Naryaviridae and Nenyaviridae) infect Entamoeba parasites, while a third (Vilyaviridae) infects Giardia duodenalis. The trio supplements five CRESS virus families already known to infect eukaryotes, extending the CRESS virus host range to protozoa. Phylogenetic analysis implies CRESS viruses infecting multicellular life have evolved independently on at least three occasions.Metagenomic techniques have enabled genome sequencing of unknown viruses without isolation in cell culture, but information on the virus host is often lacking, preventing viral characterisation. High-throughput methods capable of identifying virus hosts based on genomic data alone would aid evaluation of their medical or biological relevance. Here, we address this by linking metagenomic discovery of three virus families in human stool samples with determination of probable hosts. Recombination between viruses provides evidence of a shared host, in which genetic exchange occurs. We utilise networks of viral recombination to delimit virus-host clusters, which are then anchored to specific hosts using (1) statistical association to a host organism in clinical samples, (2) endogenous viral elements in host genomes, and (3) evidence of host small RNA responses to these elements. This analysis suggests two CRESS virus families ( Metagenomics allows virus genome discovery, but the viral hosts are often not identified. Here, Kinsella et al. use recombination events between virus genomes, statistical association of viruses to hosts in clinical samples, and analysis of endogenous viral elements in host genomes to identify probable hosts of three CRESS virus families. A decade of metagenomic studies have shown that viruses with circular Rep-encoding single-stranded DNA genomes (CRESS viruses) are highly diverse and pervasively distributed4, yet currently, the majority of known CRESS virus genetic diversity falls outside established families with characterised hosts5. Five CRESS virus families have experimentally confirmed eukaryotic hosts: Bacilladnaviridae, Circoviridae, Geminiviridae, Genomoviridae, and Nanoviridae6, respectively infecting diatoms7, vertebrates9, plants10, fungi11 and plants12. Unclassified lineages of metagenomically identified CRESS diversity exist in at least six further clusters labelled CRESSV1 through CRESSV6, and a multitude of chimeric species difficult to place phylogenetically13.Determining hosts of viruses is integral to understanding their medical or ecological impact. This is particularly challenging for virus species discovered using metagenomic sequencing, since samples such as stool or environmental matrices contain diverse potential hosts17. Classically, virus–host relationships are determined via recognition of host disease, followed by virus isolation in cell culture. Since this is impractical for metagenomically identified viruses, case-control studies are used to reveal associations between viruses and disease. Importantly though, this does not confirm the host; for example, the CRESS virus family Redondoviridae is associated with human periodontal disease and critical illness18, but it remains unknown whether the viruses infect humans or a separate host, itself associated with or causing the observed clinical outcomes.Unclassified CRESS viruses are frequently found in human and non-human primate stool samples, generating interest into their host specificity and potential impact on healthSmacoviridae, a CRESS virus family previously assumed to infect eukaryotes, were recently suggested to infect archaea19 on the basis of CRISPR spacer sequences matching a smacovirus inside the genome of an archaeon. Similarly, virus genomes can integrate into host genomes, leaving endogenous viral elements, identification of which reveals historical infections21. Searches for endogenous viral elements related to CRESS viruses have revealed integrations into the genomes of eukaryotes, for instance, sequences related to the replication-associated protein (Rep) of Geminiviridae, major global crop pathogens, are integrated in the tobacco genome22.Genomic evidence of virus–host interactions can directly establish links between species. For instance, the Entamoeba histolytica and Giardia duodenalis23, important human pathogens belonging to distantly related genera24. The Rep-like elements could imply that the parasites host CRESS viruses, however, the sequences do not belong to a known family3. One proposed alternative hypothesis is that that they were gained from bacterial plasmids directly23, which are thought to be the ancestors of CRESS virus Rep genes25. Compatible with this, no sequence related to a capsid protein (Cap) has been found integrated in Entamoeba or Giardia genomes. While several studies have discussed or attempted to identify an association between CRESS viruses and gut parasites28—none has been found to date—and indeed no CRESS virus is known to infect any protozoan. Here we provide evidence that the parasite genera Entamoeba and Giardia are hosts of CRESS viruses, introducing a framework for host determination of metagenomically sequenced viruses that can be widely applied.Rep-like sequences are found in the genomes of the protozoan gut parasites MT293410.1–MT293429.1). The 20 sequences included 18 complete genomes covering all untranslated regions, and these had a genome organisation akin to known CRESS viruses, with a conserved nonanucleotide motif at an apparent replication origin, and open reading frames that aligned to viral Rep and Cap genes  were enriched for viruses using the VIDISCA method, metagenomically sequenced, and bioinformatically analysed to identify unknown CRESS viruses. We used sequence assembly of short reads in combination with inverse PCR and Sanger sequencing to determine 20 full-length CRESS virus coding sequences . We observed that all 21 of the samples containing one of the CRESS viruses were also positive for either Entamoeba or Giardia , therefore we hypothesised that the viruses infected one or both of the parasites. To test the possible host role of other gut protozoa , we carried out further parasitological typing on the 21 virus-positive samples (see “Methods”). We found these taxa were absent from all, or a majority of the 21 samples—implying they are not hosts of the viruses  aligned inside an 11.6 kilobase (kb) contig from E. dispar (AANV02000527.1) with 100% query coverage and 84% nucleotide identity  aligned inside a 15.2 kb contig from G. duodenalis (AHGT01000120.1) with 99% query coverage and 73% nucleotide identity. As the only known examples of parasite endogenous viral elements containing both the Rep and Cap viral genes, they cast doubt on the hypothesis that Rep-like elements in protozoal genomes were derived from bacteria23. Since CRESS virus integration is likely mediated by the Rep protein during viral genome replication in the host nucleus29, the elements directly implicate Entamoeba and Giardia as hosts.In order to identify endogenous viral elements related to the identified CRESS viruses, we aligned all 20 coding sequences to GenBank databases, namely the non-redundant nucleotide  from E. histolytica strain KU27, isolated in Japan in 2001, was also found in strain HM-3:IMSS, isolated in Mexico in 1972 , and three independent assemblies of strain HM-1:IMSS, isolated in Mexico in 1967 , since this was generated using a combination of conventional short-reads and nanopore long-reads30. The latter technology vastly improves the scaffolding and repeat-resolution of assemblies, and confirmed the presence of endogenous viral elements within host sequence, even resolving a 10 kb-long tandemly repeated element not previously detectable in assemblies relying on short-read technology alone  the lineage including Geminiviridae and Genomoviridae, (2) the Circoviridae, and (3) the Nanoviridae. The Nenyaviridae are nested within the CRESSV2 cluster, suggesting these viruses may also infect protozoa.Virus alignments to endogenous viral elements in parasite genomes already suggested that distinct viral lineages with independent virus–host relationships were present among the sequences. We, therefore, resolved the relationships of the exogenous viruses by building a maximum-likelihood phylogenetic tree of the Rep protein. Sequences extracted from Rep-like endogenous viral elements in ies Fig. . Protein18. Genera infecting the same host genus were assigned a Greek number and named with reference to the host (ent for Entamoeba and gia for Giardia) (Supplementary Table Naryaviridae were thus divided into two genera (Protoentvirus and Deuteroentvirus), Nenyaviridae into two (Tritoentvirus and Tetartoentvirus), and Vilyaviridae into three . Although the viruses display large intra-family sequence diversity, the families do share distinctive features: Naryaviridae and Nenyaviridae genomes have sense open reading frames, while Vilyaviridae genomes have either ambisense or antisense open reading frames. Nucleotide usage measured by GC-content varies within each of the three families, but Naryaviridae and Nenyaviridae have on average 37% and 42% respectively, while the Vilyaviridae have a high 59%. The GC-contents of Naryaviridae and Vilyaviridae respectively represent low and high extremes among eukaryotic CRESS viruses. Since a positive association between host nucleotide usage and virus nucleotide usage has previously been observed among single-stranded DNA bacteriophages34, we hypothesised that this also underlay the observed distribution. To test this, we modelled the GC-content of CRESS virus lineages against those of known or proposed hosts using linear regression  and G. duodenalis , respectively. A positive association was found between virus and host nucleotide usage , consistent with the proposed virus–host relationships. The association may be due to codon usage bias, wherein virus codon usage is constrained by host transfer RNA availability35. Despite the positive association, exogenous viruses from the three families did have a higher GC-content than their hosts by an average of 12.6%, suggesting the existence of additional selection pressure on GC-content counter to that of transfer RNA mediated protein translation efficiency. In contrast with exogenous viruses, endogenous representatives of each family had a reduced GC-content, in some cases closely resembling that of the host  with no detectable nucleotide sequence similarity, while the Rep genes were closely related . Recombination between viruses occurs during genome replication within the host, and evidently the host range of a virus dictates its potential recombination partners36. Detection of recombination between viruses can therefore be used to group together viruses into virus–host clusters.During genomic analysis of the CRESS viruses we observed a striking bimodal genome length distribution in both dae Fig. . BLAT alted Fig. . The twoRep and Cap genes were found in close proximity in the protozoal genome . We visualised gene swapping between lineages by linking proteins extracted from the same genome across the two phylogenies, and this uncovered clear evidence of recombination of genetic modules between the Naryaviridae and Nenyaviridae. Members of these Rep families possessed either CCA1 (averaging 467 amino acid residues) or CCA2 (averaging 180 amino acid residues), with all four possible Rep and Cap gene combinations represented. Importantly, while evidence of recombination was also visible within the Vilyaviridae, they always possessed CCA3, therefore no evidence for recombination between Vilyaviridae and members of either the Naryaviridae or Nenyaviridae was found. The data strongly support the proposed host-range of the viruses, specifically Naryaviridae and Nenyaviridae sharing the same host, with Vilyaviridae infecting a separate one. Further, they provide a practical framework to identify virus–host clusters in an unbiased way with no a priori knowledge of the potential host required.To investigate recombination among the identified CRESS viruses, we constructed maximum-likelihood phylogenetic trees of Rep and Cap protein sequences from the three viral families, also including endogenous viral elements if ome Fig. . Since CEntamoeba and Giardia parasites collectively; however, evidence from endogenous viral elements and patterns of recombination among discovered viruses suggested that Naryaviridae and Nenyaviridae infect Entamoeba, while Vilyaviridae infect Giardia. We, therefore, tested the statistical associations of the families to their specific proposed host in human samples using Pearson’s chi-squared test, grouping Naryaviridae and Nenyaviridae together because of recombination between their genomes. Across all 374 study subjects, Naryaviridae and Nenyaviridae were strongly associated with Entamoeba parasites , but not with Giardia , while Vilyaviridae were strongly associated with Giardia . Vilyaviridae were also positively associated to Entamoeba, however at a greatly reduced significance compared to Giardia . This result is likely explained by Entamoeba coinfections in all 3 Vilyaviridae positive samples; indeed, Entamoeba coinfection was found in 73% of all Giardia positive samples (Table Nenyaviridae or Naryaviridae virus infections was 13% among Entamoeba cases (18 of 138), while Vilyaviridae had a prevalence of 27% among Giardia cases (3 of 11). The observed association between the viruses and their hosts in stool enabled a preliminary investigation into the biogeographic distribution of the three families. We mapped reads from public metagenome datasets derived from faecally polluted wastewater or primate stool to our viral genomes. We found reads from Naryaviridae, Nenyaviridae, and Vilyaviridae were detectable in the datasets examined, sourced from localities across North and South America, Europe, Africa, and Asia  was discovered in 198638, and the Vilyaviridae represent the second group of viruses. The discovery of viruses infecting Entamoeba and Giardia—collectively responsible for 300 million human disease cases annually39—should precipitate investigation of their potential impact on the clinical outcome of parasite infection. It is understood that only a subset of Entamoeba and Giardia infections result in symptomatic disease41, however, not all the factors underlying case variation are resolved. For example, E. histolytica interactions with gut bacteria are thought to play a role in pathogenesis42, but the effects of viruses are unexplored. As viruses can modulate parasite pathogenicity directly or indirectly via interaction with human immunity, they may result in parasite hypovirulence11 or hypervirulence43.Here we report three CRESS virus families, 44, however, in instances of hosts intractable to culture, high-throughput methods must rely on viral genome sequences alone. Machine-learning algorithms trained on viral sequences with known hosts offer one possible approach45; however, due to their reliance on conserved sequence signals between training and test data, they will suffer from increasingly coarse prediction for divergent viruses. As we show, construction of viral recombination networks provides direct and unbiased biological evidence of shared hosts among virus genomes, even when individual genes are highly divergent or non-homologous. Given the highly consequential roles protozoa play in global health and ecosystem processes, deciphering additional unknown virus–host relationships among them is imperative.A large proportion of recognised virus genomes are divorced from their biological hosts. Targeted virus discovery from potential host taxa has a vital role to play in resolving this47. Criteria for inclusion in the study were proven HIV-1 infection and being aged 18 years or older. Cohort 2: Stool samples of 85 HIV-1 positive and 95 HIV-1 negative men having sex with men (MSM) as part of the ACS, a prospective cohort study among HIV-positive and HIV-negative MSM, initiated in 198448. Studies were approved by the Medical Ethics Committee of the Amsterdam University Medical Center, the Netherlands (MEC 07/182). Written informed consent of each participant was obtained at enrolment of both cohorts.The 374 human subjects analysed here were from two cohorts. Cohort 1: stool samples of 194 HIV-1 infected individuals not on active antiretroviral therapy, who visited the out-patient clinic at the Amsterdam Medical Center in 1994 and 1995, as part of a study on unexplained diarrhoea49 and reverse transcription was done using non-ribosomal hexamer primers designed to avoid mammal rRNA sequences50. This was followed by second strand synthesis and a cleanup via phenol/chloroform extraction and ethanol precipitation. Library preparation for the two cohorts varied from this point, since two different sequencing technologies were used. For cohort 1 standard VIDISCA library preparation was carried out51. Briefly, double-stranded DNA was digested with Mse1 restriction enzyme, and sequencing adapters were ligated to sticky ends. Libraries were amplified before size selection of fragments between 200 and 600 bp, quantification, and pooling. Sequencing was then done on an IonTorrent PGM instrument. For cohort 2, double-stranded DNA was fragmented to an average length of 400–500 bp, sequencing adapters were ligated, and libraries were amplified before sequencing with Illumina MiSeq instruments (150 bp paired end)52. Sequence reads associated with this study have been deposited in the European Nucleotide Archive (ENA) under study accession PRJEB35571.At collection, faecal samples were suspended 1:3 in broth containing penicillin, streptomycin, and amphotericin B, and stored at −80 °C until processing. Sample suspension (150 µl) was transferred to a reaction tube and centrifuged (10 min at 5000 g) to pellet solid matter and cellular debris. Supernatant was treated with 20 µl TURBO DNase  for 30 min at 37 °C (to remove naked DNA). Nucleic acids were extracted using the Boom method53. Briefly, non-rRNA reads were identified using SortMeRNA v2.154 and made non-redundant using CD-HIT v4.755. Non-redundant reads were then aligned to viral proteins using UBLAST56, and false positives were reduced via BLASTn57 alignment of putative viral matches to the GenBank non-redundant nucleotides. Outputs were visualised with KronaTools v2.758 and inspected to identify candidate CRESS virus reads. Two genomes were amplified via inverse PCR, the sequences of which were determined using Sanger sequencing (accessions MT293412.1 and MT293415.1). All primers are reported in Supplementary Table 59. Further putative CRESS virus hits were manually curated or completed with Sanger sequencing, and were then used in subsequent searches. The process resulted in a final count of 20 CRESS virus coding sequences, 18 of which were complete genomes.Sequence reads from cohort 1 were analysed to discover viruses60. Reads mapping to multiple references were reassigned to their single most-likely reference using the PathoID module of PathoScope v2.0.761. High-depth Illumina sequencing is prone to barcode swapping within flow cells, which may result in false positives; therefore, for cohort 2 a cutoff was imposed for a sample to be regarded as positive. Specifically, virus reads from Entamoeba-infecting or Giardia-infecting families had to make up at least 0.05% of sample reads . In addition to the sequencing-based approach described, any PCR positive samples were also included.To determine a final list of samples regarded as virus positive, sequence reads from each cohort were mapped to the 20 virus coding sequences using BWA-MEM v0.7.1762 and best hits were recorded. DNA secondary structure surrounding the putative nonanucleotide origin motif was assessed using MFOLD63 to confirm it was situated on a predicted stem loop. Circularity of viruses was confirmed by visual inspection of genomes and mapped reads, specifically reads that overlapped with both the beginning and end of genome sequences. To confirm that viral DNA was protected by a capsid, supernatant was first passed through a filter with 1200 nm pores, then 200 nm , followed by treatment with TURBO DNase . Subsequently viral nucleic acid was extracted with the Boom method, and PCR was carried out. To compare CRESS virus GC-content with that of their hosts, the Virus–Host DB64 was used in conjunction with the GenBank genomes resource to compile this information for virus–host pairs.Virus protein sequences extracted from open reading frames were queried against the Reference Proteome database with pHMMERE. histolytica (GCF_000208925.1) and G. duodenalis (GCF_000002435.1). Predicted mRNA databases were first curated using identity searches to remove sequences derived from endogenous viral elements and ribosomal RNA. Hits were also filtered to allow only those with a minimum alignment of 50 nt at over 95% nucleotide identity to their respective subject sequence. The possibility of barcode swapping in cohort 2 Illumina data led us to impose a cutoff for a sample to be called as positive; specifically, the parasite sequence reads as a percentage of the total reads had to be greater than the lower quartile value. For a selection of samples from cohort 1, confirmatory testing was done with E. histolytica and E. dispar diagnostic qPCRs, in addition to Entamoeba generic PCR combined with Sanger sequencing of amplicons. Due to generally low read counts observed for Giardia, all 21 virus-positive samples were subjected to a confirmatory Giardia diagnostic qPCR. The prevalence of Giardia infection among our cohort participants was 2.94% (11 of 374), and the prevalence of Entamoeba infection was 36.90% (138 of 374). Our participants were 93% MSM, and these Giardia and Entamoeba frequencies are concordant with previously reported data from this demographic . To confirm that other protozoa were not the viral hosts, the 21 virus-positive samples were tested for additional parasites: Dientamoeba, Cryptosporidium, and Blastocystis were tested by diagnostic qPCR, while Endolimax, Chilomastix, Pentatrichomonas, and Retortamonas 18S rRNA sequences were analysed in the same manner described above.Faecal samples from cohort 1 were examined by light microscopy for the presence of intestinal parasites (with both direct smears and concentrations using the Ridley technique). From both cohorts, sequence reads were mapped using BWA-MEM to parasite ribosomal RNA reference sequences, with aligning sequences then queried against the GenBank non-redundant nucleotide database. Reads with the best hit to a parasite ribosomal RNA reference, and a minimum alignment of 50 nt at over 95% nucleotide identity was retained as hits. Hits were aligned to diagnostic parasite reference sequences to type the parasite species where possible. Sequence reads were also mapped using BWA-MEM to predicted mRNA sequences from parasite genomes, specifically Entamoeba and Giardia using BLASTn. Nucleotide and protein sequences of hits were extracted and manually curated to use in subsequent analyses. Comparison between independent assemblies of E. histolytica and G. duodenalis  was done using BLASTn of endogenous Rep gene elements from each genus against each assembly, recording the best aligning hit. Pairwise comparisons between sequences were all performed using BLAT via the MAFFT online server66. Available genome assemblies from relatives of Entamoeba and G. duodenalis were also analysed for the presence of elements, specifically Mastigamoeba balamuthi (GCA_902651635.1), Spironucleus salmonicida (GCA_000497125.1), Trichomonas vaginalis (GCA_000002825.1), and G. muris (GCA_006247105.1); however, none of these assemblies contained elements belonging to the Naryaviridae, Nenyaviridae, or Vilyaviridae. To assess read coverage across E. histolytica contig NW_001915013.1, raw sequencing reads were downloaded from the TraceDB  and ENA . BWA-MEM was used to map reads to the complete reference contig, followed by visualisation of coverage using CodonCode Aligner v9.0.1. Easyfig v2.2.567 was used to visualise pairwise identity between G. duodenalis contigs VSRU01000012.1 and AHHH01000265.1. To identify evidence of an RNA interference response against endogenous viral elements, BWA-backtrack60 was used to map E. histolytica AGO2-2 associated small RNAs from ENA project PRJNA18707032 against contigs containing elements from the E. histolytica RefSeq genome assembly (GCA_000208925.2). Prior to mapping, sequencing adapters were trimmed using BBDuk (http://jgi.doe.gov/data-and-tools/bb-tools/), and sequences over 40 nt and under 15 nt were discarded. Reads mapping with zero sequence mismatches were retained, and coverage of contigs was calculated using the SAMtools mpileup utility68. Positions of endogenous viral elements and small RNA coverages were visualised for a selection of contigs using Circos v0.69-869.CRESS virus genomes were aligned to GenBank databases: the non-redundant nucleotide using BLASTn, the non-redundant protein using BLASTx, and the whole-genome shotgun contigs of 13, with the addition of the Redondoviridae18, five viral sequences found during BLASTn searches of the GenBank non-redundant nucleotide database, and our CRESS virus sequences (both exogenous and endogenous viruses). Rep proteins were aligned using MAFFT v766 with the L-INS-i option leaving gappy regions unaligned. The resulting alignment was trimmed using trimAl v1.470 set to gappyout. Maximum-likelihood phylogenetic analysis was performed using RaxML v8.2.971 with the PROTCATGTR substitution model and automatic bootstopping, which stopped rapid bootstrap searching after 350 replicates. Treefiles were visualised using Figtree v1.4.4 (https://github.com/rambaut/figtree/releases). The same methods were applied for phylogenetic analysis of the three Rep protein families in isolation, as well as their corresponding Cap proteins. For delimitation of Rep genera, pairwise comparison was carried out using the online tool SIAS (available at: http://imed.med.ucm.es/Tools/sias.html), with the denominator set to mean length of sequences.Phylogenetic analysis of the Rep protein utilised a previously compiled chimaera-free datasetPRJNA16901072 were from Maiduguri (Nigeria), Kathmandu , Bangkok (Thailand), and San Francisco (USA); project PRJNA7062373 samples were from Addis Ababa (Ethiopia), Barcelona (Spain), and Pittsburgh (USA); project PRJNA32230174 was from Tallahassee (USA); project PRJNA43474475 was from Cincinnati (USA); and project PRJNA38583176 was from Sheboygan (USA). Macaque stool from project PRJNA29933214 was from the California National Primate Research Center (USA). Human stool from project PRJNA41804426 was from Caracas (Venezuela) and remote villages in South-East Venezuela; and project PRJEB952477 was from Uganda. A further site was annotated based on a public virus genome (LC406405.1) which clustered within the Vilyaviridae, sampled from a cat in Japan78.Data to estimate the global distribution of parasite-infecting CRESS viruses was obtained from a number of public metagenomes and mapped using BWA-MEM to virus genomes. Wastewater samples from ENA project Further information on research design is available in the Supplementary InformationReporting Summary


Out-of-pocket prescription drug costs are rapidly rising, particularly for insulin, which is a life-saving drug used by 3.1 million diabetics on Medicare. High out-of-pocket costs place an accentuated financial strain on older adults with diabetes, many of whom have low incomes, and may impede medication adherence, leading to poor health outcomes. The Medicare Part D Low-Income Subsidy (LIS) program limits drug co-pays to under $8.50 per prescription and caps out-of-pocket drug costs for lowest-income recipients , resulting in pronounced differences in out-of-pocket costs for those with marginally different incomes. Using detailed income data from the Health and Retirement Study linked to Medicare claims (2008-2016), we employed a regression discontinuity (RD) design to isolate the effects of differences in out-of-pocket costs at eligibility thresholds for the LIS. Diabetic beneficiaries whose income exceeded the LIS eligibility threshold had lower Part D spending  and adherence to oral antidiabetic drugs . We conducted secondary analyses at the eligibility threshold for Medicaid, as individuals whose income exceeds the eligibility limit for Medicaid (100% of FPL in most states) are significantly less likely to receive the LIS. Above the Medicaid eligibility threshold , annual spending on insulin was $395 lower (p=0.002) and proportion of insulin use was 6% lower (p=0.04). These results suggest low-income Medicare beneficiaries who are not shielded from out-of-pocket costs via the LIS are particularly sensitive to drug costs. Policy proposals to limit out-of-pocket costs could improve medication adherence to high-cost drugs for vulnerable beneficiaries.


Current localization techniques used in breast conserving surgery for non-palpable tumors show several disadvantages. Magnetic Seed Localization (MSL) is an innovative localization technique aiming to overcome these disadvantages. This study evaluated the expected budget impact of adopting MSL compared to standard of care.Standard of care with Wire-Guided Localization (WGL) and Radioactive Seed Localization (RSL) use was compared with a future situation gradually adopting MSL next to RSL or WGL from a Dutch national perspective over 5 years (2017–2022). The intervention costs for WGL, RSL and MSL and the implementation costs for RSL and MSL were evaluated using activity-based costing in eight Dutch hospitals. Based on available list prices the price of the magnetic seed was ranged €100-€500.The intervention costs for WGL, RSL and MSL were respectively: €2,617, €2,834 and €2,662 per patient and implementation costs were €2,974 and €26,826 for MSL and RSL respectively. For standard of care the budget impact increased from €14.7m to €16.9m. Inclusion of MSL with a seed price of €100 showed a budget impact of €16.7m. Above a price of €178 the budget impact increased for adoption of MSL, rising to €17.6m when priced at €500.MSL could be a cost-efficient localization technique in resecting non-palpable tumors in the Netherlands. The online calculation model can inform adoption decisions internationally. When determining retail price of the magnetic seed, cost-effectiveness should be considered. Magnetic Seed Localization could be a cost-efficient alternative to RSL and WGLIn determining the price of MSL, cost-effectiveness should be consideredThe online calculation model can inform adoption decisions on an institutional levelBreast-conserving surgery (BCS) for non-palpable tumors requires appropriate localization technologies to resect the malignancy effectively ,2. CurreRSL however has a considerable disadvantage as its radioactive nature requires adherence to strict nuclear safety regulations ,14. ThisA recent study compared WGL with MSL and concluded that WGL was equally effective as MSL. SeveralThis study aims to inform the adoption decision of MSL by evaluating the financial impact of gradually adopting MSL as a localization technology for guiding breast conserving tumor excision in the Netherlands health system compared to standard of care (SoC) by means of a Budget Impact Analysis (BIA) incorporating treatment and implementation costs. Secondly, a threshold analysis was conducted to estimate the maximum price level for MSL to become the most cost efficient technology. Finally, the BIA model was made available in a tool to enable translation of the results to other countries or specific hospital settings .For the analysis, the BIA framework of the International Society for Pharmacoeconomics and Outcomes Research was followed . The anaIn WGL, a metal wire with a hooked tip is placed in the lesion at the radiology department. This placement needs to be performed on the same day as surgery which complicates scheduling of the surgery. Intraoperatively, the surgeon removes the lesion guided by the wire.,22With the use of RSL, a small radioactive iodine-125 seed is placed in the lesion by the radiologist. The timing of the placement of the seed in the Netherlands is flexible, and not limited to a few days before surgery. Intraoperatively, a gamma probe providing continuous audible feedback is used by the surgeon to detect the seed. After surgery, the Iodine-125 seed must be removed from the excised specimen in the pathology lab to be safely disposed. We foundWith the use of MSL, a magnetic seed is placed in the lesion by the radiologist. The signal will not decay over time, therefore the timing of placement is flexible . IntIn 2017, 17,207 patients were diagnosed with BC (including DCIS) . The NatIn 2017, RSL and WGL were used in respectively 21% and 79% of the BC patients, due to hospital differences [The potential future uptake of RSL and MSL in 2022 was estimated by experts working in the Netherlands Cancer Institute (NKI) where MSL is used in a research setting next to RSL . Since, The classic diffusion theory by Rogers was used to estimate the adoption speed of RSL and MSL ,34. The In the Netherlands, reimbursement for the use of localization techniques during breast cancer surgery is part of a budget allocated for a specific combination of diagnosis and treatment. Therefore, specific costs for using a localization technology apart from e.g. hospital stay, are not specified. By means of Activity Based Costing (ABC) costs for using the localization technologies were estimated. This method takes into account all activities consumed within a process and allocates costs to the resources required for these activities .Clinical process per localization technology. First the processes had to be drafted for using WGL, RSL and MSL. The processes were evaluated by clinical expert interviews in eight hospitals in 2017 , “real-time” observations, literature and hospital treatment protocols. Since MSL was only used in one Dutch hospital, the MSL process was based on interviews held in that institute (NKI-AvL). The expert interviews assessed also the implementation process for RSL  and MSL . In drafting the processes, the incidents associated with the use of radioactive seeds were included in the process. Based on literature, the duration of seed placement 45 min) and surgery (90 min) were assumed to be similar between the localization techniques 5 min and and migrCost calculation. To calculate the costs of each process step, the Dutch manual for cost calculations was used [was used . Personnwas used . Those cwas used . The matwas used ), sterilAlthough the equipment used in WGL and RSL were already bought and will be used for several procedures, we included the equipment costs to have a fair comparison to MSL. Equipment costs for RSL, WGL and MSL were based on actual acquisition costs from the participating hospitals. Since the gamma probe and contamination monitors, essential for using RSL, are also used in other procedures these costs were partly taken into account: 50% and 30% respectively. Finally, overhead was calculated over all costs except over the intervention and material costs to avoid double counting, using a general percentage of 44% [The intervention costs included in the BIA model were: €2617, €2834, €2662 (without magnetic seed) for WGL, RSL and MSL respectively. The additional costs per patient receiving NACT in WGL and MSL were €146, and the implementation costs for MSL and RSL were: €2794 and €26826 respectively. These costs and details on the analysis are presented in To perform the analysis, Microsoft Excel version 2010 was used. The BIA compares the total intervention costs of the localization technologies used per year plus the yearly implementation costs of the hospitals that are expected to transfer to a different technology for both SoC and SoC with MSL. To calculate the yearly treatment costs, the yearly BC incidence was multiplied by the percentage of patients receiving localization during surgery and by the yearly uptake percentages of the localization technologies. These numbers were multiplied by the costs per localization technology including the additional costs for the proportion of patients receiving NACT. The future costs for 2018 and later were corrected using an average inflation ratio based on the Dutch inflation ratios of the previous five years .The model structure and input parameters were based on several assumptions and therefore associated with a level of uncertainty. To evaluate the impact of our assumptions, deterministic sensitivity analyses (DSA) were conducted.First, a one-way sensitivity analysis was conducted on the results of the cost analysis for RSL and MSL to identify the parameters with the highest influence. Upper and lower limits of 20% were used varying the for instance the number of patients per hospital, equipment costs and duration of placing the marker. Second, a one-way sensitivity analysis was conducted on the BIA results for the year 2022 with fixed magnetic seed costs of €200. Also upper and lower limits of 20% were used to check the influence of several input parameters. For example: implementation costs, treatment costs, and the percentage of patients receiving NACT. Finally, three alternative diffusion estimates were tested: a constant uptake of MSL, changing the adoption speed of MSL , and changing the initial uptake of RSL in 2017 to (1) 30% RSL, 70%WGL and (2) 40% RSL and 60% WGL.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For all the interviewees  we asked their permission to record the interviews and additionally we checked the information retrieved from the interview with each interviewee.The results of the BIA model are shown in The duration of the excision and seed placement, the costs of the magnetic seed and the overhead percentage drove the intervention costs of RSL and MSL the most . UncertaThe different diffusion estimates incorporated in the DSA had a small impact on the budget impact . A constThe results of the BIA indicated that adoption of MSL in the Dutch healthcare system could be cost-saving due to the lower implementation costs for MSL (€2974) compared to RSL (€26826). However, to maintain this advantage after implementation phase: (1) the costs of using MSL per patient should not be substantially higher than those for RSL or (2) response monitoring with MRI should be enabled when using MSL in NACT patients or (3) the use of MSL should result in improved clinical outcomes compared to WGL and RSL.To the best of our knowledge this is the first BIA on localization techniques in which the intervention costs of localization techniques have been evaluated in detail, including the additional activities related to using radioactive material. The results of the cost analysis could inform the decision to transfer from WGL to either MSL or RSL on a national or hospital level. The decision to adopt one of the technologies may be further supported by additional factors such as the improved resource allocation and impact of a localization method on logistics ,4,6. BesThe BIA results are mainly driven by the intervention costs . The cosThe main strength of the current analysis is the detailed insight in the costs of all three localization techniques, based on data from 8 Dutch hospitals. Our results can be used and adjusted on a hospital and country level to guide the decision to adopt RSL or MSL using the general model . As we wThe main limitations in this study are the assumptions regarding uptake of various techniques for 2022, and the early stage of our analysis. As the present analysis evaluates a new technology still in development and subject of clinical trials, the results from the cost-analysis related to MSL (treatment and implementation costs) are uncertain and of potentially limited applicability. The impact of the implementation costs on the budget for 2017 to 2022 could be underestimated, due to allocation of the implementation costs over 5 years and the majority of hospitals were assumed to adopt a new technology in the final two years. Other limitations were: (1) the selection of the hospitals, as this could have biased the cost-analysis results. Although we incorporated all types of hospitals  and hospitals located in different areas of the country, this could limit the generalizability of the budget impact analysis. (2) Not being able to incorporate the logistical hurdles when using WGL and therefore the intervention costs of WGL were underestimated. This however would not have altered the conclusions as the benefits are similar for MSL and RSL compared to WGL. A final limitation (3) is the main assumption that the efficacy of MSL and RSL is similar to WGL. Future comparative studies should verify whether this is truly the case. If clinical benefit is expected these factors should be incorporated in this analysis or a cost-effectiveness analysis should be performed.Our present analysis shows that MSL could be a new cost-efficient localization technology in guiding resections of non-palpable breast cancer tumors in the Netherlands. When the costs to use MSL are significantly higher than those for using RSL and WGL, the lower implementation costs for MSL will not outbalance these higher intervention costs. Manufactures should consider cost-effectiveness when determining retail price of the magnetic seed.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(XLSX)Click here for additional data file.


Apis mellifera) is the parasitic mite Varroa destructor, largely because it vectors lethal epidemics of honeybee viruses that, in the absence of this mite, are relatively innocuous. The severe pathology is a direct consequence of excessive virus titres caused by this novel transmission route. However, little is known about how the virus adapts genetically during transmission and whether this influences the pathology. Here, we show that upon injection into honeybee pupae, the deformed wing virus type-A (DWV-A) quasispecies undergoes a rapid, extensive expansion of its sequence space, followed by strong negative selection towards a uniform, common shape by the time the pupae have completed their development, with no difference between symptomatic and asymptomatic adults in either DWV titre or genetic composition. This suggests that the physiological and molecular environment during pupal development has a strong, conservative influence on shaping the DWV-A quasispecies in emerging adults. There was furthermore no evidence of any progressive adaptation of the DWV-A quasispecies to serial intra-abdominal injection, simulating mite transmission, despite the generation of ample variation immediately following each transmission, suggesting that the virus either had already adapted to transmission by injection, or was unaffected by it.The main biological threat to the western honeybee ( It is a highly plastic and adaptive trait, particularly through the virus, subject to selection at multiple levels of host organization  with different possible outcomes at the different organizational levels, including levels where the interests of virus and host may be aligned, for example as a weapon in ecological competition with more susceptible hosts4. Consequently, virulence evolution is equally varied, resulting in all types of host-virus relationships ranging from parasitic host exploitation by the virus through mutualism to enslavement of the virus in function of the host’s interests, including genetic integration of viral and host genomes5. The main mechanism for virulence evolution is through the virus genome and in particular its quasispecies, i.e. the population of virus genome variants generated by the high rates of mutation and recombination associated with RNA replication6. Through the quasispecies, a virus can leverage its small physical genome into a highly variable sequence space  with which to respond dynamically to adaptive challenges, through a combination of natural selection and complementation8. Consequently, it is the quasispecies as a whole that is both the target and the result of natural selection9. The genetic composition of the quasispecies can therefore be visualized as a cloud10, whose shape shifts depending on which groups of variants are favoured at any one time or place, and whose adaptive potential resides as much in its overall diversity as in the specific capacities of individual variants11.The requirement of viruses for a living host in order to replicate axiomatically sets limits to the amount of damage a virus can do to a host at either individual or population level. Virulence is the quantitative expression of such damage13. A potent novel transmission route that bypasses the barriers limiting virus distribution and replication at multiple organizational levels, such as vector-mediated transmission, therefore represents a major destabilizing factor affecting the core parameters for virulence evolution, in the conflict between the virus and the host for control of the primary character, and genetic composition, of the virus. Here we investigate aspects of this fundamental conflict for the relationship between honeybees (Apis mellifera) and deformed wing virus (DWV), one of the many viruses infecting honeybees and other hymenoptera15.Of the many factors affecting virulence evolution, the most important is transmissionVarroa destructor16, largely because it vectors lethal epidemics of honeybee viruses18 that, in the absence of this mite, are relatively innocuous16. The principal varroa-transmitted virus is currently DWV14. There has long been a lively debate19 as to whether the severe pathology caused by DWV is primarily due to genetic adaptation by the virus towards increased virulence22 or due to excessive virus titres25 precipitated by this potent, novel transmission route28. Of course, these two alternatives are not mutually exclusive. It has been conclusively shown that DWV is directly responsible for the deformed wing symptoms30; that these symptoms are directly related to both the amount of virus and the transmission route25, and that the different major DWV variants  have different replicative capacities in both honeybees and V. destructor33, as well as different relationships with honeybee pathology at individual bee34 and colony level35. What is less well known is how the virus adapts genetically during these transitions and transmissions.The main current biological threat to the western honeybee is a recently acquired exotic parasitic mite, V. destructor expansion front in Sweden, with a confirmed complete absence of mites and no known prior contact with bees from mite-infested regions36. This discovery allowed us to examine a possibly primordial DWV quasispecies  and to test certain hypotheses37 for DWV adaptation during the early establishment of V. destructor infestation, specifically whether the DWV quasispecies would adapt genetically to this new and highly potent transmission route during serial transmission and whether these genetic changes had any bearing on DWV pathology, independent of the known effects of DWV titre25 and transmission route39. The results presented here concern primarily the evolution of the DWV quasispecies during serial transmission. The relationship between DWV quasispecies composition and DWV pathology will be presented separately.It is in this context that the following experiments were conducted. In 2008, we discovered several DWV-infected colonies from Leksand, well beyond the 36 were placed in quarantine in autumn 2009 and moved to Uppsala in summer 2011 for experimentation. A single DWV symptomatic adult from one of these colonies was used as the original source inoculum. Mite-mediated transmission was simulated by microinjection of controlled amounts of virus into pink-eyed honeybee pupae40, which were either left to complete development to adults or sacrificed after 24 hours to provide the inoculum for the next transmission  makes it furthermore impossible to either confirm or deny a possible Leksand origin for the T0 inoculum, and thus its primordial status. The question of whether a naïve, primordial DWV quasispecies would adapt genetically to varroa-mediated transmission therefore remains unanswered.The symptomatic and asymptomatic bees at each transmission had similar DWV titres, which were at least 5 orders of magnitude larger than for the mock-inoculated control bees Fig.  while se8) to be represented. However, the DWV quasispecies analysed in these experiments was grouped around only one of the major DWV strains (DWV-A44), and therefore involves a narrower range of genetic variability and more refined resolution of effects than quasispecies consisting of multiple major strains and their recombinants35. Although the DWV consensus sequence remained constant and near-identical to the T0/T13 consensus sequence throughout this first wave of transmissions and adult progeny symptomatic adult bees. The DWV quasispecies in each sample was represented by a matrix of Single Nucleotide Polymorphism (SNP) frequencies along the entire DWV genome, which allows all the major types of variation found in viral quasispecies , hints at a more complex host-pathogen relationship than mere pathological exploitation and perhaps other, more mutualistic77 roles for DWV to bee life at some level of organization  or context , as one of the predicted possible outcomes of host-pathogen co-evolution79. Since these viruses are part of a shared pathosphere involving other bees, wasps and bumblebees78, the question also has relevance outside the beekeeping community78.These experiments also provide a possible mechanistic explanation for those instances where adaptation to transmission route36 were transferred in September 2009 from Leksand to an isolated forest area near Forsmark , at that time also still beyond the varroa front. When Varroa destructor was detected in Forsmark in 2009, the colonies were relocated in March 2010 to a quarantine apiary in Grötlingbo Nature reserve on Gotland , 4.8 km from the nearest registered bee colonies, since they could no longer be moved back into varroa-free areas on mainland Sweden or its archipelagos. One year later, in June 2011, the colonies were brought back to Uppsala for experimentation. The DWV starting inoculum (T0), prepared as described below, was obtained from a single symptomatic adult honeybee from one of these colonies. By the end of 2011 both colonies had acquired varroa. A Varroa destructor-free and DWV-free honeybee colony from Utö , an island in the Swedish archipelago that has never been infested with V. destructor, was used to obtain the experimental worker pupae for serial transmission. Throughout the experiments, control worker pupae from this colony tested negative for DWV by RT-qPCR.The two surviving colonies in the Leksand study40. At each transmission passage, around 15 pink-eyed, white-bodied pupae were each microinjected laterally in the abdomen, in between the 2nd and 3rd integuments, with 2 µL DWV inoculum obtained from the previous passage, using a 50 µl microsyringe  and very thin, 30 gauge disposable needles. The needles were replaced between individual pupae. The pupae were incubated on Whatman filter paper in disposable petri dishes in an incubator at 35 °C and 80% relative humidity40. After a 24 hour incubation, a single random pupa was selected as the DWV inoculum for the next serial passage, while the remaining pupae were incubated a further 6 days to complete their development into adult bees. All collected bees were stored at −80 °C until further use. This procedure was repeated for 14 serial transmissions per trial, and for two independent trials. For technical controls, ten pink-eyed pupae were microinjected with just 2 μL PBS at each transmission. From the serial transmission assays detailed above, all the serial 24-hour inocula pupae and at least one of the corresponding adult transmission progeny were selected for DWV quasispecies analysis through IonTorrent sequencing.The serial transmission experiments were conducted during the summer of 2011, according to the experimental design shown in Supplementary Fig. The 24-hour pupal samples were homogenized at room temperature in 500 µL PBS buffer , of which 100 µL was used for RNA extraction and the remainder stored at −80 °C until further use. The 7-day adult bee samples were homogenized in 200 µL TN buffer , of which 50 µL was used for RNA extraction and the remainder stored at −80 °C. RNA was extracted from these 24-hour and 7-day sample homogenates using NucleoSpin® RNA Kit  and was eluted into 50 μL nuclease-free water. The approximate RNA concentration was determined by NanoDrop. Reverse transcription was performed in 20 µL reactions containing approximately 2.5 µg RNA, 10 mM dNTPs, 100 µM random hexamer primers and 400 units SuperScript™ III RT . The resulting cDNA was purified using the High Pure PCR purification Kit , eluted into 100 μL elution buffer  and stored at −80 °C until further use.23, as was the Apis mellifera β-actin mRNA as an internal reference gene for quality control and data Normalization80  and strain B (DWV-B); Sacbrood virus – SBV; Slow bee paralysis virus – SBPV; Bee Macula-like virus - BeeMLV) were quantified by qPCR of the cDNA using previously described protocols and primers44. PCR was performed using the high fidelity KAPA HiFi™ HotStart kit , to minimize the introduction of PCR-derived errors. The reactions contained approximately 0.2 µg cDNA, 10 mM dNTPs, 0.3 µM of each primer and 0.5 units of KAPA HiFi™ HotStart DNA polymerase in 25 µL volumes. The reaction was processed in a ECO™ Real-Time PCR machine  with the following qPCR cycling profile: 2 min at 95 °C, followed by 35 cycles of [20 sec at 98 °C - 20 sec at 56 °C – 3 min at 72 °C], followed by 2 min at 72 °C. The fragments were purified using the High Pure PCR purification Kit , and the fragment sizes were confirmed by electrophoresis through a 1.2% agarose gel, stained in freshly prepared GelRedTM for 30 min and visualized under UV light. Although the diagnostic DWV assays detected very low amounts of DWV in some of adult bees developing from mock/PBS-inoculated pupae, the DWV whole-genome amplification assays failed for the Control samples, except for the 694 bp Lp fragment.The whole DWV genome was amplified from one symptomatic adult bee and (where possible) one asymptomatic adult bee from each of the first 9 serial transmissions, as well from the corresponding 24-hour serial transmission inocula, each of which was also derived from a single bee. The DWV genome was amplified in four overlapping sections covered by primer-pairs F27/B1806, F1725/B3095, F3018/B7295 and F7243/B10101 . Library size and concentration were assessed by a Bioanalyzer High Sensitivity Chip (Agilent Technologies) and the Fragment Analyzer system . The DNA samples were pooled together in sets of nine, followed by template preparation using the Ion PGM™ Template OT2 400 Kit on the Ion OneTouch™ 2 system (ThermoFisher). Samples were then sequenced on the Ion PGM™ System with Ion PGM™ Sequencing 400 Kit on Ion 316v2 chips (ThermoFisher) aimed at a 400 bp read length.First, the DWV whole-genome PCR fragments of the T0 and T13 24-hour inocula Fig.  were seq82 and an in house script. This resulted in a numerical file for each sample containing the frequencies of each nucleotide or a deletion at each position of the DWV genome between nucleotides 10 and 10,120 of the mapping reference sequence for all samples, as well as the sequencing coverage across the DWV genome (average/max/min per sample = 8952×/14292×/2772×), thus capturing the full genetic complexity of the DWV quasispecies83. These files form the basis for the data analyses. Statistical analyses were conducted in R unless stated otherwise.The IonTorrent sequence reads were first filtered by bar-code, to identify the sample origin, and then mapped to reference sequences AY292384 (DWV-A), AY251269 (DWV-B) and CEND01000001 (DWV-C) using Tmap included in TorrentSuite 3.6.2 with LifeTechnologies recommended parameters. Variability data and consensus sequences were created using mpileup from SAMtools (v.0.1.8)84 and saved in NEXUS format. Molecular evolutionary analyses were conducted using DnaSP (v5)85 and MEGA. Genetic variability from the coding region (polyprotein) within inocula and 7-day sample populations was estimated as nucleotide diversity (π)86, consisting of the average number of nucleotide differences per site between all pairs of analyzed sequences, using DnaSP. Similarly, Tajima’s D87 and Fu and Li’s D and F88 were used to test for neutrality and to infer positive  or negative (purifying) selection as implemented by DnaSP.The DWV consensus sequences of the 23 NGS sequenced samples were, with the exception of 3 individual SNPs, identical among each other  and the number of non-synonymous substitutions per non-synonymous site (dN) between inocula and 7-day sample populations using the Nei and Gojobori’s method89 with MEGA in order to reject the null hypothesis of strict neutrality (Ho: dS = dN) and infer, as well, the balance between neutral mutations, negative selection and positive mutations acting on the DWV quasispecies.The Codon-based Z test of Selection was performed calculating the number of synonymous substitutions per synonymous site , which are subsequently used by discriminant analysis (DA) to assign the samples to the different genetic clusters by maximizing between-group variance and minimizing within-group variance. The input for the DAPC consisted of frequencies of polymorphic SNPs (columns) for each of the 23 virus quasispecies populations (rows). The data for sample 8AS was based on a reduced genome, since the sequence coverage for the last 1,302 nucleotides at the 3′ end of the genome was below the ≥100x cut-off value for nucleotide frequency calculations  to identify the optimal number of genetic clusters that describe the data. The optimal K is associated with the lowest BIC value91.The clustering analysis of the genetic variation between the sequenced samples was performed using Discriminant Analysis of Principal Components (DAPC), a model-free method implemented in the adegenet package for RNPs Fig. . The secNPs Fig. . The seq93. Nei’s genetic distance86 between the 23 DWV quasispecies samples was calculated from the SNP frequencies of the 1,212 characters in the DPC data matrix using the programme ‘Gendist’. This distance matrix was used to construct a phylogenetic topology using the Neighbour-Joining algorithm in the programme ‘Neighbour’ and the consensus tree was built using the programme ‘Consense’. Statistical support for the branches in the consensus topology was provided by 1,000 replicate bootstraps of the data, using the programme ‘Seqboot’. The phylogenetic analysis describing the relationship between the DWV samples from the current experiment and other biogeographic isolates from Sweden and Gotland93 relative to a selection of DWV-A94 isolates representing Europe and the Middle East95, North and South America96 and East Asia100 was inferred using the Maximum Likelihood method and the Tamura-Nei model of evolution101, as implemented by MEGA-X85, using the DWV-B48 and DWV-C44 reference strains as outgroup sequences. The statistical confidence of each of the nodes was determined by bootstrap analysis involving 500 replicates. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated resulting in a total of 694 positions in the final dataset.The phylogenetic analyses of the SNP frequency data were conducted with various programmes implemented by PHYLYP (v.3.65c)Supplementary Figures Tables.


Bovine viral diarrhea virus (BVDV) is a cause of substantial economic loss to the cattle industry worldwide, and there are currently no effective treatment or preventive measures. Bovine enterovirus (BEV) has a broad host range with low virulence and is a good candidate as a viral vaccine vector. In this study, we explored new insertion sites for the expression of exogenous genes in BEV, and developed a recombinant infectious cDNA clone for BEV BJ101 strain expressing BVDV E0 protein.pro was inserted in the GpBSK-BEV plasmid at the 2C/3A junction by overlapping PCR. Subsequently, the optimized full-length BVDV E0 gene was inserted to obtain the recombinant infectious plasmid GpBSK-BEV-E0. The rescued recombinant virus was obtained by transfection with linearized plasmid. Expression of BVDV E0 in the recombinant virus was confirmed by PCR, western blotting, and immunofluorescence analysis, and the genetic stability was tested in MDBK cells over 10 passages. We further tested the ability of the recombinant virus to induce an antibody response in mice infected with BVDV and immunized them with the recombinant virus and parental strain.A recognition site for the viral proteinase 3CThe rescued recombinant virus rBEV-E0 was identified and confirmed by western blot and indirect immunofluorescence. The sequencing results showed that the recombinant virus remained stable for 10 passages without genetic changes. There was also no significant difference in growth dynamics and plaque morphology between the recombinant virus and parental virus. Mice infected with both recombinant and parental viruses produced antibodies against BEV VP1, while the recombinant virus also induced antibodies against BVDV E0.A new insertion site in the BEV vector can be used for the prevention and control of both BEV and BVDV, providing a useful tool for future research on the development of viral vector vaccines. Enterovirus in the family Picornavirus. The virion is spherical, icosahedral, non-encapsulated, and has a diameter of 25–30 nm. The viral genome is a non-segmented single-stranded positive-stranded RNA with a total length of about 7.5 kb. It can directly translate a polyprotein as mRNA and undergoes a series of degradation steps to produce four structural egg proteins (VP1–VP4) and seven non-structural proteins , among which the viral protease 3Cpro recognizes and cleaves a characteristic amino acid sequence (ALPQG) within exposed and flexible structural domains [Bovine enterovirus (BEV) was first reported in 1957 and has been prevalent in major cattle breeding countries and regions worldwide, including the United States, Canada, the United Kingdom, Germany, Poland, Pakistan, and China . Infecti domains , 13.BEV is generally considered to be non-virulent or of low virulence, not highly pathogenic, resistant to acidic environments, and can infect animals through the intestinal tract, making it a good candidate for a vaccine vector. Despite substantial progress in the development of recombinant and chimeric human enteroviruses such as poliovirus and EV-71 virus , relativFlaviviridae and the genius Pestivirus, is the cause of bovine viral diarrhea (BVD). BVD is a complex disease with various clinical manifestations and is considered one of the main threats to the cattle industry worldwide. BVDV not only infects cattle but also infects sheep, goats, pigs, deer, and other ruminants with a wide host range [Bovine viral diarrhea virus (BVDV), a single-stranded positive-strand RNA virus belonging to the family st range . Since tst range , which cIn this study, we used the BEV BJ101 strain as a viral vector to express exogenous genes by inserting the BVDV E0 gene between the genes encoding the non-structural proteins 2C and 3A. We compared the replication, infection, and biological characteristics of the rescued recombinant virus after insertion to those of the parental virus, and evaluated the expression of E0 protein and its ability to induce an immune response in vivo.Viral RNA was extracted from Madin-Darby bovine kidney (MDBK) cells  infected with the BEV BJ101 strain (maintained in our laboratory) using an RNA extraction kit . The first-strand cDNA of the virus was synthesized with the PrimeScript™ II 1st Strand cDNA Synthesis kit .NotI enzyme cleavage site was added to the 5′ end and an XbaI enzyme cleavage site was added to the 3′ end. The whole gene sequence was connected to the pUC-57 plasmid vector and named pUC57-E0.Mammalian codon optimization was carried out on the gene sequence of BVDV E0 (GenBank: EU709763.1) and the optimized sequence is shown in Additional File In accordance with the strategy schematically outlined in Fig. The synthesized cDNA was used as a template for subsequent polymerase chain reaction (PCR) amplification. The genome sequence of BEV BJ101strain (GenBank: MG650158.1) was divided into three fragments, fragment A, B and C, containing nucleotides 1–3334, 3301–6269, and 6213–7409, respectively, for amplification. Three pairs of specific primers  were designed to amplify the three fragments, and cloned into the modified GpBlueScript (−) vector  present in the original plasmid. An HA tag was added between the NotI and XbaI restriction sites to prevent the proximity of the two enzyme cleavage sites from affecting the efficiency of double enzyme cleavage. Finally, the GpBSK-BEV plasmid containing NotI and XbaI enzyme cleavage sites was obtained through a T4 ligase  connection.The NotI and XbaI enzymes , respectively. Subsequently, T4 ligase was used for overnight ligation at 16 °C, and the product was transferred into the competent cells of Escherichia coli JM109 . Positive clones were sent to Genewiz  for sequencing, and the correct plasmid was named GpBSK-BEV-E0.The pUC57-E0 and GpBSK-BEV plasmids were digested with EcoRI restriction enzyme . The enzyme digestion system comprised 10 μg recombinant plasmid, 40 μL of 5× CutSmart buffer, 8 μL EcoR I-HF, and RNase Free ddH2O up to 200 μL. Enzyme digestion was performed at 37 °C for 3 h, and the restriction enzyme was inactivated at 65 °C for 20 min. Linear plasmids were recovered from the agarose gel and purified by ethanol precipitation.The recombinant plasmid GpBSK-BEV-E0 was linearized by the 2. Viruses were observed at 12-h intervals and harvested until cytopathic effects (CPE) were visible.Linearized GpBSK-BEV-E0 was transfected in BSR-T7 cells  using a transfection kit  according to the manufacturer’s instructions. In brief, BSR-T7 cells were inoculated into a six-well plate until covering 80% of the bottom of the wells, and then washed with phosphate buffered saline (PBS). Two milliliters of Dulbecco’s modified Eagle’s medium  containing 2% fetal bovine serum  was added to each well, and 200 μL of jetPRIME buffer and 2 μg DNA were added to the sterilized 1.5-mL tube and mixed gently. Subsequently, another 4 μL of jetPRIME was added, mixed gently, centrifuged at low speed for 10 s, and left to react at room temperature for 10 min. The mixture was then added to a six-well plate and cultured in the cell incubator at 37 °C and 5% COSpecific detection primers of BEV (BEV-F/R) and BVDV E0 (E0-F/R) were designed Table  and applThe rescued recombinant virus rBEV-E0 was also detected by western blot according to standard procedures. In brief, MDBK cells were infected with the 10th generation recombinant virus and parental virus, and incubated for 12 h. Viral samples were loaded on a 12% polyacrylamide gel, transferred to nitrocellulose membranes, and then blocked with 5% skim milk in Tris-buffered saline with Tween 20 for 2 h at 37 °C. The membranes were then incubated with anti-BJ101-VP1 monoclonal antibody (mAb) 4B2 and anti-BVDV E0 polyclonal antibody  at 37 °C for 1 h followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody . Detection was performed using an enhanced chemiluminescence kit .The rescued recombinant virus rBEV-E0 and parental virus were transfected in MDBK cells in 96-well plates. After 24 h, the cells were fixed with ice-cold anhydrous ethanol at 4 °C for 30 min, followed by the addition of 0.3% Triton 100 for 15 min. Anti-BEV VP1 mAb and anti-BVDV E0 PcAb (1:1000) were added and the cells were further incubated at 37 °C for 1 h, followed by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody  in the dark. The results were observed under a fluorescence microscope.2 for 96 h. The plaques of moderate size were selected by staining with 0.1 g/L neutral red solution.MDBK cells in the six-well plate were infected with the 10th generation recombinant virus and parental virus by 10-fold gradient dilution at 37 °C for 2 h. After removing the unabsorbed virus, the cells were overlaid with 2 mL of 1.5% low-melting agarose containing 2% FBS in DMEM and cultured at 37 °C in 5% CO2 for 1 h. After adsorption, the cells were washed with PBS and added to DMEM containing 2% FBS. Infected cells were harvested at 3, 6, 12, 24, 36, and 48 h after infection, and then frozen and thawed three times. The 50% tissue culture infective dose (TCID50) of the virus was calculated according to the Reed-Muench method [The growth curves of the recombinant virus and parental virus in MDBK cells were compared. In brief, the MDBK cells in 96-well plates were infected at a multiplicity of infection of 1 with the 10th-generation rescued virus or parental virus and incubated at 37 °C in 5% COh method  and eachThe stability of the rescued recombinant virus was determined by PCR and sequencing. RNA was extracted from MDBK cells infected with the 3rd, 5th, 7th, and 10th generation recombinant viruses. RNA was then reverse-transcribed into cDNA and used as a template for PCR detection with primers  and at 7-day intervals after immunization.Twenty-four BALB/c female mice  were randomly divided into four groups of six mice each. The test group was intraperitoneally injected with 102SO4. The optical density at 450 nm (OD450) cutoff values were determined for analysis.The blood samples were separated into serum, and indirect enzyme-linked immunoassay (ELISA) was used to detect anti-BEV VP1 and anti-BVDV E0 antibodies in the immunized mice. The 96-well ELISA plates were coated with VP1 and E0 proteins at 100 ng/mL each and incubated overnight at 4 °C. The wells were blocked with PBS and Tween containing 1% bovine serum albumin for 2 h at 37 °C. Serum samples serially diluted two-fold starting at a dilution of 1:500 were added and incubated at 37 °C for 1 h, followed by the addition of HRP-conjugated goat anti-mouse secondary antibody. Finally, 3,3′,5,5′-Tetramethylbenzidine (TMB) was added for color development, and the reaction was terminated by the addition of 2 mol/L HP value of < 0.05 was considered significant.Statistical analysis was performed using GraphPad Prism 6 software . Data were analyzed using two-way analysis of variance (ANOVA). Data are shown graphically as the geometric mean of the fold change plus the standard error of the mean. A All animal experimental procedures were approved by the Animal Care and Use Committee of the Institute of Animal Sciences of Chinese Academy of Agricultural Sciences.After generating recombinant infectious BEV-E0 cDNA clones (GpBSK-BEV-E0) following the strategy outlined in Fig. As shown in Fig. Western blots showed a band for VP1 in the MDCK cells infected with both the parental and recombinant viruses Fig. a, whereaIndirect IFA confirmed these results, as the samples infected with recombinant virus or parental virus all reacted with anti-VP1 mAb. The samples infected with the recombinant virus only reacted with anti-E0 PcAb, and the uninfected samples did not respond against either anti-VP1 mAb or anti-E0 PcAb Fig. . These rN = 7) showed a plaque morphology similar to that of the parental virus , and the difference is not significant . We also found that the recombinant virus could be rescued using the recognition site for 2A (LTTLG) (data not shown), suggesting that the 2C/3A junction in BEV BJ101 is suitable for insertion of an exogenous antigen, and the used hydrolysis sites for 2A/3CThe prevalence of antibodies against BEV-E was reported to be generally lower than the prevalence of antibodies against BEV-F in cattle populations . TherefoWe successfully constructed and rescued the recombinant virus rBEV-E0, and demonstrated that insertion of the exogenous gene BVDV E0 at the 2C/3A protein junction did not affect the biological characteristics of the recombinant virus and induced an effective immune response in vivo. This recombinant virus can offer a useful tool for the simultaneous prevention and control of BEV and BVDV, as well as in further research to find appropriate insertion sites for other exogenous genes or markers. Therefore, rBEV-E0 provides an ideal technical platform for the subsequent insertion of exogenous genes, research on the mechanism of action, and further development of viral vector vaccines.Additional file 1. The Optimized sequence of BVDV-E0 (Length: 681 bp)Additional file 2: Figure S1. GpBlueScript(−) Vector Figure S2 Modified GpBlueScript(−) VectorAdditional file 3: Table S1. The raw data of the anti-BEV VP1 antibodies detection in experimental mice Table S2. The raw data of the anti-BVDV E0 antibodies detection in experimental mice


Background: People with Parkinson’s disease (PD) present deficits of the active range of motion (ROM), prominently in their trunk. However, if these deficits are associated with axial rigidity, the functional mobility or health related quality of life (HRQoL), remains unknown. The aim of this paper is to study the relationship between axial ROM and axial rigidity, the functional mobility and HRQoL in patients with mild to moderate PD. Methods: An exploratory study was conducted. Non-probabilistic sampling of consecutive cases was used. Active trunk ROM was assessed by a universal goniometer. A Biodex System isokinetic dynamometer was used to measure the rigidity of the trunk. Functional mobility was determined by the Get Up and Go (GUG) test, and HRQoL was assessed with the PDQ-39 and EuroQol-5D questionnaires. Results: Thirty-six mild to moderate patients with PD were evaluated. Significant correlations were observed between trunk extensors rigidity and trunk flexion and extension ROM. Significant correlations were observed between trunk flexion, extension and rotation ROM and GUG. Moreover, significant correlations were observed between trunk ROM for flexion, extension and rotations (both sides) and PDQ-39 total score. However, these correlations were considered poor. Conclusions: Trunk ROM for flexion and extension movements, measured by a universal goniometer, were correlated with axial extensors rigidity, evaluated by a technological device at 30°/s and 45°/s, and functional mobility. Moreover, trunk ROM for trunk flexion, extension and rotations were correlated with HRQoL in patients with mild to moderate PD. The decreased amplitude of joint movement is one of the characteristic symptoms in Parkinson’s disease (PD) ,2. This One of the possible explanations regarding these ROM deficits is the presence of an intrinsic muscle stiffness at the local level ,10,11. RAlthough these hypotheses seem plausible, limited research has been developed to explore the relationships between these clinical phenomena. Given the importance of trunk mobility and the relationship between axial rigidity with risk of falls ,19 and aThe objective measurement of rigidity is difficult and complicated in the clinical context. Instrumentalized tools with high costs and complex procedures are needed in order to obtain reliable and quantitative data , since clinical scales and methods are subjective in their nature and present biases . For exaNevertheless, in clinical practice, conventional goniometry assessment is used by therapists to evaluate the active and passive ROM of the trunk due to its advantages, such as ease of use, cost and intra and inter-rater reliability . If the On the other hand, axial rigidity seems to be correlated with the functional mobility and HRQoL, as our research team have suggested in previous works ,18. TherThereby, our principal aim is to analyse the existing relationships between the ROM of the trunk, in all its movements, and axial rigidity, in patients with PD. Secondarily, the relationships between the ROM of the trunk and the functional mobility and HRQoL were explored.A cross-sectional study was conducted at a Motion Analysis Laboratory located at a University Department. Non-probabilistic sampling of consecutive cases was used. The study was approved by a local Ethical Committee, and informed consent was obtained from each subject in accordance with the 1964 Declaration of Helsinki. All patients gave their informed consent prior to their inclusion in the study. All assessments were performed by the same investigator expert in movement disorders. The same environmental conditions were maintained during all assessments to limit the influence of external factors.All patients were recruited from a Neurology Service , with a diagnosis of idiopathic PD .The inclusion criteria were as follows: (1) stages I-III of the Hoehn and Yahr scale, (2) no diseases that affect the trunk and lower limbs, (3) stable on anti-Parkinsonian medication. The study exclusion criteria were as follows: (a) the diagnosis of diseases other than PD; (b) patients with dementia, established by a Mini-Mental State Examination (MMSE)  score ≤ All tests were performed by the same rater within 1–3 h of the administration of anti-Parkinsonian medication. All patients were assessed by the Hoehn and Yahr scale and the Unified Parkinson’s Disease Rating Scale-Motor Scale (UPDRS-III)  to evaluA universal goniometer was used. The measurement of the active ROM of the trunk was performed according to the method described by Norkin and White  and CherFor trunk rotation, PD patients were instructed to place in sitting position with their feet on the floor, and knees and hips in 90° of flexion. The goniometer was positioned with the axis fixed in the transverse plane at the level of T1-T2, following the measure recommendations  Figure .For trunk lateral flexion, PD patients began the test in an upright standing position, with the knees completely extended. Then, upon a verbal command from the examiner, they did slow and gradual lateral flexion movements in a maximum amplitude, where the goniometric measurement was made. To evaluate lateral flexion, the first sacral vertebra was taken as the axis fixed reference point. The stationary arm of the universal goniometer was pointed perpendicular to the floor, while the mobile arm remained pointing to the spinous process of the seventh cervical vertebra  Figure .The active ROM was measured three times for trunk flexion, extension, lateral flexion (both sides) and rotation (both sides) movements, by the same examiner. An average of the measurements of each movement was used for the analysis. At the end of the measurement, the participant was instructed to return to the starting position where the universal goniometer was removed and repositioned before the following measurement. Participants were allowed to rest while the examiner entered the measurement into a spreadsheet.Trunk extension-flexion component of a Biodex System isokinetic dynamometer was used to measure the rigidity of the trunk. Subjects were instructed to rest their body comfortably against the back of the trunk dynamometer component. The investigator then measured the anatomical zero, which was then used as a reference point to measure trunk muscle tone over a range of 50°, from 30° trunk extension to 80° flexion (S: 30-50-80) ,10.All subjects were examined prior to the test to ensure that they had the appropriate passive range of trunk movement. The continuous passive mode of Biodex System with three angular speeds  was used to assess trunk muscle tone. To ensure subject safety, the Biodex dynamometer was calibrated using the “speed calibration” programme. If subjects felt any discomfort during the test, the examiner could stop the machine immediately by pressing a safety switch. During the test, the subject’s trunk was passively moved into flexion then extension, and muscle resistance was recorded over the range. For each speed in every movement, one practice trial was given, and five trials were recorded with the patient being instructed to relax. Each trial was followed by 30 s of rest. To minimise possible errors due to the inertia of the subject’s weight, the resistance measurements of the first trial were discarded, and the measurements from the remaining four trials were analysed. This protocol was validated in previous studies by our research team ,9,10.The overall trunk extensors rigidity and trunk flexors rigidity were expressed as work done (Nm°/Body Weight) (W/BW). The resistance to passive trunk flexion represented trunk extensor muscle tone, while passive trunk extension represented trunk flexor muscle tone.The Get Up and Go (GUG) test is a measure of functional mobility in PD patients. For the assessments, the patient is asked to stand up from a standard chair and walk a distance of 3 m, turn around and walk back to the chair and sit down again. Mobility is rated on a 5-point scale, ranging from “normal” to “severely abnormal” according to the observation of a clinician .HRQoL was assessed with the 39-item Parkinson’s Disease Questionnaire (PDQ-39) and with the Euro-Quality of life-5 dimensions (EuroQol-5D). PDQ-39 is a specific instrument to assess HRQoL in PD and comprises 39 questions related to its frequency in the previous month. Scores ranging from 0 to 100 and higher scores indicate worst HRQoL .The EuroQol-5D is a generic HRQoL questionnaire. It is a self-administered instrument and includes a visual analogue scale on which patients rate their own health between 0 and 100. Higher scores indicate better HRQoL .The Statistical Package for the Social Sciences (SPSS) program was used. Spearman correlation coefficient (r) was used to establish the relationship between the ROM of the trunk, axial muscle stiffness, functional mobility and HRQoL. Correlation coefficients of 0.00 to 0.49 were interpreted as poor, from 0.50 to 0.79 as moderate and ≥0.8 as excellent . p valueThirty-six PD patients participated in the study . The mean age of the sample was 62 ± 11 years. The left side was the side most affected in 40% of the sample, while the right side was the most affected in the 60% of the sample. All of them were classified between I–III in the Hoehn and Yahr scale and reached 80%–100% scores in the Schwab and England Scale. Mean values of disease duration and disease severity are described in Mean values of the ROM of the trunk, the rigidity of the trunk, functional mobility and HRQoL are described in r = −0.687, p = 0.005; r = −0.534, p = 0.042; r = −0.510, p = 0.006; r = −0.410, p = 0.005, respectively). No other significant correlations were observed.Correlations between the ROM of the trunk, axial rigidity and GUG are described in Significant correlations were observed between the ROM of the trunk for flexion, extension and rotation (both sides) and GUG scores .Statistically significant correlations were found between ROM and PDQ-39 total score only for trunk flexion, extension and rotations for both sides . These cGoniometry is one of the most extended methods for the assessment of the ROM by physical therapists, due to its ease of use, cost effectiveness and reliability . To our People with early PD exhibit less axial ROM compared with healthy controls ,37,38. TIn our study, we have found significant correlations between extensors rigidity at specific movement velocities (30°/s and 45°/s), and flexion and extension ROM deficits. These findings could be due to the fact that mostly of the ADL are performed in a specific trunk ROM, not total, and under speed movements considered until 45°/s. However, this contrasts with the evidence that people with PD exhibit axial ROM deficits in rotation, as measured by a triaxial dynamometer . In our In short, our results showed only a partial relationship between trunk ROM and axial rigidity. These findings could be used in future studies to determine improvements in axial rigidity in a post-treatment period, using of a simple and feasible ROM assessment, as suggested in previously published studies about the effects of deep brain stimulation in trunk movements in PD patients ,43.To determine risk of falls, three-dimensional computerized analysis may be considered the gold standard because it provides objective data about kinematic and kinetic parameters. Nevertheless, customized instrumentation is needed; the equipment is expensive; it takes longer time and requires complex data analysis, and it is not always available in clinical practice ,45; therIn our study, the ROM of the trunk measured by goniometry was partially related to functional mobility assessed by the GUG in a sample of mild to moderate PD patients. Our results are consistent with other studies that have found ROM deficits in PD fallers in comparison with PD non-fallers and healthy controls, in other body parts such as the hip joint. Besides, these studies have used instrumentalized methods with three-dimensional motion analysis techniques .Our findings suggest that an association exists between functional mobility and ROM of the trunk for the flexion, extension and rotation (both sides) movements. Furthermore, this association is stronger for the rotation movements (both sides). These associations make sense since these movements are used by the patient to perform the GUG. Thus, we did not find associations between the GUG and the lateral flexion ROM, probably because this test does not demand to the patient to make these movements and ROM for lateral flexions were not highly affected at this stage of the disease in our sample. However, to recuperate and/or maintain this specific articular joint amplitude should be an essential objective in the physical therapy programs for PD. Other instrumentalized methods could find associations between the functional mobility and the lateral flexion ROM, so more research is needed in this field.Axial impairments related to gait disorders, postural instability  and the In our study, the ROM of the trunk for flexion, extension and rotation movements for both sides were correlated with the PDQ-39 total score. These findings are possibly due to most functional tasks involving a trunk flexion and rotation components, such as dressing, walking, eating, transfers, bathing or turning in bed ,52. HoweThis study was limited by its relatively small sample size. Future studies should have a larger sample group. Our results cannot be extrapolated to other PD stages of the disease or during the “off” phase of the medication cycle. Further studies could also assess the relationship between the ROM of the trunk and axial rigidity at additional angular velocities, using other dynamic postural control tests, and examine the relationship between axial ROM impairments and HRQoL in PD patients with higher disease severity. Another limitation was the use of a non-objective evaluation of the risk of falls. Future studies should record fall incidents through longitudinal researches ,54. FinaTrunk ROM for flexion and extension movements, measured by a universal goniometer, were correlated with axial extensors rigidity, evaluated by a technological device at 30°/s and 45°/s and functional mobility. Moreover, trunk ROM for flexion, extension and rotations were correlated with HRQoL in patients with mild to moderate PD. However, the strengths of these associations were considered poor to moderate. These findings highlight specific opportunities for a free conventional trunk ROM measure to serve as a complementary evaluation in clinical practice.