cm), and taken to field water capacity for 15 days before sowing. The soil field water capacity was calculated previously.The experiment was a two-way random design with three biochar rates, 0 g kgPhaseolus calcaltus were sterilized in 3% H2O2, washed three times, then planted in pot with 6 seeds in each pot. The pots were watered with 20 mL Rhizobia solution for three times during the first three weeks after seeding. The relative soil water contents were kept around 70–80% of the field water capacity through adding water. The pots were placed in a glass greenhouse with temperature ranging from 20 °C to 25 °C, and relative humidity ranging from 65% to 75%. One week after seed sprouting, three seedlings were kept in each pot.Seeds of 2 concentration) were measured by using LI-6400XT Portable Photosynthesis System . The plants were in their branching stage.Two months after seed sprouting, one day before harvest, leaf photosynthesis parameters  were carefully collected at a depth of 5–10 cm from the pot. After soil sampling, the remaining soils with roots were carefully put into a big basin filled with tap water, shaken carefully, until the roots were separated from the soil. The roots were collected on a 1 mm sieve and cleaned free of soils. The soil solutions in the basin were also sieved (1 mm) to collect the abscised nodules and fine roots. Nodules were separated manually with tweezers from the roots, and the nodule numbers were counted. The shoot biomass was also washed two times in clean tap water. Then, the plants, roots and nodules were dried at 75 °C for 48 h, and weighed . The pla2SO4 until the solution color turned to transparent, then nutrients were analyzed by methods of About 0.4 g plant sample was digested in 5 ml 18.4 M H2SO4 and then analysed for TN by Kjeldahl method  2Fe(SO4)2 ⋅ 6H2O (Dried soils (1.00 g) were digested in 5 mL Hl method . Dried sl method . Dried sgen (AN) . Dried srocedure . Dried sr method . Soil to2 ⋅ 6H2O .−1 KCl and exchangeable Al concentration was determined by Eriochrome cyanine R spectrophotometric method (−1 BaCl2 and then 0.01 mol L−1 (1/2 MgSO4), and then measured the amount of added cation that was retained (Dried soils (0.200 g) were extracted with 1 mol Lc method . Dried sretained . Soil pHretained  .4 released in the hydrolysis reaction of urea  and microbial biomass nitrogen (MBN) was measured by the fumigation-extraction procedure . Ten graMBC = (A − B) ÷ 0.38A: extractable organic C in fumigated.B: extractable organic C non-fumigated soil.0.38: The conversion factor for microbial biomass carbon.MBN = (C − D) ÷ 0.45C: extractable organic N in fumigated.D: extractable organic N non-fumigated soil.0.45: The conversion factor for microbial biomass nitrogenP = 0.05. The relationship between the soil pH and the plant growth, nutrient concentration and uptake, and soil chemical and biological parameters was fitted to simple linear model or quadratic model using SigmaPlot 10.0 .Data were subjected to a two-way ANOVA analysis using SPSS version 17 software , to analyze the effects of lime and biochar and their interactions on soil and plant parameters. One way ANOVA analysis was further applied to analyze the effects of biochar and lime application on soil and plant parameters. Significance was tested according to the least significant difference (L.S.D) test at −1 (L0) to 6.30 at 1.5 g lime kg−1 (L2) without biochar application. Within each lime rate, biochar application, particularly at 1.5 g kg−1, significantly increased the pH. The concentrations of soil exchangeable Al reduced significantly with increased lime rates and biochar rates. The concentrations of AP increased at B2 under L0 condition, at B1 and B2 under L1 condition, and at B1 under L2 condition. Biochar application significantly increased the concentrations of AK and AN and overall higher concentrations of AK and AN were observed at B2 than B1. Without biochar addition, the concentrations of soil AP at L0 were significantly higher than those at L1 and L2; while the concentrations of soil AN and AK changed insignificantly with lime application.Soil pH and the concentrations of soil exchangeable Al and available nutrients were significantly influenced by lime and biochar applications and their interactions . Lime apy = 72.78 − 6.10x; R2 = 0.529; P < 0.0001)  . Also, a 0.0001) .P < 0.05). However, the ratio of MBC/MBN changed insignificantly, excepting a higher ratio at B2 under L1 condition. Biochar application increased the urease activity under all lime rates, with significance only at B2. The urease activities also increased with increased lime rates (P > 0.05). Biochar application increased the dehydrogenase activity under all lime rates except for B1 at L0, with significant changes being observed only at L2. Lime application showed no significant influence on dehydrogenase activity at B0.The contents of MBC and MBN and soil enzyme activities were significantly influenced by lime and biochar applications and their interactions . The MBCCorrelation analysis indicated that the MBC, MBN, MBC/MBN, and the activities of urease and dehydrogenase were positively correlated with soil pH –1H.2 concentration of plants in limed soils were significantly higher than those in un-limed soils. Biochar application increased the net photosynthesis rate at B2 under L0 and L2, increased transpiration rate at B2 under L2, with the other photosynthesis parameters unchanged.There was no significant interaction between lime and biochar applications on parameters of leaf photosynthesis . Lime apy =  − 6.75 + 2x; R2 = 0.569; P < 0.0001)  .P > 0.05). The plant failed forming nodule at B0 and B1 under L0 conditions.Lime significantly increased the nodule number, dry weight and mean nodule weight . The bioLime application significantly influenced the concentrations of TN and Al in plants, whereas biochar significantly influenced the concentrations of TP and Al, and there existed significant interactions between lime and biochar . BiocharBoth lime and biochar application significantly influenced the uptake of N, P, K and Al . BiocharCorrelation analysis indicated that the soil pH was positively correlated with the concentrations of plant N and the uptake of N, P and K . A quadr3+ concentration, and correct nutrient deficiencies in acid soils , the soil pH increased from 5.49 to 6.30 and the soil exchangeable Al concentration reduced by 69.28%, reducing from 3.39 to 1.04 cmol kg−1 (Al saturation from 39.69% to 12.18%). However, lime application reduced the concentrations of soil AP, which in turn might negatively influence the plant growth. Correlation analysis also indicated that the concentration of soil AP decreased with increased soil pH and increased with increased soil exchange Al concentration. It has been reported that lime could decrease soil P sorptivity, and thus increase soluble P and the P uptake by plant  but not at low lime rates or control. It suggested that lime might be better in improving the growth of P. calcaltus in the tested acid soil when compared with biochar addition. Using global-scale meta-analysis, As a green manure, obtaining optimum aboveground biomass is the prerequisite to achieve high fertilizer efficiency . Acid so−1. This might be the main reason why no further growth improvement could be observed under low lime rates. Under high lime rate (L2), the soil exchangeable Al concentrations reduced to lower than 1.11 cmol kg−1, with its concentration reduced by 27.76% at B1 and 68.68% at B2, which might alleviate the limitations of Al3+ on plant growth. Surprisingly, the highest plant biomass was observed at B1 but not at B2 under high lime rate (L2), and the plant P concentrations was also higher at B1 than at B2 under L2. One possible reason might be that high biochar addition (B2) under high lime rate (L2) might further enlarge the nutrients imbalance, which might offset the positive influence on plant growth induced from reducing soil exchangeable Al concentrations. For example, in the current study, biochar application significantly increased the ratio between the concentrations of AK and AP, mainly attributed to the increased potassium release into soil through biochar. + into soils, which might be exchanged with the Ca2+ in soil, causing alteration of soil ion ratios, and finally leading to lower yields  and L1 (reduced by 19%), where the soil exchange Al concentrations were all larger than 1.64 cmol kgr yields . In a 3-In the current study, high biochar addition significantly increased the leaf net photosynthesis rate with transpiration rate unchanged under all lime rates. This was inconsistent with the responses of biomass to biochar application in un-limed and low limed soils. One possible reason might be that the increase of photosynthesis during daytime might be offset by relatively higher respiration rate during evening in biochar added soils .P. calcaltus in acid soils, finally providing agronomic management decisions in planting legume green manures in acid soils with varying soil exchangeable Al concentrations  uptake was positively correlated with soil pH and negatively correlated with soil exchangeable Al concentration. In tropical clay soil in Zimbabwe, application of sludge biochar significantly improved the maize growth, biomass production and nutrient uptake when compared to the unamended control , no further reduction could be observed in plant aluminum content when biochar was added, and a higher amount of aluminum uptake was also observed in biochar added soils, though soil exchange Al was significantly reduced. This implied that P. calcaltus might tolerate or need certain amount of aluminum in soils to grow normally. In a review, 3+ could have a beneficial or toxic effect, depending on its concentration, chemical form, and the plant species and growing conditions. This might also explain why higher shoot biomass was observed at B1 but not at B2 under high lime rate (L2), where the soil exchangeable Al concentration reduced to 0.33 cmol kg−1 when compared to the 0.75 cmol kg−1 at B1.Both biochar and lime significantly reduced the concentrations of aluminum in P. calcaltus was severely limited in acid soils. Both lime and biochar application could reduce soil exchangeable Al concentrations and increase soil pH, improving soil nutrient availability and microbe activities, plant nutrient uptake, nodulation, and plant growth. Compared with liming, application of sole biochar could not achieve higher shoot biomass. High lime rate  incorporated with low biochar rate  could obtain higher shoot biomass, nutrient uptake, and nodule number when compared with high lime rate and high biochar rate. Long term field experiment is still needed to clarify the lime and biochar rates associated with planting P. calcaltus in specific acid soils, providing appropriate agronomic management decisions.The growth of 10.7717/peerj.6346/supp-1Data S1Data of all parameters with four replicates.Click here for additional data file.


This study shows that only a single introduction of SINV into a new geographical area is required for spread and establishment, provided that the requisite vector(s) and reservoir(s) of epizootological and epidemiological importance are present. Furthermore, we present the first report of recombination between two strains of SINV in nature. Our study increases the knowledge on new introductions and dispersal of arboviruses in general and of SINV in particular. Bird-hosted viruses have the potential to be transported over large areas of the world and to be transmitted in distant geographical regions. Sindbis virus (SINV) is a mosquito-borne alphavirus that is locally amplified in a bird-mosquito enzootic cycle and distributed all over the Old World and Australia/Oceania. Sindbis virus genotype I (SINV-I) is the cause of disease outbreaks in humans in South Africa as well as in northern Europe. To trace the evolutionary history and potential strain-disease association of SINV-I, we sequenced 36 complete genomes isolated from field material in Europe, as well as in Africa and the Middle East, collected over 58 years. These were analyzed together with 30 additional published whole SINV-I genomes using Bayesian analysis. Our results suggested that SINV-I was introduced only once to northern Europe from central Africa, in the 1920s. After its first introduction to Sweden, it spread east and southward on two separate occasions in the 1960s and 1970s. Another introduction from central Africa to southern/central Europe seems to have occurred, and where these two introductions meet, one recombination event was detected in central Europe. In addition, another recombinant strain was found in central Africa, where the most divergent SINV-I strains also originated.IMPORTANCE This study shows that only a single introduction of SINV into a new geographical area is required for spread and establishment, provided that the requisite vector(s) and reservoir(s) of epizootological and epidemiological importance are present. Furthermore, we present the first report of recombination between two strains of SINV in nature. Our study increases the knowledge on new introductions and dispersal of arboviruses in general and of SINV in particular. Viruses hosted by birds have the potential to be transported over large areas of the world, connecting countries and regions that are well separated in climate and habitat. The occurrence of human disease from vector-borne bird viruses is dependent on the availability and abundance of competent vectors in proximity to the bird hosts and humans in different regions. Thus, these viruses need to be able to replicate in various vector and host species, as the geographic distribution often spans tropical as well as subtropical or temperate regions, with unique sets of mosquito and bird species.Togaviridae), e.g., Sindbis virus (SINV) and Western equine encephalitis virus (WEEV), and flaviviruses (Flaviviridae), e.g., West Nile virus (WNV), Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), St. Louis encephalitis virus (SLEV), Rocio virus (ROCV), and Usutu virus (USUV)  –5. In ad regions , 6–8. Huegions 6–.SINV is a positive single-stranded RNA virus that has a wide distribution throughout the Old World. It has a genome size of about 11.7 kb and has two open reading frames (ORFs) that encode four nonstructural (nsP1 to -4) and five structural  proteins, respectively . Based oSINV-I is the only genotype that has been associated with outbreaks of human disease, and it was first isolated from mosquitoes in Cairo, Egypt, in 1952 . OutbreaThe first cases of Sindbis fever in Fennoscandia were reported from Sweden in the 1960s, and later, this disease was shown to be associated with a strain of SINV-I isolated from mosquitoes in Edsbyn, Sweden . Cases oTo further elucidate the evolutionary history of SINV-I and its dispersal patterns, we have sequenced and analyzed the complete genomes of 36 new strains, in addition to 30 previously sequenced publicly available strains, from various vector and host species, spanning 10 countries and 58 years.The phylogenetic tree of the complete coding sequence of SINV-I contained 64 strains (2 strains were excluded due to recombination [see below]) and showed that the strains from central Africa were basal in each clade. According to the parsimony principle , this indicates that SINV-I originated in central Africa and diverged into two main clades with well-supported values  . Clade AMK045245), suggesting that SINV-I was first introduced from central Africa to Sweden, with subsequent circulation there, and then further dispersed from Sweden to other parts of northern, eastern, and central Europe   did not have any temporal structure . However, the data set including sequences from clades A and B showed a temporal signal  and was subsequently used to estimate the evolutionary rate of clades A and B. Following model testing, the evolutionary history of clades A and B of SINV-I was reconstructed with a strict molecular clock mode and a coalescent exponential population demographic model. The evolutionary rate for this data set is 5.45 × 10 × 10−5) . The tim × 10−5) . Notably × 10−5) . Hence, We also performed the same analysis on 163 sequences of the partial E2 alignment , and theThe SINV-I genome was highly conserved, with an average nucleotide similarity of 96.5% . The similarity of the nonstructural protein genes nsP1 , nsP2 , nsP3 , and nsP4  showed similar levels compared to the structural protein genes capsid , E3 , E2 , 6K/TF , and E1 , with an average nucleotide similarity ranging from 95.4% (nsP3) to 97.5% (E1).A comparison of nucleotide and amino acid differences showed that the highest degree of observed divergence was found in clade D . Within KY616988) and H7_Culex_pipiens_Germany_2013 (GenBank accession number MG779534) have undergone recombination , songthrush (Turdus philomelos), and fieldfare (Turdus pilaris) sometimes exceeds 70% in Sweden  that most likely underwent a recombination event. This is, to our knowledge, the first time that a recombination event has been detected for SINV-I in nature. A previous recombination event between a Sindbis-like virus and Eastern equine encephalomyelitis virus (EEEV) in South America has had a large impact on the alphavirus genus, giving rise to WEEV, Highlands J virus, and Fort Morgan virus , 36. TheCulex torrentium, is decreasing southward in Europe  according to the manufacturer’s instructions. cDNA was synthesized using the RevertAid H minus first-strand cDNA synthesis kit  in a 20-μl mixture containing 2 μl of random hexamers (10 pmol/μl), 2 μl 10 M deoxynucleoside triphosphate (dNTP), and 5 μl RNA. The presence of SINV RNA was confirmed by quantitative PCR (qPCR) as described previously .HM147984) as the reference sequence . All sequences, 66 in total, were then aligned using MAFFT with default settings, followed by manual refinement using AliView  website  the complete open reading frames (ORFs), (ii) the single genes , (iii) complete and partial E2 sequences, and (iv) the complete ORFs excluding recombinant sequences. The best-fit evolutionary nucleotide substitution model following jModelTest analysis, GTR+F+I+G4 , was used in all phylogenetic analyses . PotentiMF409177), was used to root the tree. All computations were run using the CIPRES computational cluster (http://www.phylo.org/index.php/). Finally, trees were viewed and edited using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).To reconstruct the evolutionary history of the SINV-I complex, Bayesian phylogenetic trees of the complete ORFs and separated genes  of SINV-I were constructed by employing MrBayes v.3.2.6 . BayesiaA number of studies have previously used partial E2 sequences for creating SINV phylogenies , 22, 57.MK045224 to MK045259.All newly sequenced strains have been deposited in GenBank under accession numbers


Coguvirus. Molecular and phylogenetic analyses presented here support the classification of CiVA, and likely of other two recently described nsRNA viruses infecting plants, in this new genus. By showing that the evolutionary origin of the MP of all the putative coguviruses likely differs from that of their respective RdRp and NP, this study also provides evidence of a likely modular genome evolution for these viruses. Moreover, phylogenetic data support the proposal that, during the evolutionary history of nsRNA viruses, the plant-infecting viruses most likely emerged from an invertebrate-infecting ancestor several times as independent events. CiVA was identified in a field sweet orange tree not showing any obvious symptom and was graft-transmitted to sweet orange, grapefruit, rough lemon and Dweet tangor indicator plants that did not developed symptoms. The capacity of infecting citrus hosts of several species was also confirmed by a preliminary survey that identified orange, mandarin, clementine and lemon trees as natural hosts of CiVA in several fields of southern Italy, again without any obvious association with specific symptoms.A new RNA virus has been identified from a sweet orange tree in southern Italy. This virus, tentatively named citrus virus A (CiVA), has a bipartite genome composed of (i) a negative-stranded (ns) RNA1, encoding the viral RNA-dependent RNA polymerase (RdRp), and (ii) an ambisense RNA2, coding for the putative movement protein (MP) and nucleocapsid protein (NP), with the two open reading frames separated by a long AU-rich intergenic region (IR) adopting a hairpin conformation. CiVA genomic RNAs and the encoded proteins resemble those of the recently discovered citrus concave gum-associated virus (CCGaV). This CCGaV, a nsRNA virus associated with the ancient citrus concave gum disease, has been proposed as the representative member of a new genus tentatively named  Bunyavirales to contain nine families . Here, we report a new nsRNA virus infecting citrus with molecular features and phylogenetic relationships with CCGaV, which strongly suggest that it is a prototype of a second species in the proposed genus Coguvirus. The results of preliminary field surveys in southern Italy documented the presence of this new virus, tentatively named citrus virus A (CiVA), in citrus trees of several species and in several citrus-growing areas.Based on high-throughput sequencing (HTS) many new negative-stranded (ns) RNA viruses, most of which infecting invertebrates , have befamilies . Recentlfamilies . CCGaV hhropodes . ConsequLeaf tissue, collected on 2016 from a non-symptomatic sweet orange tree (cv. Tarocco grafted on sour orange) grown in a commercial orchard in Southern Italy, was used for generating a cDNA library of small RNAs (16–30 nt). The library was generated from total nucleic acids (TNA) extracted with phenol-chloroform from leaves and sequenced (run 1 × 50) on an Illumina Genome HiScan Analyzer by Fasteris custom service  as reported previously .de novo assembled (k-mer 15–17) using the Velvet Software 1.2.08 (Supplementary Table S1). Amplicons were gel-purified, cloned and sequenced by Sanger Sequencing Custom Service  according to standards protocols  and the complete amino acid sequences of movement and nucleocapsid proteins , were built using MEGA7 . Multiplal Omega , then Tral Omega  was usedCitrus sinensis (L.) Osbeck, cv. Madame vinous], grapefruit (C. paradisi Macf.), rough lemon (C. jambhiri Lush), and Dweet tangor (C. reticulata Blanco x C. sinensis). At least three plants of each species were graft-inoculated. Mock-inoculations were performed using bark tissues harvested from a certified virus-free Tarocco plant, maintained in screen-house at University of Bari.Bioassays were performed by grafting bark tissues from the CiVA-infected tree to several indicator plants, including sweet orange , grapefruit (C. paradisi Macf.), rough lemon (C. jambhiri Lush) and Dweet tangor (C. reticulata Blanco x C. sinensis). Visual inspections carried out periodically up to 1 year after graft-inoculation, did not reveal the appearance of any leaf alteration or virus-induced symptom.Following graft-inoculations of bark tissues from the CiVA-infected source onto indicator plants, the virus was successfully detected, using the newly developed RT-PCR assay, on all grafted indicator plants. In contrast, the mock-inoculated plants tested negative to CiVA infection. These results proved that CiVA is graft-transmissible to sweet orange [C. clementina Hort. Ex Tan.) and mandarin (C. reticulata Blanco) trees from different surveyed orchards in Campania region  were assayed by RT-PCR, a total of 15 samples tested positive for CiVA infection, with at least one tree of each assayed host species being infected by the virus (Table 1). The trees tested in this survey were also assayed to investigate and confirm the association of CCGaV with CG disease (Table 1). In addition, 22 lemon (C. lemon L.) trees from Apulia region  were also assayed, three of which tested positive to CiVA infection. Altogether these data, besides confirming the distribution of the virus in several citrus fields in southern Italy, extended the natural host range of CiVA, first identified in sweet orange, clementine, mandarin and lemon trees.When samples from 71 citrus trees of different species, including sweet orange, clementine ( disease . InteresCoguvirus)  . Given tCoguvirus.Interestingly, in the phylogenetic trees inferred using RdRp, MP and NP the recently reported WCLaV-1 and WCLaV-2 always cluster together with CiVA and CCGaV. In contrast to CiVA and CCGaV, WCLaV-1 and WCLaV-2 have been reported to have tripartite genome composed exclusively of negative sense RNAs . HoweverPhenuiviridae  the number of the genomic components (at least three in phenuiviruses), (ii) the absence in CiVA and CCGaV  of genes coding for glycoproteins, which are instead encoded by phenuiviruses, (iii) the presence in the putative NP encoded by CiVA and CCGaV  of a N-terminal region with conserved structural elements absent in all the other phenuiviruses , and (iv) a putative MP  phylogenetically related to members of the genus Ophiovirus (family Aspiridae) (Tenuivirus)  , rather uivirus) . Some ofuivirus) . RelatioCoguvirus with the homologous proteins of ophioviruses , which are nsRNA viruses distantly related to bunyaviruses, is worth of note. This finding, showing that the MPs of all putative members of the genus Coguvirus likely have an evolutionary origin differing from those of their respective RdRp and NP, support a modular genome evolution  which, in the phylogenetic trees generated with RdRp and NP proteins, appears to be the closest relative among the known extant viruses . A similar situation is also observed with the plant-infecting tenuiviruses and Wuhàn horsefly virus that infects insects, as shown in the RdRp phylogenetic tree and in previous studies (From an evolutionary point of view, the phylogenetic link of CiVA MP and all the other members of the tentative genus volution . Indeed,volution . Based o studies , thus sh studies .Infectivity of CiVA was ascertained by graft-transmission and its distribution in Southern Italy assessed by a preliminary survey that identified the virus in about 20% of the tested samples. Interestingly, this virus was neither associated with symptoms in the inoculated indicator plants nor with CG symptoms in the field, thus supporting that CiVA and CCGaV differ from a biological point of view. However, due to the limited extension of the survey, the possible association of CiVA with symptoms and, in particular, its relationship with some ancient citrus diseases of unknown origin, such as cristacortis and impietratura , demand FDS, BN, DA, and MS conceived the project. FDS and BN supervised the project and wrote the manuscript. BN, SZ, MM, FDS, and DA performed the experiments and analyzed the data. All authors revised the manuscript.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.


MV is highly sensitive to process conditions, and a substantial fraction of the virus is lost during current purification processes. We therefore conducted forced degradation studies under thermal, pH, chemical, and mechanical stress to determine critical process parameters. We found that MV remained stable following up to five freeze–thaw cycles, but was inactivated during short-term incubation (< 2 h) at temperatures exceeding 35 °C. The infectivity of MV declined at pH < 7, but was not influenced by different buffer systems or the ionic strength/osmolality, except high concentrations of CaCl2 and MgSO4. We observed low shear sensitivity (dependent on the flow rate) caused by the use of a peristaltic pump. For tangential flow filtration, the highest recovery of MV was at a shear rate of ~5700 s−1. Our results confirm that the application of forced degradation studies is important to identify critical process parameters for MV purification. This will be helpful during the early stages of process development, ensuring the recovery of high titers of active MV particles after purification.Oncolytic measles virus (MV) is a promising treatment for cancer but titers of up to 10 One ps per mL ,13, whils per mL ,15,16,17To our knowledge, only we and a research group from Croatia investigate DSP unit operations for oncolytic MV . Our DSPWe therefore conducted FD studies to determine critical parameters that reduce the infectivity of MV and therefore limit the yield of infectious particles during purification. We focused on the DSP-relevant parameters of repetitive freeze–thawing, temperature, composition of the feed (serum-containing and serum-free medium), osmolality, ionic strength, buffers, inorganic salts, additives, Ph, and shear stress. We also determined the isoelectric point (pI) of MV (the point at which the net charge is zero), allowing us to avoid aggregation and to adjust the surface charge in order to optimize DSP steps that rely on electrostatic interactions. The FD studies are a valuable starting point to develop a purification strategy and build the basis of an efficient downstream process.−1)  either in serum-containing medium (DMEM-HG supplemented with 10% (v/v) fetal calf serum, both from Biochrom, Cambridge, UK, and 10 mM HEPES) or serum-free medium  [g in a Heraeus Multifuge X1R . The MV titer was established by measuring the infectivity of the virus using the 50% tissue culture infective dose (TCID50) method [The MV strain MVvac2 GFP (P) was propagated in Vero cells , as previously described . MVvac2 MA, USA) . At the ) method  and conv) method ,24.The clarified MV suspension was divided into 1-mL aliquots in 1.5-mL tubes, which were frozen at –80 °C for at least 2 h. The tubes were then thawed by incubating for 15 min in a water bath at room temperature. This procedure was repeated for 1, 3, 5, and 10 cycles, with some aliquots removed for testing after cycles 1, 3, 5, and 10. The experiments were conducted in triplicates. The infectivity of the virus was determined as above, with the untreated MV suspension as a positive control.We incubated 1-mL aliquots of the clarified MV suspension for 1 or 2 h at 15, 27, 35, 45, 50, 55, and 65 °C using either a WNB 22 water bath  or a Thermomixer compact heating plate . Following the incubation period, the aliquots were immediately frozen at –80 °C to stop any further deterioration. The experiments were conducted in triplicates. The infectivity of the virus was determined as above, with the untreated MV suspension as a positive control.The clarified MV suspension was diluted 1:10 with 20 mM Tris-HCl (pH 7.5–9.0) or 20 mM citrate-phosphate buffer (pH 3.0–7.0) in steps of 0.5 along the pH scale for the used buffers. Experiments were conducted at room temperature and in triplicates. The infectivity of the virus was determined as above immediately after mixing the virus suspension with the buffer or after incubation for 1 h at room temperature followed by freezing at –80 °C. A suspension of MV diluted 1:10 in 20 mM Tris-HCl (pH 7.4) was used as a positive control.The size distribution of the MV particles was investigated by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 device  and a low-volume cuvette . The suspension was stored on ice and mixed with buffer immediately before the initial measurement, and the measurement was repeated after incubation for 1 h at room temperature.−1; 16.8 ± 1.5 mS cm−1) was diluted 1:10 with sucrose or glucose solutions for omsolality measurements, and with sodium chloride or potassium chloride solutions for ionic strength measurements prepared in Milli-Q water . The adjusted ranges were ~40–1100 mOsm kg−1 and ~3–62 mS cm−1. Aliquots were incubated for 24 h at 4 °C before freezing at –80 °C. The ionic strength was measured indirectly by the conductivity, using a SevenGo Duo Pro device . Osmolality was determined by using a Type 6 osmometer . The experiments were conducted in triplicates. The infectivity of the virus was determined as above with an MV suspension, diluted 1:10 in PBS as a positive control.The clarified MV suspension (441 ± 48 mOsm kg2HPO4/NaH2PO4), and citrate-phosphate buffer. In each case, the pH was set to 7.4 to avoid pH-dependent effects. We also tested the following additives, all of which were prepared in 100 mM Tris-HCl (pH 7.4): NaCl (up to 2 M), MgSO4 and CaCl2 (both up to 1.5 M), and L-arginine (up to 0.2 M). For all buffers and additives, clarified MV suspensions were diluted 1:10 in 1.5-mL tubes and were incubated for 24 h at 4 °C before freezing at −80 °C. The experiments were conducted in triplicates. The infectivity of the virus was determined as above with an MV suspension diluted 1:10 in PBS as a positive control.The following buffers were tested at two concentrations (20 mM and 100 mM prepared in Milli-Q water): Tris, HEPES, phosphate buffer (Na−1) for a maximum of 2 h. We took 1-mL samples after 1 and 2 h, which were frozen at −80 °C, and the infectivity of the virus was determined as above, with the untreated suspension as a positive control. When the membrane was fitted, the experiments were carried out in independent triplicates with closed permeate valve and the membrane was cleaned with 1 M NaOH.The effect of shear stress on MV was evaluated using the Sartoflow Slice 200 Benchtop System  fitted with a Tandem 1082 pump head and Phar Med #15 tubing, with and without a Sartorius 100-nm polyether sulfone (PES) membrane (filtration area 0.02 m²). For each run, 50 mL of the clarified virus suspension was loaded in the feed reservoir and pumped at different flow rates , μ is the dynamic viscosity (N m−2 s) and −1). The shear rate is dependent on the geometry of the flow channel. For a tube with a circular flow area, the shear rate is dependent on the volumetric flow rate Q (m3 s−1) and the tube inner radius Shear stress can be calculated using the viscosity of a fluid and the shear rate, as shown in Equation (1)Based on the shear rate, the resulting shear stress can be calculated using Equation (3)B is the width of the flow channel (m) and H is the thickness of the flow channel (m). The shear stress can then be determined using Equation (5)For a rectangular geometry such as the flow channel of a flat sheet membrane, the shear rate can be calculated using Equation (4)n = 3, three replicates). The three replicates were used to calculate the mean and standard deviation. To compare the mean values, a t-test (two sample test with known variances) was performed.Each experiment was carried out three times (p < 0.05) by approximately one order of magnitude , whereas incubation at 55 °C completely inactivated the virus within 30 min (p < 0.05). Our results therefore showed that all DSP steps for the purification of MV can be performed at room temperature without reducing the stability of the virus.The thermosensitivity of MV has been addressed during upstream production by setting a fermentation temperature that balances host cell activity with the retention of MV infectivity . In contp < 0.1 and pH 6.8: p = 0.05 ;The pH is an important DSP parameter, because it can be adjusted to influence the surface charge of MV in the feed stream to facilitate separation based on electrostatic interactions, as long as the virus remains stable. We found that MV was completely inactivated at pH values below 6.0, and that infectivity fell by 0.5 log values within the slightly acidic pH range 6.0–7.0 after 1 h of incubation in contrast to 0 h  compared to the average size. This empirically determined pI was very close to the theoretical value.Particle aggregation is most likely to occur if the DSP buffer matches the pI of the virus, so we investigated this phenomenon by DLS b. The tyThe effects of buffer ionic strength and osmolality were tested to replicate the changing conditions encountered during DSP. We therefore tested the stability of MV in the presence of two salts often used in chromatography elution buffers (NaCl and KCl) and two sugars often used in cell growth medium and/or in formulation buffers (glucose and sucrose). We found that neither variations in ionic strength a nor varDSP typically requires the use of several different buffers, so we also tested the stability of MV in four common buffers at two different concentrations while maintaining the pH at a constant value of 7.4. The four buffers were Tris, HEPES, phosphate, and citrate-phosphate, and each was tested at 20 and 100 mM. We found that MV remained stable in all four buffers at both concentrations, indicating the virus is broadly compatible with the buffer conditions widely used during DSP .2 and MgSO4. The loss of infectivity was strongest in the case of CaCl2, resulting in log reduction values of ~0.6 at 0.75 M (p < 0.05) and ~3.7 at 1.5 M (p < 0.05). The presence of MgSO4 resulted in log reduction values of ~0.8 at 0.75 M (p < 0.05) and ~1.25 at 1.5 M (p < 0.05). NaCl and L-arginine had no effect on the infectivity of MV, even at high concentrations.We also tested a range of additives that are commonly used in DSP and/or that enhance the stability of vaccines and other virus preparations . We obseDSP involves various forms of mechanical stress, which are imposed by the pumps and tubes that move the feed stream through the unit operations and also by the unit operations themselves as the feed stream passes through various columns and membranes. We therefore investigated the effect of shear stress on the infectivity of MV by separately testing the basic DSP setup (pumps and tubes) and the complete system including a tangential flow filtration (TFF) module.−1) and observed only a slight flow-rate-dependent decrease in the infectivity of MV compared to the reference (p = 0.05 for 250 mL min−1 and 2 h). Only laminar flow conditions were present within the range of flow rates we tested (Re = 172–862 and µ (20 °C) = 1.28 ± 0.03 mPas assuming the density of water at 20 °C is ~998 kg m−3). The maximum flow rate of 250 mL min−1 generated a shear rate of 383.8 s−1 , but the loss of active MV particles declined as the flow rate increased from 50 to 150 mL min−1  to determine the effective shear rate . Based oThe average shear stress on MV particles in the rectangular flow channel of flat-sheet membranes can therefore be calculated by including the dynamic viscosity of water, as shown in Equation (7)−1 (50 mL min−1) to 9470 s−1 (250 mL min−1), and the effective average shear stress within the membrane module ranged from 2.4 Pa (1894 s−1) to 12.1 Pa (9470 s−1).The shear rates therefore ranged from 1894 sFreeze–thaw stress is often induced between the upstream and the downstream process or during DSP, and it is important to understand the effect of temperature fluctuations on viruses because protein denaturation occurs at the ice–liquid interface . Rapid f2 TCID50 mL−1), representing a log reduction value of at least 4. This probably reflects the denaturation of protein receptors displayed on the virus envelope [Temperature is always an issue in biopharmaceutical manufacturing, particularly in large-scale processes where the unit operations take much longer (hours) compared to processes in the laboratory. Temperature sensitivity is a critical parameter during upstream production because the optimum temperature must balance the sensitivity of the product against the productivity of the cell line . Former envelope . Previouenvelope , and MV envelope . Other eenvelope ,39. The The pH sensitivity of MV defines the DSP working pH range. We found that MV started to lose infectivity as soon as the buffer pH fell below 7.0, and complete inactivation was observed at pH 6.0, supporting the results of earlier studies ,18. ThesAutographa californica nucleopolyhedrovirus (a baculovirus) has a pI of 5.4, Moloney murine leukemia virus (a retrovirus) has a pI of ~6, and the modified vaccinia Ankara virus (poxvirus) has a pI in the range 2.3–5.0 depending on the antigen it displays [The pH of DSP buffers must also be adjusted to avoid virus aggregation, which is promoted if the buffer pH matches the pI of the virus and its overall surface charge is therefore zero. It is advantageous to know the pI of the virus in order to develop appropriate DSP steps, and accordingly we predicted a value of 6.7–6.8 based on the average pI of the virus surface proteins adjusted according to their relative abundance. A value of ~7 has previously been reported in a technical note, based on isoelectric titration . Interesdisplays . The pI displays ,44. The displays . This ma4 and CaCl2 caused the inactivation of MV. The negative effect of CaCl2 has been reported before [4 has a protective effect on MV at 50 °C [4 has at most a minimal negative effect at room temperature [4 appears to protect MV from heat inactivation but carries an inherent ability to inactivate the virus. NaCl is typically used as an eluent in ion exchange chromatography or as a high-salt diafiltration buffer, and we found that not even high concentrations (up to 2 M) affected the infectivity of MV, in agreement with previous experiments [Buffer osmolality and ionic strength are interrelated factors that can affect the infectivity of viruses, for example, by promoting/inhibiting interactions with charged surfaces and by promoting water flux into or out of the virus particle ,40,45. Wd before , but earat 50 °C , that evat 50 °C , or thatperature . Therefoeriments .We also tested L-arginine, which is used as a stabilizer in vaccine formulations and is known to prevent the aggregation of proteins ,50,51. LMechanical stress during DSP reflects a combination of shear stress caused by pumps that move the feed stream between different unit operations and shear stress that occurs within units such as filter modules, where the liquid is forced through narrow channels. It is important to investigate the effect of such factors on product quality, and indeed this is recommended by ICH guidelines .2 in the tubes, which is very low compared to computational fluid dynamics (CFD) models of other pumps . This s−1, but adsorption was inhibited at higher flow rates. However, increasing the flow rate too much also increased the shear stress, to a maximum of 12.1 N/m2 in the membrane module at a flow rate of 250 mL min−1. We therefore found that a flow rate of 150 mL min−1 was optimal, eliminating non-specific adsorption without significantly reducing the infectivity of the recovered virus. Similar optimal TFF flow rates were also reported for Influenza A virus [TFF with PES membranes is widely used as virus purification method, but the PES substrate tends to adsorb proteins (including those on the surface of enveloped viruses) by non-specific binding, even though PES has a low non-specific binding capacity compared to other materials. Accordingly, we found that the PES membrane adsorbed MV at a low flow rate of 50 mL min A virus  and bacu A virus . Purific2/MgSO4), and shear stress. We found that MV was rapidly and completely inactivated at temperatures exceeding 40 °C, even in the presence of serum, indicating that the purification of MV from the culture medium should begin with a cooling step immediately after harvesting. However, we saw no evidence of inactivation at temperatures below 35 °C in incubations lasting up to 2 h, suggesting that all DSP steps can be carried out at room temperature within this time frame. It is therefore unnecessary to cool the entire DSP to 4 °C. Importantly, our results indicated a narrow DSP working pH range of 7.0–9.0 for MV, reflecting its relatively high pI of ~6.8, ruling out several unit operations that require acidic buffers. We also found that MV is shear sensitive, indicating that each DSP unit operation should be evaluated carefully to determine the conditions that reduce shear stress. For the purification of MV by TFF, the optimal flow rate was 150 mL min−1—this prevented the non-specific adsorption of the virus but limited the shear rate in the membrane module to ~5700 s−1. The identification of these critical parameters will facilitate the development of an effective DSP strategy for MV and supports the use of FD studies as an integral part of early-stage process development for each virus candidate. This approach will ensure product quality and reduce losses during product purification.We have shown that FD studies are useful for the establishment of optimal DSP conditions for the purification of enveloped viruses, specifically MV. FD studies are a starting point to develop a purification strategy and build the basis of an efficient downstream process. The purification of MV is challenging, because many factors contribute towards the inactivation of the virus. The treatment of cancer using oncolytic MV requires a large number of infectious particles, so it is necessary to understand the critical process parameters during the early stages of process development. We found that several process parameters were not critical  and sugar), which offers a degree of flexibility during process development. However, we also identified critical process parameters that significantly affected the infectivity of MV: temperature, pH, the presence of certain salts (CaCl


Nanomaterials have extensive applications in the life sciences and in clinical diagnosis. We have developed magnetic nanoparticles with high dispersibility and extremely low nonspecific binding to biomolecules and have demonstrated their application in chemical biology . Recently, the excellent properties of nanobeads have made possible the development of novel rapid immunoassay systems and high-precision technologies for exosome detection. For immunoassays, we developed a technology to encapsulate a fluorescent substance in magnetic nanobeads. The fluorescent nanobeads allow the rapid detection of a specific antigen in solution or in tissue specimens. Exosomes, which are released into the blood, are expected to become markers for several diseases, including cancer, but techniques for measuring the absolute quantity of exosomes in biological fluids are lacking. By integrating magnetic nanobead technology with an optical disc system, we developed a novel method for precisely quantifying exosomes in human serum with high sensitivity and high linearity without requiring enrichment procedures. This review focuses on the properties of our magnetic nanobeads, the development of novel biosensors using these nanobeads, and their broad practical applications. Magnetic materials have been used as magnetic storage systems, sensors, and shielding systems. Ferrite particles are frequently used as magnetic materials in the life sciences owing to their biocompatibility . For insVarious types of chromatography, such as ion-exchange or gel-filtration chromatography, have been used for protein purification. Affinity chromatography is an effective technique, but efficient purification is limited by the nonspecific binding of proteins, insufficient chemical stability, and physical properties, such as dispersibility. We initially developed affinity latex beads (SG beads) as a carrier for affinity chromatography . As showN,N-dimethylformamide, tetrahydrofuran, ethyl acetate, 1,4-dioxane, toluene, and dichloromethane, without the structural destruction of beads. Thus, FG beads have magnetic properties in addition to superior stability in organic solvents compared with SG beads. Using FG beads, we have identified target proteins for several low molecular weight compounds, including drugs [http://www.magneticnanoparticle.jp/jp/htdocs/index.html).To develop an automated system for affinity purification, we further developed highly functionalized magnetic beads (FG beads) with characteristics superior to those of SG beads. We found that compounds containing carboxyl or thiol groups strongly bind to magnetic iron oxide (ferrite); therefore, we used these compounds as “adapter molecules” for the modification of the ferrite surface. We prepared ferrite particles with diameters of 35–40 nm and coupled them to adapter molecules , 21. Theng drugs , 23, toxng drugs –26, metang drugs –29, and ng drugs . Notablyng drugs –33. Thusng drugs . FG beadIn addition to their use for affinity purification, we applied FG beads in the development of novel biosensors. Biomarkers in biological fluids provide important information regarding disease progression and prognosis –37. VariTo prepare fluorescent particles, fluorescent substances are generally immobilized on the surface of polymer particles by covalent or affinity binding, such as biotin–avidin coupling . However3(TOPO)2), for incorporation into FG beads. Eu(TTA)3(TOPO)2 was mixed with FG beads in an acetone solution for swelling, and the solvent was removed by quick vacuum drying and added to an aqueous solution for shrinking. In this way, we successfully incorporated Eu(TTA)3(TOPO)2 into the polymer layer of FG beads [FG beads are tolerant to several organic solvents; however, we found that the FG bead expands or shrinks according to the type of solvent. In particular, the polymer structure of FG beads swells in acetone and returns to its original configuration in water. The fluorescent substances yttrium (Y), cerium (Ce), europium (Eu), and terbium (Tb), which are rare earth elements, each have peculiar fluorescence wavelengths and can control excitation wavelengths by forming chelate complexes. Eu has been used for labeling with nucleic acids or antibodies . We usedFG beads  2 is shifted by changes in conditions, such as the pH and buffer, and is suppressed by the addition of quenchers, such as ethylenediamine tetraacetic acid (EDTA). However, the dispersion of FF beads in micellar solution with various pH values and EDTA did not affect the fluorescence intensity of FF beads [5 molecules of Eu(TTA)3(TOPO)2 in a single particle, as determined by inductivity coupled plasma optical emission spectroscopy, and this enabled us to observe a single particle using a normal fluorescence microscope. In addition, FF beads did not cause density extinction, as is typical of fluorescent molecules. These findings indicated that FF beads can be used to detect time-resolved fluorescence with high sensitivity and low background noise. Furthermore, several ligands can be conjugated to the surface of the beads because the surface is not covered with fluorescent molecules. On the basis of these properties, we developed an immunoassay using FF beads.The fluorescent FG beads (FF beads) emitted a strong red-orange color under ultraviolet light; when the beads were collected with a magnet, the fluorescence was drawn to the magnet Fig. , right. FF beads . There wIn conventional immunoassays, several processes are time-consuming, such as the antigen–antibody reaction or signal amplification step by an enzymatic reaction. When antibody-labeled fluorescent magnetic beads are collected or concentrated magnetically on an antigen-coated plate, the antigen–antibody reaction will be accelerated. Furthermore, the target antigen can be detected by the direct measurement of the fluorescence of beads. Therefore, we expected FF beads to be suitable for a rapid fluorescence-based immunoassay. Accordingly, using FF beads, we evaluated a sandwich immunoassay system with magnetic collection. Figure Using this system, we attempted to detect brain natriuretic peptide (BNP) , which iWe also verified the magnetically promoted sandwich immunoassay system for the detection of a 33–34-kDa glycoprotein enzyme, prostate-specific antigen (PSA) , which iWe further applied the magnetically promoted immunoreaction system to the analysis of pathological tissue sections . For tisIn addition to the development of immunoassays using FF beads, we recently developed a novel system for quantifying the absolute number of exosomes by integrating the magnetic nanobead technology with the optical disc system. Exosomes are cell-secreted membranous vesicles of around 100-nm diameter , 60. WheSeveral methods have been developed to detect exosomes, and these can be divided into two general categories: particle-counting methods and labeling-detection methods Table . NanoparFor the accurate detection of the number of exosomes expressing specific marker proteins, we have developed a novel device to quantify specific exosomes with high accuracy by combining optical disc technology with nanobead (FG bead) technology, named “ExoCounter” . In thisR2), for the ExoCounter system was superior to that for ELISA or flow cytometry. The ExoCounter system allows quantification of exosomes derived from clinical samples that include colorectal cancer, breast cancer, or lung cancer cells using FG beads conjugated to antibodies against exosomal or cancer-related proteins. In particular, CD9/HER2 double-positive exosomes were observed in the sera of patients with colorectal cancer and breast cancer cells to a greater extent than in those with other malignancies.To evaluate the properties of the ExoCounter system, we analyzed exosomes isolated from the colorectal cancer cell line Colo1. Exosomes were detected only when exosome-specific antibodies were used, indicating that the ExoCounter system has high selectivity Fig. d. In addSince it is difficult to directly quantify exosomes in crude samples, such as sera, by conventional methods, a preenrichment procedure is generally necessary for reliable results. We found that the ExoCounter system can detect the exact number of exosomes in human serum without any enrichment procedures. On the basis of this result, we attempted to detect disease-specific exosomes in the sera of patients with and without cancer obtained from the Biobank Japan Project and the sera of healthy donors obtained from the Tohoku University Tohoku Megabank Organization using the ExoCounter system. The numbers of CD9/HER2 double-positive exosomes were significantly increased in the sera of patients with breast cancer or ovarian cancer compared with other samples, suggesting that HER2-expressing exosomes were specifically released from the lesions of breast or ovarian cancer Fig. e. Despithttp://healthcare.jvc.com/exosome/).In addition to exosomes secreted in cancer patients, other types of specific exosomes are found in patients with various diseases. For instance, exosomes expressing L1 cell adhesion molecule or neural cell adhesion molecule include disease-associated proteins, such as β-amyloid and α-synuclein, and may be related to neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease , 83. In In conclusion, by applying our magnetic nanobead technology, we successfully developed a rapid immunoassay system and a quantification system for the absolute number of exosomes, beginning with affinity purification. The development of these systems was possible because of the excellent properties of the beads, such as their strong paramagnetism, physical stability, low nonspecific adsorption, and uniform dispersibility with a nanosized diameter. Further analyses are expected to lead to the development of novel diagnostic systems based on our high-performance magnetic nanobeads.


Anopheles malaria vector populations.Mosquitoes are colonized by a large but mostly uncharacterized natural virome of RNA viruses, and the composition and distribution of the natural RNA virome may influence the biology and immunity of Anopheles mosquitoes were sampled in malaria endemic forest village sites in Senegal and Cambodia, including Anopheles funestus, Anopheles gambiae group sp., and Anopheles coustani in Senegal, and Anopheles hyrcanus group sp., Anopheles maculatus group sp., and Anopheles dirus in Cambodia. The most frequent mosquito species sampled at both study sites are human malaria vectors. Small and long RNA sequences were depleted of mosquito host sequences, de novo assembled and clustered to yield non-redundant contigs longer than 500 nucleotides. Analysis of the assemblies by sequence similarity to known virus families yielded 115 novel virus sequences, and evidence supports a functional status for at least 86 of the novel viral contigs. Important monophyletic virus clades in the Bunyavirales and Mononegavirales orders were found in these Anopheles from Africa and Asia. The remaining non-host RNA assemblies that were unclassified by sequence similarity to known viruses were clustered by small RNA profiles, and 39 high-quality independent contigs strongly matched a pattern of classic RNAi processing of viral replication intermediates, suggesting they are entirely undescribed viruses. One thousand five hundred sixty-six additional high-quality unclassified contigs matched a pattern consistent with Piwi-interacting RNAs (piRNAs), suggesting that strand-biased piRNAs are generated from the natural virome in Anopheles. To functionally query piRNA effect, we analyzed piRNA expression in Anopheles coluzzii after infection with O’nyong nyong virus (family Togaviridae), and identified two piRNAs that appear to display specifically altered abundance upon arbovirus infection.Anopheles vectors of human malaria in Africa and Asia are ubiquitously colonized by RNA viruses, some of which are monophyletic but clearly diverged from other arthropod viruses. The interplay between small RNA pathways, immunity, and the virome may represent part of the homeostatic mechanism maintaining virome members in a commensal or nonpathogenic state, and could potentially influence vector competence.The online version of this article (10.1186/s12864-019-6034-1) contains supplementary material, which is available to authorized users. Anopheles mosquitoes are the only vectors of human malaria, which kills at least 400,000 persons and causes 200 million cases per year, with the greatest impact concentrated in sub-Saharan Africa and South-East Asia [Anopheles mosquitoes also transmit the alphavirus O’nyong nyong , which is the only arbovirus known to employ Anopheles mosquitoes as the primary vector [Anopheles [Anopheles mosquitoes harbor a diverse natural virome of RNA viruses [ast Asia . In addiy vector . A recen viruses . These vAnopheles viruses have been discovered by isolation from cultured cells exposed to mosquito extract, serology, specific amplification and sequencing, and more recently, deep sequencing and de novo assembly [Anopheles, there are probably many still unknown. Because Anopheles mosquitoes are not thought to be important arbovirus vectors, relatively little research has been done on their viruses. In contrast, culicine mosquitoes in the genera Aedes and Culex transmit multiple arboviruses such as dengue virus  Zika virus , chikungunya virus  and others, but do not transmit human malaria.assembly . AlthougAnopheles mosquitoes captured in forest ecologies in central and northern Cambodia and eastern Senegal. The collection sites are considered disease emergence zones, with high levels of fevers and encephalopathies of unknown origin.Here, we assembled small and long RNA sequences from wild Anopheles viruses because persistent exposure to ISVs, rather than the relatively infrequent exposure to arboviruses such as ONNV, has probably been the main evolutionary pressure shaping Anopheles antiviral immunity. Anopheles resistance mechanisms against arbovirus infection may be particularly efficient, given their low level of known arbovirus transmission despite highly anthropophilic feeding behavior, including on viremic hosts. Nevertheless, ONNV transmission indicates that arbovirus transmission by Anopheles is at least possible, so it is worth asking why transmission by Anopheles is apparently restricted to a single known virus. Identifying the complement of natural viruses inhabiting the Anopheles niche will help clarify the biology underlying the apparent inefficiency of arbovirus transmission by Anopheles, and may suggest new translational tools to decrease arbovirus transmission by the more efficient Aedes and Culex vectors.It is important to study Anopheles sequences from the Barcode of Life COI-5P database . Mosquito species composition of sample pools was estimated using sequences of transcripts from the mitochondrial cytochrome c oxidase subunit 1 (COI) gene, which were compared with  rufipes . In the  vectors –12. Elev vectors , and theOrthomyxoviridae, Rhabdoviridae and Phenuiviridae, ssRNA positive-strand viruses of the families Virgaviridae, Flaviviridae and Bromoviridae, dsRNA viruses of the family Reoviridae and multiple unclassified viruses of both ssRNA and dsRNA types   formed a monophyletic group that includes Xincheng Mosquito Virus and Shungao Fly Virus. Finally, 10 novel virus assemblies  formed a monophyletic group that includes Beaumont Virus and a virus from Culex tritaeniorhynchus within the Rhabdoviridae family  gene annotated in the 115 virus sequences, along with RdRP peptide sequences of related virus sequences from GenBank. This allowed the placement of 44 of the 115 assembled viruses in phylogenetic trees, revealing clusters of highly related viruses in the wild es) Fig. . In addiily Fig. a.Fig. 2We characterized the degree of completeness of the virus assemblies to determine whether they contain full or almost full viral genome sequences, and whether predicted peptides are likely to be functional. Many of the viral contigs are too diverged from each other and from reference viruses in the phylogenetic tree to align informatively at the nucleotide level, and reliable sequence comparisons were only possible at the peptide sequence level. We translated nucleotide sequences for the novel viral contigs and compared them to the translated nucleotide database using TBLASTX. An example of this analysis is shown for viral contigs homologous to Xincheng virus Fig. b. CloselThis combined nucleotide and peptide-based analysis was applied to the 115 novel viral contigs. A total of 195 open reading frames (ORFs) were annotated among the 115 viral contigs, an average of 1.7 ORFs per viral contig . Among these, the majority were associated with Cluster 4 virus assemblies (1219 unclassified contigs) and to less extent with Cluster 1 (309 unclassified contigs). Both clusters were characterized by a strong bias towards reads from a single strand (positive for Cluster 1 and negative for Cluster 4).To evaluate how specific these latter profiles of 1219 and 309 contigs are for virus-related sequences, we designed a reconstruction control experiment using the same small RNA size profiling and clustering analysis as above, but instead using 669 RNA contigs known to map to the mosquito reference assembly, thus strictly of host origin. As above, contigs with at least 100 small RNA sequence reads were used. Five hundred sixty-one of these mosquito contigs could be grouped with small RNA size profiles of virus contigs (Spearman correlation> 0.9), most of them (98.21%) with Cluster 4 (78.6%) and Cluster 1 (19.6%) profiles.Aedes mosquito cells, piRNAs can probably mediate responses to arboviruses or ISVs [Anopheles mosquitoes express annotated piRNAs from genomic piRNA clusters [Drosophila and other arthropods [Aedes, which are largely ISV-derived, mainly map only to the virus strand antisense to the viral ORF [piRNAs are endogenous small noncoding RNAs that ensure genome stability by protecting it from invasive transposable elements such as retrotransposons and repetitive or selfish sequences . In addi or ISVs –23. Anopclusters , 25. Thethropods , 26. Notiral ORF .An. coluzzii, about half of annotated piRNAs display a strong or exclusive strand bias [Drosophila. Until the current study, Anopheles piRNAs have not previously been examined for relatedness to ISVs. Overall, these small RNA results are probably most consistent with an interpretation that RNA profile Cluster 1 and Cluster 4 detect strand-biased piRNAs derived from the natural ISV virome of wild Anopheles. On that interpretation, the above 561 contigs mapping to host that share the Cluster 1 and Cluster 4 RNA profiles are most likely also piRNAs, but instead derived from endogenous host RNA templates. Previous results showed that most An. coluzzii piRNAs are derived from long-terminal repeat retrotransposons and DNA transposable elements [Anopheles piRNA production, and indicate that further work is warranted on Anopheles piRNA. Our results also suggest the possibility that piRNAs may be involved in Anopheles response to viruses, a phenomenon found for only Aedes among a wide range of arthropods [Anopheles were not tested.In and bias , which ielements . Our curthropods , but AnoAnopheles piRNAs could be involved in response or protection to virus infection has not been previously examined or reported to our knowledge. To examine this possibility, we challenged An. coluzzii mosquitoes with the alphavirus ONNV by feeding an infectious bloodmeal, and sequenced small RNAs expressed during the primary infection at 3 d post-bloodmeal. Mosquitoes fed a normal bloodmeal were used as the control condition.The potential that An. coluzzii candidate piRNA genes located in 187 genomic piRNA clusters [p-value = 5e-5, adjusted p-value = 6.7e-3, reference genome sequence coordinates AgamP4:UNKN:19043685:19043716; and locus XLOC_012762, Cuffdiff test statistic = 2.39, p-value = 9.5e-4, adjusted p-value = 0.046, reference genome sequence coordinates AgamP4:UNKN:13088289:13088321).The small RNAs were mapped to previously annotated clusters , and expp-value< 2.2e-16; and locus XLOC_012762, Chi-squared = 75.78, df = 1, p-value< 2.2e-16).Differential abundance was confirmed by quantifying small RNAs mapping to the two candidate piRNAs using the Integrative Genomics Viewer. By this test also, both candidate piRNAs displayed lower normalized read counts in ONNV infected samples as compared to uninfected controls  and Cheav Rov village, Kampong Chhnang province (sample pools Cam5–01 and Cam10–01). The majority of inhabitants are engaged in forest-related activities  and may spend the night in forest plots during the harvest period. Vegetation varies from evergreen forest to scattered forest, and the dry season typically runs from November to May and the rainy season from June to October. In Senegal, sampling sites were located in the department of Kedougou in southeastern Senegal. Kedougou lies in a transition zone between dry tropical forest and the savanna belt, and includes the richest and most diverse fauna of Senegal. Recent arbovirus outbreaks include CHIKV in 2009–2010, yellow fever virus in 2011, Zika virus in 2010, and DENV in 2008–2009.Anopheles spp. at the collection site (non-Anopheles were not retained) were immediately transferred into RNAlater stabilization reagent kept at 4 °C, and then returned to the laboratory and stored at − 80 °C until RNA extraction.Permission to collect mosquitoes was obtained by Institut Pasteur Cambodia from authorities of Ratanakiri and Kampong Chhnang, and by Institut Pasteur Dakar from authorities of Kedougou. Wild mosquitoes visually identified as www.fasteris.com). Long RNA libraries from the eight mosquito pools were made from total RNA depleted of ribosomal RNA by treatment with RiboZero . Libraries were multiplexed and sequenced on a single lane of the Illumina HiSeq 2500 platform  by the paired-ends method (2 × 125 bp), generating on average 36 million high-quality read pairs per library. Small RNA libraries with insert size 18–30 nt were generated from the same eight mosquito pools as above, multiplexed and sequenced in duplicate  in two lanes of the Illumina HiSeq2500 platform  by the single-end method (1 × 50 bp) generating on average 34 million reads of high-quality small RNA reads per library.Total RNA was extracted from four pools of mosquitoes from each of Senegal and Cambodia  using the Nucleospin RNA kit (Macherey-Nagel) following the supplied protocol. Library preparation and sequencing steps were performed by Fasteris  extracted from the Barcode of Life database [Anopheles species with at least 100 reads and 1% frequency in the sample pool were represented as pie charts using the ggplots2 R package.Quality-filtered long RNA read pairs were mapped with SortMeRNA  against database . 98% ideProcessed reads from each country  were combined and de novo assembled using different strategies for long and small RNA libraries. Small RNA reads were assembled using the Velvet/Oases pipeline  using a Contigs produced by parallel assembly of Cambodia and Senegal processed reads were filtered in order to remove trans-self chimeric sequences using custom shell scripts, and the resulting contigs were merged with cd-hit-est   and double strand RNA (dsRNA) viruses using MAFFT v7.055b with the E-INS-i algorithm . IndepenIn order to recruit contigs of potential viral origin from the pool of unclassified transcripts, we use the approach of Aguiar . This apAn. coluzzii with ONNV, library preparations, and sequencing were described [An. gambiae PEST AgamP4 genome assembly using STAR version 2.5 with default parameters [An. coluzzii were designated in [Infection of escribed . Brieflyrameters . The resrameters , publiclnated in  as eitheAn. gambiae PEST AgamP4 genome assembly in the Integrative Genomics Viewer version 2.5 [Independent confirmation of the Cuffdiff analysis was obtained using BAM and BAI indices generated using Bowtie 2 version 2.3.0 from the above small RNA sequence files of ONNV infected and uninfected samples. These generated files were analyzed with the sion 2.5 . We quansion 2.5 .Additional file 1:Table S1.Anopheles mosquito taxa represented in the collections from Senegal and Cambodia, as detected by comparison to Anopheles sequences from the Barcode of Life COI-5P database. Data corresponds to pie charts of Anopheles taxa by country and sample pool depicted in Fig. Additional file 2:Table S2. List of 115 assembled virus contigs displaying sequence similarity to known virus families. (XLSX 11 kb)Additional file 3:Table S3. Predicted open reading frames, peptide annotation and functional status of 115 assembled virus contigs displaying sequence similarity to known virus families. (XLSX 56 kb)Additional file 4:Figure S1. Small RNA size profiles (A) and coverage profiles (B) of 15 novel virus assemblies with classic RNAi processing pattern. Virus assemblies shown are in Fig. Additional file 5:Figure S2. Small RNA size profiles of high-quality assemblies left unclassified by sequence similarity grouping. Thirty-nine unclassified contigs that meet the same quality criteria as the 115 classified contigs (non-redundant and > 500 nucleotides), and with at least 100 small RNA sequence reads. The 39 contigs display strong association by small RNA profile Cluster 2 and Cluster 3 Additional file 7:Classified Virus Sequences. Sequence assemblies and annotations for the final 115 novel virus contigs non-redundant contigs > 500 nucleotides, classified by sequence similarity to known viruses. In EMBL flat sequence file format. (ZIP 208 kb)Additional file 8:Unclassified Virus Sequences. Sequence assemblies for 39 non-redundant contigs > 500 nucleotides, unclassified by sequence similarity to known viruses. The small RNA profile matched a pattern of classic RNAi processing of viral replication intermediates, suggesting they are undescribed viruses. In fasta text sequence file format. (FASTA 38 kb)


The nine human herpesviruses are some of the most ubiquitous pathogens worldwide, causing life-long latent infection in a variety of different tissues. Human herpesviruses range from mild childhood infections to known tumour viruses and ‘trolls of transplantation’. Epstein-Barr virus was the first human herpesvirus to have its whole genome sequenced; GenBank now includes thousands of herpesvirus genomes. This review will cover some of the recent advances in our understanding of herpesvirus diversity and disease that have come about as a result of new sequencing technologies, such as target enrichment and long-read sequencing. It will also look at the problem of resolving mixed-genotype infections, whether with short or long-read sequencing methods; and conclude with some thoughts on the future of the field as herpesvirus population genomics becomes a reality. There are nine currently recognised human herpesviruses. All share the classic herpesvirus life cycle of causing primary infection  before establishing latency within particular cell subsets, awaiting periods of reduced host immunity to reactivate and go on to infect new hosts. Humans carry herpesviruses in three subfamilies: the alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae. Within and between these subfamilies, the human herpesviruses have tropisms for a range of tissues  and have genomes of varying sizes . Some of the human herpesviruses have strong associations with specific diseases , while others are less clearly linked to disease in adulthood. All of the human herpesviruses were sequenced at the whole-genome level over a period of around 15 years , and folThis review is not intended to be an exhaustive history of the sequencing of herpesviruses or a detailed picture of the entire field of herpesvirus genomics. Instead, I hope to provide vignettes of where genomics for each virus currently stands, and interesting future directions herpesvirus genomics may take.The human herpesviruses range in length from ~125kbp (varicella-zoster virus (VZV)) to ~235kbp ). This makes their genomes much less amenable to over-lapping PCR or rolling-circle amplification , which aAfter the sequencing and publication of the reference sequences of the nine human herpesviruses, there was gradual accrual of further genome sequences for the next ~25 years. In 2011, there were in total 29 whole human herpesvirus genomes in GenBank . The B95I argue that the changes to sequencing herpesvirus genomes that have transformed this area of science can be related to three factors over the last decade: the change from Sanger (dideoxy) to high-throughput sequencing (HTS), most notably Illumina short-read technology; the use of target-enrichment technologies ; and theThese changes have been especially important for sequencing directly from clinical samples. In cultured virus isolates, it is possible to generate high concentrations and relatively large volumes of viral genome material for sequencing, which can be further amplified in over-lapping PCR amplicons  or which can provide material to be sequenced directly. Both approaches increase the signal  to noise (host genome) ratio of a herpesvirus sequencing project. However, for some herpesviruses there are well-known problems of rapid genomic adaptation to culture, most notably in HCMV . This isTarget enrichment technology such as IDT, MyBaits or SureSelect DNA and RNA oligonucleotide baits have driven our ability to sequence from clinical samples, avoiding the problem of culture artefacts in sequence data . By bindThe final change which has favoured herpesvirologists  is the falling cost of sequencing itself . The lowHumans carry two simplex viruses, herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2), unlike our closest living relatives chimpanzees, bonobos and gorillas which have a single, oral simplex virus species each . Both huHerpes simplex virus 1 is the ancestral human HSV ,29. OrigThe HSV-1 strain which provided the reference sequence was isolated in 1972, and its genome was sequenced via Sanger sequencing of plasmids, later supplemented by Illumina sequencing of viral DNA . Since tHigh-throughput sequencing has allowed researchers to elucidate the histories of commonly used HSV-1 strains KOS63 and KOS79, which were isolated independently from the same individual who had experienced a dual-strain infection . HSV-1 gHSV-1 sequencing studies have also shed light on the debate over direct metagenomic versus culture enriched versus target enriched sequencing discussed earlier in this paper. Low-passage cultured HSV-1 isolates were compared to paired virus from the same patient sequenced directly from clinical material. Cultured virus isolates underwent minimal change, which has allayed worries that HSV-1 undergoes significant and rapid changes in cell culture at low passage numbers .Appropriately for the virus which is classically thought of as the cause of genital herpes, even if that epidemiological pattern is changing, the reference sequence for HSV-2 (strain HG52) was isolated from a woman with genital herpes. The virus was sequenced from cloned plasmids, with later Illumina sequencing (as with the HSV-1 RefSeq) .HSV-2 was originally a chimpanzee oral pathogen than jumped the species barrier around 1.6 million years ago , perhapsVaricella-zoster virus is typically a childhood infection, spread by exposure to virus particles through coughing, skin to skin contact or aerosolized virus particles from the skin rashes caused by primary infection  or reactivation  . There hVZV has relatively low genomic diversity compared to other herpesviruses ,47, whicHuman cytomegalovirus has a rich sequencing history. Following the plasmid-library sequencing of AD169, sequencing methods have moved through bacterial artificial chromosome (BAC) and fragment cloning ,52, sequThe state of the HCMV sequencing landscape was recently and extensively reviewed elsewhere . The fieThe virus sequences generated to answer clinical questions can also be used to tackle questions such as the relationship between mixed-genotype infection, its role in drug resistance ,61 and iDespite their similar names, human herpesviruses 6A (HHV6A) and 6B (HHV6B) are sufficiently different at the level of sequence similarity, immunological reactivity and perhaps disease association to be recognised as distinct species . Both 6AComparisons between these viruses, including both iciHHV6A/B and naturally circulating strains, have suggested that 6A is three-fold more diverse than 6B . It remaHHV6A genomics is slowly catching up to fellow betaherpesvirus HCMV . The iniRoseolovirus diversity and history. HHV6B is most closely associated with the childhood disease roseola infantum (exanthema subitem or ‘sixth disease’) )Streptococcus pneumoniae [Where large numbers of herpesvirus genomes are available, as for EBV, HCMV and HSV-1 and -2, virologists increasingly have the ability to compare patterns of diversity and associations of particular genotypes with specific populations at a genomic scale . Genome-eumoniae , hepatiteumoniae  and humaHuman herpesviruses are also finally entering the ancient DNA era , with thFinally, it is hoped that the geographic representation of particular regions, both in the origin of sequences and the location of research groups with the technology and expertise to conduct herpesvirus sequencing, will continue to expand. Neither patients nor researchers can afford for whole continents to miss out on the insights which genomics can contribute .


The experience with a diagnostic technology based on rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) analyses, and direct or deep sequencing (Circomics) over the past 15 years is surveyed for the plant infecting geminiviruses, nanoviruses and associated satellite DNAs, which have had increasing impact on agricultural and horticultural losses due to global transportation and recombination-aided diversification. Current state methods for quarantine measures are described to identify individual DNA components with great accuracy and to recognize the crucial role of the molecular viral population structure as an important factor for sustainable plant protection. Only inAlphasatellitidae now including two subfamilies  [The molecular biology of geminiviruses, nanoviruses, and their satellites has been reviewed previously ,13,14,15litinae) .A literature survey on the Web of Science for the years 2004 to 2018 yielded 146 papers in which RCA was mentioned explicitly for geminiviruses, mastreviruses, begomoviruses, curtoviruses, and nanoviruses ,159,160.TM cards have been used to avoid any risk of virus spread [In particular for tropical and subtropical countries, where most geminiviruses and nanoviruses are pests, an important prerequisite for reliable virus detection is the collection of plant material and the preservation of its DNA. The simplest way to do this is by harvesting fresh young leaves without any sign of necrosis, if possible, and placing them immediately between two sheets of blotting paper to allow for slow drying at room temperature. The samples are stored dry at room temperature in the dark to avoid hydrolytic DNA base deamination and oxidation and they may be analyzed several years later for RCA/RFLP without any loss of resolution ,26,72,96s spread , but theClipping off a small disk from a dried leaf, e.g., with an Eppendorf tube lid, yields sufficient material for several RCA/RFLP analyses. Extraction of nucleic acids based on the cationic detergent cetyltrimethylammonium bromide (CTAB) is reliable for most plants, even those which are technically recalcitrant due to mucilage and other secondary plant components . IncludiFor an optimal usage of the RCA reaction, it is necessary to consider some basic prerequisites and peculiarities of geminiviruses and nanoviruses . In a leThe primary product of RCA is a complex mixture of ssDNA and dsDNA, building a huge network due to the high processivity and strand-displacement activity of the Phi 29 polymerase . MultiplHpaII and Sau3A. For a genome size of 2500–5000 nt, enzymes that recognize 4 nt yield a sufficient number of fragments to differentiate genomic components, as well as different viruses in general [Depending on the virus to be detected, appropriate restriction enzymes have been used, mostly  general . The res general ,184. Slo general . In suchThe power of resolution may be enhanced by Southern blot hybridization using component specific probes, which also visualize the multitude of variants in the pool of viral DNAs and/or incompletely cut RCA products ,96.Begomovirus, Curtovirus, Mastrevirus)  .MluI) (A bias towards an enhanced T frequency over the expected frequency has to be noted for begomoviruses (30.1%) and curtoviruses (31.4%), possibly the result of a high deamination rate leading to C > T transitions, as observed experimentally ,186. ConMluI) . This seMluI) . The disMluI)  is mostl2 = 0.991) and led to an error for fragment size determinations below 3%  values) can be obtained in the central part of a gel, but for the complete gel, a sigmoid curve gives a better fit ,72 Figu. The invbelow 3%  in a larbelow 3% .The appropriate standard fragments in each gel are absolutely necessary to reach this degree of accuracy. The use of RCA/RFLP products of sequenced viruses for comparison has been found superior to other marker restriction fragment mixtures. If a specific virus is suspected in the plant, the RCA products of this virus retrieved from cloned material may be sufficient and easy to compare for calibrating the analysis. If unknown viruses have to be compared to the electronic database, a broad range of fragment sizes for the standards is desirable. The RCA products of plasmids may be useful, or Abutilon mosaic virus (AbMV) DNA may be chosen, since this virus is available worldwide in botanical and market gardens . AbMV isAn incorrect assignment of a band in the gel to a fragment size is tested most easily by way of graphical evaluation . The whoIf digestion with the restriction enzyme is complete for each component, the band intensities of the proper genomic fragments should follow a consistent molar series for each component. This can be graphically checked by scanning a lane by ImageJ software and judging the peak heights decline. Double and multiple bands for fragments with similar molecular weight can be identified, as well as products from D-DNAs, which can also be identified if samples from separate plants are compared, since they are usually different in different individual plants ,54,105. a priori knowledge. When viruses are imported into a new country, such as tomato yellow leaf curl virus (TYLCV) in the Americas [The major advantage of RCA, in comparison to PCR and immunodetection, is its ability to detect circular DNA molecules without any Americas  or NorthAmericas  and squaAmericas , they caAmericas ,96,190. Americas ,43. ScreAlthough the plain RCA products can be generally regarded as safe with reference to natural spread, it is possible to raise infectious viruses by biolistic inoculation of the RCA-amplified DNA to test plants under laboratory conditions ,184,186.Sau3A (or MluI) and insertion into BamHI (or EcoRI) sites of the plasmids is generally applicable for all genomic components without a need to search for proper restriction enzymes. The versatile small agrobacterial plasmid pGreen [in planta delivery by stem inoculation or leaf infiltration with many viruses, aSats and plants [If infectious DNA is to be cloned in bacterial plasmids, RCA products are digested by restriction enzymes at limited concentration or incubation time ,57. Fragd pGreen  has beend plants ,124,179,If some sequence information is known to deduce a specific primer, and if the plant under investigation is infected by a single virus, direct sequencing of the RCA product is the technique of choice to obtain the whole sequence of the virus components by primer walking ,184, in For practical purposes, such as the identification of known viruses, direct sequencing of RCA products across the short segment of the most variable intergenic region in the virus component will be satisfactory for most applications. With the decreasing costs of commercial sequencing, this approach will outperform the RFLP analyses for cases of known conserved primer sequences, e.g., within the coat protein or Rep gene. The information thus obtained is much more precise and can be easily compared to the electronic databases.Arabidopsis thaliana to determine the shifts in the population structure and the influence of single host genes on the replication of the virus [−4 to 10−3 were noticed in all these data sets, with preferences for deamination and oxidative changes of the nucleobases. In addition, high levels of D-DNAs were detected which had been overlooked before, since they form a heterogeneous population not represented in discrete bands in the RFLP analyses, but in background smears only. Raising elevated levels of DI-DNAs may be important as a means for cross-protection. However, D-DNAs may have ambivalent effects on symptoms and viral multiplication [For fundamental research, Circomics extends the view to the complexity of DNA virus variation and the unexpected molecules generated in response to different plants and plant lines. A better understanding of the viral population structure will have many implications for resistance breeding, irrespectively of whether a conventional method or genetic engineering is used. It is important to note that viruses may be regarded as highly polyploid entities. An error-prone lifestyle is, therefore, not only possible because wild type (wt) versions can complement mutants in a cell, but it is also advantageous for the viral evolution, because a reservoir of potential survivors for changing environments is created before it is needed. Following this basic concept, begomoviruses  and their aSats were investigated in various T-DNA insertion lines of he virus ,180,186.he virus , or whenlication ,105, as E. coli as a fusion protein [In spite of some progress in engineering resistance in plants against geminiviruses and nanoviruses, quarantine measures, including early detection of invading viruses, are still the method of choice to combat epidemics worldwide. Educational training of farmers and plant protection staff to enable early eradication remains the key remedy. The RCA technology described here is simple and can be applied cost-effectively, in particular for tropical and subtropical countries with low budgets. Although commercial kits may be still too expensive for many applications in agriculture in these countries, an alternative exists, since the core enzyme, the Phi29 DNA polymerase, can be easily expressed in  protein . For mor protein .


Hepatitis delta virus (HDV) is the smallest known RNA virus, encoding a single protein. Until recently, HDV had only been identified in humans, where it is strongly associated with co-infection with hepatitis B virus (HBV). However, the recent discovery of HDV-like viruses in metagenomic samples from birds and snakes suggests that this virus has a far longer evolutionary history. Herein, using additional meta-transcriptomic data, we show that highly divergent HDV-like viruses are also present in fish, amphibians, and invertebrates, with PCR and Sanger sequencing confirming the presence of the invertebrate HDV-like viruses. Notably, the novel viruses identified here share genomic features characteristic of HDV, such as a circular genome of only approximately 1.7 kb in length, and self-complementary, unbranched rod-like structures. Coiled-coil domains, leucine zippers, conserved residues with essential biological functions, and isoelectronic points similar to those in the human hepatitis delta virus antigens (HDAgs) were also identified in the putative non-human viruses. Importantly, none of these novel HDV-like viruses were associated with hepadnavirus infection, supporting the idea that the HDV–HBV association may be specific to humans. Collectively, these data not only broaden our understanding of the diversity and host range of HDV, but also shed light on its origin and evolutionary history. Deltavirus, is the smallest known RNA virus and results in chronic or fulminant hepatitis in humans co-infected with hepatitis B virus (HBV). The HDV genome has a set of distinctive features including a covalently closed, circular, single negative-stranded RNA genome that forms a viroid-like self-complementary, unbranched rod-like structure of about 1.7 kb in length (Hepatitis delta virus (HDV), a member of the genus n length . An impon length . The virin vivo transmission. This was traditionally thought to be provided by the HBV envelope protein  were re-screened against the NCBI non-redundant protein (nr) database using Diamond blastx at an e-value cut-off of 1e-5 , based on the similarities to previously described HDV genomes. To further validate our HDAg predictions, we compared sequences using the protein domain search tools, including HHpred  and Phyre2  of HDV-like contigs were predicted using Geneious R11 . These ORFs were first annotated using a reverse PSI-BLAST  search ag HHpred  . Five sets of PCR primers  across diverse animal hosts, screening for the presence of divergent HDV-like agents. These data were generated from previous (Schedorhinotermes intermedius), which we designate the ‘termite HDV-like’ agent; (2) a mixture of fish —‘fish HDV-like’; and (3) two amphibians, the Asiatic toad (Bufo gargarizans)—‘toad HDV-like’, and the Chinese fire belly newt  – ‘newt HDV-like’. The RNA sequencing results of the rRNA depleted libraries from newt, toad, fish, and termite libraries resulted in 5,545,902, 4,266,161, 11,064,877, 68,094,815, 366,319,352, and 431,345,357 paired reads, which were assembled into contigs, ranging from 9,687 to 639,393 contigs per library  signal sequence (5′-AAUAAA-3′) upstream of each putative HDAg. In addition, we utilized alternative homology-based tools (HHpred and Phrye2) to define protein domains. Similar to the reverse PSI-BLAST, all the top scoring hits using tools for our putative HDAgs matched the human delta antigen protein. The HHpred results showed a probability over 94 per cent for the HDAg . In the case of Phrye2, the putative HDAgs all hit template c12a9B as the best-match, with more than 89 per cent confidence, denoting the oligomerization domain of HDAg . HoweverPhylogenetic analysis of the HDAgs returned a tree topology that is broadly congruent with the evolutionary relationships of the hosts, as expected if there were long-term virus-host co-divergence . HoweverPost-translational modifications of HDAgs include lysine acetylation, arginine methylation, serine, and threonine phosphorylation, which are important for modulating HDV function and the viral cycle , and theThe predicted coiled-coil domains of putative HDAgs, that facilitate multimerization and replication, were found at N-terminal sites overlapping with the coiled-coil region of known HDAgs . The putThe complete tHDV genome from termite libraries, which is the most divergent HDV-like agent in this study, was validated by the amplification of five overlapping PCRs products across eight termite RNA libraries . The fivStrikingly, none of these newly described HDV-like agents were associated with co-infecting hepadnaviruses, which has been regarded as central to the biology of human HDV. Instead, a number of other viruses were present in the relevant sequencing libraries . These iUsing a meta-transcriptomic approach we describe novel HDV-like agents from invertebrates and vertebrates. These results build on the recent discovery of bird and snake HDVs that lacked any evidence for co-infecting hepadnaviruses, but were infected with influenza and arenaviruses, respectively . It therWhile all the novel HDV-like circular agents identified here are highly divergent compared to existing human HDVs and recently identified bird and snake viruses at the level of primary sequence, they retain important HDV-defining genomic features including a small circular genome and unbranched rod-like RNA structures. Similarly, despite their sequence divergence, conserved HDAg domains are readily identifiable and the putative HDAgs also demonstrate similar promoter structures, amino acid properties, and conserved post-translational residues.Our knowledge of HDV has been limited to human infections and its interaction with HBV. Consequently, it has been commonly assumed that the evolutionary origins of HDV lie with humans. For example, it has been proposed that HDV may have originated as an escaped human gene  and that