Ilarvirus.Virus discovery based on high-throughput sequencing relies on enrichment forvirus sequences prior to library preparation to achieve a sufficient number ofviral reads. In general, preparations of double-stranded RNA or total RNApreparations treated to remove rRNA are used for sequence enrichment. We usedvirus-specific antibodies to immunocapture virions from plant sap to conductcDNA synthesis, followed by library preparation and HTS. For the four potatoviruses PLRV, PVY, PVA and PYV, template preparation by virion immunocaptureprovided a simpler and less expensive method than the enrichment of total RNA byribosomal depletion. Specific enrichment of viral sequences without anintermediate amplification step was achieved, and this high coverage ofsequences across the viral genomes was important to identify rare sequencevariations. Using this approach, the first complete genome sequence of a potatoyellowing virus isolate  was determined in this study. PYVcan be confidently assigned as a distinct species in the genus High-throughput sequencing (HTS) has become a central element of virus discoveryresearch, as it provides global insight into the virus composition of a (plant)sample , 2. The One possibility for obtaining a large number of viral reads is to start with thespecific extraction of small RNAs. Sequencing of virus-derived small RNA (smRNA) iswidely used to detect viruses with both RNA and DNA genomes , 3, 1, 2Theoretically, the genomes of RNA and DNA viruses can be analyzed with the genomicand/or mRNA sequences found in total RNA preparations , 14, 1. The replication of plant RNA viruses involves dsRNA intermediaries, which aremolecules that are otherwise very rare in plants. Because dsRNA is very stable andcan be easily purified, the obtained dsRNA consists almost entirely of virus RNA inthe sense and complementary sense direction and can serve as an ideal template forcDNA synthesis and HTS ,16, 1. Other enrichment methods for virus sequences correspond to particular virus genomecharacteristics. Plant viruses with polyadenylated RNA genomes can be enriched bythe purification of plant mRNA , 25, 1. potato leafroll virus ,potato virus S  and potato virus Y . Todemonstrate the suitability of the IC-HTS method, we determined the complete genomesequence of potato yellowing virus (PYV), an unassigned species in familyBromoviridae for which no genome sequence was available.Here, we present a method for capturing virions with virus-specific antibodies. Withthis method, random cDNA synthesis is directly performed using RNA fromimmunocaptured (IC) virions. We assessed the IC template preparation method for theHTS analysis of Solanum tuberosum L. var. ‘Bamberger Hörnchen’, collectedin 2013) and were kindly provided by Dr. Volker Zahn . The PLRV isolate (PLRV-136) was separated from PVS andPVY by sequential aphid transmission via the intermediate hostD. stramonium and back transfer to potatoplants (‘Bamberger Hörnchen’). The potato yellowing virus isolate DSMZ PV-0706was maintained at the Plant Virus Department in D.stramonium. This virus isolate was originally obtained froma pepino (Solanum muricatum) fruit purchased in a supermarketin Braunschweig  in 2001 and was kindly provided by Dr.D.E. Lesemann . The geographic origin of the fruit was notspecified.The three viruses PLRV, PVS and PVY were present in a potato sample (W13-136)originating from a potato field in Lower Saxony in Germany .Antisera against PLRV (DSMZ AS-0741), PVA (DSMZ AS-0535), PVM (DSMZ AS-0273), PVS(DSMZ AS-0547), PVY (DSMZ AS-0343), PVX (DSMZ AS-0126) and PYV (DSMZ AS-0599)and their respective positive controls were taken from the stock of the DSMZPlant Virus Department. All ELISA tests were performed following DSMZ standardprocedures to capture virus-specific polyclonal antisera. Plant extracts were prepared bygrinding fresh leaves (at 1:20 w/v) and freeze-dried leaf material (at 1:50 w/v)in standard ELISA sample extraction buffer . For each IC reaction, a 1.5 ml Eppendorf tubewas used, and 2 variants for coupling the magnetic beads with the target IgG andantigen (IC-1 and IC-2) were tested. For variant IC-1, 50 μl of purified beadswas mixed with 1 ml of virus-infected plant extract and 4 μg of IgG. Thismixture was incubated by shaking at 4°C overnight. For variant IC-2, 50 μl ofpurified beads was mixed with 4 μg of IgG diluted in 1 ml of sample extractionbuffer without a virus-infected sample and incubated for 70 min at 37°C.Subsequently, the Dynabeads precoated with the IgGs were washed, and 1 ml ofvirus-containing plant extract was added, followed by overnight shaking at 4°C.For Library-03, all three antisera were mixed at a ratio of 1:1:1 (v/v/v) for atotal of 4 μg/μl IgG.Following the overnight incubation described above, the beads of IC-1 and IC-2were washed with Dynabead washing buffer according to the manufacturer’sinstructions. The bound virions were eluted from the beads with 69.5 μl of waterand transferred to PCR tubes. Subsequently, 3 μl of random octamer primers (100pmol/μl) and 0.5 μl of RiboLock RNase Inhibitor (Thermo Fisher Scientific) wereadded. After a denaturation step at 99°C for 2 min and immediate cooling on ice,27 μl of master mix  was added for cDNA synthesis, andthe reaction was incubated at 45°C for 1 h, followed by inactivation at 70°C for10 min. The synthesized cDNA was purified using a NucleoSpin Gel and PCRClean-up kit (Macherey-Nagel). For library preparation, all subsequent stepswere performed as described for the total RNA templates. As described above, thelibraries were pooled and analyzed as paired-end reads.S. tuberosum v4.03, downloadfrom Phytozome 12) reads were removed from the potato samples; for the daturasamples, only the chloroplast (NC_018117)-related reads were subtracted. De novoassembly was performed with the Geneious assembler (medium-lowsensitivity/fast). The first 1000 contigs (starting with the greatest number ofreads related to the respective contig) were compared by local Blastn searchesagainst plant virus and viroid reference sequences, followed by Blastp searchesagainst the plant virus protein reference database (NCBI download 11.06.18).Sequence analysis and alignments were carried out using the BLAST webserver(http://blast.ncbi.nlm.nih.gov/blast.cgi) and Clustal Omega. Phylogenetic treesfor PYV were inferred with 1,000 replicates of the neighbor-joining procedure,applying default settings using MEGA 6 [Sequence reads retrieved from the MiSeq platform were screened for plant virussequences using Geneious 11.1.4 software. In the first step, all trimmed reads(error probability limit 0.01) from a library were mapped to the reference plantvirus and viroid database (NCBI download 11.06.18) with a sensitivity of amaximum of 20% mismatches per read. Plant-related reads were removed before denovo assembly to reduce the number of nonspecific reads. Chloroplast (NC_008096)and chromosome  antiserum,and sequencing results revealed hits to three genome components of correspondingilarviruses .These rPolerovirus, showing theclosest nucleotide BLAST hit to PLRV isolate PBI-6  originating from potato in India.The full-length genome sequence of PLRV from W13-136 (accession number MH937415)was assembled from the final sequence obtained from the de novo assembly(Library-01) and from the sequencing of RT-PCR products from 5’- and 3’-RACEreactions for the same RNA extract. The full-length genome sequence of PLRV(5883 nt) represented a typical The PVY from the mixed infected W13-136 sample (accession number MH937417) wasassembled de novo from Library-01 reads with a complete genome sequence of 9703nt. The top nucleotide BLAST hit  was to PVYisolate F65 (accession number KX184818) from potato from Israel.S. tuberosum from Hungary.The complete genome sequence of the PVS isolate (accession number MH937416) wasdetermined to be 8485 nt. PVS W13-136 presented the typical genome organizationof carlaviruses. The top GenBank hit found by BLAST analysis was to PVS isolate89.249 from Ilarviruscoat proteins. Overall, a genomic organization typical of ilarviruses could beinferred. When compared to the sequences available in GenBank, all threecomponents showed the top nucleotide BLAST hits to Fragariachiloensis latent virus (FCILV) isolate CFRA 9087 , a member of subgroup 4 in the genusIlarvirus (family Bromoviridae). Thepairwise identity values ranged from 72% to 80%  polymerase for all fourribonucleotides to synthesize homopolymers prior to 3’ RACE. Analysis of the 3’RACE sequences revealed a stretch of identical 100 nt sequences among the threeRNAs, with only minor differences in the terminal 170 nt. The lengths of the PYVgenome component were determined to be 3467 nts for RNA1 (MH937418), 2567 ntsfor RNA2 (MH937419) and 2375 nts for RNA3 (MH937420) . RNA1 an% to 80% . When th% to 80% .D. stramonium tissue infected with PYV isolatePV-0706 was investigated. The different settings and sequencing results aresummarized in Two library preparation methods, one starting with rRNA-depleted total RNA,followed by cDNA synthesis, and the other starting with IC, followed by cDNAsynthesis, were compared in three different settings. In the first setting,fresh potato leaf material of isolate W13-126, infected with PLRV, PVY and PVS,was used. In the second setting, fresh or frozen potato leaf material ofPLRV-136 was analyzed, and in the third setting, freeze-driedIn the first setting, when the libraries were generated from fresh leaf materialof virus isolate W13-136, two independent repetitions (Library-01 andLibrary-02) prepared from total RNA were compared with a library prepared by theIC method. For the libraries based on RNA, in both cases, approx. 0.1% of thereads mapped to a PLRV reference sequence. In contrast, the library prepared viathe IC method (Library-03) resulted in 0.9% of reads mapping to PLRV . A similIn the second setting, the libraries were prepared from plant material harboringvirus isolate PLRV-136 stored at -20°C. Library-04, prepared from total RNA,exhibited 0.05% viral reads, whereas enrichment via IC (Library-05) resulted in0.8% of PLRV-specific reads. When fresh leaf material was used for IC, the viralhits for PLRV reached 1.3% (Library-06), and IC from ELISA sap stored at -20°Cresulted in 2.5% , whereas the library prepared by ICreached only 0.9% . When usThe focus of this study was to compare strategies for enriching plant extracts forvirus sequences prior to HTS. For this purpose, a potato sample that was mixedlyinfected with PVY, PVS and PLRV and a plant virus sample (PV-0706) containing anisolate of PYV  wereanalyzed. The genome sequences of all virus isolates were assembled from cDNAlibraries prepared from total RNA or from IC templates. The obtained PYV, PVS andPLRV sequences showed only marginal sequence variations compared to alreadypublished virus sequences, with nt identities ranging from 98% to 99%.Solanum muricatum) of unknown geographic origin purchased ina German supermarket and was propagated on D.stramonium. The partial sequences available  were almost 100% identical to our newly determinedgenome sequences. From these data and the complete genome sequence determined inthis study, PYV can be confidently assigned as a distinct species in genusIlarvirus that is most closely related to FCILV. Theavailability of genome sequences will allow the further development ofsequence-based detection and identification methods, which is important because PYVis listed in EPPO Annex A1, comprising pests recommended for regulation asquarantine pests of the EU (https://www.eppo.int/QUARANTINE/listA1.htm) [PYV was first found in potato plants from Peru and was initially described as Andeanpotato yellowing virus without providing sequence information. The virus was laterrenamed potato yellowing virus , 30, 31.etal. 2016, the enrichment efficiency achieved through the IC of dsRNA isbetween 31–74% of reads of viral origin. However, the possibility of enriching viraldsRNA by using an antibody depends on whether the virus infections result inconsiderable quantities of dsRNA intermediates. This might not be the case for allviruses and result in an underrepresentation of virus-derived sequences in HTS.Blouin et al., 2016, proposed the use of a PCR enrichment step toaccumulate sufficient DNA for library preparation [In this study, we report a new method for enriching viral templates for thepreparation of HTS libraries by using immunocapture (IC) to trap virions in apull-down assay. The maximum enrichment achieved via the IC method usingvirus-specific antibodies was 40%, compared to a virus purification protocol thatachieved 89% enrichment for viral sequences . Howeverparation . This PCOther viral enrichment strategies for HTS such as the enrichment of polyadenylatedRNA for potyviruses, RCA for circular DNA viruses or the use of small RNA extractsresult in percentages of approximately 10%, 60% or 30% of viral reads, respectively, 1, 36. In our study, HTS libraries from IC preparations and from rRNA-depleted total RNAwere compared for four virus species . Based oWith the IC method, we provide an advantageous way to efficiently enrich viralsequences prior to library preparation for HTS. This method is an option forenriching specifically for viruses, and when several antibodies are combined, abroader spectrum of species can be covered. However, the enrichment efficiencydepends on the antibodies used and may be affected by virion stability.


Pseudomonas aeruginosa is a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating how P. aeruginosa uses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicate P. aeruginosa and its viruses evolve in both a broad global population and in isolated human “islands.” These data set the stage for exploring metapopulation dynamics occurring within and between isolated “island” populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains. Pseudomonas aeruginosa have been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution of P. aeruginosa and its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark . We show that new spacers are not added to or deleted from CRISPR arrays over time within a single patient but do vary among patients in this data set. We compared assembled CRISPR arrays from this data set to CRISPR arrays extracted from 726 additional publicly available P. aeruginosa sequences to show that local diversity in this population encompasses global diversity and that there is no evidence for population structure associated with location or environment sampled. We compare over 3,000 spacers from our global data set to 98 lytic and temperate viruses and proviruses and find a subset of related temperate virus clusters frequently targeted by CRISPR spacers. Highly targeted viruses are matched by different spacers in different arrays, resulting in a pattern of distributed immunity within the global population. Understanding the multiple immune contexts that P. aeruginosa viruses face can be applied to study of P. aeruginosa gene transfer, the spread of epidemic strains in cystic fibrosis patients, and viral control of P. aeruginosa infection.Viruses that infect the widespread opportunistic pathogen IMPORTANCEPseudomonas aeruginosa is a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating how P. aeruginosa uses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicate P. aeruginosa and its viruses evolve in both a broad global population and in isolated human “islands.” These data set the stage for exploring metapopulation dynamics occurring within and between isolated “island” populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains. Pseudomonas aeruginosa genomes have revealed a dynamic variable genome replete with horizontally transferred elements, many of which are proviruses and virus-like elements  immunity  and spacers (B) per strain in strains from the clone-corrected Copenhagen data set. Download Copyright © 2018 England et al.2018England et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00075-18.8TABLE S1P. aeruginosa strains and spacer sequences used in this study. Download Table S1, XLSX file, 0.3 MB.Copyright © 2018 England et al.2018England et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To look for evolution in CRISPR arrays within a patient, we compared assembled arrays from longitudinal samples. Of the 34 longitudinally sampled patients with any change in the spacer content in their CRISPR arrays over time, we find this variation results from spacer deletion in only two patients, with no examples of spacer addition . Most st10.1128/mSystems.00075-18.2FIG S2FIG S2, EPS file, 1.0 MB.Related array groups containing patient-unique arrays. RAGs are demarcated by thick black lines. Each box in an array represents a spacer; boxes of different colors represent variant spacers. White boxes represent identical spacers shared among arrays within a RAG. The leader end of each array is to the left. Observations outside Copenhagen patients and prevalence within each Copenhagen patient  are shown below each array. RAGs showing evidence of within-patient deletions are marked with an asterisk. RAGs unique to the Copenhagen patient population are marked with a ‡. Download Copyright © 2018 England et al.2018England et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the related array groups, or RAGs). In total, we found 15 RAGs in the Copenhagen population  contain known CRISPR repeats. We identified 3,152 unique spacer sequences in 729 unique arrays that differ by at least one spacer. A rarefaction curve of spacer sequences reveals that despite a broad sampling of P. aeruginosa sequence data, it is unlikely that all spacers in the population have been observed . Two examples of this are highlighted in P. aeruginosa viruses into clusters. We gathered 92 sequenced viruses along with six characterized proviruses integrated in the genome of epidemic strain LESB58 .We compared all spacers in our library to the viral data set. In total, 1,172 spacers (37.2%) match these viruses with up to four mismatches across the length of the spacer, and with an appropriate protospacer-adjacent motif (PAM). Remarkably, 1,980 spacers (62.8%) match no viruses in this data set, indicating that CRISPRs are sampling a genome space of viruses and other elements that are not included in this data set. Of the 98 virus sequences, 46 (46.9%) contained a protospacer matched by at least one spacer. Unmatched viruses include all members of clusters 02, 04, 05, 06, 09, 11, 12, 16, and 17, along with singletons 20, 21, 23, 24, 26, 27, 31, and 32 . With th10.1128/mSystems.00075-18.5FIG S5t test, P = 6.127e−07). The center line of the box represents the median; the upper and lower lines mark the first and third quartiles, respectively. Whiskers extend to 1.5× the interquartile range; points outside this range are shown as black dots. Download FIG S5, EPS file, 0.4 MB.Spacers target temperate viruses. (A) Number of protospacers per base pair of sequence in 97 sequenced viruses categorized by lifestyle. (B) Number of protospacers per base pair is higher in temperate viruses than in lytic viruses , showing as expected that viruses with similar genomes share more protospacers.To see if protospacers were more likely to be shared between closely related strains, we compared shared protospacers in pairs of viruses to the proportion of their genomes that align . We found a positive relationship between PLA and shared protospacers  Heat map of proportion length aligned (PLA) between viral genomes. Genomes are grouped by cluster (colored bars below/left). (B) Protospacers shared between virus clusters. Heat map indicates the proportion of protospacers shared between each cluster pair. Cluster lacking protospacers are omitted. (C) Genome similarity (PLA) correlates with proportion of protospacers shared between virus strains. Download Copyright © 2018 England et al.2018England et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the t test, P = 0.83 between superspacers and normal spacers found in arrays).Spacer matches are typically not unique to one virus; spacers match 1 to 13 viruses with an average of 2.75 viruses . While t10.1128/mSystems.00075-18.7FIG S7P. aeruginosa viruses. (A) Count of viruses matched per spacer. (B) Number of spacers matching each viral cluster. Download FIG S7, EPS file, 0.4 MB.Spacer matches to Copyright © 2018 England et al.2018England et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00075-18.10TABLE S3P. aeruginosa CRISPR arrays. Download Table S3, XLSX file, 0.02 MB.Superspacers identified in Copyright © 2018 England et al.2018England et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.05). This breadth of targeting is not primarily due to individual spacers targeting multiple clusters, as evidenced by the small number of superspacers observed , or proportion of hosts that lack CRISPR immunity, for each virus  and f. TP. aeruginosa CRISPR spacer sequences in both a small, highly sampled CF patient population and a broad sampling of the global P. aeruginosa population. Diversity in the Copenhagen samples reflects that of the global population. Change of CRISPR arrays within a patient over time is limited; however, we observe divergence and recombination of CRISPRs in global data. Comparing these spacers to known P. aeruginosa viruses reveals differential targeting of related viral groups by CRISPRs, with distributed immunity to highly targeted viruses emerging in the global population.Here, we have identified a large, diverse pool of P. aeruginosa in these patients and may have limited our ability to capture within-patient CRISPR evolution; however, we were still able to identify numerous strains with related CRISPRs across patients. Publicly available P. aeruginosa genomes present the issue of misassemblies. As these genomes were largely assembled without specific focus on CRISPR regions, missing or misordered spacers are possible due to the repetitive nature of CRISPR arrays, and in most cases sequence reads are not available to facilitate the careful assembly of CRISPR regions used on the Copenhagen data. Despite these possibilities, we still find numerous CRISPR arrays identical to Copenhagen arrays in non-Copenhagen strains . Assembled genomes of 24 P. aeruginosa strains described in reference AWYJ00000000 to AWZG00000000). Assembled genomes of 388 strains described in reference PRJNA264310). All other complete and draft-stage P. aeruginosa genomes were retrieved from the NCBI Nucleotide database in September 2014 . To establish spacer order, repeats were identified on sequence reads, and 12-nucleotide “chunks” of DNA flanking each repeat were identified using fuzznuc  (An established panel of seven markers  was usedginosa/)  against ginosa/)  using thP. aeruginosa were downloaded from the NCBI Nucleotide database on June 23, 2015, totaling 92 unique viruses. Six previously identified proviruses from P. aeruginosa LESB58 were added using genomic coordinates from reference Genomes of all viruses identified as infecting Protospacers in virus genomes were identified via BLASTn of spacer sequences. The parameter “-task blastn-short” was used due to short query length. A minimum E value of 0.01 was used to capture incomplete and imperfect matches, allowing up to four mismatches over a full-length match. PAMs were identified and partial-length matches were extended to cover the full spacer length using clDB , and theTo assign viruses to clusters, all virus genomes were compared using BLASTn (E < 0.001). For each pair of genomes, the proportional length alignment (PLA), or total length aligned by BLAST over the length of the query, was calculated and used as our measure of viral similarity. MCL  was usedPopulation distributed immunity (PDI) was calculated on a per-virus basis using all possible pairs of hosts. For each host-host pair, if each host has a spacer matching the virus which is not present in the other host, PDI is 1; else, PDI is 0. At the population level, PDI is then averaged across all host-host pairs. Criteria for matching spacers and protospacers are as described above. Individual distributed immunity (IDI) is measured as a count of spacers in a host matching a virus. At the population level, IDI is averaged across all hosts. The susceptibility index (SI) is the number of host-virus pairs where the host is not immune to the virus divided by the total number of host-virus pairs. Immunity is defined as a spacer-protospacer match as described above.All statistical tests were performed in R versions 3.2.2 to 3.2.4 . Games-H


Although several cases of hepatitis B virus reactivation have been described in patients with a history of hepatitis B virus infection while undergoing treatment for hepatitis C virus infection with direct acting antivirals, the question of whether hepatitis B virus surface antigen immune-escape mutations might play a role has not been addressed so far.We report a case of hepatitis B virus reactivation in a Caucasian patient infected with hepatitis C virus during treatment with sofosbuvir and velpatasvir. A 50-year-old man with a genotype 1a hepatitis C virus infection was considered for therapy. His serological profile was hepatitis B virus surface antigen-negative, hepatitis B virus core antibody-positive, hepatitis B virus surface antibody-negative, and anti-hepatitis D virus-positive. The detection of hepatitis B virus deoxyribonucleic acid (DNA) indicated active viral replication during the direct acting antiviral treatment that spontaneously returned to undetectable levels after treatment completion. Starting from week 12 after the end of treatment, hepatitis B virus surface antibody titers and hepatitis B virus e antibody developed. Sequencing analysis revealed the hepatitis B virus genotype D3 and the presence of two relevant immune-escape mutations (P120S and T126I) in the major hydrophilic region by analyzing the S region.We speculate that the presence of the hepatitis B virus surface antigen mutations, endowed with the enhanced capability to elude the immune response, could play a role in hepatitis B virus reactivation. This observation confirms that occult hepatitis B infection should also be carefully monitored, through surveillance of the hepatitis B virus viral load before and during direct acting antiviral treatment of hepatitis C virus. Chronic infection with either hepatitis B virus (HBV) or hepatitis C virus (HCV) is responsible for a high morbidity and mortality burden worldwide. Since these viruses share common transmission routes, HBV and HCV coinfection is not rare, and it is estimated that 1–15% of patients with HCV infection might be coinfected with HBV . OBI, deA Caucasian 50-year-old man, in a stable relationship and employed, was considered for HCV treatment at our center. In 1996, during a hospitalization for jaundice, he received a diagnosis of acute hepatitis B/hepatitis D virus (HDV) infection. At that time, an HCV infection genotype 1a was also diagnosed, which had never been treated. The patient had a hystory of previous use of injected heroin and inhaled cocain, and he was in opiate substitution therapy (OST) with buprenorphine. Except for this, he did not use other drugs. He smoked tobacco, reported a previous alcohol abuse, and at the first assessment he stated that he drank 1–2 drinks a day. His medical history was notable for a sinus tachycardia and he underwent inguinal hernioplasty and appendectomy. No liver disease was documented in his family history.His serological profile at admission to our center was HBsAg-negative, HBV core antibody (anti-HBc)-positive, HBV surface antibody (anti-HBs)-negative, and hepatitis D virus antibody (anti-HDV)-positive.On presentation, he did not report any symptoms and denied previous episodes of ascites, hematemesis, melena, hepatic encephalopathy, and vomiting. A physical examination excluded signs of hepatic decompensation; in particular, it did not reveal ascites, splenomegaly, leg swelling, jaundice, and spider angiomas. The remainder of the examination was normal.In the HCV pre-treatment assessment, he had F0–F1 fibrosis stage (Metavir score) evaluated by transient elastography (median liver stiffness 6.9 kPa) and an abdominal ultrasound excluded the presence of HCC and signs of portal hypertension. Laboratory analysis showed that alanine aminotransferase (ALT) was 51 IU/L, aspartate aminotransferase (AST) was 52 IU/L, and glucose 108 mg/dL. The count of white cells and platelets, and levels of hemoglobin, creatinine, alpha-fetoprotein, and electrolytes were normal; liver function tests were normal. HCV VL was 7,014,213 IU/ml and no HCV resistance-associated substitutions were found. His FIB-4 score was 2.13. Other laboratory test results are shown in Table In October 2017, he started once daily sofosbuvir/velpatasvir (400/100 mg) without ribavirin (RBV) for 12 weeks. HCV ribonucleic acid (RNA) was 37 IU/ml at week 4, below the limit of quantification (< 12 IU/ml) at week 8 and undetectable at the end of treatment (EOT)  or a reversion to an HBsAg-positive status in individuals who are HBsAg-negative and has been extensively reported in patients treated with immunosuppressive agents and/or intensive chemotherapy [To the best of our knowledge, we are the first to report a case of HBVr characterized by the emergence of viral strains with immune-escape mutations in a patient coinfected with HCV with isolated anti-HBc positivity and on a successful treatment with the latest generation of DAAs sofosbuvir and velpatasvir. HBVr is characterized by a sudden increase in HBV replication (> 2 logotherapy , but recotherapy  showed totherapy –14 and ootherapy  and may otherapy , patientet al. [An element of great interest is that, to the best of our knowledge, this is the first report of HBVr characterized by the emergence of viral strains carrying immune-escape mutations in a patient coinfected with HCV with isolated anti-HBc positivity and on successful treatment with the latest generation of DAAs. Immune-escape mutations in HBsAg are clinically relevant since their presence, by enabling the evasion of the adaptive immune response, can promote both viral replication  and chroet al.  describeTo conclude, we speculate that the presence of the antigen S mutations, endowed with enhanced capability to elude immune response, could play a role in HBVr. This observation confirms that OBI should also be carefully monitored, through the surveillance of HBV VL, before and during DAA treatment of hepatitis C. This approach could enable early identification of HBVr and find a pre-existing detectable HBV viremia in the context of OBI, which may be at higher risk of reactivation. Further studies are warranted to confirm the hypothesis regarding the role of HBsAg immune-escape mutants in predisposing HBVr during DAA regimens for CHC.


We analyze how shear stress and collision angle, the angle at which the beads collide with the pillars, affect the angular location of beads captured on the pillars. We also find that the fraction of captured beads as a function of distance (γ) in the channel is affected by rm. Using γ, we derive the probability of capture per collision with the pillar (ε). We show that ε is linearly related to rm, which is analogous to the expression level of proteins on cell surfaces. Although demonstrated with beads, this assay can next be expanded with cells, thus paving the way for a rapid antigen expression test.Antigen expression is an important biomarker for cell analysis and disease diagnosis. Traditionally, antigen expression is measured using a flow cytometer which, due to its cost and labor intensive sample preparation, is unsuitable to be used at the point-of-care. Therefore, an automatic, miniaturized assay which can measure antigen expression in the patient could aid in making crucial clinical decisions rapidly. Such a device would also expand the use of such an assay in basic research in biology. In this paper, we present a microfluidic device that can be used to measure antigen expression on cells. We demonstrate our approach using biotin-neutravidin as the binding pair using experimental and computational approaches. We flow beads with varying biotin surface densities ( Antigen expression on cells can be used as a marker for disease/infection diagnosis. For example, it has been reported that CD64 expression on neutrophils can be used as a biomarker for early diagnosis of bacterial infection.et al. reported a spiral microfluidic device for separating physically similar human umbilical vein endothelial cells and human microvascular endothelial cells based solely on the difference in CD31 expression.Zhang and Pappas have reported a microfluidic device for estimating the surface expression ratio of cell types.rm) in a microfluidic channel which has cylindrical obstacles functionalized with neutravidin. Adhesive interaction between the beads and the pillars captures the beads. First, we will observe how an interplay between the shear stress and the collision angle, the angle at which beads collide with the pillars, determines the angular location of a captured bead on a pillar. Thereafter, we show that the fraction of beads captured as a function of length (γ) can be used to obtain the probability of capture per collision (ε), which varies linearly with rm (r2 = 0.99). Although demonstrated with beads, this assay can just as easily be extended to estimate antigen expression on cells.In this paper, we demonstrate a simple microfluidic technique which can be used to optically measure the expression level of proteins on particles and cells. Central to our assay is an immuno-capture technique which has been shown to specifically capture cells of interest.μl/min. We tested 6 populations of beads with varying surface densities of biotin (rm) . Therefore, the surface density of biotin molecules can be accurately determined by analyzing the spatial profile of captured beads.In order to quantify this influence, we extract the probability of capture per interaction with a pillar, denoted by file Eq.  to the erm. This hypothesis is verified in Fig. γ increases slowly for rm = 0.8 × 1017/m2 compared to rm = 14.6 × 1017/m2. According to Eq. ε when the flow rate increases .We hypothesized in the Interplay Between Collision Angle section that bead capture would be spread far apart for beads with lower μm so that the gap between adjacent pillars should be set at 20–26 μm so that they travel in a “zigzag” fashion.Although this technique for measuring surface density has been demonstrated with beads, we believe that it could be applied to the measurement of antigen expression on cells too, for example, measurement of CD64 on neutrophils. In order to take into account the variability in the size of neutrophils, the channel has to be designed in such a way that all the cells of interest travel in a straight line. For example, neutrophils' size varies between 10 and 15 It is a well-known fact that antigen expression varies within a cell population. However, a limitation of this technique is that it measures only the mean surface density of molecules of cells. In a cell population with varying antigen expression, the profile of captured cells would resemble the profile of a cell population in which all the cells express the antigen equal to the mean antigen expression in the said cell population. Technical advancements in the technique might allow researchers to extract the range of antigen expression in a cell population. However, for diagnostic purposes at point-of-care, mean antigen expression on cells, like CD64 on neutrophils, still provides valuable insight into the disease state of a patient.ε, which predicted the surface density of biotin accurately (2r = 0.99). Although this technique has been demonstrated with beads, it can just as easily be extended to cells. It is easy to implement as it requires commonly available laboratory instruments such as syringe pumps and a bright-field microscope or a cell phone camera. Hence, this technique can prove to be an inexpensive, rapid, and label-free way to estimate antigen expression on cells.We presented a microfluidic technique that can be used to measure the antigen expression or surface density of molecules on particles. We began with a geometry of pillars in which beads travel predictably and its trajectory repeats every three rows. Next, we observed that the angular distribution of captured beads around a pillar broadens as the surface density of biotin increases. This showed that the capture of beads is affected progressively more by the collision angle as compared to the shear stress. Next, we observed that the spatial profile of the captured beads is strongly affected by the surface density of biotin molecules. From this spatial profile, we obtained the probability of capture per collision, Ethics approval is not required for this study.P, mediated by bond formation between receptors and ligands has the formrm is the surface density of receptors (biotin), lm is the surface density of ligands (neutravidin), cA is the area of interaction between the biotinylated bead and the neutravidin coated pillar, and a(f/n)K is the binding affinity. It is equal to the ratio of forward and reverse rate coefficients for the formation and breakage of the nth bond, respectively. This equation has been used in the literature to estimate the probability of capture.f, is equally shared by n bonds, where n is the smaller number of molecules available on two surfaces for bond formation, i.e., n = min . In this experiment, lm is less than rm . So, n can be replaced by cmlA in the above equation. This equation predicts that the probability of adhesion increases linearly with receptor density (rm). As the flow rate increases, the dislodging force increases, while the probability of capture decreases.The equation that governs the probability of particle capture, Δx and the chance of capture per encounter with a pillar is ε, then, a bead will encounter x/Δx pillars at a distance x, and the probability of capture γ can be expressed asn encounters is (1 − ε)n, and subtracting it from 1 gives the probability of capture; here, n = x/Δx]. This model, however, does not take into account the bead-bead interaction. Through our experiments, we will acquire γ as a function of x. Thereafter, we fit the experimental data to this model to estimate ε. ε is expected to be linearly related to rm  chemistry. The step-wise schematic for preparing the beads is shown in Fig. S2 . The full protocol can be found in the supplementary material. To confirm the presence of biotin on the beads, they were incubated with fluorescently labelled streptavidin  for 2 h at room temperature. After discarding the aspirate, the beads, suspended in 1× PBS , were analyzed using a flow cytometer. Figure Beads with varying surface densities of biotin were prepared by linking Amine-PEGrm). We made 6 populations of beads with rm = 0.8 × 1017/m2, 1.1 × 1017/m2, 2.4 × 1017/m2, 5.9 × 1017/m2, 10.5 × 1017/m2, and 14.6 × 1017/m2. The number of biotin molecules consumed in the reaction was determined by subtracting the number of unreacted molecules from the total number of molecules supplied at the start of the reaction. The number of unreacted molecules was estimated by using a Fluorescence Biotin Quantitation Kit . The standard curve for estimating the biotin concentration in the solution is provided in the supplementary material. The mean fluorescence intensity varies linearly with rm [Fig. The absolute concentration of biotin on the surface of the beads was also determined. The number of molecules consumed in the reaction was divided by the number of beads to obtain an estimate of average biotin molecules on each bead. The estimated average number of biotin molecules is divided by the surface area of a bead to obtain the surface density of biotin  by using standard soft-lithography. The full protocol for channel fabrication has been described elsewhere.μl of 1× PBS.For bead capture experiments, channels were incubated with 100 μg/ml neutravidin  and stored at 4 °C overnight to functionalize PDMS with neutravidin.μl of 1× PBS.To prevent non-specific binding, the channel was incubated with 1% BSA for 1 h at room temperature to block any interaction. To remove the unreacted molecules, chips were washed by flowing 100 The bead capture experiment was performed by using beads and flow rates described above in microfluidic channels functionalized with neutravidin. A total of 20 000 beads were injected in the channel for each experiment. All bead populations were assayed in triplicates. After the bead capture experiment, the still images of the channel were acquired with a 10× objective. Under this magnification, 24 images were required to cover the area where beads were captured. The images were then stitched together, and the physical location of each bead was determined using custom written macros in FIJI.To record the trajectories of beads around the pillars, the beads in the buffer were injected at flow rates described above in a channel blocked with BSA. The motion of beads was recorded at 2000 frames/s using a high speed camera . To obtain the coordinates of beads in each frame of the movie, a macro was written in FIJI. The coordinates of all the beads in all the frames were exported to an excel file for further analysis. We used the particle tracking code developed by John Crocker and Eric WeeksWe used COMSOL 5.1 to simulate the hydrodynamic focusing of particles and estimate the shear stress in the channel. All the simulations were performed in the Microfluidics Module using appropriate boundary conditions.supplementary material for the protocol for fabricating beads with varying biotin surface densities, characterization of the surface density of biotin on beads and neutravidin on PDMS, and the effect of the flow rate on shear stress and capture probability.See


This systematic review aimed to summarise and critically appraise the evidence for the effect of bodily illusions on corticomotoneuronal excitability.Five databases were searched, with two independent reviewers completing study inclusion, risk of bias, transcranial magnetic stimulation (TMS) reporting quality, and data extraction. Included studies evaluated the effect of an illusion that altered perception of the body (and/or its movement) on excitability of motor circuitry in healthy, adult, human participants. Studies were required to: use TMS to measure excitability and/or inhibition; report quantitative outcomes ; compare the illusion to a control or active comparison condition; evaluate that an illusion had occurred .B-mediated intracortical inhibition in motor cortex), with the opposite occurring  when the fake hand was not embodied prior to observing movement (“other-action”). Visuomotor illusions manipulating agency had conflicting results, but in the lower risk study, illusory agency over movement resulted in a relative decrease in corticomotoneuronal excitability. Last, an illusion of a missing limb reduced corticomotoneuronal excitability.Of 2,257 studies identified, 11 studies (14 experiments) were included, evaluating kinaesthetic illusions (n = 5), a rubber hand illusion (RHI) paradigm (n = 5), and a missing limb illusion (n = 1). Kinaesthetic illusions (induced via vision/tendon vibration) increased corticomotoneuronal excitability. Conflicting effects were found for traditional, visuotactile RHIs of a static hand. However, embodying a hand and then observing it move (“self-action”) resulted in decreased corticomotoneuronal excitability and increased silent period duration  and had a high risk of bias, kinaesthetic illusions and illusions of embodying a hand (and seeing it move), had consistent effects. Future investigations into the role of embodiment and the illusion strength on corticomotoneuronal excitability and inhibition are warranted. Illusions that alter one’s perception of the body by manipulating sensory or motor input are a useful way to explore the relationship between body perception and the neurophysiological regulation of our body . There iThe excitability of the pathway between the motor cortex and the muscle  can be measured using transcranial magnetic stimulation (TMS). A magnetic pulse is delivered to M1 through the scalp and the stimulus-evoked response in the target muscle is measured using surface electromyography (EMG), with the amplitude of this response termed the motor evoked potential MEP;  8]. Res. Res8]. The most common bodily illusions involve the use of unisensory  or multisensory input  to induce an altered perception of the body part  or of its movement. For example, kinaesthetic illusions use unisensory input to induce a feeling that the body part is moving when it is not. Kinaesthetic illusions can be induced via visual stimuli–i.e., by viewing limb movement (of another person or via mirrored reflection) in a first-person perspective. The mirror neuron system  is hypotIn contrast, the rubber hand illusion (RHI) is induced by synchronous visuotactile input–stroking both a rubber hand  and the real, hidden hand  to manipThe RHI can also be induced using synchronous visuomotor input–moving your real hand and seeing a rubber hand move at the same time to influence body ownership and also the sense of agency (i.e. the sense that the actions we perform are our own). It is proposed that the brain has an internal prediction model whereby an efference copy of a motor command is created and is compared to actual sensory input resulting from the movement , 31. If without additional sensory stimuli ); GABAA-mediated short-interval intracortical inhibition ; intracortical facilitation . To be included, studies were required to have compared the effect of bodily illusions on excitability measures to that of a control condition/group , or to have made an active comparison . Studies were also required to have evaluated that a body illusion actually occurred . Studies in which both clinical samples and healthy control samples were recruited were considered eligible if the healthy control data were provided separately.Studies were included if they evaluated the effect of an illusion that altered perception of the body (and/or body movement) by measuring corticomotoneuronal  or corticocortical  excitability during the illusion using single- or paired-pulse TMS in healthy, adult (>18 years of age), human participants. Specifically, studies were required to have evaluated at least one of the following outcomes: corticomotoneuronal excitability (single-pulse MEP area or amplitude); Gamma-Aminobutynic acid [GABA]B-mediated intracortical inhibition (i.e. silent period duration) was measured at <100 ms , or if the illusion did not alter the perception of a body part . All animal studies were excluded. Case studies  were also excluded.Studies were excluded if there was no control or comparison condition, if adjunctive interventions known to influence corticomotoneuronal excitability were used , if GABAThis study used an established definition of a bodily illusion . That isTwo independent reviewers screened potential studies by title and abstract to remove obviously irrelevant studies. Following this, full texts of potentially eligible studies were retrieved and formally assessed by the independent reviewers for eligibility (on Covidence) using the above criteria. The eligibility criteria were piloted by the two reviewers on three studies to ensure comprehensiveness, understanding, and agreement.The same two independent reviewers assessed the risk of bias of included studies using a custom-designed, piloted tool, which evaluated the presence of selection bias, detection bias, blinding, statistical bias, reporting bias, performance bias, attrition bias, and other forms of relevant bias (e.g. control for confounding variables). See The established TMS quality checklist was used to evaluate the quality of reporting of TMS methods . StudiesA customised, piloted data extraction form was used by the same two independent reviewers to extract the following data: participant demographics , study design (within- versus between-group comparison), type of illusion , control condition/group used , assessment method of body perception change. The TMS testing parameters that were extracted included: the cortical testing site(s), including the cerebral hemisphere for TMS application; the target muscle for electromyography measurement for MEPs (e.g. FDI or Abductor Digiti Minimi [ADM]); the number of stimuli provided/MEPs measured; and the stimulus intensity (% of active/resting motor threshold/other). For paired-pulse paradigms, the interstimulus interval (e.g. 2 or 3 ms) and the stimulus intensity of the conditioning and the test pulse was also extracted.For all TMS outcomes, measures of central tendency  and variability  were extracted for all provided time points, including pre-intervention, during intervention, and post-intervention, for each group/condition. If insufficient data were provided, authors were contacted a maximum of three times to retrieve this data.Included studies were grouped according to type of bodily illusion evaluated, the comparison condition/group used, and the type of excitability outcome evaluated. Illusion categories were defined based on the potential of the illusion methodology to impact corticomotoneuronal excitability, not based on the percept altered. For example, the RHI can be induced using visuotactile input  or visuomotor input . A moving vs stationary hand will clearly have a differential impact on corticomotoneuronal excitability, thus precluding direct comparison of these illusions, despite evaluating similar perceptual constructs. Additionally, kinaesthetic illusions induce a feeling of movement without actual movement occurring. This perception of movement can be induced through tendon vibration or through watching movement in a first-person perspective. Similarly, both types of kinaesthetic illusions may have different impacts on corticomotoneuronal excitability.Effect sizes were calculated for each study comparison when possible. For within-subject analyses, Cohen’s d for dependent samples was used and based on body illusion and corticomotoneuronal excitability data from our lab (in preparation), a within-subject correlation of 0.6 was used. For between subject analyses, Cohen’s d for independent samples was calculated. For significant main effects or interactions in between group analyses, the F-statistic (and group size) were used to calculate Cohen’s d. A meta-analysis, using a random effects inverse variance approach (Revman 5.2 software) to calculate the pooled mean difference , was considered when two or more studies evaluated a similar illusion, compared with a similar control group/condition, and evaluated the same TMS outcome. The Grading of Recommendations, Assessments, Development and Evaluations (GRADE) assessment of level of evidence was considered if meta-analyses could be performed. The GRADE analysis considers features such as risk of bias, consistency, precision, directness, and publication bias. However, rating of the feature ‘consistency’ requires consideration of heterogeneity statistics from meta-analyses, therefore if meta-analyses are not possible, the only possible rating of level of evidence is “very low” and such conclusions can be drawn without the scale application.The search resulted in 2,257 citations and hand searching retrieved a further 50 potentially relevant studies . After title and abstract screening, 2252 studies were clearly ineligible resulting in retrieval of full text for 55 studies. After formal eligibility assessment of full text studies, 11 studies (14 experiments) were included in this review . Data weExperimental study designs were used in all included studies , with 10 experiments using a within-subject study design and two using a between-subject comparison. One study had a combination of within and between group data, but for analyses purposes, these data were combined and a between group analysis  was performed . Eleven a priori sample size calculation . Fu. Futh fiduring the illusion while adding in an additional condition where the participant watched the rubber ball rotation task from a third-person perspective and also comparing to a rest condition (measured pre- and post-experiment). Consistent with the study above [rd-person perspective–differed from rest). Further, paired pulse stimulation was used to evaluate the effect of the illusion on short-interval intracortical inhibition  and intracortical facilitation  within the motor cortex in both experiments [Noijama et al (2015)  also used non-immersive virtual reality where participants watched a video of a ball rotation task (first-person perspective) to create an illusory sense of movement . In expedy above , there weriments . There wConcerns with randomisation and analysis in Kaneko et al (2007) question whether increases in corticomotoneuronal excitability seen with the kinaesthetic illusion vs baseline resting levels were due to the illusion or due to a confounding factor, such as time. However, that the illusion and non-illusion conditions (order randomised) differed in the study by Kaneko et al (2007) and that similar effects on corticomotoneuronal excitability were shown by Noijma et al (2015) (order randomised) increases confidence in these results. Aoyama et al’s (2012) finding suggest that such effects of illusory movement on corticomotoneuronal excitability are not limited to the upper limb, but also extend to the lower limb. That the DF illusion was more intensely experienced than the PF illusion and that only the DF illusion altered corticomotoneuronal excitability suggests a link between illusion strength and influence on the motor cortex. However, concerns with randomisation in Aoyama et al (2012) suggest that this result requires replication.Noijma et al (2015) showed that visual kinaesthetic illusions may have differential effects on inhibition and facilitation within the motor cortex. While there was no significant effect of illusory movement on SICI, the effect sizes for some comparisons were moderate in size (d = 0.34–0.49), which raises the possibility that it may be underpowered. However, that effects of illusory movement on corticomotoneuronal excitability are specific to the muscle consistent with the movement illusion is supported by both Kaneko et al (2007) and Aoyama et al (2012). Clearly further work is needed to elucidate such findings, namely with studies using assessor blinding, and robust (and preferably pre-registered) randomisation procedures and statistical analyses. Despite small sample sizes, the effect sizes seen were generally moderate to large.Two studies used high frequency tendon vibration to induce an illusory sense of limb movement , 52. DatMancheva et al (2017) provided Flexor Carpi Radialis (FCR) tendon vibration to induce an illusory feeling of wrist extension . CorticoNaito et al (2002) evaluated the effect of an illusory sense of wrist flexion using tendon vibration of the wrist Extensor Carpi Ulnaris (ECU) tendon, but with both hands placed together, palm on dorsum  . CorticoTaken together, the above findings suggest that illusory limb movement (induced by tendon vibration) has muscle and condition-specific effects–stimulation of the motor area of the antagonist muscle to the illusory movement direction was found to elicit increased corticomotoneuronal excitability in illusion versus non-illusion control conditions. In contrast, condition-specific effects do not exist in the muscle receiving the tendon vibration. That is, while tendon vibration elicits increased corticomotoneuronal excitability for its respective muscle’s motor area, it does so regardless of the test condition. Further studies are clearly needed, particularly given that the present studies were at high risk of bias for statistical methods, with many of the primary analysis findings not reported , and smIt is interesting that in Mancheva et al (2017) only one of the illusion conditions–high amplitude vibration with eyes open but no vision of the hand–elicited increased corticomotoneuronal excitability in the motor area of the muscle (ECR) consistent with the direction of illusory movement. The other illusion condition–high amplitude vibration with eyes closed–did not elicit increased corticomotoneuronal excitability, despite inducing a feeling of illusory movement. This result may be explained by the influence of vision–during low amplitude vibration of the FCR tendon (no illusion), ECR MEP area was reduced (versus rest) only when the eyes were closed; this suppression did not occur in either of the eyes open conditions. It is possible that such suppression with eyes closed counteracted any facilitatory effects of the visual illusion. These findings suggest a role for both vision itself and illusory movement influencing excitability of the motor cortex and its descending projections to the muscle.B-mediated intracortical inhibition within the motor cortex .Five studies  evaluated the effect of a rubber hand illusion (RHI) on corticomotoneuronal excitability, with one study also evaluating the strength of GABAThree studies evaluated the effect of a traditional RHI where synchronous visuotactile input is provided  , 48, 49.della Gatta et al (2016) found a significant decrease in raw MEP amplitude (mean ± SD) in the RHI condition (i.e. synchronous stroking: (0.53±0.33mv) compared with the asynchronous control condition  and the baseline condition   in partiSchutz-Bosbach et al (2006) applied the principles of the traditional RHI to a paradigm that used the experimenter’s hand in place of a rubber hand . This study also evaluated the effect of movement observation (the experimenter moving their hand) when the participants embodied the experimenter’s hand (synchronous stroking: ‘self action’) and when they did not (asynchronous stroking: ‘other person action’). When the participants embodied the experimenter’s hand and observed the experimenter’s hand moving (synchronous ‘self action’), the FDI MEP amplitude normalised to baseline (mean +/- SEM) was suppressed (1.46±0.23) relative to observation of a static hand . When participants did not embody the experimenter’s hand (asynchronous ‘other action’), and observed it moving, the FDI MEP amplitude normalised to baseline increased (1.44 ±0.10) relative to observation of a static hand  .While not directly calculated in the paper , the FDIB-mediated intracortical inhibition within the motor cortex) and MEP amplitude while participants maintained isometric tonic contraction of the FDI [B-mediated intracortical inhibition within the motor cortex. These effects were reversed in the asynchronous stroking condition: when subjects viewed hand actions (not attributed to themselves), mean cortical silent period duration was significantly shorter (153±11ms) than when subjects observed a static hand . Lastly, silent period duration in the synchronous stroking hand action condition was significantly longer (162±11ms) than the asynchronous stroking hand action condition . There were no differences in cortical silent period between the synchronous  and asynchronous static hand conditions , although the synchronous condition (RHI) differed from baseline (p = 0.042), while the asynchronous condition did not (p = 0.12). Because the silent period was the primary outcome measure for this study, the experimental set-up resulted in 5.9% of the MEPs not being measurable (because the top and/or bottom part of the MEP was missing). Therefore, the lack of difference seen in FDI MEP amplitudes between conditions is taken with caution.Schutz-Bosbach et al (2009) evaluated the effect of the RHI on the cortical silent period  decreases corticomotoneuronal excitability [That conflicting results were seen for corticomotoneuronal excitability during the static visuotactile RHI merit discussion. First, such differences may reflect differences in RHI paradigm , but are most likely due to differences in TMS protocol  and seeing that embodied hand move (while your hand is still static) were largely consistent between two studies , 49. Vierelative decrease in corticomotoneuronal excitability vs conditions in which agency wasn’t present. However, it is noted that the illusion of agency condition had an absolute increase in excitability versus baseline levels . Further, there was a positive correlation between delay and MEP amplitude normalised to baseline (r = 0.40): the greater the temporal delay, the greater the normalised MEP amplitude . Last, delay had no influence on corticomotoneuronal excitability of a control muscle (ADM) that was unrelated to the executed/observed movement (p = 0.69).Two studies evaluated a motor version of the RHI that involved participants moving their own hands. Weiss et al (2014) (n = 12), evaluated excitability when active movement of the participant’s finger was paired with either synchronous visual input  or asynchronous visual input  . The visAdditionally, Weiss et al (2014) considered the influence of explicit recognition of delay in ambiguous situations  . When coIn Karabanov et al (2017), a customised experimental set-up was used where the participant’s left hand was positioned underneath (but not touching) two gloved rubber hands . One of While the two study results find conflicting effects of altering agency, differences in study size and TMS testing paradigm may underpin the differences seen here. It is plausible that Karabanov et al’s finding of no effect of agency or ownership on corticomotoneuronal excitability  may be aa priori sample size estimates, combined with moderate-to-large, but non-significant statistical results, raise the possibility that this study was underpowered.One study created an illusion of a missing or amputated arm . It was This review found a limited number of studies (n = 11) evaluating the effect of bodily illusions on corticomotoneuronal excitability. All studies evaluated corticomotoneuronal excitability, while two experiments also evaluated intracortical inhibition and one experiment evaluated intracortical facilitation in the motor cortex. All included studies had a high risk of bias, with low to moderate quality of TMS reporting. Despite these limitations, there was evidence that perception of illusory movement of your own limb  increased corticomotoneuronal excitability, specifically in the illusion condition and for the muscle group corresponding to the direction of the movement illusion. While tendon vibration itself increased corticomotoneuronal excitability for the muscle being vibrated, this occurred regardless of the condition.B-mediated intracortical inhibition within the motor cortex). In contrast, not embodying a hand and then seeing it move (other-action) has a facilitatory effect: it increases corticomotoneuronal excitability and decreases inhibition . Such results are supported by visuomotor RHI findings showing increased corticomotoneuronal excitability as a function of increasingly delayed observed movement (becoming more ‘other-action’ than ‘self-action’). Conflicting results were found for the traditional visuotactile static RHI although differences seen likely reflect variations in methodology. Last, a single study found that an illusion of an amputated limb reduces corticomotoneuronal excitability. Together these findings have important theoretical implications.Interestingly, the opposite effect of illusory movement was seen when the limb was disowned. In visuotactile RHI paradigms, embodying a hand and then seeing it move (illusory self-action) has an inhibitory effect: it reduces corticomotoneuronal excitability and increases inhibition  [This review found evidence that, regardless of the mode of induction (vision or tendon vibration), kinaesthetic illusions that induce the feeling of limb movement increase corticomotoneuronal excitability. That such changes are due to the illusion (and not merely sensory input) are supported by both past work and the present findings. First, the changes in corticomotoneuronal excitability seen here were specific to the muscle whose action was consistent with the direction of illusorily induced movement (‘illusion agonist muscle’), regardless of mode of induction , 50–52. That corticomotoneuronal excitability changes reflect the illusion induced and not merely the sensory input applied are supported by previous neuroimaging work. For example, distinct brain activation patterns (measured using fMRI) are seen when one is experiencing a kinaesthetic illusion compared with merely watching that same movement . In addiThe mechanisms underlying kinaesthetic illusions induced via tendon vibration or via vision are clearly different, in so far as that they involve unique initiating neural inputs. Therefore, that both types of kinaesthetic illusions have similar effects on excitability suggests that the unique sensory representations of limb movement–i.e., a proprioceptive representation and a visual representation of movement–can each similarly influence excitability of the motor cortex and its projection to the muscle. These findings suggest involvement of higher-order multisensory areas that integrate input from vision and proprioception, such as the premotor cortex , 62 and/The present findings also raise the possibility that kinaesthetic illusions may have specific effects on numerous mechanisms underlying motor cortex function. Increases in corticomotoneuronal excitability during illusory movement (via tendon vibration) were seen concurrent with increases in facilitation (ICF), but in the absence of changes in inhibition (SICI) . Past woincreased corticomotoneuronal excitability, consistent with real movement, disowning your real hand and then seeing the embodied hand move (illusory self-action) resulted in a relative decrease in corticomotoneuronal excitability  compared with asynchronous stroking . The MEP amplitude and silent period findings are therefore consistent with a picture of facilitation of the motor system when observing others’ actions (asynchronous visuotactile) and a relative reduction in corticomotoneuronal excitability when the same observed actions are illusorily attributed to their own body (synchronous visuotactile). The best available evidence  suggested study , providenot be your own, corticomotoneuronal excitability increases. For example, when agency over a moving finger was present, MEP amplitudes were increased from baseline levels, but when agency was broken via visual delay of finger movement, MEP amplitudes were significantly higher than during the agency condition [not disowning your real hand and then seeing the rubber hand move (other person action) also results in increased corticomotoneuronal excitability [This review found evidence from both visuotactile and visuomotor RHI paradigms that when movement is perceived to ondition . Such eftability , 49. Howtability , 33), thtability . Similartability . Togethetability ) supportOf interest are preliminary findings that explicit awareness of agency as induced by illusions might influence the excitability of the motor cortex and its projections. Specifically, in ambiguous situations of small visual delay between seen and actual movement, corticomotoneuronal excitability (via MEP amplitudes) was relatively reduced for trials in which visual input was judged as corresponding (agency) versus non-corresponding (no agency) to actual movement . This raa priori. The search strategy used was sensitive and all processes of the review  were completed by two independent authors. Dual assessment by independent reviewers and hand searching of the reference lists of all full text studies, reduces the risk of missing potentially eligible studies. Additionally, the majority of studies adequately described the illusion, allowing for future replication testing.This review used gold-standard methodology for systematic reviews , 45 withThe conclusions of this review are limited in that all included studies had a high risk of bias and none were of high-level evidence . Sample sizes of included studies were typically small , which increases the risk of spurious findings. Furthermore, the wide variety of illusions, comparison conditions, and TMS methodologies used prevented meta-analyses.Given that the present review focussed on the effect of illusions on corticomotoneuronal excitability in healthy volunteers, the current findings clearly have very limited generalisability to clinical populations for therapeutic use. However, that use of non-invasive brain stimulation paradigms to alter corticomotoneuronal excitability have shown evidence of positive effect for movement and pain –11 suggeReplication studies that use high-quality methodology to evaluate the effect of bodily illusions on corticomotoneuronal excitability are needed. Specifically, the most common forms of bias found by this review were a lack of blinding of participants/researchers and use of small sample sizes. Addressing these methodological issues will reduce bias, thus allowing stronger conclusions. Given the consistent effect of kinaesthetic illusions (induced by vision or tendon vibration) on increasing corticomotoneuronal excitability, future studies could extend use of these illusions to clinical conditions in which altered corticomotoneuronal excitability is present to determine if similar effects to those in healthy volunteers are seen. Additionally, it is known that individual differences exist in the degree to which illusions are experienced–for example, some people do not experience the RHI . SpecifiB-mediated intracortical inhibition within the motor cortex relative to observing other-action , which increased excitability and reduced inhibition. That RHI effects on corticomotoneuronal excitability differ than those resulting from kinaesthetic illusions of movement suggests that embodiment has unique effects on corticomotoneuronal excitability. Lastly, a reduction in corticomotoneuronal excitability was found during an illusion of a missing limb. The findings are preliminary and high-quality research is needed to better understand the mechanisms underlying perceptually-induced changes of the motor system.This systematic review found very low-level evidence for the effect of bodily illusions on corticomotoneuronal excitability, given high risk of bias and low overall numbers of eligible studies. Despite this, kinaesthetic illusions consistently increased corticomotoneuronal excitability specific to the muscle group undergoing the movement illusion. Conflicting effects were found for a static RHI. However, an illusory feeling of self-movement, via embodying a fake hand and watching it move, consistently decreased corticomotoneuronal excitability and increased GABAS1 Checklist(DOC)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluate our multiplexed amplicon approach, PrimalSeq, to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We develop an experimental protocol and computational tool, iVar, for using PrimalSeq to measure virus diversity using Illumina and compare the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.The online version of this article (10.1186/s13059-018-1618-7) contains supplementary material, which is available to authorized users. RNA viruses, including HIV, influenza, West Nile, and Zika, pose significant threats to public health worldwide. Part of this burden stems from their ability to rapidly evolve within hosts . GeneratIn many cases, however, accurately measuring intrahost RNA virus diversity using deep sequencing remains a significant challenge. Multiple factors, such as virus titer, sample preparation, sequencing errors, and computational inferences, can bias measures of genetic diversity –16. MoreWe previously developed a multiplex primer design tool  coupled to a laboratory protocol  to sequence RNA viruses directly from clinical samples in a way that is cheap, accurate, and scalable under resource-limited conditions . VersionIn this study, we benchmarked PrimalSeq for sequencing diverse virus populations, highlighting its limitations and providing recommendations for accurately measuring intrahost single-nucleotide variants (iSNVs) from both Illumina and Oxford Nanopore data. We used these results to develop comprehensive laboratory protocols and a computational tool (iVar), and further tested PrimalSeq to characterize Zika virus populations generated from cell culture, mosquito, non-human primate, and human clinical samples. We demonstrate the utility of PrimalSeq for other viruses by designing an amplicon scheme for West Nile virus and measuring genetic diversity from field-collected mosquitoes and birds. Our data show that virus diversity can be significantly impacted by the experimental and biological systems, and we provide a framework to uncover the underlying mechanisms. PrimalSeq and iVar provide a scalable platform for viruses other than Zika and West Nile that can be applied to discover ecological, epidemiological, and immunological drivers of virus evolution in a variety of systems.primal.zibraproject.org), a multiplex primer design tool for amplicon-based sequencing of RNA virus genomes directly from clinical samples [We previously developed Primal Scheme . We then made two sets of mixed virus populations: 1) altering the ratios of mixed viruses, while keeping the overall input concentration constant at 1000 virus RNA copies  independently three times and sequenced each replicate to a minimum coverage depth of 1000× using the Illumina MiSeq. We measured iSNV frequency at each site and calculated the mean iSNV frequency of all iSNVs within an amplicon to estimate the computed virus #2 haplotype frequency  individually three times and sequenced each on the Illumina MiSeq. We limited our analysis to only those amplicons with perfect primer matches. Within these regions, we analyzed 54 sites with expected 10% iSNVs (true positives) and 4173 sites that were invariable in our mixed virus population. We considered any iSNVs detected > 0.1% frequency at the invariable sites as false positives. We found that on average, 631 of the 4173 expected invariable sites 16%) had false positive iSNVs at a > 0.1% frequency cutoff. We observed false positive iSNVs on every amplicon but found that they were unevenly distributed across the 250 nucleotide long Illumina reads , for iSNV measurement accuracy, we generated triplicate sequencing libraries from our Mix10% virus population (1000 RNA copies). The triplicate amplicon libraries were sequenced using both Illumina MiSeq and Oxford Nanopore MinION platforms, while the metagenomics libraries were sequenced using the MiSeq Fig. a. We fouTo further explore Oxford Nanopore sequencing for measuring intrahost virus diversity, we examined if we could (1) differentiate between mixed genotypes within a virus population and (2) computationally remove false positive iSNVs. Though the mean iSNV frequencies measured from Nanopore were not significantly different from Illumina Fig.  and we cFirst, using a reference database containing virus #1 and #2, plus two divergent Zika viruses , we determined if we could differentiate between virus haplotypes in a mixed population. We found that 92.38% of the aligned reads mapped to virus #1 and 7.35% mapped to virus #2, the roughly expected 90%:10% proportions (0.27% of reads mapped erroneously to haplotypes not present in the mixture). Overall, the results indicate that nanopore sequencing reads are useful for identifying highly divergent haplotypes within a mixture—as might be expected for some co-infections —despite To attempt to differentiate between true and false positive iSNVs, we limited our analysis to regions only covered by perfect primer matches and analyzed 54 true positive and 4173 true negative sites, as we did above for the Illumina data  to accurately analyze data from amplicon-based sequencing  pring Fig. c and 4c,ing Fig. d.Fig. 6EOur computational package, iVar, contains functions broadly useful for viral amplicon-based sequencing that cannot be accomplished using currently existing tools. We programmed iVar in C++ with minimal dependencies, and created the following functions to accurately call intrahost variants and generate virus consensus genomes from sequencing data across multiple replicates: 1) trimming of primers and low-quality bases, 2) consensus calling of virus sequences, 3) intrahost variant calling of iSNVs, insertions, and deletions, followed by a filtering step that uses variants called across multiple replicates to exclude false positives, and (4) identification of mismatches in primer sequences and exclusion of the corresponding reads from alignment files. We also created two pipelines using iVar to call intrahost variants from samples with or without known reference sequences, and prepared with or without technical replicates . When using iVar in combination with PrimalSeq, from our empirically-derived data we found that the following guidelines produced robust and reproducible results: (1) only call intrahost variants detected in two or more technical replicates greater than 3% frequency  or infeAedes aegypti (Aag2) cells  plasma  or vertebrate hosts (humans or birds), though the mosquito samples were more variable .For multi-sample comparisons of genetic diversity, omit genome regions amplified with primers that contain iSNVs within the binding sites.Understanding how RNA viruses evolve within hosts can help lead to breakthroughs in medicine and biology. Rapid developments in sequencing technologies are facilitating more research into these areas, yet usage of unvalidated tools and systematic biases can dramatically limit the utility of the results –16. To aSeveral factors can alter the accuracy of measuring intrahost virus diversity. In particular, we found that input virus concentrations, sequencing coverage depths, and primer mismatches can have profound effects on iSNV estimations. Using the recommendations above, however, we could consistently and accurately detect iSNVs at 3% frequency and higher. We predict that the lower limit of iSNV detection can be improved with a higher effective sampling depth  .Given no primer mismatches to the virus sequences, we found that measures of iSNVs frequencies from PrimalSeq were nearly as accurate as an untargeted metagenomics approach . BecauseFalse iSNV calls significantly influence measurements of intrahost virus diversity . We foun2 method (where template and complementary strands of each fragment are sequenced) [The ease and portability of Oxford Nanopore technologies, particularly the MinION, are revolutionizing the way we sequence viruses, including its use in near real-time outbreak tracking , 28, 37.quenced) , tandem quenced) , 51 or uquenced) .For viruses that utilize multiple hosts, like mosquito-borne viruses, being able to compare results from many samples types is critically important. A lack of standardization, however, means that the field does not yet have a consensus to whether the mosquito vector or the vertebrate host contributes the most to virus genetic diversity –62. The primal.zibraproject.org) [We demonstrate that PrimalSeq can accurately measure intrahost virus genetic diversity if properly validated. We benchmarked our highly multiplexed and streamlined amplicon-based sequencing method using a series of experiments with mixed virus populations, developed an all-inclusive computational analysis tool (iVar), and showcase its utility by measuring intrahost virus diversity from cells, mosquitoes, primates, birds, and humans. Furthermore, using our free online primer designer, Primal Scheme (ect.org) , PrimalSZika virus RNA from isolates PRVABC59  and FSS13025  were quantified by qRT-PCR (as previously described ). The coAe. aegypti) and primates  fetal bovine serum, l-glutamine, sodium bicarbonate, and antibiotics (penicillin and streptomycin). Aag2 and HeLa cells were incubated with 5% CO2 at 27 °C and 37 °C, respectively.Aag2 (derived from  embryos ) and HeLAe. aegypti mosquitoes were infected with Zika virus as previously described [10 Vero plaque-forming units of Zika virus (PRVABC59). At 14 days post infection, individual mosquitoes were collected and homogenized. Viral RNA was extracted from 50 μL of mosquito homogenate using the using the MagMax Viral RNA Extraction Kit and eluted 50 μL of elution buffer . Indian origin rhesus macaques (Macaca mulatta) were inoculated subcutaneously with 3 log10 Vero plaque-forming units of Zika virus (PRVABC59) and plasma was collected 5 days post infection, as described [escribed . In brieescribed , 70. RNAAe. aegypti mosquitoes were collected using BG-Sentinel mosquito traps (Biogents AG) and sorted into pools of up to 50 females per trap. The pooled mosquitoes were stored in RNAlater (Invitrogen), RNA was extracted using the RNAeasy kit (Qiagen), and Zika virus RNA was quantified by qRT-PCR [Clinical and entomological samples were collected during the 2016 Florida Zika virus outbreak  to compa qRT-PCR . RNA fro qRT-PCR , it is hCulex quinquefasciatus mosquitoes (up to 50 per trap) and dead American crows were collected by the San Diego County Vector Control Program during 2015. RNA was extracted using the RNAeasy kit (Qiagen) and screened for the presence of West Nile virus RNA using standard qRT-PCR.7 to 100 copies per reaction). The Zika virus RNA standards were constructed by PCR amplifying a 848 bp segment of the Zika virus NS5 protein coding region (genome positions 8644 to 9492) with the following primers: forward containing a T7 promoter region  and reverse primer . The amplified cDNA was converted into RNA to be used as standards using the Invitrogen MEGAscript T7 Transcription Kit.Zika virus RNA copies were quantified using a qRT-PCR assay targeting the NS5 protein coding region of the genome using the BioRad One-step qRT-PCR for probes kit. In a 20-μL reaction, 2 μL of virus RNA was added to 10 μL of iTaq universal probes reaction mix, 0.5 μL of iScript RT, 6 μL of nuclease-free water, 0.5 μL of the forward primer , 0.5 μL of the reverse primer , and 0.5 μL of the 6-FAM labeled probe . The reactions were then amplified on a thermocycler with the following conditions: 50 °C for 10 min, 95 °C for 3 min, and followed by 40 cycles of 95 °C for 10 s and 57 °C for 10 s (fluorescence read at the end of the 57 °C step). To calculate the number of virus template copies using standard curves, we include 10-fold dilutions of partial Zika virus RNA genomes spanning the primer sites  was reverse transcribed into cDNA using Invitrogen SuperScript IV VILO (20 μL reactions). Virus cDNA (2 μL) was amplified in 35× ~ 400 bp fragments from two multiplexed PCR reactions using Q5 DNA High-fidelity Polymerase (New England Biolabs) using the conditions previously described . For thehttp://grubaughlab.com/open-science/amplicon-sequencing/ [https://andersen-lab.com/secrets/protocols/ [A detailed protocol for our amplicon-based sequencing methods can be found in Additional file uencing/  and httpotocols/ . Virus aEscherichia coli DNA ligase and polymerase (New England Biolabs). The cDNA was purified by Agencourt AMPure XP beads (Beckman Coulter) prior to library preparation using Nextera XT (Illumina) following the vendor’s protocols, but with less reagents. Specifically, for tagmentation (12.5 μL reaction), we concentrated our cDNA to 4 μL using a DNA speedvac and used 5 μL of Tagment DNA Buffer  and 1 μL of Amplicon Tagment Mix (one fifth recommended). After incubation, the reaction was stopped using 2.5 μL of Neutralize Tagment Buffer . The libraries were indexed and amplified using one half of the Nextera PCR reagents and primers in a 25-μL reaction. Agencourt AMPure XP beads (Beckman Coulter) were used for the final purification step (purified twice at a ratio of 0.7:1 beads to sample). The libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and Bioanalyzer (Agilent). Paired-end 251 nt reads were generated using the MiSeq V2 500 cycle kit. The paired-end reads were aligned to a provided reference genome using BWA [We followed the general outline of a previously developed protocol for untargeted sequencing of the mixed viral populations . In briesing BWA , the reasing BWA , and iSNsing BWA . No othegithub.com/andersen-lab/ivar [github.com/andersen-lab/ivar [github.com/samtools/htslib) and Awk (cs.princeton.edu/~bwk/btl.mirror/). Awk is generally available on most unix based operating systems and HTSlib has only one dependency—zlib (zlib.net/). The output of `mpileup` command from the widely used SAMtools [We developed an open source software package to process virus sequencing data and call iSNVs from technical replicates, iVar , and detailed documentation can be found at lab/ivar . The toolab/ivar . The verlab/ivar . iVar waSAMtools  was usedSAMtools . The paigithub.com/andersen-lab/paper_2018_primalseq-ivar [iVar was validated against existing tools. . A total of 2.4 million reads were generated which after alignment and trimming covered 95.66% of the reference genome (Genbank KX087101). For the purposes of assigning to genotypes , reads were assigned to individual strains using BWA-MEM [github.com/nickloman/zika-isnv [github.com/pysam-developers/pysam). Only predicted variants with more than 10 supporting forward and 10 supporting reverse reads were considered. The logistic regression model was trained and tested under a 10-fold cross validation scheme using the train function with the parameters method = “glm” and family = “binomial” from the caret (github.com/topepo/caret/) library in R. Class probabilities for the ROC curve were captured from the same function and plotted using ggplot2 (github.com/tidyverse/ggplot2).Using the same PCR amplicons used for amplicon-based Illumina sequencing, we sequenced three replicates of the mixed Zika virus population  using the Oxford Nanopore GridION sequencer. Native, 1D barcode libraries  were prepared according to previously published methods , with th BWA-MEM  against  BWA-MEM . Allele ika-isnv ). This sVirus iSNVs > 3% were used to genetically characterize the populations derived from in vitro, in vivo, and field samples Figs.  and 7. Fp is the iSNV frequency and the mean Sn from all evaluated sites within the virus genome was used to determine the population complexity Additional file 2:Figure S1.), sensitivity of measuring intrahost diversity at 5% , metagenomic sequencing coverage depth , and validation of iVar for intrahost single-nucleotide variant calling , consensus calling , and trimming . (PDF 7109 kb)iSNV frequencies calculated using pseudo replicates