Vaccinium corymbosum L.) is cultivated near wild V. corymbosum as well as closely related species in Florida, USA. The expansion of blueberry cultivation into new areas in Florida and deployment of new cultivars containing viruses can potentially increase the diversity of viruses in wild and cultivated V. corymbosum. In this study, viral diversity in wild and cultivated blueberries (V. corymbosum) is described using a metagenomic approach. RNA viromes from V. corymbosum plants collected from six locations (two cultivated and four wild) in North Central Florida were generated by high throughput sequencing (HTS) and analyzed using a bioinformatic analysis pipeline. De novo assembled contigs obtained from viromes of both commercial and wild sites produced sequences with similarities to plant virus species from a diverse range of families . In addition, this study has enabled the identification of blueberry latent virus (BlLV) and blueberry mosaic associated ophiovirus (BlMaV) for the first time in Florida, as well as a tentative novel tepovirus (blueberry virus T) (BlVT) in blueberry. To the best of our knowledge, this is the first study that compares viral diversity in wild and cultivated blueberry using a metagenomic approach.Southern highbush blueberry (interspecific hybrids of  Vaccinium spp. and the adjacent Florida’s blueberry production areas could serve as a reservoir for a diverse assemblage of viruses in these species, thus causing spillover and spillback between the wild and cultivated hosts [Vaccinium spp. is the lack of virus screening prior to the use of native, wild blueberries in the development of new SHB cultivars.The identification of new plant viruses using viral metagenomics has been described in numerous publications ,2. Whileed hosts . AnotherBlunervirus) [Soymovirus) [Vaccinium corymbosum in Florida using a metagenomic approach; and (2) identify and characterize novel and known viral sequences in the blueberry viromes. We showed that analysis of viromes determined from double-stranded RNA (dsRNA)-enriched samples using metagenomics is an excellent tool to explore viral diversity of RNA viruses of all genome types ). Metagenomic analysis of the RNA viromes has shown that viral diversity in the wild V. corymbosum is found to be greater than that in the cultivated species, with considerable overlap and unique genera represented in both species. This study has also led to the discovery of two known blueberry viruses, BlLV  and BlMaV (genus Ophiovirus), that are new to Florida, as well as a tentative novel tepovirus (BlVT) that has never been reported in blueberry. In addition, this research is the first to show that BlMaV can be found in wild highbush, V. corymbosum.Fifteen species of viruses have been recorded in highbush, lowbush, and rabbiteye blueberries around the world, including RNA and DNA viruses from eight known and two unassigned genera . Only twervirus)  and bluemovirus) . Since vn = 120) with and without virus-like symptoms were randomly collected from wild and cultivated blueberries  in central Florida. The wild blueberry samples were collected from four locations in Florida, USA: (1) O’leno State Park, High Springs, a wild site not adjacent to commercial blueberries; (2) Morning Side Nature Center, Gainesville, a wild site adjacent to residences; (3) Interlachen, a wild site neighboring commercial blueberry production; and (4) Island Grove, another wild site neighboring commercial blueberry production  [−5. Scaffolds with similarity to plant viruses were organized by family, genus, and species according to the 2016 Virus Taxonomy Release of the International Committee on Taxonomy of Viruses (ICTV) website  (Accessed on 10 March 2017) for approved virus species. Scaffolds with hits to virus species not yet approved by ICTV were assigned to its corresponding genus and family based on the information provided in the NCBI Taxonomy Database (https://www.ncbi.nlm.nih.gov/taxonomy) (Accessed on 25 April 2017). Scaffolds with sufficient genome coverage were directly used for genome analysis or subjected to another round of assembly using the Geneious assembler built in Geneious 9.1.6 to produce longer scaffolds. These scaffolds were mapped against the corresponding reference genome using Geneious mapper in Geneious 9.1.6 to observe the genome coverage. The scaffolds and the reference sequences were compared by alignment to predict the open reading frames (ORFs) and coding sequences (CDS). Reads were aligned to published viral sequences and scaffolds in a reference-based mapping approach using Bowtie2 to obtain complete viral genomes for known and novel viruses, respectively [Reads from the RNA libraries were analyzed according to the virome analysis pipeline illustrated in dex.cgi)   . Multiplmp) region, and the 3′ UTR was used to detect the virus in the RT-PCR using pooled dsRNA extracts of V. corymbosum  in a reaction mixture containing 2.5 mM MgCl2, 0.5 mM dNTP mix, 0.5 µM of each forward and reverse primer, and 0.625 U Taq DNA Polymerase  using the following cycling conditions: initial denaturation at 94 °C for 3 min, 35 cycles of 94 °C (30 s), 62 °C (45 s), and 72 °C (1 min 10 s), and a final extension at 72 °C for 10 min. The RT-PCR reaction was carried out in the Eppendorf AG 22,331 Hamburg Mastercycler . Amplified fragments of the PCR products (~1 kB) were visualized on 0.8% agarose gel and used as an inset in cloning reaction with pGem-T easy cloning kit . The clones were sequenced by the Sanger method at Eurofins MWG Operon LLC . The presence of the novel tepovirus in twenty V. corymbosum samples in five cultivars collected from Island Grove was screened using the same RT-PCR assay described above.A set of primers enabling the amplification of the whole CP region of the de novo assembled complete genome of the putative novel tepovirus was designed based on the movement protein gene  and 43 (0.007%) putative plant virus scaffolds with matches to known viral sequences from the wild and cultivated V. corymbosum, respectively. The percentage of putative plant virus scaffolds with sequence similarity to known viruses was calculated from the total scaffolds in both wild and cultivated viromes varied between locations, ranging from 0.60 to 1.86%. The highest and lowest number of putative plant virus scaffolds obtained from the viromes of wild V. corymbosum were identified from Interlachen and Island Grove, respectively. The viromes of cultivated V. corymbosum, however, contained a similar number of putative plant virus scaffolds.The RNA viromes generated from wild and cultivated of reads . De novoV. corymbosum viromes produced sequence similarity to plant virus species from a total of 25 viral genera, representing a total of 17 viral families , Gainesville (9), and Island Grove (2)  b. The hirymbosum . Each viV. corymbosum from Interlachen compared to Island Grove produced a higher number of scaffolds with matches to plant virus sequences with 7 and 5 viral genera, respectively  were identified in viromes of both wild and cultivated V. corymbosum collected from Interlachen. Scaffolds with the highest sequence similarity to unclassified viruses and ophioviruses were identified in viromes of both wild and cultivated V. corymbosum from Island Grove. The viromes of V. corymbosum collected from Interlachen at the wild site produced scaffolds with similarity to virus species from ten genera, while the cultivated site produced scaffolds with similarity to virus species from four genera. Similarly, viromes of V. corymbosum collected from Island Grove contained scaffolds closely related to virus sequences belonging to two viral genera exclusively identified in wild sites and five viral genera exclusively identified in cultivated sites.Viral populations between the viromes of V. corymbosum RNA viromes. The viruses were: blueberry latent virus (BlLV), Amalgavirus; blueberry mosaic associated ophiovirus (BlMaV), Ophiovirus; blueberry red ringspot virus (BRRV), Soymovirus; a putative new species closely related to prunus virus T (PrVT), Tepovirus; and tobacco mosaic virus (TMV), Tobamovirus  in the corresponding virus sequences with varying frequencies  and two (1132–2401 nt in length) scaffolds with high sequence similarity to BlLV. Complete BlLV genome was further obtained by reference-based mapping approach using published reference genome (NC_014593). Mapping of reads from Interlachen to the reference genome showed that 258 (0.00062%) reads were aligned to the reference sequence. However, the Island Grove virome had 11× higher number of reads mapped to BlLV compared to those from Interlachen, with mapped reads of 1943 (0.007%). The average coverage of reads mapped to the reference genome was shown to be 10× for Interlachen and 75× for Island Grove. However, there were four missing nucleotides between nucleotide position 1066–1069 in the complete BlLV genome assembled from the virome of cultivated V. corymbosum at Interlachen.The viromes of cultivated V. corymbosum at Island Grove was 3432 nt, including the 5′ (167 nt) and 3′ (99 nt) UTRs. Similar to the genome organization of other published sequences of BlLV, the genome encodes for two ORFs that were partially overlapped by 314 nt [The size of the genome assembled from the virome of cultivated y 314 nt . A putaty 314 nt ,18.V. corymbosum with other published sequences as shown by the high percentage of pairwise identity (>99%)  a. Phylogy (>99%) a.V. corymbosum from Island Grove (cultivated) and High Springs, respectively, were used to assemble the three complete RNA segments of BlMaV. However, only the RNA3 segment was assembled from the virome of wild V. corymbosum from Interlachen. The percentage and average coverage of reads in the viromes of Island Grove and High Springs that were mapped to each assembled BlMaV RNA segment is shown in Six (1376–2429 nt in length) and twenty scaffolds (519–7946 nt in length) obtained from the viromes of The length of each RNA segment assembled from each virome varied, although the size of corresponding ORFs were similar when compared to the reference sequence . The comEach RNA sequence obtained from the virome was compared to the reference sequence (KJ704366–KJ704368) by pairwise local alignment. RNA1, 2, and 3 produced approximately between 80–81, 80, and 83% of nucleotide identity, respectively . PairwisA single scaffold of 8392 nt in length was assembled from the virome of the Island Grove cultivated site, representing the complete genome of BRRV (Accession number: MZ004932). The percentage and average coverage of reads mapped to the BRRV scaffold in Bowtie2 were 0.95% and 4500×, respectively. Similar to the genome organization of other published sequences, the scaffold of BRRV encoding for eight ORFs: ORF I (movement protein), A, B, C, IV (coat protein), V (reverse transcriptase), VI , and VII. Though, there were some differences in the length of ORFs I, C, RT, and VII, as well as the length of the whole-genome, which was the longest when compared to other isolates .Whole-genome pairwise comparison using SDT showed that the BRRV scaffold shared the highest nucleotide identity (97%) to the published sequence of BRRV isolates from the Czech Republic (HM159264) and Poland (JN205460) c. In addApart from finding known viruses discussed so far, a total of 10 scaffolds, with the greatest length of 2504 nt and represented by 0.04% of the total mapped reads (225× average coverage), from the virome of the Island Grove cultivated site had the closest sequence similarity to PrVT in the BLASTx analyses. Mapping of these scaffolds against the reference sequence PrVT isolate Aze239 (NC_024686) , which wTepovirus in the Betaflexiviridae. The genome of 7200 nt contained three overlapping ORFs encoding for the RNA-dependent RNA polymerase (RdRp) (5457 nt), MP (1146 nt), and CP (663 nt). The genome also contained the 5′ (109 nt) and 3′ (264 nt) UTRs. The RdRp and MP region overlapped by 89 nt (bases 5463 to 5566), whereas the MP and CP overlapped by 350 nt (bases 6274 to 6623). The differences between the length of BlVT and PrVT in the RdRp, MP, and CP regions were 120, 6, and 3 nt, respectively (Primers were designed in the movement protein (MP) ORF and 3′UTR regions of the draft genome to obtain the whole CP for validation and genome completion of the new virus by PCR. The sequence from the cloned fragment (1010 bp) showed more than 96% pairwise identity to the viral genome draft. Alignment of sequence obtained by the Sanger method to the draft genome allowed us to assemble the complete CP sequence of the new virus species, tentatively named as blueberry virus T (BlVT) (Accession number: MZ054240). This virus had genome organization similar to the previously described sequences of members from the genus ectively .Betaflexiviridae using SDT indicated that BlVT shared the highest identity to the RdRp (55%) and CP (64%) of PrVT . The presence of scaffolds with the highest sequence match to viruses from a total of 23 genera indicated that the population of viruses in this host are greatly diverse. This is in agreement with the findings of diverse viral species in previous studies of various wild plant species through viral metagenomics [V. corymbosum native to Florida, as shown by the number of viral genera, which is almost twice more than those of cultivated viromes. Several conditions could contribute to the greater diversity of viruses in the viral population of the wild V. corymbosum compared to the cultivated ones. These conditions include the presence of different wild Vaccinium species and other plant communities within and surrounding the areas, the occurrence of vectors, and abiotic factors at these locations. In addition, the presence of diverse plant communities within and surrounding the wild V. corymbosum may act as a virus reservoir, thus increasing the heterogeneity in the population of viruses due to their movement between these plants. The occurrence of insects as virus vectors in the wild area may be another important factor that contributes to the greater virus diversity in the wild V. corymbosum by facilitating the movement of viruses between plants. On the other hand, the movement of viruses in the cultivated plants may be restricted due to the implementation of agricultural management practices, which could contribute to the lower number of viral genera in this host.Without doubt, plant viral metagenomics has contributed to our understanding of viral populations, as well as unravelling the etiology of viral diseases in various plant species. This is supported by the exponentially increased discovery of novel plant viruses that have been thoroughly described ,2. In thgenomics ,23,24,25V. corymbosum in Florida has enabled the reconstruction of ten consensus sequences of five virus species, including BlLV, BlMaV, BRRV, TMV, and a putative new species closely related to PrVT. Although the complete genome of TMV was detected in four out of six viromes, TMV was not included for further sequences analysis since it is well known that tobamoviruses are frequently found in various environments due to their high stability [Metagenomic analysis of the viromes of wild and cultivated species of tability . In addiV. corymbosum. Pairwise sequence comparison between the whole genome of BlLV from Florida with other published isolates showed more than 99% nucleotide identity. This finding was expected since it was previously reported that BlLV has a very stable population structure, with less than 0.5% diversity among partial and complete sequences of isolates from Japan and the US [The complete genomes of BlLV, an amalgavirus, were recovered from viromes of cultivated d the US ,27,28. PV. corymbosum virome at Interlachen. The presence of scaffolds closely related to BlMaV in all viromes of wild V. corymbosum suggests that the occurrence of this virus in wild blueberry might be common. Pairwise comparison using the NP regions of BlMaV isolates assembled from this study  and those from Arkansas and Japan indicated that the isolate from Island Grove and Interlachen had 14% sequence divergence from the rest of the isolates. This result was supported by a previous study which found that BlMaV had low genetic diversity among isolates, as shown by 13% nt divergence in the NP regions [V. corymbosum at High Springs in order for diversification to occur.Scaffolds with high homology (96–97%) to an ophiovirus, BlMaV, were identified in all viromes except the cultivated  regions . In contV. corymbosum at Island Grove. The detection of BRRV in the RNA virome is not surprising since it produces RNA/DNA intermediate during its replication cycle, thus making it possible to form dsRNA complex at some point. The genome organization and ORFs of BRRV isolate from Island Grove are similar to the recently published BRRV genomes identified from the cultivar “Emerald” in Florida [The analysis of the RNA virome additionally allowed for the assembly of a complete BRRV genome, a dsDNA virus, from the virome of cultivated  Florida . OverallTepovirus belonging to the family Betaflexiviridae. Partial resequencing completely validated the CP region of the de novo assembled scaffold obtained by metagenome analysis. Pairwise comparison of the RdRp and CP of the putative novel virus with the corresponding proteins of selected members representing different genera in the family Betaflexiviridae demonstrated that this novel virus was distantly related to other members of the respective viral family, though higher sequence homology was observed with PrVT. Furthermore, pairwise nucleotide comparison showed that the putative novel virus only shared 61 and 65% identity with the RdRp and CP of PrVT, respectively, which fall well below the currently accepted species demarcation criteria in the genus Tepovirus, described as having less than 72% nt identity between the RdRp and CP genes [Betaflexiviridae as well as statistically supported phylogenetic grouping further suggested that this novel virus, proposed as BlVT, should be considered as a new species in the genus Tepovirus. The biological information regarding BlVT is still lacking due to the unknown vector as well as the limited knowledge on the spread of this virus in the field. Although molecular screening of BlVT in twenty V. corymbosum samples collected from Island Grove, Florida, indicated a 15% virus incidence, the infection of BlVT still could not be associated with specific virus symptoms at this point due to mixed viral infections in the plants. Hence, the primers developed in this study could potentially be used for detection of BlVT in blueberry or related crops in the future.In addition to the detection of known viruses of blueberry that are new to Florida, this study also led to the discovery of a novel virus that is closely related to PrVT, a member in the genus CP genes . Phyloge


In this registry study, we aimed to identify the efficacy of HID G-PB HSCT (HID-PBSCT) for acute leukemia (AL) patients in first complete remission (CR1). Also, we reported the outcomes for the use of G-PB grafts in comparison with the combination of G-BM and G-PB grafts in HID HSCT recipients. Sixty-seven AL patients in CR1 who received HID-PBSCT were recruited at Institute of Hematology, Peking University. Patients who received haploidentical HSCT using the combination of G-BM and G-PB harvests in the same period were enrolled as controls (n=392). The median time from HSCT to neutrophil and platelet engraftment was 12 days  and 12 days , respectively. The 28-day cumulative incidence of neutrophil and platelet engraftment after HSCT was 98.5% and 95.5%, respectively. The cumulative incidences of grade II–IV and grade III–IV acute graft-versus-host disease (GVHD) were 29.9% (95%CI 18.8–40.9%) and 7.5% (95%CI 1.1–13.8%), respectively. The cumulative incidences of total and moderate-severe chronic GVHD were 54.9% (95%CI 40.9–68.8%) and 17.4% (95%CI 6.7–28.0%), respectively. The cumulative incidences of relapse and non-relapse mortality were 13.9% (95%CI 5.4–22.5%) and 3.4% (95%CI 0–8.1%), respectively. The probabilities of overall survival (OS) and leukemia-free survival (LFS) were 84.7% (95%CI 74.7–94.7%) and 82.7% (95%CI 73.3–92.1%) respectively. Compared with the HID HSCT recipients using the combination of G-BM and G-PB grafts, the engraftments of neutrophil and platelet were both significantly faster for the G-PB group, and the other clinical outcomes were all comparable between the groups. In multivariate analysis, graft types did not influence the clinical outcomes. Overall, for the patients with AL CR1, G-PB graft could be considered an acceptable graft for HID HSCT recipients. This study was registered at  Allogeneic hematopoietic stem cell transplantation  is the most important curative option for patients with acute leukemia (AL). The graft was one of the critical factors for allo-HSCT. Both peripheral blood (PB) and bone marrow (BM) harvests could be used as the graft sources, and cord blood cells could also be used as the graft source. Many studies had compared the clinical outcomes between patients using PB and BM grafts. In patients with human leukocyte antigen identical sibling donors (ISDs), engraftment was faster –3, the rAnother important HID HSCT regimen was “Beijing protocol”, which proposed by Peking University Institute of Hematology and based on antithymocyte globulin (ATG) . “BeijinIn the present registry study, we aimed to identify the clinical outcomes of HID PBSCT in AL patients in CR1. We also aimed to compare the clinical outcomes between G-PB alone and G-BM plus G-PB in HID HSCT recipients.https://clinicaltrials.gov (NCT03756675). The recipients receiving HID HSCT using the combination of G-BM and G-PB harvests  in the same period were collected as controls.Sixty-seven AL patients in CR1 who received HID PBSCT were recruited in this prospective study at the Peking University People’s Hospital between November 1, 2018, and February 29, 2020. All cases were treated according to the protocol registered at The inclusion criteria: 1) patients aged 2–60 years old; 2) in AL CR1; 3) donors refused the donation of BM; and 4) patients agreed to receive haploidentical PBSCT Figure 1The primary endpoint was engraftment rates as defined by neutrophil recovery and platelet recovery. The secondary endpoints include acute graft-versus-host disease (aGVHD), chronic GVHD (cGVHD), relapse, non-relapse mortality (NRM), leukemia-free survival (LFS), and overall survival (OS).2 for 2 days, busulfan 3.2 mg/kg for 3 days, cyclophosphamide 1.8 g/m2 for 2 days, rabbit anti-thymoglobulin 2.5 mg/kg for 4 days, and semustine 250 mg/m2 orally for one dose; or (2) cytarabine 4 g/m2 for 2 days, busulfan 3.2 mg/kg for 3 days, cyclophosphamide 1.0 g/m2 for 2 days, fludarabine 30mg/m2 for 5 days, rabbit anti-thymoglobulin 2.5 mg/kg for 4 days, and semustine 250 mg/m2 orally for one dose. Five patients received total body irradiation (TBI)-based conditioning. The immunosuppressants included cyclosporine A (CsA), mycophenolate mofetil (MMF), and short-term methotrexate (MTX). G-CSF was administered subcutaneously to patients at 5 ug/kg per day from day +6 until myeloid recovery  including class I  and class II  HLA antibodies. Immunoglobulin anti-HLA reactivity in the serum was tested with a bead-based screening assay. Briefly, we used the LABScreen Mixed kit , which simultaneously detects class I and class II antibodies with microbeads coated with purified class I and class II HLA antigens. For HLA antibody-positive samples with a median fluorescent intensity (MFI) >500, DSAs were further tested using a LABScreen Single Antigen Kit (One Lambda). Above a cut-off value of MFI ≥2000 was considered positive. Patients with positive DSA received rituximab before transplantation, and the co-infusion of umbilical cord blood .9/L, and platelet engraftment was defined as the first of 7 consecutive days that platelets ≥20×109/L and transfusion independence. Relapse was defined as BM blasts >5%, or extramedullary manifestation. NRM was defined as death without evidence of leukemia. OS was the period between the date of HSCT and death. LFS was the period between the date of HSCT and relapse or death in remission. GVHD was diagnosed and graded according to internationally accepted criteria , gender, donor age (<30 years vs. ≥30 years), underlying disease (AML vs. others), diagnosis to transplant (≤6 months vs. >6 months), HLA mismatching (1 locus vs. ≥2 loci), donor-recipient gender matching , ABO compatibility, CD34 count , CD3 count , and graft source (G-PB vs. G-BM+G-PB). Testing was two-sided at the P<0.05 level. Statistical analysis was performed on SPSS software , and R software (version 2.6.1) (http://www.r-project.org).The last follow-up date was September 1, 2020. Survival was estimated with Kaplan-Meier outcome curves. The cumulative incidences of engraftment, relapse, GVHD were calculated in the completing risk model. The chi-square test, or Fisher’s exact test was used for categorical variables. The non-parametric tests  were used for continuous variables. The multivariate Cox model was performed to determine the impact of potential prognostic factors on clinical outcomes. Factors included in the regression model were patient age  and 12 days  after HID PBSCT, respectively. The 28-day cumulative incidence of neutrophil engraftment after HSCT was 98.5% (95%CI 95.1–100%), and the 100-day cumulative incidence of platelet engraftment after HSCT was 95.5% (95%CI 90.1–100%) after HID PBSCT.A total of 15 and five patients showed grade II and grade III aGVHD after HID PBSCT, respectively. The 100-day cumulative incidences of grade II–IV and grade III–IV aGVHD after HSCT were 29.9% (95%CI 18.8–40.9%) and 7.5% (95%CI 1.1–13.8%), respectively.A total of 23, 9, and 2 patients showed mild, moderate, and severe cGVHD after HID PBSCT, respectively. The cumulative incidences of total cGVHD and moderate to severe cGVHD at 1 year after HSCT were 54.9% (95%CI 40.9–68.8%) and 17.4% (95%CI 6.7–28.0%), respectively.A total of 57 patients showed CMV-DNA after HID PBSCT, and 1 of them developed CMV diseases. The 100-day incidences of CMV-DNA viremia and CMV disease after HID PBSCT were 85.1% (95%CI 76.3–93.8%) and 1.5% (95%CI 0–4.4%), respectively.A total of five patients showed EBV-DNA viremia, and 2 of them developed posttransplant lymphoproliferative disorders (PTLD) after HID PBSCT. The 100-day cumulative incidences of EBV-DNA and PTLD was 6.0% (95% CI 0.3–11.7%) and 3.0% (95%CI 0–7.1%), respectively.At the last follow-up, 9 patients experienced relapse with a median time of 126 days  after HID PBSCT. The 1-year cumulative incidence of relapse after HID PBSCT was 13.9% (95%CI 5.4–22.5%). In multivariate analysis, female donor/male recipient (FDMR) combination was the only independent prognostic factor for relapse .At the last follow-up, three patients experienced NRM with a median time of 212 days  after HID PBSCT. The causes of death were summarized in The median follow-up among survivals was 341 days (range 177 to 662 days) after HID PBSCT. The probability of OS and LFS at 1 year after HID PBSCT was 84.7% (95%CI 74.7–94.7%) and 82.7% (95%CI 73.3–92.1%), respectively. In multivariate analysis, FDMR combination was the only independent prognostic factor for OS  and LFS .The characteristics between the patients in the G-PB alone group and G-BM plus G-PB group were summarized in P<0.001; platelet: 12 days  versus 15 days , P=0.006]. However, all the other outcomes were comparable between the groups  effect in different transplant settings , 36, andOur previous study showed inferior results after PBSCT on advanced-stage leukemia, as compared to that receiving HID HSCT using G-BM plus G-PB . DiffereThis study was not a randomized designed trial. Thus, it would be premature to derive conclusions regarding the superiority of PBSCT over HID using G-PB plus G-BM in patients with AL in CR1, and these results should be further confirmed by prospective randomized trials.In summary, this study confirmed the safety and efficacy of HID PBSCT in patients with AL in CR1, and it also suggested that hematopoietic recovery for those using G-PB grafts was faster comparing with those using G-BM plus G-PB grafts, and other clinical outcomes were all comparable between the groups. While BM harvest needed the hospitalization of the donor, trained physicians, and specialized equipment, PBSCs were more convenient and were easy to be collected. For patients with AL in CR1, the G-PB grafts could be used as a reasonable alternative to G-BM plus G-PB grafts in HID HSCT. In the future, these results should be further confirmed by prospective randomized trials.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board of Peking University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.XDM and XJH designed the research. Y-RM analyzed the data and wrote the manuscript. All authors provided patient data. All authors contributed to the article and approved the submitted version.This work was supported by National Key Research and Development Program of China (grant number 2017YFA0104500), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (grant number 81621001), the Key Program of the National Natural Science Foundation of China (grant number 81930004), the Capital’s Funds for Health Improvement and Research (grant number 2018-4-4089), CAMS Innovation Fund for Medical Sciences (CIFMS) (grant number 2019-I2M-5-034), the Science and Technology Project of Guangdong Province of China (grant number 2016B030230003), Peking University Clinical Scientist Program (grant number BMU2019LCKXJ003), and the Fundamental Research Funds for the Central Universities.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.


Tuberous sclerosis complex (TSC), a multisystem genetic disorder, affects many organs and systems, characterized by benign growths. This German multicenter study estimated the disease-specific costs and cost-driving factors associated with various organ manifestations in TSC patients.A validated, three-month, retrospective questionnaire was administered to assess the sociodemographic and clinical characteristics, organ manifestations, direct, indirect, out-of-pocket, and nursing care-level costs, completed by caregivers of patients with TSC throughout Germany.TSC2 were reported in 46% of patients, whereas 14% were reported in TSC1. Mean total direct health care costs were EUR 4949  per patient over three months. Medication costs represented the largest direct cost category , with mechanistic target of rapamycin (mTOR) inhibitors representing the largest share . The cost of anti-seizure drugs (ASDs) accounted for a mean of only EUR 260 (5%). Inpatient costs  and ancillary therapy costs  were also important direct cost components. The mean nursing care-level costs were EUR 1163  over three months. Total indirect costs totaled a mean of EUR 2813  for mothers and EUR 372  for fathers. Multiple regression analyses revealed polytherapy with two or more ASDs and the use of mTOR inhibitors as independent cost-driving factors of total direct costs. Disability and psychiatric disease were independent cost-driving factors for total indirect costs as well as for nursing care-level costs.The caregivers of 184 patients  submitted questionnaires. The reported TSC disease manifestations included epilepsy (92%), skin disorders (86%), structural brain disorders (83%), heart and circulatory system disorders (67%), kidney and urinary tract disorders (53%), and psychiatric disorders (51%). Genetic variations in This study revealed substantial direct (including medication), nursing care-level, and indirect costs associated with TSC over three months, highlighting the spectrum of organ manifestations and their treatment needs in the German healthcare setting.Trial registration: DRKS, DRKS00016045. Registered 01 March 2019, http://www.drks.de/DRKS00016045.The online version contains supplementary material available at 10.1186/s13023-021-01899-x. First study to measure both direct and indirect costs of children with TSC and their caregivers.Mean total direct costs were estimated to be EUR 4949 for three months.Medication, especially mTOR inhibitors, and hospitalization are major direct cost components.Total indirect costs were higher for mothers (mean EUR 2813 for three months) than fathers (EUR 372).Total costs are driven by the number of TSC manifestations and affected organ systems.Tuberous sclerosis complex (TSC) is a rare, multisystem, genetic disorder that affects up to 1 in 5,000 individuals worldwide. Until recently, the prevalence of TSC was underestimated due to incomplete penetrance and considerable inter-individual phenotypic variability among those affected by TSC . The cliTSC1 or TSC2  pathway, resulting in the abnormal production of end products, which ultimately promotes tumorigenesis ), the patients and society as a whole. The cost categories that were included in this analysis included direct health service costs covered by the statutory health insurance and as patients’ and caregivers’ out-of-pocket (OOP) expenses, nursing care-level costs covered by the statutory care insurance, and further informal care not covered by any statutory insurance, and indirect costs. Costs were evaluated according to the German recommendations for performing economic evaluations related to healthcare .Direct health service costs, which included inpatient stays, outpatient visits, medicines , medical aids, healthcare professional visits, emergency transportation, diagnostic studies, specific diets, patients’ co-payments, rehabilitation costs, private transport costs, and co-payments for therapies, were drawn from the literature and standard reference sources for Germany and were estimated as previously described , 25. DruOOP expenses (co-payments) were reported by respondents. For situations in which supply-side utilization estimates were not available , and for those expenditures outside of formal healthcare settings , the reported costs were listed as OOP expenses and added to total direct healthcare costs. For instances in which supply-side cost estimates were calculated according to resource utilization , OOP expenses were considered to be accounted for, and were not added to total direct costs to prevent double accounting.In Germany, care insurance payments are determined by the patient care grade, which ranges from Level 1 to 5 on the Pflegegrade scale (which categorizes the need for care), and determines the basis for the care allowances that are paid by the German statutory care insurance “Pflegeversicherung” . Care grThe “grade of disability” is assigned in the German social system to people with disabilities who are entitled to certain monetary and social compensations for their disadvantages. The grade of disability quantifies the type and severity of a disability, upon which these compensations depend. Grade of disability is classified by an independent medical professional (“Versorgungsamt”) and if applicable can vary between 20 and 100, in steps of ten.Productivity losses associated with the need to care for TSC patients were calculated for caregivers of working age  using the human capital approach for days off, quitting work, and reductions in working hours. Productivity losses due to TSC among adolescent patients of working age  were considered in terms of days off for working adolescents and the inability to work among those who were completely unable to work or attend school. A mean gross wage of EUR 44,964 in 2019  was assuSome questionnaire items were collated into groups when presenting the results, as follows: ‘ancillary costs’: physiotherapy, speech therapy, occupational therapy, acupuncture, hippotherapy, and other ancillary costs; ‘healthcare professionals’: neurologists, general practitioners, orthopedic surgeons, child psychiatrists, alternative medicine practitioners, homeopathy, dietitians, and other specialists; and ‘diagnostic studies’: electroencephalography (EEG), blood tests, magnetic resonance imaging (MRI) or computed tomography (CT) scans, X-rays, and other diagnostic studies.The reported data and the questionnaire are available to qualified researchers upon reasonable request.p < 0.05. The relationship between patient characteristics and TSC-related costs was investigated using multivariate regression. Total direct, total indirect, and nursing care-level costs were regressed on a set of clinical variables that were selected following univariate analyses and according to evidence from previous cost-of-illness studies for TSC [Statistical analysis was performed using IBM SPSS Statistics, version 26 . The variables of interest were summarized using the mean, median, and standard deviation (SD). For cost data, the 95% confidence interval (95% CI) was calculated using the bootstrap-corrected and accelerated method, considering the fact that most cost variables are highly skewed . Compari for TSC , 32, 33.TSC1, whereas in 85 patients (46.2%), variations were observed in TSC2 (TSC1:TSC2 ratio 1:3.4), and five patients (2.7%) suffered from a polycystic kidney disease with tuberous sclerosis (PKDTS), which is a contiguous gene deletion syndrome. Most children lived with both parents at home . Forty percent (n = 74) of patients attended a school with a special focus on learning, and mental and physical development, whereas the majority of small children attended kindergarten . Among the respondents, mothers had a mean age of 41 years, and fathers were older, on average, with a mean age of 43 years. The occupational situations of parents revealed that 70% of mothers (n = 128) and 91% of fathers (n = 167) were employed. Additional sociodemographic and clinical characteristics and affected family members are presented in Table One hundred and eighty-four caregivers of children and adolescents with TSC completed the questionnaire. The mean patient age was 9.8 years , and 48.4% (n = 89) of patients were females. TSC was diagnosed at a mean age of 1.3 years , and the first symptoms of TSC were observed at a mean age of 0.8 years . In 34 children (18.5%), a diagnosis of TSC was suspected before birth based on the findings of ultrasound examinations. In 25 patients (13.6%), variations were identified in The mean total direct costs were calculated at EUR 4949  per study participant for the reported 3-month study period, and details are presented in Table p < 0.001). The detailed costs and daily dosages of the various ASDs are listed in Table Costs for mTOR inhibitors  were the primary direct cost components with a mean of EUR 2309 , which was greater than the costs associated with ASDs (synonymous to anticonvulsants or antiepileptic drugs) which were associated with a mean cost of EUR 260 . The patients used a mean number of 1.8 ASDs . The five most frequently prescribed ASDs included oxcarbazepine , vigabatrin , lamotrigine , valproate , and levetiracetam . ASD monotherapy was prescribed to 32.6% (n = 60) of all participants and was associated with lower costs than ASD polytherapy, comprising two or more ASDs  of the included children and adolescents were admitted at least once to a hospital because of TSC during the 3-month study period. Overall, 49 admissions were reported, with a mean length of stay of 6.04 days . Seizures were the reported reason for 23 admissions, whereas 22 admissions were due to diagnostics, and four additional admissions were associated with other TSC-related causes.Ancillary treatments, such as occupational therapy, physiotherapy, and speech therapy were prescribed to 116 participants (63%), and with costs as high as EUR 407 per 3 months, representing 8.2% of total direct costs . In addition, EUR 125 in the 3-month study period were directly paid by the families for therapies.Sixty-five percent (n = 120) of patients were categorized as meeting the care levels defined by the “Pflegebedürftigkeits scale” . Approximately 3% (n = 5) of patients did not meet the Level I–V criteria but were reported as being in need of care according to their caregivers. Only 32% of patients were reported as not in need of care. The costs for nursing care were calculated as EUR 1163  per 3-month period and EUR 4652 annually, assuming that care is provided by family members. Parents reported that they incurred further costs for informal care, with a mean of EUR 20.8, and supervision, with a mean of EUR 27.6 per 3-month period. In total, 123 patients (66.8%) had a severely disabled pass between 70% and 100% (maximum = 100%). No disability or disability ≤ 60% were identified in 33% (n = 61) of patients.All parents were of working age, and lost work time was recorded separately for mothers and fathers. In total, 111 parents reported that they had changed their working situation or remained out of work due to TSC in their child. Total indirect costs were calculated at a mean of EUR 3184  over three months, or EUR 12,736 annually. Twenty-four mothers  reported that they quit working, 49 mothers reduced their working hours , and 41 mothers missed days of work during the last three months due to TSC . Mean productivity costs over three months were estimated at EUR 1466, associated with mothers quitting work (EUR 122 for fathers); EUR 1109, associated with mothers’ reduced working hours (EUR 86 for fathers); and EUR 238 associated with mothers’ lost workdays (EUR 163 for fathers). The total mean maternal indirect costs totaled EUR 2813  over three months, or EUR 11,252 annually, whereas the total indirect costs for fathers were calculated at EUR 372  over three months, or EUR 1488 annually. These details are provided in Table Twenty-five (13.6%) of the adolescents included in this study were older than 16 years of age. Five of them were working, two were unable to work or attend school due to TSC, and 18 were in school, at university, or in vocational training. The mean indirect costs were calculated at EUR 1002  per 3 months, which were due to 12 days off work due to TSC in one adolescent and the two individuals who were unable to work or attend school.To identify potential cost-driving factors, we performed univariate analyses of total direct, total indirect, and nursing care-level costs, and a number of demographic and clinical patient characteristics. The indirect costs of mothers and fathers were considered together. Polytherapy with two or more ASDs, the use of mTOR inhibitors, TSC manifestations, such as epilepsy, structural brain disorders, psychiatric and cardiac disease, and disability, were all associated with increased total costs according to the univariate analyses, which are detailed in Table Overall, total direct, total indirect, and nursing costs increased as the number of affected organ systems increased Table .2) of the total variance. Younger age and psychiatric disease were independent cost-driving factors for total indirect costs . Psychiatric disease and disability were independent cost-driving factors for nursing care-level costs .Multiple regression analyses revealed that polytherapy with two or more ASDs and the use of mTOR inhibitors were independent cost-driving factors for total direct costs. After applying a Bonferroni correction for eight comparisons, the significance threshold for the p-value was set to 0.00625, and the variables were able to explain 53%  and hospitalization  and were impacted far less by ASD use  . mTOR inPotential limitations associated with the questionnaire used in this study include recall bias regarding the three-month-old events, which might result in incomplete and underestimated costs. However, a validation of three-months recall for all items and of twelve-months recall regarding high-impact events like hospitalization or emergency calls with a prospective diary covering three months showed an excellent overlap in patients with Dravet syndrome, another developmental and epileptic encephalopathy . FurtherMedical and care-related expenditures among patients with TSC are high and appear to be driven by the severity of disease manifestations. In the long term, high direct costs might be outweighed by the preventive potential and multi-organ benefits of newer therapies. Productivity losses represent a major source of costs and may be addressed by improving socio-medical support systems and therapeutic interventions. Efforts should be focused on reducing work absenteeism and the rate at which caregivers stop working entirely while maintaining the quality of care provided to children and adolescents with TSC.Additional file 1.Supplementary Table 1. Direct costs related to TSC manifestations.


Further genome walking allowed for the characterization of full coding sequences of all the viral proteins . We tentatively named the virus “Mundri virus” (MUNV) and classified it as a novel virus species based on the high divergence from other known viruses . Phylogenetic analysis revealed that MUNV forms a monophyletic clade with several human-infecting tibroviruses prevalent in Central Africa. A bioinformatic machine-learning algorithm predicted MUNV to be an arbovirus (bagged prediction strength (BPS) of 0.9) transmitted by midges (BPS 0.4) with an artiodactyl host reservoir (BPS 0.9). An association between MUNV infection and Nodding syndrome was evaluated in a case–control study of 72 patients with Nodding syndrome (including the index case) matched to 65 healthy households and 48 community controls. No subject, besides the index case, was positive for MUNV RNA in their plasma. A serological assay detecting MUNV anti-nucleocapsid found, respectively, in 28%, 22%, and 16% of cases, household controls and community controls to be seropositive with no significant differences between cases and either control group. This suggests that MUNV commonly infects children in South Sudan yet may not be causally associated with Nodding syndrome.A divergent rhabdovirus was discovered in the bloodstream of a 15-year-old girl with Nodding syndrome from Mundri West County in South Sudan. Nodding syndrome is a progressive degenerative neuropathy of unknown cause affecting thousands of individuals in Sub-Saharan Africa. The index case was previously healthy until she developed head-nodding seizures four months prior to presentation. Virus discovery by VIDISCA-NGS on the patient’s plasma detected multiple sequence reads belonging to a divergent rhabdovirus. The viral load was 3.85 × 10 Nodding syndrome (NS) is a severe progressive degenerative neuropathy affecting children and young adults in remote areas in Sub-Saharan Africa. NS has largely been neglected, and although thousands of individuals are affected today, the etiology remains unknown, and no preventative or treatment options are available . One hypTo explore a potential novel viral cause of NS, we performed virus-discovery assays using VIDISCA-NGS on the plasma and cerebrospinal fluid of children with new-onset NS. VIDISCA-NGS allows unbiased detection of known and unknown RNA and DNA viruses in a plethora of clinical specimen, and it was previously used to discover viruses such as human coronavirus NL63  and NtweThe index case and all other study subjects participated in the case–control study of the South Sudan Nodding Syndrome Study project . Cases wVIDISCA-NGS was performed on 220 μL CSF pooled 1:1 with Universal Transport Medium  and on 110 µL EDTA plasma as previously described . Brieflyhttps://www.ncbi.nlm.nih.gov/genbank) (accessed on 19 January 2021). Open reading frames (ORFs) were identified and translated into amino acid sequences by NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffinder) (accessed on 19 January 2021). Amino acid sequences from structural proteins were aligned to those from other rhabdovirus RefSeq sequences from GenBank using all available alignment methods on M-Coffee  (Thermo Fisher Scientific). An overnight culture of the transformed bacteria was inoculated into Luria Broth (LB) medium, supplemented with 1% glucose and carbenicillin (10 µg/mL). The culture was grown to exponential phase prior to inoculation with 0.5 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 5 h. Recombinant proteins were purified with Ni-NTA (Qiagen), and concentration was measured with a Nano-Drop (Thermo Fisher Scientific). ELISA was performed as described .Mansonella perstans microfilaria in a blood smear. Hemoglobin measurement, analysis of complete blood count, urine dipstick and microscopy on blood, skin snip and cerebrospinal fluid were all normal. An older brother also met the case-definition for probable NS; an older sister was healthy. At 6 and 12 months after the initial examination at Lui Hospital, the patient was brought to the hospital for the study follow-up. At 6 months, the patient reported generalized convulsions and had developed further weight loss (BMI 16.4). At 12 months, the clinical presentation remained unchanged.In August 2018, a 15-year-old girl was evaluated at the NS study clinic at Lui Hospital, Mundri East County, South Sudan, for clinical and laboratory evaluation for study purposes. The patient met the case definition  of proba3 RNA copies/mL. No viral reads could be detected in the CSF VIDISCA-NGS library, nor could viral RNA be detected in the patient’s CSF by RT-qPCR; the viral load was undetectable in her plasma at the 12-month follow-up.Plasma and cerebrospinal fluid were analyzed by VIDISCA-NGS to identify known and unknown viruses. VIDISCA-NGS identified 19,505 sequence reads in the patient’s plasma and 26,233 in her CSF. Several reads from the patient’s plasma belonged to a divergent rhabdovirus which prompted further genomic characterization by Illumina sequencing and genome walking on low-coverage regions. Accordingly, the complete coding sequence of the genome could be characterized . An RT-qRhabdoviridae, genus: Tibrovirus, The virus genome encodes for eight viral proteins: a nucleocapsid (N), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), a large protein , and three accessory proteins , 65 matched household controls , and 48 matched community controls. Using an RT-qPCR to detect MUNV RNA, all samples remained negative except for the index case.p = 1.3 × 10−6, Mann–Whitney U test). Accordingly, a seropositivity threshold was defined at two standard deviations above the Dutch median antibody concentration: 0.127 AU/mL , nor between the NS cases and the community controls . Comparing absolute antibody concentrations, we found a marginally significant higher concentration of antibodies in the NS cases than in the community controls , yet no difference was found between the NS cases and the household controls .We also developed a serological assay detecting anti-nucleocapsid IgG antibodies. The index case had high levels of MUNV antibodies at presentation compared to a Dutch control sample, which marginally decreased at the 12-month follow-up . To defip = 0.13, linear regression, p = 0.33, logistic regression) amongst the South Sudanese children. There was no clustering of seropositive samples amongst children living in the same household . Lastly, there was no association between M. perstans microscopical positivity and MUNV seropositivity .Age was not significantly associated with antibody concentration  and Culicoides  is a divergent rhabdovirus discovered in the bloodstream of a child with new-onset Nodding syndrome. Rhabdoviruses are negative-sense RNA viruses known to infect a wide range of plants, vertebrates, and invertebrates. Only few rhabdoviruses infect humans, but when they do, they may cause severe central nervous system (CNS) infection, such as rabies virus and Chandipura virus . Within Rhabdoviridae—a family of viruses known to cause CNS infections in humans—and was detected in a child with an encephalopathy of unknown cause, we evaluated a potential causal association. We determined whether infections with MUNV were more common in patients with NS than healthy children living in the same household (household controls) or living in the same community (community controls). Using RT-qPCR, no additional MUNV RNA positive cases or controls could be detected besides the index case. Moreover, we were unable to detect MUNV RNA in the CSF of the index case, arguing against a CNS infection. Next, as MUNV may only have caused a temporary infection, or may no longer have been detectable in the bloodstream at the time of sample collection, we also investigated evidence of prior infection by serology. No significant difference in seroprevalence was found between the NS cases and the household controls, nor between the NS cases and the community controls. While we cannot exclude cross-reactivity with other rhabdoviruses—as seen between EKV1 and the rabies virus [Since MUNV belongs to es virus —our resuMansonella perstans, for which the index case was also positive. This co-infection of pathogens with a common vector suggests that individuals exposed to one are at increased risk of infection by the other. Although we found no association between MUNV seropositivity and Mansonella perstans infections, this may be a result of temporality and still be true for acute infections. Interestingly, several studies have found an association between NS and filarial infection (both with Onchocerca volvulus and Mansonella perstans), although proof of causality is still lacking [A machine-learning algorithm predicted MUNV to be an arbovirus transmitted by midges with an artiodactyl host reservoir. Midges are also known to transmit many other pathogens, including filaria such as  lacking . ConsequThe discovery of MUNV in the bloodstream of a human, which was not predicted to be a host reservoir, suggests that humans are accidental and potential dead-end hosts ,17. GiveThe ability to detect novel viruses has greatly improved with the decreased costs of metagenomic next-generation sequencing. It is estimated that more than half a million yet-to-be-discovered viruses with zoonotic potential are circulating in mammals and birds. This argument has been used to support the Global Virome Project, aiming to characterize these viruses before they become zoonotic . However


Viruses cause a big fraction of economically important diseases in major crops, including tomato. In the past decade (2011–2020), many emerging or re-emerging tomato-infecting viruses were reported worldwide. In this period, 45 novel viral species were identified in tomato, 14 of which were discovered using high-throughput sequencing (HTS). In this review, we first discuss the role of HTS in these discoveries and its general impact on tomato virome research. We observed that the rate of tomato virus discovery is accelerating in the past few years due to the use of HTS. However, the extent of the post-discovery characterization of viruses is lagging behind and is greater for economically devastating viruses, such as the recently emerged tomato brown rugose fruit virus. Moreover, many known viruses still cause significant economic damages to tomato production. The review of databases and literature revealed at least 312 virus, satellite virus, or viroid species (in 22 families and 39 genera) associated with tomato, which is likely the highest number recorded for any plant. Among those, here, we summarize the current knowledge on the biology, global distribution, and epidemiology of the most important species. Increasing knowledge on tomato virome and employment of HTS to also study viromes of surrounding wild plants and environmental samples are bringing new insights into the understanding of epidemiology and ecology of tomato-infecting viruses and can, in the future, facilitate virus disease forecasting and prevention of virus disease outbreaks in tomato. Solanum lycopersicum L.) is one of the most economically valuable fruit or vegetable crops worldwide, valued at 93.9 billion US dollars in 2018, with yield estimated at 180.8 million tons in 2019 , including unknown ones, became possible due to the rapid decrease in high-throughput sequencing (HTS) costs  costs  and the S) costs . This ciS) costs . CurrentS) costs . HTS is S) costs . Each meS) costs  and shouS) costs . On the S) costs .Both classical (Sanger) sequencing and HTS are regularly used for discovery of new viruses in tomato; however, the usage of the latter is evidently increasing in the past few years. In the first part of this review, we discuss the discovery of 45 novel virus species in tomato within the 2011–2020 period and contextualize the role of HTS in these findings. Moreover, a post-discovery characterization of new viruses represents an important step toward understanding their biological and/or economical relevance ; thus, wReview of the past and recent discoveries of viruses in tomato shows that tomato is currently associated with at least 312 different viral species, which is likely, according to our knowledge, the largest recorded number known for any cultivated plant. Among those, many known and several recently discovered viruses cause significant economic damage in tomato production in different parts of the world. In the second part of this review, we focus on important tomato viruses, which caused significant economic losses in tomato production in the past decade and new virus discoveries in tomato, for which limited or no knowledge about potential impact on tomato health is available.Iflaviridae  . Majoritaviridae , Phenuiviviridae , Kitaviraviridae , and Genoviridae . SpecifiSolanum nigrum, named Solanum nigrum ilarvirus 1 (SnIV1) (Capulavirus (Kitaviridae), named tomato fruit blotch virus (ToFBV), was discovered by HTS (Iflaviridae), first detected in Australia using HTS (MK517476) (Fourteen new tomato viruses or virus satellites were detected using HTS in recent years . As an e (SnIV1) . Three b (SnIV1) . A novelulavirus . In Itald by HTS . Tomato sing HTS , was alsK517476) . The infPotyviridae), potato virus H, potato virus S, potato virus M (Betaflexiviridae), and turnip yellows virus (Luteoviridae) were reported for the first time in tomato (Tospoviridae) and Lindernia anagallis yellow vein virus (Geminiviridae) were associated with tomato for the first time , electron microscopy, and targeted diagnostics. Virus characterization is a time- and resource-intensive task that often requires efforts from different institutions, sometimes at an international scale. A framework for biological characterization of viruses discovered by HTS was proposed to facilitate this part of the research pipeline . We usedOur review showed that, e.g., for majority (>60%) of the HTS-based new virus discoveries, whole genome sequence was determined, primers for detection of the virus with PCR were designed, local survey of prevalence was done, and co-infection with other viruses was checked . CompariSpatially, crop lands are always in conjunction with the wild or urban ecosystems bound by an agroecological interface, which is characterized by active biological interactions and exchange of materials, including pests and pathogens . NeverthSolanaceae family highlights the importance of studying the virome of both wild and cultivated plants to fully understand the plant virus impact on the agroecological scale , for which there was a strong evidence for a likely spillover from tomato to S. nigrum. On the other hand, a possible biological and ecological barrier was proposed for a spread of broad bean wilt virus (known to be able to infect both hosts) from S. nigrum to tomato, since high incidence and diversity of this virus was observed in S. nigrum; however, the virus was not detected in tomato in this study. Moreover, discovery of Euphorbia caput-medusae latent virus by HTS in South Africa in wild plant Euphorbia caput-medusae, which was shown to be infective in tomato and Nicotiana benthamiana, led to the establishment of a new genus, Capulavirus, in the family Geminiviridae , were studied as possible effective ecological “samplers” of plant viruses. HTS was employed in a recent study of honeybee-assisted surveillance in Australia to investigate the presence of plant viruses, and cucumber green mottle mosaic virus was detected on bees in several states of Australia before its subsequent detection in diseased plant material  and associated satellites. On the other hand, a greater richness of tomato viruses is found for RNA viruses, for which 84 known species are classified in 18 families. Finally, eight viroids, from a single family, are known to infect tomato , tomato torrado virus (ToTV), tomato leaf curl New Delhi virus (ToLCNDV), and ToBRFV . On the Begomovirus (Geminiviridae) is the biggest and one of the most studied genera of plant viruses, comprising 162 known species infecting tomato. Seven viral species belonging to other genera from the family Geminiviridae are also known to infect tomato . Begomoviruses have monopartite or bipartite genomes  identified from wild tomato relatives are used to breed for tolerant varieties against ToLCNDV, TYLCV, and other begomoviruses and multi-gene resistance is currently considered to increase the level of resistance and prevent its breakdown  are often associated with begomoviruses . They caIn the last 10 years, several novel begomovirus species were described in different parts of the world . From thS. nigrum was shown to harbor four known begomoviruses associated with tomato yellow leaf curl disease. Through infectivity assays, tomato severe rugose virus was shown to be infective and harbored in common weeds, such as Nicandra physaloides, Datura stramonium, and Sida sp., which are found surrounding the tomato-growing areas in Brazil  are among the most environmentally stable viruses that remain viable for a long time outside of their host plant in various environmental media   . A positr, soil) . They arr, soil) . Six tobatically .The most recent virus outbreaks with significant losses in tomato production worldwide were connected to the emergence of two new tobamovirus species, ToMMV  and ToBRTm-22 resistance in some tomato cultivars  is currently composed of 26 species and is the only plant-infecting genus in the order Bunyavirales . This insect vector remains a global problem due to inefficient insecticidal control  and Sw-5 (in tomato). This further worsened the economic losses caused by TSWV in tomato and pepper, which were previously estimated at more than 1 billion US dollars annually (Tomato spotted wilt orthotospovirus resistance-breaking (RB) strains were first discovered in the early 2000s, and since then, they are occurring sporadically in many regions causing significant damages. These strains overcome TSWV resistance regulated by hypersensitivity genes: annually . Currentannually .Among other orthotospoviruses, important viruses reported in tomato in the past decade are tomato chlorotic spot virus (TCSV), groundnut ringspot virus (GRSV), capsicum chlorosis virus (CaCV), and tomato yellow ring virus (TYRV) . TCSV waPotyvirus (Potyviridae) is one of the largest genera of plant-infecting viruses, with 183 recognized species , pepper mottle virus (PepMoV), and pepper veinal mottle virus (PVMV). PepMoV infection in tomatoes in Hawaii resulted in unmarketable fruits with mottling symptoms  and, recCucumovirus, family Bromoviridae) is an important tomato-infecting virus. Cucumoviruses have a spherical virion encapsidating a tripartite (+)ssRNA genome. Some CMV strains encapsidate subviral RNAs termed satellite RNAs (satRNAs)  . In natut tomato . CMV hast tomato . Severalom Spain , and othom Spain . Due to om Spain , CMV becOutbreaks of CMV infection in tomato-growing areas were reported, and some of these were associated with satellite RNAs (satRNAs) that induce systemic necrotic symptoms, referred to as lethal necrosis . This diTomato aspermy virus (TAV) is another cucumovirus that naturally infects tomato. TAV was recently reported in tomato samples from China; however, its economic impact was not recorded . TAV wasBromoviridae family. Detections of these viruses were reported in the United States , causing necrotic yellows disease on tomato (Tomato necrotic spot virus (ToNSV) and the novel species named tomato necrotic streak virus (TomNSV) are tomato-infecting ilarviruses within d States , 2019. B. nigrum . However. nigrum . Moreove. nigrum . Outbrea. nigrum  and rese. nigrum . Lastly,n tomato  and pelan tomato .Potexvirus (Alphaflexiviridae) and has an unsegmented (+)ssRNA genome coated in a flexuous rod virion. PepMV was first described in Peru, and since its first detection in tomato in Netherlands in 1999, it quickly spread in Europe (Solanum muricatum)  , and it ricatum) . In tomaricatum) . PepMV iricatum)  and was ricatum) , South Aricatum) , Spain aricatum) , and Serricatum) .S. muricatum) and wild Solanum spp.; (2) the European EU-tomato (EU) strain; (3) the American US1 strain; (4) the Chilean-2 (CH2) strain; and (5) the PES strain, described in wild tomato populations in Peru  the Peruvian (LP) strain, originally found infecting pepino  are whitefly transmitted viruses with bipartite genome composed of two (+)ssRNA genome segments that are separately coated in filamentous virions  can propagate inside either a leafhopper, an aphid, or mite vector or a plant. Rhabdoviruses are composed of large bacilliform virions encapsulating an (−)ssRNA genome of around 13–15 kbp. There are currently three known species of rhabdoviruses that infect or are associated with tomato: the newly discovered TYMaV in genus Cytorhabdovirus ssRNA genomes encapsidated in an icosahedral virion (Nepovirus and ToTV and tomato marchitez virus (ToMarV), both from genus Torradovirus. TBRV was recently reported infecting tomatoes in Poland showing chlorotic and/or necrotic ringspots and was found to be associated with eight satellite RNAs, but no assessment about economic losses were made  named olive latent virus 1 (OLV1); however, its economic impact has not been assessed  was discovered in field tomatoes that exhibited severe leaf mosaic and blistering symptoms  , a double-stranded (ds) RNA virus, was first detected in tomato plants in United States and Mexico exhibiting stunting of the growing tips, fruit discoloration, and reduced fruit size (Southern tomato virus (STV) , eight are known to infect tomato. Among these viroids, potato spindle tuber viroid (PSTVd) and tomato chlorotic dwarf viroid (TCDVd) are the most prevalent and economically important ssRNA molecules, around 246–401 bp long, and are the smallest entities known to infect any living organisms. They are known to infect only plants . They camportant . Severalmportant . In ordemportant . Since tmportant .New discoveries and studies on emergence of tomato viruses in the recent decade contributed to our understanding of the tomato virome, its diversity, virus ecology, and epidemiology. Tomato is likely associated with the highest number of viruses and viroids known for any plant species to date, currently amounting to at least 312 known species. Because of the absence of resistant varieties for some viruses, global spread of insect vectors, emergence of RB viral strains, increased global movement of plant materials, such as seeds, and high stability and environmental persistence of some viruses, viral diseases in tomato cause massive yield and economic losses. Begomoviruses and associated satellite viruses represent the most numerous group of tomato viruses. ToLCNDV and TYLCV are two of the most important begomoviruses, with ToLCNDV currently prevailing as an emergent and damaging species worldwide . On the The increasing knowledge on the virome of tomato, the environment, and the surrounding wild plants contributes to better understanding of virus disease emergence and epidemiology of viruses associated with tomato. Moreover, an increase in viral sequence information in databases can enable more rapid development of targeted diagnostic tests. A tailored approach in studies of plant virus diseases is needed because each crop species or family has its distinctive virome . At the MPSR and DK conceptualized the review topic. MPSR did the data mining and analysis and wrote the first draft of the manuscript, including the table, figures, and 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.


Totiviridae family have been described in crustacean hosts. However, there has been no characterization of the most newly discovered putative viruses beyond description of their genomes. In this study, two novel double-stranded RNA (dsRNA) virus genomes were discovered in the Atlantic blue crab  and further investigated. Sequencing of both virus genomes revealed that they each encode RNA dependent RNA polymerase proteins (RdRps) with similarities to toti-like viruses. The viruses were tentatively named Callinectes sapidus toti-like virus 1 (CsTLV1) and Callinectes sapidus toti-like virus 2 (CsTLV2). Both genomes have typical elements required for −1 ribosomal frameshifting, which may induce the expression of an encoded ORF1–ORF2 (gag-pol) fusion protein. Phylogenetic analyses of CsTLV1 and CsTLV2 RdRp amino acid sequences suggested that they are members of two new genera in the family Totiviridae. The CsTLV1 and CsTLV2 genomes were detected in muscle, gill, and hepatopancreas of blue crabs by real-time reverse transcription quantitative PCR (RT-qPCR). The presence of ~40 nm totivirus-like viral particles in all three tissues was verified by transmission electron microscopy, and pathology associated with CsTLV1 and CsTLV2 infections were observed by histology. PCR assays showed the prevalence and geographic range of these viruses, to be restricted to the northeast United States sites sampled. The two virus genomes co-occurred in almost all cases, with the CsTLV2 genome being found on its own in 8.5% cases, and the CsTLV1 genome not yet found on its own. To our knowledge, this is the first report of toti-like viruses in C. sapidus. The information reported here provides the knowledge and tools to investigate transmission and potential pathogenicity of these viruses.The advancement of high throughput sequencing has greatly facilitated the exploration of viruses that infect marine hosts. For example, a number of putative virus genomes belonging to the  With the wide application of next generation sequencing (NGS), a huge number of virus genomes have been described from studies of metagenomes and viromes. How to best use the massive amount of data produced by NGS remains a fundamental challenge. For instance, an increasing number of toti-like virus sequences have been revealed by metagenomic studies, but further characterization and investigation are lacking . IdeallyTotiviridae family have a non-segmented, double-stranded RNA (dsRNA) genome, with two open reading frames (ORFs) encoding the putative capsid protein (Cp) and the RNA dependent RNA polymerase (RdRp). Most virions in this family are isometric with no projections, and are ∼40 nm in diameter  in freshwater crayfish (Cherax quadricarinatus) and infectious myonecrosis (IMN) virus (IMNV) in the Pacific white shrimp (Litopenaeus vannamei). CGV was the first totivirus identified in crustaceans and caused high morbidity and mortality in infected juvenile crayfish , which causes disease and mortality in C. sapidus, is more prevalent in blue crabs at higher latitudes  revealed all virions are ~40 nm in diameter, suggesting that either the two viruses are in similar size or that only one of the viruses produces virions. Pathology caused by the viruses was revealed by histology. Additionally, probe-based real-time reverse transcription quantitative PCR (RT-qPCR) assays were developed to screen and quantify totivirus infections in large numbers of C. sapidus across a climatological gradient.Here, we report the discovery and characterization of two novel toti-like virus genomes that co-infect Blue crabs were collected from coastal states of the United States, including Massachusetts (MA), Rhode Island (RI), New York (NY), Maryland (MD), Delaware (DE), North Carolina (NC), Texas (TX), and Louisiana (LA) between the years 2009 and 2021 . A portig centrifuge washes with 500 μl 75% ethanol were carried out. Resulting RNA pellets were dissolved in 50 μl 1 mM EDTA and stored at −80°C. Process control samples (muscle from frozen smelt) were extracted before and after sets of tested crab samples to monitor for cross contamination between each sample. RNA quality and concentration were determined by NanoDrop™ spectrophotometry . The dsRNA content of RNA extractions was revealed by electrophoresis on 1.0% agarose TBE gel and stained with ethidium bromide. The isolated dsRNA was agarose gel purified with the NucleoSpin® Gel and PCR Clean-Up Kit .Crab dissections were performed with sterile wooden rods and single-use razor blades on a bench cleaned with ELIMINase™. After the external cuticle was cleaned with ELIMINase™, approximately 50 mg of muscle and hypodermis was dissected from a walking leg and homogenized in 1.0 ml of homemade Trizol , with a Callinectes sapidus toti-like virus 1 (CsTLV1) and Callinectes sapidus toti-like virus 2 (CsTLV2), collected from Agawam River, MA, United States was used for cDNA synthesis with barcoded octamers (5’-GGCGGAGCTCTGCAGATATC-NNNNNNNN-3’) with the M-MLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit . The resulting cDNA was amplified by PCR using the barcode primers (5’-GGCGGAGCTCTGCAGATATC-3’). Amplification was achieved through 40 cycles of 95°C for 5 s (denaturation), and 60°C for 30 s , followed by 72°C for 30 s (elongation). PCR products of 250–500 bp were obtained by agarose gel purification with a NucleoSpin® Gel and PCR Clean-Up Kit . The DNA library was constructed using the NEBNext R Ultra™ DNA Library Prep Kit for Illumina  following manufacturer’s instructions . The library was sequenced in a 2 × 250 paired-end configuration on the Illumina MiSeq platform with a MiSeq Reagent kit v3 .Purified dsRNA of a single crab infected with both de novo assembly . Dotknot . The clean reads were collected and used for assembly  with def Dotknot  was used Dotknot .E. coli Poly(A) Polymerase . Poly (A)-tailed dsRNA was used for RACE First-strand cDNA synthesis of each terminus using 5′- or 3′- CDS primers as described by the manufacturer . Then, 5′-RACE and 3′-RACE PCR amplification was performed with viral gene specific primers (GSP) and universal primers (UMP), and then a nested PCR was performed with gene specific primers short (GSPS) and universal primer short  5′/3′ Kit  with purified dsRNA as the initial template. The 3′ poly(A) tailing of RNA was performed at 37°C for 30 min using ort UPS;  using AdHelminthosporium victoriae virus 145S  was used for the outgroup of the ML tree. Branch support values greater than 0.5 were shown in the tree. The accession numbers of the proteins and the corresponding virus names and acronyms are shown in Predicted RdRps from all reference sequences, and closest homologues from NCBI were aligned with RdRps of newly identified viruses in this study using MAFFT 7.0  with an C. sapidus, a probe-based RT-qPCR assay was developed with primer pairs designed to detect a 193-bp region of CsTLV1 genome and a 183-bp region of CsTLV2 . Plasmids containing the targeted region were used as templates to synthesize each strand of the viral RNA standards by T7 or Sp6 RNA polymerase, respectively . Viral RNAs were then quantified and annealed into dsRNA on ice, and serially diluted in 25 ng per μl yeast tRNA carrier. Standard curves were generated by RT-qPCR amplifications of a 10-fold dilution series of synthesized dsRNA containing 10–10e6 genome copies per μl. The qPCR cycling contained qScript® Virus 1-Step ToughMix®  in 10 μl reactions with 0.25 μM each primer and 0.25 μM each probe for both genomes. To anneal PCR primers to dsRNA, primers and extracted RNA were combined, heated to 95°C for 5 min, and then cooled to 4°C prior to being added to the reverse transcriptase and Taq polymerase reaction mixture. Reverse transcription and amplification conditions were 50°C for 10 min (reverse transcription) followed by 1 min at 95°C (reverse transcriptase inactivation and template denaturation). Amplification was achieved through 40 cycles of 95°C for 10 s, and 61°C for 30 s. Gene target copies were then calculated as copies per mg of crab muscle, and samples with greater than 100 copies per mg were recorded as CsTLV1 and CsTLV2 positive, which was based on empirical observations of cross contamination in process control RNA extractions.To screen CsTLV1 and CsTLV2 infections in f CsTLV2  simultanp ≤ 0.05. To determine whether CsTLV1 and CsTLV2 infections were correlated with sex, crab size or latitude, binomial (infected vs. non-infected) generalized logistic regression models (GLM) were conducted . Akaike’s information criterion (AIC) was used to choose the best GLM to determine, which factors best correlate with CsTLV1 and CsTLV2 infection  with CsTLV1 and CsTLV2 confirmed and quantified by RT-qPCR were dissected with muscle, gill and hepatopancreas tissues removed for further examination for virus presence. These crabs were also tested by RT-qPCR  uranyl acetate for 1 h. Specimens were then washed and dehydrated using 30, 50, 70, 90, and 100% ethanol in series. After dehydration, specimens were embedded in Araldite-Epoxy resin  at 4°C overnight. Tissue fragments were then trimmed into ~1 mmIn a search for viral dsRNA in blue crabs, RNA extracted from leg muscle analyzed on agarose gels. The RNA of 31% of sampled of crabs (9 of 29) harvested from the Agawam River, MA in the summer of 2008, showed two prominent dsRNA bands, termed dsRNA-S and dsRNA-L. The apparent molecular weights of the two dsRNA segments were ∼6.5 and ∼7.5 kbp, respectively .Total dsRNA, containing both dsRNA-S and dsRNA-L was sequenced with NGS. Assembly of trimmed and quality filtered reads  resulted in two contigs for each CsTLV1 and CsTLV2, at 383- and 120-fold average coverage, respectively. The longest contig was 4,016 nucleotides (nt) in length. The 5′ and 3′ untranslated regions (UTRs) of both genomes were obtained by RACE PCR and Sanger sequencing, to reveal two toti-like genome sequences of 6,444 and 7,421 nt, and designated as CsTLV1 and CsTLV2, respectively. Each genome contained two ORFs (ORF1 and ORF2) encoding Cp and RdRp proteins, respectively . GenomicPlasmopara viticola lesion associated totivirus-like 5, Hubei toti-like virus 5, and Beihai sesarmid crab virus 7 . CsTLV1 RdRp amino acid sequences showed >33% identity with the corresponding predicted RdRp of Beihai barnacle virus 15, Ahus virus, Parry’s Creek toti-like virus 1, and Diatom colony associated dsRNA virus 17 genome type A . CsTLV2  virus 7 . A searc virus 7 . Sequenc virus 7 , that al virus 7 .Totivirus, and Artivirus which is a proposed genus that includes IMNV and IMNV-like viruses (A maximum likelihood phylogenetic tree was used to show the relationships between CsTLV1 and CsTLV2 and other selected totivirids. As shown in  viruses . CsTLV1 The probe-based RT-qPCR assay consistently detected as few as 10 copies of the target when tested on a dilution series of synthesized dsRNA standards . Efficien = 496) but not the lower latitudinal Atlantic states  and Gulf states . CsTLV1 and CsTLV2 infections in C. sapidus were detected in all three estuaries sampled in MA: Agawam River, Acushnet River, and Falmouth (C. sapidus sampled from MA (n = 198) varied from 11.8%  to 30.6% , and CsTLV2 RNA prevalence ranged from 12.6 to 36.7%. In crabs from RI, CsTLV1 prevalence was 27.6% and CsTLV2 prevalence was 32.8%. Viral RNA was detected in crab specimens collected from Moriches Bay, Shinnecock Bay, and Napeague Bay in Long Island, NY (n = 133) with an average prevalence of 42.1 and 43.6% for CsTLV1 and CsTLV2, respectively. CsTLV1 RNA was detected in Georgica Pond, NY, with low prevalence in 2012 , but not in 2013, 2020, or 2021, and CsTLV2 RNA was detected in 2012  and 2013 , but not in 2020 (n = 33) or 2021 (n = 37).The prevalence of CsTLV1 and CsTLV2 was investigated by RT-qPCR in 875 crabs from the US Atlantic and Gulf of Mexico coasts. CsTLV1 and two infections were detected in the northern states  of CsTLV2 positive specimens . The dsRp < 0.01), in which latitude and CW were significant fixed effects (p < 0.01). The reduced model, including only the association between CsTLV1 and CsTLV2 infection and latitude, sex, or CW, reinforced that latitude and CW were the significant factors correlated with CsTLV1 and CsTLV2 prevalence , and CW showed a negative association with CsTLV1 and CsTLV2 prevalence . In both full and reduced models, the association between CsTLV1/CsTLV2 prevalence and crab sex was not significant (p > 0.1).A binomial (infected vs. non-infected) generalized linear model (GLM) was used to test whether latitude, crab size, or sex could predict CsTLV1 and CsTLV2 infection status . The 415C. sapidus muscle, gill, and hepatopancreas tissues  were revealed  and Helminthosporium victoriae virus 145S  co-infect the pathogenic fungus Helminthosporium victoriae. HvV145S has never been found alone but is always associated with HvV190S virus. HvV145S was originally thought to be the cause of the diseases, however, a recent study suggested that HvV190S alone is the cause of diseases, and the co-infection is not required . Therefore, further systematic and comprehensive studies on the virome of C. sapidus, including CsTLV1 and CsTLV2, at different geographical locations are urgently needed for a better understanding of the virus ecology and epidemiology with the host habitat expansion.The significant correlation between CsTLV1 and CsTLV2 infections and latitude has also been identified in another blue crab dsRNA virus-CsRV1, which also showed significantly higher prevalence at higher latitudinal locations compared to lower latitudes . Howevern waters , made C.C. sapidus were characterized with NGS, and shown to be associated with virus particles and histopathology. Based on their genomic organizations, phylogenetic relationships, and conserved motifs, the viruses are tentatively named CsTLV1 and CsTLV2, and proposed to be members of two new genera in the family Totiviridae.In conclusion, two putative viral dsRNA sequences in https://www.ncbi.nlm.nih.gov/genbank/, OL456199, OL456200.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: ES and MZ designed the experiments and analyzed the results and drafted the manuscript. MZ, LX, and HB performed the experiments. ES, MZ, LX, and HB revised the paper. All authors contributed to the article and approved the submitted version.This work was supported by NSF Division of Ocean Sciences—Biological Oceanography awards 1658466 (ES), Maryland Sea Grant—Graduate student research support grant . MZ and LX were supported by awards from the China Scholarship Council.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.