Giardia have adapted to colonizing different hosts, most notably the intestinal tract of mammals. The human-pathogenic Giardia species, Giardia intestinalis, has been extensively studied at the genome and gene expression level, but no such information is available for other Giardia species. Comparative data would be particularly valuable for Giardia muris, which colonizes mice and is commonly used as a prototypic in vivo model for investigating host responses to intestinal parasitic infection. Here we report the draft-genome of G. muris. We discovered a highly streamlined genome, amongst the most densely encoded ever described for a nuclear eukaryotic genome. G. muris and G. intestinalis share many known or predicted virulence factors, including cysteine proteases and a large repertoire of cysteine-rich surface proteins involved in antigenic variation. Different to G. intestinalis, G. muris maintains tandem arrays of pseudogenized surface antigens at the telomeres, whereas intact surface antigens are present centrally in the chromosomes. The two classes of surface antigens engage in genetic exchange. Reconstruction of metabolic pathways from the G. muris genome suggest significant metabolic differences to G. intestinalis. Additionally, G. muris encodes proteins that might be used to modulate the prokaryotic microbiota. The responsible genes have been introduced in the Giardia genus via lateral gene transfer from prokaryotic sources. Our findings point to important evolutionary steps in the Giardia genus as it adapted to different hosts and it provides a powerful foundation for mechanistic exploration of host–pathogen interaction in the G. muris–mouse pathosystem.Diplomonad parasites of the genus  Raw DNA and RNA sequence reads are archived at NCBI Sequence Read Archive (SRA) under accession numbers SRR8858297–SRR8858305.https://giardiadb.org/giardiadb/).This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number PRJNA524057. The version described in this paper is version VDLU00000000.1. The genome sequence and annotations generated in this project have been integrated into GiardiaDB  and parstinalis  and S. smonicida ) relativG. muris cysts purified from the faeces of infected mice  and assembled a high-quality draft genome using sequences obtained by PacBio and Illumina technologies. In addition, we generated RNA-Seq data for gene prediction and gene-expression analyses with total RNA extracted from cysts, recently excysted cells (excyzoites) and trophozoites isolated from the small intestine of infected mice .We extracted DNA from freshly excysted G. muris draft genome consists of 59 contigs spanning 9.8 Mbp, which is notably smaller than the G. intestinalis WB genome  that they were not readily resolved using pulsed-field gels, and are named accordingly from 1 to 5 from largest to smallest in size  in the assembly was estimated to be 0.016 % , equival0.026 %, . Distrib0.026 %, .G. muris . We found that the average number of domains in G. muris orthologues was not significantly different from G. intestinalis , indicating that protein domain loss is not the cause of protein shortening.The B genome  do not mG. muris genes except for an enrichment of A residues around the start codon , which resembles observations in G. intestinalis [G. muris genome was also apparent at the 3′ end of genes where the putative polyadenylation signal, which is similar to the one described in G. intestinalis [G. intestinalis have specific promoter elements [G. muris, with one notable exception of cyst-wall protein 3 , similar to G. intestinalis infection in mice [We could not identify any universal, conserved promoter motifs shared by all stinalis . The strstinalis , is overelements . Most ofrotein 3 . Encystar motifs , similarstinalis , suggest in mice .G. intestinalis contain introns, with only eight known cis-spliced and four trans-spliced genes (five trans-introns) [cis- and no trans-introns were identified in the parasitic diplomonad S. salmonicida [, whereas the free-living fornicate K. bialata has on average seven cis-introns per protein encoding gene [G. muris maintains homologues to the eight cis-spliced G. intestinalis genes, but has only three retained introns . All four trans-spliced genes in G. intestinalis have homologues in G. muris with conserved splicing motifs . Mining genes with similar motifs did not reveal additional intron-containing genes in G. muris. Similar to G. intestinalis [trans-spliced genes in G. muris preserve a similar cleavage motif TCCTTTACTCAA  as the RNA processing sequence motif [G. muris, and cis-introns seem to be easier to lose than trans-introns.Very few genes in introns) . Similarmonicida , whereasing gene . G. muristinalis  all the ce motif . Thus, wG. intestinalis are characterized as cysteine-rich proteins with frequent CXXC motifs and a conserved C-terminal transmembrane (TM) domain followed by a cytoplasmic pentapeptide . We identified 265 VSP homologues in G. muris. Their C-terminal pentapeptide  differed slightly from that in G. intestinalis. However, the cysteine and arginine residues in the pentapeptide, which are known to be post-translationally modified in G. intestinalis, are conserved [G. muris VSP TM domain show conservation to the TM domain of G. intestinalis VSPs . Most G. muris VSPs contain the conserved GGCY motif present in most G. intestinalis VSPs (Table S1c). Since bona fide VSPs need signal peptides (SPs) at the N-terminus to guide VSPs to the parasite surface, we divided this group into two subgroups; proteins with predicted SPs are called VSPs, whereas VSP proteins without SPs are referred to as pseudogenized VSPs (ψVSPs). The 26 complete VSP genes (16 unique at 98 % identity to each other) are mostly located chromosome-centrally (Variant specific-surface proteins (VSPs) in onserved . In addientrally , Table 2entrally . The intentrally . There aentrally  interspeentrally .n=17) in G. muris, herein defined as having >3 ψVSPs genes . Strikingly, nine out of the ten ends of the main contigs have a ψVSP array at or close to the ends of the chromosomes (telomere-adjacent) containing a total of 131 genes (Table S1c). The only main contig that ends without an array has a cluster of two ψVSPs close to the chromosome terminus. We found a single ψVSP array consisting of 12 genes in a chromosome position that was non-telomere adjacent (on chromosome 5)  are found in linear arrays (osome 5) . The ψVSG. muris to investigate their evolutionary dynamics. The phylogeny revealed relaxed clustering of ψVSP genes originating in each linear array . However, the internal linear array on chromosome 5 represents a noteworthy exception showcasing a very recent gene duplication event. Interestingly, the great majority of full-length VSPs (23/28) are clustered in the phylogeny, including the VSPs in the scrambled ψVSP arrays, despite these genes being distributed in physically separate chromosomal locations across all five primary scaffolds . The few non-clustered VSP genes in the phylogeny that are not part of pairs or clusters are found directly adjacent to ψVSPs genes. The relaxed clustering of VSPs and ψVSPs suggested that these genes might be undergoing periodical recombination or gene conversion.We constructed a phylogeny of all the VSPs and ψVSPs in The VSPs and ψVSPs showed distinct expression patterns. Essentially all the ψVSP genes were non-transcribed in the three surveyed life-stages . VSPs, on the other hand, showed on average higher expression with one or a few loci displaying dominant expression in the different life-stages .G. intestinalis, high cysteine membrane proteins (HCMPs) [G. muris genome were named HCMPs after the corresponding gene family in G. intestinalis. They are found spread-out on the five chromosomes  , with 10 (HCMPs) . Many ar (HCMPs) . The HCM (HCMPs) . The 34 omosomes .G. intestinalis outside the VSPs and HCMPs are the NEKs [G. muris . Among the NEK orthologues, we found that 65 % of them consists of NEKs that in G. intestinalis are predicted to be catalytically ‘dead’  [G. muris having the largest with 104 members and the one in G. intestinalis having 79 members. ARPs show a similar evolutionary stability with 132 conserved 1 : 1 orthologues between species . G. muris shows a major species-specific expansion of 91 genes whereas the largest expanded clusters in G. intestinalis amounts to two groups of 15 genes each. The partly shared domain-structure of NEKs and ARPs prompted us to investigate their relationship by a network analysis employing reciprocal blastp (1e-05 cutoff). The two groups are not recovered as clearly separated clusters but form partially overlapping networks . The larger numbers of genes in these multigene families in G. muris compared to G. intestinalis and their evolutionary dynamics are intriguing given the otherwise streamlined features of the G. muris genome, perhaps suggesting that they have unique roles in adaptation to their murine hosts.The largest multigene families in the NEKs  and ankythe NEKs . There aG. muris , Table 3stinalis . We clasfined in ). This pe (71 %)  indicatiG. intestinalis is not known to possess classical virulence factors, such as enterotoxins, but several genes are important for colonization of the host and thus for pathogenesis. These include genes for motility [G. muris is very similar to G. intestinalis apart from several fragmented alpha-tubulins . The adhesive disc is a unique cytoskeletal structure of Giardia parasites essential for attachment of the trophozoite in the small intestine, but is missing in other fornicates [G. muris trophozoites [G. intestinalis. The vast majority (82 of 85) of G. intestinalis disc proteins [G. muris (Table S1d); 12 were NEK kinases and 27 were ARP-1 proteins. Two of the three G. intestinalis disc proteins not found in G. muris (Table S1d) localize to a structure in the G. intestinalis disc on top of the ventral groove, but this structure is missing in the G. muris disc [G. intestinalis infections .motility , the adhmotility , secretemotility , and cysmotility  and the motility  that undrnicates . The firhozoites , but morproteins  were alsris disc , suggest-tubulin  are highGiardia virulence [G. muris genome, compared to 96 proteins identified in an identical search in G. intestinalis WB (Table S1e). The largest family of proteases in G. muris, with 15 members, are papain-like cysteine proteases (C1A family). This protein family is also the largest protease group in G. intestinalis with 21 members [Giardia and Spironucleus, although we also found evidence for lineage-specific gene loss and expansion in G. muris . The most highly expressed cysteine protease of G. muris in vivo is the closest homologue to the highest expressed protease in G. intestinalis WB [Proteases are important virulence factors in many pathogens and cysteine-protease activities have been suggested to play a role in irulence . We iden members . Severalnalis WB .G. muris compared to 98 pathways detailed in G. intestinalis WB; four pathways were unique in G. muris and eight in G. intestinalis WB. Even though the overall metabolism is highly similar between G. muris and G. intestinalis WB, the genes for several specific enzymes and their putative reactions show distinct differences. Thus, 18 unique reactions (14 enzymes) were predicted in G. muris and 25 in G. intestinalis WB (10 enzymes)  . SeveralG. muris compared to G. intestinalis. Fructokinase and mannose 6-phosphate isomerase enable G. muris to use fructose and mannose 6-phosphate unlike G. intestinalis. In both cases, the enzymes were acquired from bacteria via lateral gene transfer . Curiously, the G. muris gene for mannose 6-phosphate isomerase is found next to a bacterial transcriptional regulator/sugar kinase gene. Phylogenetic analyses show that G. muris mannose 6-phosphate isomerase and the transcriptional regulator/sugar kinase gene clusters deep in the Bacteroidetes group. This gene arrangement is observed in bacteria of the genus Alistipes, whose genomes harbour the most similar homologues, supporting the notion that a single event of lateral gene transfer best explains the origin of these genes in G. muris . Both genes have the A-rich initiator that precedes the start codon in most G. muris genes and are expressed in G. muris trophozoites (Table S1b).The potential utilization of carbohydrate sources for glycolysis is different in G. intestinalis upon depletion of primary carbon sources [Spironucleus and Trichomonas but has been lost in G. muris. Another notable metabolic difference to G. intestinalis WB is the lack of pyrophosphate-dependent pyruvate phosphate dikinase (PPDK) that leaves a single, less energy efficient, route from phosphoenol pyruvate to pyruvate via pyruvate kinase in G. muris.The utilization of glycerol for ATP synthesis via glycerol kinase has been suggested in  sources . This enG. muris is predicted to synthesize coenzyme A from pantothenate, employing a bifunctional phosphopantothenoyl decarboxylase-phosphopantothenate synthase. The same pathway is described in S. salmonicida [G. intestinalis  pathway that enables the use of arginine as an energy source and at the same time, reduces the available free arginine in the environment, preventing nitric oxide (NO) production in host cells [G. intestinalis trophozoites and the main scavenger enzyme for NO in G. intestinalis is Flavohemoprotein [G. muris  and NEKs (dark red arrows). The homologous regions on the three chromosomes likely originated via two duplication events.We noticed that several of the bacterial derived genes that are shared with Nucleotide substitutions have accumulated since the duplication events and in-frame stop codons  proteins are innate immune defense proteins that bind to lipopolysaccharide and display potent killing activity against Gram-negative bacteria by increasing membrane permeability. Beyond this basic function BPI proteins might also act as effectors in controlling mutualistic symbioses . Homologstinalis , but it G. muris encodes tryptophanase, an enzyme that metabolizes tryptophan to pyruvate with concomitant release of indole and ammonia. While pyruvate can be utilized in energy metabolism, indole and its metabolites have been shown to affect gut microbiota composition, possibly by interfering with quorum-sensing systems, and might be able to influence host health [G. intestinalis .t health . PhylogeG. muris: Tae4 and quorum-quenching N-acyl-homoserine lactonase. The Tae4 proteins are wide-spread amidases that were first described in association with the T6SS system effector Tae4 in Salmonella Typhimurium [d-glutamyl-mDAP (dl-bond) of Gram-negative bacteria [S. Typhimurium [G. muris, quorum-quenching N-acyl-homoserine lactonase was present in the common ancestor of G. muris and G. intestinalis and lost in G. intestinalis WB and P15 .Two more proteins with potential importance for microbiota interactions are encoded in himurium . The Taebacteria , and is himurium . Quorum-himurium . Our phyG. muris has an even more compact genome than G. intestinalis, whose genome is already known to be highly streamlined [G. muris appears to fall into this category as it encodes no known classes of mobile elements and repetitive elements are mostly confined to telomeric contexts. The shortness of intergenic regions in G. muris ranks among the most extreme recorded for any eukaryote, even shorter than Microsporidia, which are known as the most compact and reduced eukaryotic genomes [G. muris to G. intestinalis indicates many frequent small-scale genome rearrangements that often favours a more efficient gene packing in G. muris, thus allowing shorter intergenic regions. This evidence of gene shuffling and the fact that there is very little evidence of genome degradation would argue for optimization of growth as the driving force of G. muris genome streamlining.Our data shows that eamlined . Genome eamlined . G. muri genomes . The gloG. muris trophozoites have not been grown axenically in vitro, which has hampered exploration of its genome, gene regulation and metabolism [G. muris as an in vitro model system for the human parasite G. intestinalis and other intestinal protozoan parasites [G. muris and G. intestinalis that might indicate avenues to successful strain axenization. Most of these differences are represented by instances of lateral gene transfer of metabolic genes or losses thereof in either G. intestinalis or G. muris. The lack of metabolic genes such as glycerol kinase and PPDK might shift the relative emphasis of metabolic pathways compared to G. intestinalis. The possible utilization of additional carbohydrates (such as mannose and fructose) and differences in amino acid utilization (via tryptophanase and arginase) and their impact on growth in vitro are interesting avenues to investigate in any future axenization efforts.tabolism , and hasarasites . We idenG. intestinalis isolates are typically poor at infecting mice. Despite this, the assemblage B isolate GS, which shares more metabolic enzymes with G. muris than the assemblage A isolate WB, is better able to establish infection in mice than WB. This suggests that the shared metabolic capacity of G. muris and G. intestinalis GS enables survival in the murine intestinal tract. Additionally, G. muris might be able to interact or interfere with intestinal Gram-positive and Gram-negative bacteria and this could be a key to establish successful infections. G. intestinalis, which lacks some of the putative microbiome modulators, such as Tryptophanase and Tae4, is dependent on reduction of the small intestinal microbiota in order to efficiently infect mice [G. muris is cleared from the murine host by secretory IgA [G. intestinalis GS [G. muris is more resistant to elimination by innate factors such as competition with the normal microbiota, or host production of reactive oxygen species and/or NO, whereas GS is more sensitive to innate factors and eliminated much faster within 1–3 weeks (while G. muris clearance requires 4–8 weeks). Future insights into the importance of innate factors in G. muris infection should be facilitated by the availability of the complete genome sequence.ect mice . G. muritory IgA , whereasnalis GS . We specG. muris that allows genetic drift and recombination to drive rapid and stealthy diversification, thus avoiding elimination by adaptive immune defenses. It was previously reported that G. muris is capable of antigenic variation and encodes VSP genes with high similarity and conserved structural features (CRGKA pentapeptide) to those G. intestinalis [G. muris VSPs in our G. muris genome. Instead, these VSP genes show much greater similarity and in one case almost absolute identity to G. intestinalis VSPs of assemblage B. Importantly, our finding that G. muris VSPs have distinctive structural features (GCRGK cytoplasmic tail) not reported in G. intestinalis VSPs to date indicates that the previously reported G. muris VSPs are likely due to contamination from a G. intestinalis assemblage B isolate.Sub-telomeric regions in parasitic protozoa often contain arrays of expanded gene families that are under positive selection by the immune system . The relstinalis . We failG. muris have previously been described in G. intestinalis [G. muris VSPs and ψVSPs revealed evidence of recombination or segmental gene conversion, as previously demonstrated in G. intestinalis [G. muris and G. intestinalis. First, G. muris encodes a low number of intact VSP loci that are located internally on the chromosomes. Second, the ψVSP arrays are almost exclusively telomere adjacent, as opposed to G. intestinalis where this tendency is not apparent [G. muris VSP repertoire resemble the antigenic variation systems of Pneumocystis spp. and Trypanosoma brucei [The linear ψVSP arrays in stinalis . Our phystinalis . Howeverapparent . These aa brucei . DespiteGiardia genomes [Giardia NEK kinases lack transmembrane domains and have been suggested to target and localize to different intracellular structures with their ankyrin repeats [G. intestinalis NEK kinases localize to cytoskeletal structures, including the flagella and adhesive disc [G. muris genome are frequently associated with these large gene families  and displays maintained gene order to the one seen in the closest related bacterial lineages . Interestingly, several lateral gene transfers were found clustered in amplified areas of the G. muris chromosome. Curiously, G. intestinalis assemblage B also maintains clustered copies of arginase and 2,5-diketo-d-gluconic acid reductase, while these genes have both been lost in the G. intestinalis assemblage A and E lineages.Lateral gene transfer is an important shaping factor in the evolution of metabolism in protists . The orirvations . Most ofG. muris infected mice [G. intestinalis have been shown to be able to degrade defensins [G. muris. The protease with the highest expression is suggested to have a role in encystation and excystation [G. intestinalis homologue is up-regulated and secreted during interactions with human intestinal epithelial cells [G. muris.Anti-microbial peptides of several classes, such as defensins and trefoil-factor 3, are up-regulated in the small intestine of ted mice . Secreteefensins . We deteystation . Its G. al cells  and it cal cells . Thus, tGiardia’s virulence traits in Giardia’s ability to infect the intestine of mammals are pre-parasitic inventions  were collected from small intestines of three C57 mice, washed once in PBS and pellet frozen at −80 °C (Biosample SAMN11231832). Viable cysts of G. muris isolate Roberts–Thomson passaged through mice were obtained from Waterborne. These cysts had been purified from faecal material using Percoll and sucrose gradients (Biosample SAMN11231833). DNA and RNA were extracted from 1×107 cysts using standard methods.In total, 4.5×108 cysts were excysted according to the procedure in Feely et al. [7 cysts. DNA for long-read sequencing was prepared from the remaining cysts as described in Methods S1.Then, 1×10y et al.   pipeline.PacBio long reads were assembled blastn  of the contigs against the NT database revealed contamination, mostly from fungi, and the contaminated contigs were removed from the final assembly, resulting in 59 final contigs.Reads were assembled with HGAP followed by consensus sequence calling with Quiver. The resulting assembly contained 317 contigs. A http://bioinformatics.net.au/software.nesoni.shtml) was used to correct mostly the indels that we have observed to cause frame-shifts in certain genes. Overall, 8 deletion, 46 insertion and 16 SNPs were corrected by Nesoni with setting –majority 0.75 based on the mapped bam file.The Illumina MiSeq reads were mapped to the PacBio assembly using BWA v0.7.12-r1039  and Nesoblastp results against NR database as well as HMMER (v3.0) search results of domain information against Pfam (v27.0). All genes were then manually examined, with RNA-Seq reads mapped   and Glimsing BWA ) as a guA detailed description of genome sequencing, annotation, protein families and synteny analyses is available in Methods S1. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number PRJNA524057. The version described in this paper is version VDLU00000000.1.BAM files were generated from mapping the RNA-Seq reads to the reference genome using BWA (v0.7.12) . CufflinG. muris and G. intestinalis WB were predicted with a combination of BlastKOALA [The metabolic pathways of astKOALA  implemenastKOALA , PathwayastKOALA  and GiarastKOALA . The difastKOALA . PathwayastKOALA  was usedastKOALA  was usedastKOALA .G. muris sequences were used as queries to retrieve at least 5000 hits with e-value <0.001, using blastp against the nr database and the organism-specific proteomes. The datasets were aligned in the forward and reverse orientation using MAFFT v6.603b [ v6.603b  and PROB v6.603b . The fou v6.603b  and trim v6.603b . Maximum v6.603b  under LG v6.603b . Branch  v6.603b  with 100 v6.603b . A detaiClick here for additional data file.Click here for additional data file.


Influenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is “fit” and able to overcome vaccine pressure. Influenza viruses are negative-sense segmented RNA viruses responsible for highly infectious respiratory disease in humans and variable pathology in important hosts such as aquatic birds and poultry, ranging from asymptomatic to fatal . The infAntigenic drift is the accumulation of mutations in HA that enables evasion of pre-existing anti-HA antibodies induced by natural infection or vaccination. Influenza viruses have at their disposal several mechanisms for driving antigenic drift. The simplest mechanism involves an amino acid substitution that directly changes the biophysical property of a cognate epitope e.g. its shape or charge, which leads directly to diminished antibody binding . A seconFor a single mutation to significantly alter antigenicity it must impose a radical change to the biophysical property of a virus; single mutations typically do this by introducing/removing an N-linked glycosylation motif, or by altering receptor binding avidity . Alone, Previously, we characterized the impact of an adsorptive mutation in H9HA which impacted antigenicity, receptor binding and viral fitness . Briefly2 without the addition of antibiotics. Virus was grown in 10-day old specific-pathogen-free (SPF) embryonated hens’ eggs and harvested at 48 h post-inoculation.Chicken Kidney (CK) cells were prepared at the Pirbright institute three days prior to use in virus replication assays in 6-well plates as previously described  and mainReverse-genetics (RG) viruses were generated from co-transfected bidirectional plasmids (pHW2000 or pCAGGs) as described previously ,18. ViruTM WX90 Ultracentrifuge. Concentrated virus was then homogenized using a glass homogenizer then spun at 1700 × g at 4°C for 5 min using a low-speed centrifuge. Virus supernatant was purified using a density gradient in 30–60% continuous sucrose at 135,000 × g at 4°C for 2 h. A virus-containing fraction within the density gradient was recovered, re-suspended in PBS and pelleted again at 135,000 × g at 4°C for 2 h using the ultracentrifuge. Purified virus was re-suspended in PBS with 0.01% Sodium Azide, aliquoted and stored at 4°C for subsequent use or at −80°C for future use. The concentration of purified virus was determined by enzyme-linked immunosorbent assay (ELISA) by targeting the influenza nucleoprotein (NP) using anti-NP monoclonal antibodies. ELISA assays of purified viruses studied here were conducted alongside ELISA assays of a reference virus of known concentration that was subsequently used to make a standard curve. Concentration of virus was expressed in picomolar (pM) for octet use [Virus from allantoic fluid was concentrated and purified for use in biolayer interferometry, western blot and vaccine preparation. The purification of a single virus utilized 450 ml of infectious allantoic fluid from embryonated hens’ eggs. Clarified allantoic fluid was concentrated by ultracentrifugation at 135,000 × g at 4°C for a total of 4 h (2 separate runs of 2 h each) using a Sorvallctet use .DK values [The receptor binding phenotype of viruses was determined by biolayer interferometry using an Octet RED biolayer interferometer  as previously described . PurifieD values ,21.In vitro virus replication kinetics were studied in MDCK and CK cells with virus at a MOI of 0.001 and 0.01, respectively. Virus was inoculated in triplicate for 1 h prior to washing with PBS and overlaying with virus growth medium -treated trypsin) for MDCK cells and CK media for CK cells . Culture supernatants were taken at 12, 24, 48 and 72 h post-infection and titrated by plaque assay in MDCK cells [CK cells .The hemagglutination assay was first used to determine virus titre and then virus was diluted to 4 HA units and used in haemagglutination inhibition (HI) assays as described previously . Assays TM (7.5%) precast protein gels (Biorad) were used to perform SDS-PAGE gel electrophoresis. Samples were prepared by denaturing at 95°C for 10 min (non-PNGaseF treated virus only) in protein loading buffer, separated by gel electrophoresis at 100 volts, then transferred to a nitrocellulose membrane soaked in Transfer buffer  for subsequent western blot using a Trans-Blot Turbo Transfer System (Biorad). After protein had transferred from the precast gel to the nitrocellulose membrane, the membrane was blocked with Blocking buffer (PBS with 0.05% Tween 20 and 5% Marvel milk powder) at 4°C for 24 h then probed with primary antibodies (anti-H9N2 (UDL-01/08) mouse monoclonal antibodies; produced in-house) diluted in antibody dilution buffer (PBS-Tween and 2% Marvel milk powder). The membrane was washed 3 times with PBS-Tween  then probed with secondary antibody (LI-COR Biosciences) diluted in antibody dilution buffer. The membrane was again washed three times with PBS-T before protein was visualized using an Odyssey CLX system (LI-COR Biosciences). A prestained SDS-PAGE protein standard was run in parallel to protein samples.SDS-PAGE and western blot were performed using purified virus to investigate band-shifting as a result of N-linked glycosylation. Mini-PROTEAN® TGXin ovo after the introduction of an N-linked glycosylation site at residues 134, 148 and 189, concentrated virus was deglycosylated using PNGase F (NewEngland Biolabs), according to manufacturer’s instructions. Briefly, 30 µg of virus was combined with 4 µl of Glycoprotein Denaturing Buffer (10x) in a total reaction volume of 40 µl and denatured at 100°C for 10 min, then chilled on ice for 2 min. Denatured virus was mixed with 4 µl of Glycobuffer 2 (10x), 4 µl of 10% NP-40 and 2 µl of PNGase F enzyme and incubated at 37°C for 18 h. Deglycosylated virus was mixed with protein loading buffer and loaded directly into a gel for SDS-PAGE and western blot.To confirm viruses were being glycosylated Chicken polyclonal antisera raised against UDL-01/08 virus was generated previously . ChickenThe use of animals to produce polyclonal antisera was conducted under the guidance and regulations of European and United Kingdom Home Office regulations under project license number P68D44CF. The ethics committee at The Pirbright Institute has approved and reviewed all work involving animals.in vitro and in vivo models  was substituted for asparagine (N). Interestingly, this substitution introduced an N-linked glycosylation motif to the HA protein. As it has been established that glycosylation can compensate for antibody escape fitness costs in other viruses, we therefore wanted to investigate the role of N-linked glycosylation in a H9N2 virus with various adsorptive mutations. To this end we expanded our study to include two additional residues prone to mutations that introduce an N-linked glycosylation motif to the HA protein and which impact virus replication in various To determine the impact of N-linked glycans on receptor-binding in three different avidity backgrounds  we conducted biolayer interferometry. In the low avidity SKP-827/16 T180A virus background, N134 had a negligible effect on binding to 3SLN(6Su), while N148 and N189 both reduced binding avidity towards 3SLN(6Su)  could only poorly inhibit the haemagglutination ability of SKP-827/16 T180 virus carrying an N-linked glycan at residue 148. This suggests N-linked glycosylation at residue 148 trumps avidity in this case. Furthermore, given the improved replication kinetics of this virus relative to non-glycosylated T180 virus it is possible that such a virus would persist – it can evade immune pressure even from high avidity antisera while retaining fitness.vice versa. Importantly, a virus containing T180 and a glycan at residue 134 or 189 still has the capacity to bind human-like receptors. Thus, we have a virus that escaped immunity, recovered viral fitness after a period of attenuation and then enhanced its capacity for zoonotic infection. Taken together these data highlight how viral fitness is a fine balance to be maintained and tuned by choice mutations, which can often be pleomorphic. To conclude, avidity and glycosylation can act together to allow efficient immune escape whilst also potentially changing receptor preference and promoting viral fitness.The use of mutations to modulate avidity or introduce an N-linked glycan are interchangeable during immune escape and compensatory mutation. More specifically, an N-linked glycan can be used to escape immunity, or it can be used to achieve viral fitness, likewise with avidity mutations. In the data presented here, it is plausible that an H9N2 virus could escape immunity through addition of an N-linked glycan at residues 134, 148 or 189 and subsequently achieve viral fitness with a mutation that modulated avidity such as with substitutions A180 T/V, or Click here for additional data file.Click here for additional data file.


High throughput sequencing technologies accelerated the pace of discovery and identification of new viral species. Nevertheless, biological characterization of a new virus is a complex and long process, which can hardly follow the current pace of virus discovery. This review has analyzed 78 publications of new viruses and viroids discovered from 32 fruit tree species since 2011. The scientific biological information useful for a pest risk assessment and published together with the discovery of a new fruit tree virus or viroid has been analyzed. In addition, the 933 publications citing at least one of these original publications were reviewed, focusing on the biology-related information provided. In the original publications, the scientific information provided was the development of a detection test (94%), whole-genome sequence including UTRs (92%), local and large-scale epidemiological surveys (68%), infectivity and indicators experiments (50%), association with symptoms (25%), host range infection (23%), and natural vector identification (8%). The publication of a new virus is cited 2.8 times per year on average. Only 18% of the citations reported information on the biology or geographical repartition of the new viruses. These citing publications improved the new virus characterization by identifying the virus in a new country or continent, determining a new host, developing a new diagnostic test, studying genome or gene diversity, or by studying the transmission. Based on the gathered scientific information on the virus biology, the fulfillment of a recently proposed framework has been evaluated. A baseline prioritization approach for publishing a new plant virus is proposed for proper assessment of the potential risks caused by a newly identified fruit tree virus. Potyvirus) causes “sharka” disease, the most devastating viral disease of stone fruit trees worldwide, which causes severe damages, estimating a total cost at 10 billion Euros worldwide in 30 years (Closterovirus) is probably the most economically important virus infecting citrus, causing a decline of sour orange rootstock, yellow seedling of lemon and grapefruit, and stem pitting in grapefruit and sweet orange  of viral disease symptoms could be challenging, due to the low titer of several viruses, their heterogeneous distribution between tissues or over time within the tree, the frequent mixed infections, the absence of universal primers for the detection of all viruses, the occurrence of symptomless infection, and the impact of the cultivar on the symptom development . AnotherThe advent of high throughput sequencing (HTS) technologies during the last decade has dramatically changed research on viral and virus-like agents. HTS technologies are a potential universal screening method for plant virus detection, allowing for the theoretical detection and identification of any known or unknown agents. Until recently, there were many fruit tree diseases with unknown etiology, although a viral origin was suspected. Nevertheless, the development of HTS technologies has drastically changed the situation . In factThe pace of virus discovery since the advent of HTS technologies also raises an important question: How do virologists address the biological characterization of new viruses with the genome sequence information as a starting point? Indeed, these new viruses identified by HTS technologies are often lacking information on their biology and the risk they can pose on fruit production. This is an important concern for carrying out a proper risk assessment. For example, a panel of experts highlighted in a recent report that for several viruses, especially those recently discovered, the pest categorization based on pest risk analysis is associated with high uncertainties due to the absence of data on biology and distribution . BiologiA decade after the first discovery of a phytovirus by HTS technologies , it is nAfter the original publication reporting the discovery of a new fruit tree virus, the high pace of virus discoveries also raises another question: Once a new virus is discovered and published, does it trigger additional experiments or surveys by the virologists to complete its biological characterization? Indeed, resources are limited for plant virologists, and the current abundance of newly identified viruses might limit their downstream biological characterization. This publication also evaluated how a recently proposed framework for the evaluation of biosecurity, commercial, regulatory, and scientific impacts of plant viruses and viroids identified by HTS technologies is fulfilled .The objectives of this publication were, therefore, to critically review and perform and in depth analysis of (1) the scientific information on the virus biology that was published together with the genome sequence when a fruit tree virus was discovered, and (2) the content of the scientific publications citing the original publication reporting the first publication of a fruit tree virus. We focus our analysis on the newly discovered viruses since 2011 and from the major fruit tree species worldwide. The gathered information was categorized, and general conclusions are provided. Overall, this analysis contributes to the establishment of a baseline and prioritization of complementary experiments to be done once a new fruit tree virus is discovered in the near future.Thirty-two fruit tree species which listed in identified host species in All the publications reporting the discovery of a new virus or viroid species from January 1, 2011 to April 1, 2020 were screened. The information provided for the characterization of the new viral species has been classified into 13 categories:–Complete genome: whole viral genome has been sequenced, including the UTR region at the 3′- and 5′-terminus.–Primer design: virus-specific oligonucleotide primers and (RT)-PCR protocol were designed for RT-PCR detection and are described in the publication or the –Genome diversity: several complete or near-complete genome sequences from different isolates were published and compared.–Gene diversity: several partial or complete gene sequences (like the polymerase or the coat protein) were sequenced and aligned.–Local survey: after the discovery, samples were collected in the same location  to evaluate the prevalence of the virus.–Large scale survey: after the discovery, samples were collected from different locations, producing regions in one country or different countries.–Association with symptoms: whatever the scale of the survey, the sampling was carried out on symptomatic and asymptomatic trees to evaluate the association between the virus presence and symptoms.–Co-infection with other viruses: samples infected by a new virus are also infected by other known viruses which have been checked by RT-PCR.–Infectivity bioassays: inoculation of plants using an infectious clone or graft inoculation to a host from the same cultivar or different cultivars of fruit tree species.–Indicators: transmission to several herbaceous indicators were attempted, even if not successful.–Symptomatology: symptoms have been observed on the grafted plants in a greenhouse experiment or grafted plants, including host plants and indicators, which have been mechanically inoculated with sap from symptomatic samples or transfers by a natural vector.–Transmission: at least one natural vector  has been identified. It might have been used for transmission assays.–Host range: the virus has been detected on at least another plant species during a survey or successfully inoculated to another plant species (not an indicator).The peer-review publications citing one of the publications describing a newly identified fruit tree virus were also reviewed and analyzed in depth. They were retrieved based on the Scopus citations. First, the citation of each publication reporting the discovery of a new fruit tree virus was analyzed as follows: the total number of citations and number of citations per year from the initial publication. The information provided in the citing publication was classified into 18 categories in Citrus sp., 8 viruses for Ribes sp., 6 viruses for Rubus sp., 17 viruses and one viroid for other minor fruit trees. The full name and abbreviations of all these new virus species is listed in A total of 78 scientific peer-review publications describing the discovery of a new virus or viroid species in the studied fruit tree species were identified between January 1, 2011 to April 1, 2020. These publications reported 81 new virus and three new viroid species among which 13 viruses and 2 viroids for pome fruit species, 22 viruses for stone fruit species, 14 viruses for For the most identified fruit tree species, 15 viruses from apple trees and thirteen viruses from sweet orange, at the opposite only 2 from pear, nectarine and lemon trees while a single virus from pear, Japanese pear, David’s peach, Japanese apricot, plum, raspberry and, American blackcurrant. 13 viruses  identified from more than one host species in the first publications.The scientific information provided in the original publication describing a new virus is highly variable. The minimal information corresponded to a recent publication of HTS sequences gathered into contigs without any biology confirmation of the results . On the Our analysis showed that almost all of the discovered genomes . The primers were only used to confirm the virus presence in the original sample or experimentally inoculated plants for 32 new viruses. For example, the detection primers designed for CJLV and CVLV had only been used to confirm the presence of the viruses identified in the RNA-seq and sRNA libraries . The PCRMore than a single genome was sequenced for nine viruses (11%). In this case, the genome diversity was always analyzed. A maximum of 45 complete viral genomes of TFDaV were sequenced, including 17 from apple, 26 from pear and two from grapevine . On the The diversity has been analyzed at genes or partial gene levels for 15 (18%) new viruses. The comparison was based on partial genome sequences obtained by classical Sanger sequencing. The sequenced fragment ranged from a partial gene to several genes. For apricot vein clearing-associated virus (AVCaV), PCR amplicons of 330 bp from the replicase gene were obtained from two samples . In contPrunus sp.  new species. The number of tested samples were also variable, ranging from four to more than 200. For example, four sweet cherry trees maintained at the Yamagata horticultural experiment station were evaluated to the study ChVB . On the unus sp. . Tested Prunus sp. trees from China, Japan, Czech Republic, Azerbaijan, Kazakhstan, Italy, and France were screened  new species. The extent of the survey was highly variable, ranging from tens to hundreds of samples. For example, in a publication with cherry virus F (CVF), a small survey on nine cherry trees form four locations in different countries was carried out . Moreovescreened . 80 peacscreened . In totaThe sampling was carried out on symptomatic and asymptomatic trees to evaluate the association between the virus presence and symptoms in 56 (67%) species, including 15 at a local scale, 10 both at a local and large scale, and 31 at a large scale. Depending on the survey results, a high association with symptoms was found for 21 (25%) virus species: AGCaV, ApNMV, ARWV-1, ARWV-2, ARWaV-1, ARWaV-2, CCGaV, CYVCV, CiLV-C2, IrCRSaV, CiLV-N, CiYMaV, RLBV, BVBaV, BLMaV, BNRBV, BlMaV, BFDaV, MMDaV, AcCRaV, and AcEV-2. A possible latent infection had been concluded for eight (10%) species: AGV, AaLV, PrGVA, BBLV, BLSV, BVA, PeCV, and ASbLV. An association between symptoms and virus infection were confused in 27 (32%) species.The association with symptoms in 25% species based on much higher incidence in symptomatic plants than in asymptomatic plants, regardless of the scale of the survey. For example, the incidence of ApNMV was 83% in symptomatic samples and 37% in asymptomatic samples from 359 samples in a survey of mosaic-diseased apple trees from major apple-producing provinces in China . CYVCV wLatent infection was found in 10% species, and this was based on most of the positive samples that were symptomless. For example, PeCV had a high incidence in symptomless plants, while the original symptoms (vein necrosis) could not be linked to its presence . BLSV waThe association between symptoms and virus infection remained unclear in 32% species after a large-scale survey due to several reasons. The low incidence (<1%) or absence of virus  in the survey impeded any association based on sufficient number of samples. Another limitation of the association is the presence of other viruses in the infected trees. This phenomenon was reported for 12 species  and underlined the need to complete the survey on the newly identified virus with other known viruses. Symptom variability can also hamper the establishment of an association in eight species .The presence of co-infecting viruses was reported and confirmed by RT-PCR for 28 (34%) viral species. Among them, a prevalence survey of four  was carried out surveying for other co-infecting viruses. Another 24 species mentioned co-infection in the original sequenced plant but did not survey for another co-infecting virus or report the names of the co-infecting virus species.Geminiviridae and AHVd belonging to Pelamoviroid. Growth reduction was observed in apple and pear plants following biolistic inoculation with the cloned TFDaV (Infectivity bioassays in the correspondent hosts were carried out for 30 (36%) viral species. All of the bioassays resulted in positive RT-PCR detection, and 12 reported observed symptoms. Infectious clones were constructed for TFDaV, consisting of circular single-stranded DNA of family ed TFDaV . But, noed TFDaV . CiLV-C2ed TFDaV . Three ped TFDaV . Eight pSpeI-linearized and recircularized genome onto Nicotiana benthamiana (Transmission to herbaceous indicators have been attempted for 21 viruses (25%). More than one herbaceous plant species was inoculated for 13 species. Ten transmissions allowed the observation of symptoms. However, nine failed to inoculate . Two species (AGV and BVF) had successful infections, although none of the infected plants displayed visible symptoms. Among the 10 species with observed symptoms, CiLV-N and CiCSV were transmitted by mites while eight  were mechanically transmitted. Among the nine failed experiments, only TFDaV was biolistically-inoculated with the thamiana . The othSymptoms have been observed for 16 (19%) viral species. These including symptoms observed on both grafted host plants and herbaceous indicators in six , only observed on experimental host plants in six , and only on herbaceous indicators plants in four .More than one host species within the trees studied was identified for 19 (23%) viruses. 13  were naturally infected in different host species identified, and six  were successfully inoculated to another plant species in infectivity bioassays. Three species  that were identified in different host species naturally were also infectable while inoculated on another plant species . Only TFThe possible natural transmission mode and vectors were investigated for 7 (8%) viral species. Among them, potential natural vectors have been identified for five species . Four of them successfully transmitted from one host plant to another through a natural vector (mite or aphid) while one (BVA) failed with cotton aphids . Only BBSeventeen viruses (20%), including PpPV2, ApRVA, PeVD, PcVT, PCLSV, PYSaV, CJLV, CVLV, CTNGmV-1, CTNGmV-2, BVE, CuLV, CuVA, RAVA, BGMaV, PeLV, and AcV-1 lacked information regarding the biological characterization of epidemiological survey, infectivity or indicator assays, symptomatology, transmission, and host range. However, they did have genetic annotations or information regarding detection confirmation when first published.The 78 original publications of a new fruit tree virus have been cited 933 times , represeThe percentages  for eachThe other nine categories, which were not related to the biological or geographical characterization of a new virus, corresponded to 82% of all citations. Nearly half of the citations corresponded to reviews (22.9%) or the discovery of another virus (24.3%). In addition, 23% were related to the study of a known virus. The other categories represented less than 5%. Most of the citing publications were, therefore, not related to virus biology and repartition. Moreover, they did not provide useful information to better evaluate the phytosanitary risks posed by the new viruses.The detection of the new virus in another country on the same continent was reported for six species: ApNMV, CYVCV, CCDaV, CRMaV, RLBV, and PeCV. ApNMV was identified in Japan and reported from China and Korea . CYVCV wA new virus was detected in another continent for 14 species: AGCaV, AHVd, ARWV-1, ALV-1, NSPaV, CVF, ChALV, PrVF, PaLV, CVEV, BlMaV, PeVA, MBV1, and AcV-1. AGCaV, identified from Australia and Canada, was reported in Korea . AHVd haCydonia oblonga; Malus.spp; P. salicina, P. mume, P. domestica, and P. persica; Prunus species , was reported in a new natural host, sour cherry  when reported in a new country or host. ApNMV, which was first correlated with the apple mosaic symptoms in Japan, was also reported at a high incidence in apple trees with mosaic symptoms in China and Korea . AGCaV, Six publications focused on transmission related to four species: CYVCV, CiLV-C2, CiLV-N, and BNRBV. Four citing publications identified the transmission vector of CYVCV . One pubEight publications focused on genome diversity related to six species: AHVd, CYVCV, BCLCaV, BlMaV, MBV1, and AcV-1. Two publications analyzed the genetic diversity of genome sequences from variants of AHVd . GeneticSixteen publications focused on the diagnostic method for 14 species: AGCaV, NSPaV, PaLV, PeVD, CCDaV, CYVCV, CiLV-C2, CVEV, BE, BVBaV, BBLV, BLSV, BNRBV, and BlMaV. Real-time RT-PCR assays were developed for two viruses (ASGV and AGCaV) infecting pome fruit . MultiplSix publications focused on epidemiological surveys related to nine species: ApNMV, CCDaV, CYVCV, BVE, BLMaV, BBLV, BlMaV, BFDaV, and AcCRaV. A large scope epidemiological survey in China demonstrated that ApNMV was highly associated with mosaic disease in apple trees . One pubFive publications focused on co-infection synergisms between different virus or viroid species. The citing number was larger than the number of publications, while simultaneously citing several new species. For example, one publication included five new viruses in their analysis of co-infection patterns in peach  satisfied the criteria to be considered as Union quarantine pests. With the exception of the impact in the EU territory, on which the Panel was unable to conclude, 11 species  satisfied the other criteria to be considered as potential Union quarantine pests. AGCaV satisfied the criteria to be considered as Union quarantine pests with the possible exception of being absent from the EU territory or having a restricted presence and being under official control. PrGVA met the criterion of having a negative impact in the EU. For those recently discovered, the categorization is associated with high uncertainties, mainly because of the absence of data on their biology, distribution, and impact .The information provided in the first publication of a fruit tree virus and the citing publications was compared to a recently proposed framework for the efficient characterization of new phytoviruses . This frCaulimoviridae family. Even in the absence of possible integration, the presence of virions was observed by electron microscopy for 11 species, representing 13% of the publications.As shown in Regarding the second step, nearly all the original publications already sequenced the full genome (92%) and designed a diagnostic test (94%) for the new viral species. Meanwhile, only 30% performed a local epidemiological survey. This low percentage is nevertheless compensated by the fact that half of the publications completed a large-scale survey, among which 37% were only at large scale. This means that an epidemiological survey was carried out in 67% of the first publications. So, the rate of completion of this second step is also very high with the first publication.For the third step, half of the publications (42) analyzed the symptoms or host range. An analysis of symptoms was carried out for 30 species. This includes the evaluation of symptom association during the epidemiological survey for 21 species (25%); virus incidence in symptomatic and asymptomatic plants) and the observation of symptoms on inoculated plants for 16 species (19%). For eight species, both analyses were carried out. The host range was evaluated for 19 species (23%). The host range was completed by symptom analysis for seven species. The transmission mode was studied for 35 viruses (42%). It was carried out either successfully with indicator plants , host plant candidates, or other cultivars of the same species  by mechanical transmission, grafting, or possible vectors (for six species). For eight species, both indicator plants and crop species were inoculated. The seed transmission was only studied for one species (BBLV). As expected, the completion rate of the third step of the framework is, therefore, significantly lower than the two first steps.In addition, new progress was made regarding global epidemiology with the report of a new country or continent for nine viruses (11%): AcV-1, PeCV, PeVA, CVEV, CYVCV, CCDaV, PaLV, CRMaV, and NSPaV; which only tested samples collected from the discovery location in the first publications.A new host was reported for eight species, and only four of them (5%), AGCaV, ApNMV, AVCaV, and NSPaV, did not have their host range tested in the first publications. The other four had different host species identified when first published. New progress of transmission and vectors had been obtained for four species. Two of them (2%), CYVCV and BNRBV, were not included in any transmission experiments when first published. The other two, CiLV-C2 and CiLV-N, had identified possible natural vectors in the first publications. No new species progressed with symptomatology in the citing publications when compared to the first publications.This large-scale, retrospective analysis of the biological information provided by the publications reporting the discovery of new fruit tree viruses and their citing publications highlights important trends in the characterization of these new viruses with the publication of the genome sequence.First, various categories of information are always or nearly always provided: full or nearly full genome, confirmation of detection by an independent technique, and the design of diagnostics primers. Electron microscopy has been carried out in 13% of the publications. Even if this information was scientifically sound and demonstrated the presence of a viral particle, it provided limited information on the biology of the virus and the associated risks for plant health. An epidemiological survey is carried out in more than two-thirds of the publications. However, its extent was highly variable in geographical range, from only the same orchard to several continents and the number of analyzed trees, from a few to hundreds.Further biological characterization experiments  were only selectively carried out in the publications. For 20% of the discovered species, there was no information at all on the biological characterization, and the publications focused on genetic information. Biological characterization requires more resources and a longer time than genetic characterization. The association of the new species with symptoms might be limited by the low prevalence of symptomatic plants in a survey or the difficulty in reproducing symptoms experimentally. Symptom variability due to cultivar or environmental effects is also a bottleneck to associating a new virus with a disease. In addition, it can be further complicated by the presence of co-infections with other viruses in the orchards.Therefore, there is a trade-off between quickly publishing partial information and building a robust characterization of a newly identified virus before publishing. Journals have very different policies on this subject. For example, a recent publication presenting only the contigs generated without any biological confirmation has been recently accepted , while oRubus spp. was restricted to a single province in Turkey  can be shared between partners. For example, HTS data sharing allowed for a better understanding of the geographical spread of a new closterovirus . The preOur analysis also showed that, in general, the citing publications provided very little additional information on the biological characterization of a newly discovered virus, except for enlarging the geographical spread (11%) or host range (5%). The biological characterization of a newly discovered virus is therefore rarely pursued, which reinforces the need to provide as much information as possible when publishing a new virus, even though the biological characterization experiments can be time-consuming and could delay the publication of the results.Our analysis also allowed a preliminary evaluation of a published framework for the biological characterization of new plant viruses  with theAll the authors significantly contributed to the writing and editing of the manuscript. WH drafted the manuscript and created the figures. SM and SL coordinated the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


O’Callaghan  Cognitive decline is a common feature of Parkinson’s disease, and many of these cognitive deficits fail to respond to dopaminergic therapy. Therefore, targeting other neuromodulatory systems represents an important therapeutic strategy. Among these, the locus coeruleus-noradrenaline system has been extensively implicated in response inhibition deficits. Restoring noradrenaline levels using the noradrenergic reuptake inhibitor atomoxetine can improve response inhibition in some patients with Parkinson’s disease, but there is considerable heterogeneity in treatment response. Accurately predicting the patients who would benefit from therapies targeting this neurotransmitter system remains a critical goal, in order to design the necessary clinical trials with stratified patient selection to establish the therapeutic potential of atomoxetine. Here, we test the hypothesis that integrity of the noradrenergic locus coeruleus explains the variation in improvement of response inhibition following atomoxetine. In a double-blind placebo-controlled randomized crossover design, 19 patients with Parkinson’s disease completed an acute psychopharmacological challenge with 40 mg of oral atomoxetine or placebo. A stop-signal task was used to measure response inhibition, with stop-signal reaction times obtained through hierarchical Bayesian estimation of an ex-Gaussian race model. Twenty-six control subjects completed the same task without undergoing the drug manipulation. In a separate session, patients and controls underwent ultra-high field 7 T imaging of the locus coeruleus using a neuromelanin-sensitive magnetization transfer sequence. The principal result was that atomoxetine improved stop-signal reaction times in those patients with lower locus coeruleus integrity. This was in the context of a general impairment in response inhibition, as patients on placebo had longer stop-signal reaction times compared to controls. We also found that the caudal portion of the locus coeruleus showed the largest neuromelanin signal decrease in the patients compared to controls. Our results highlight a link between the integrity of the noradrenergic locus coeruleus and response inhibition in patients with Parkinson’s disease. Furthermore, they demonstrate the importance of baseline noradrenergic state in determining the response to atomoxetine. We suggest that locus coeruleus neuromelanin imaging offers a marker of noradrenergic capacity that could be used to stratify patients in trials of noradrenergic therapy and to ultimately inform personalized treatment approaches. Cognitive decline in Parkinson’s disease remains an ongoing therapeutic challenge. The mainstay dopaminergic therapies often fail to improve cognitive deficits, and in some cases can exacerbate them.,,Response inhibition deficits are a well-documented feature of Parkinson’s disease, ranging from subclinical impairments to extreme impulsive behaviours.,,The locus coeruleus-noradrenaline system modulates the stimulus detection and behavioural reorienting required for rapid action cancellation.,,,,Atomoxetine selectively inhibits presynaptic noradrenaline transporters, resulting in a 3-fold increase in extracellular levels of noradrenaline in the prefrontal cortex.,,The potential for atomoxetine to modulate locus coeruleus-noradrenaline function and improve response inhibition holds therapeutic promise in Parkinson’s disease. Previous work using stop-signal tasks in Parkinson’s disease demonstrated that atomoxetine can improve response inhibition and enhance its attendant stopping network activation.Here, we test the hypothesis that structural integrity of the noradrenergic locus coeruleus explains the variation in improvements in response inhibition following atomoxetine. This is now possible through recent developments in ultra-high field 7 T imaging of the locus coeruleus.Nineteen patients with idiopathic Parkinson’s disease were recruited via the University of Cambridge Parkinson’s disease research clinic and the Parkinson’s UK volunteer network. They met the United Kingdom Parkinson’s Disease Society Brain Bank criteria and were not demented based on Movement Disorders Society (MDS) criteria for Parkinson’s disease dementiaParticipants with Parkinson’s disease were tested across three sessions. First, they underwent MRI scanning and clinical assessment, including the Movement Disorders Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS),,On the second and third sessions, patients completed a double-blind randomized placebo-controlled crossover study, with 40 mg of oral atomoxetine or placebo. Atomoxetine 40 mg was chosen as this is at the lower end of the clinical dose range and very well tolerated,Control participants were tested in one session to provide normative data on the task, in which they underwent MRI scanning and completed the same experimental task battery as the patients. The control group did not undergo the drug/placebo manipulation. Both the patient and control groups completed a set of self- and informant-rated questionnaires to assess mood and behaviour, which are reported in detail in the We used a stop-signal task to measure response inhibition. This paradigm involves a two-choice reaction time ‘go’ task that is occasionally interrupted by a ‘stop signal’, which requires the initiated response to be cancelled . On go tet al.,Participants were given standardized instructions at the start of the experiment. They were asked to respond as ‘quickly and accurately as possible’, and were discouraged from strategically slowing down in anticipation of a stop signal.et al.We used a Bayesian parametric model of the stop-signal task to infer the latency of the unobservable stop response—the SSRT.,The model assumes that the finish times of the stop and go processes follow an ex-Gaussian distribution, which is a positively skewed unimodal distribution that is commonly used to describe reaction time data.,,,,We additionally estimated two parameters that represent the probability that the stop and go processes failed to start, referred to as ‘trigger failure’ and ‘go failure’, respectively.go-match (1.5 s), μgo-mismatch (1.5 s) and μstop (1 s), to account for slower reaction times in older age (see the We used Markov chain Monte Carlo (MCMC) sampling to estimate the posterior distributions of the parameters. The parameters were estimated hierarchically, such that parameters for a given participant were sampled from corresponding group-level distributions. We fitted this hierarchical model separately for the control group, the Parkinson’s disease group on placebo, and the Parkinson’s disease group on atomoxetine. We placed the same set of prior distributions on the group-level parameters for each of these three groups. The prior distributions were identical to those suggested by the model developers,ribution s 8–10.stop + τstop. We repeated this for each MCMC sample to approximate a posterior distribution of SSRT. This approach was also used to approximate a posterior distribution of go reaction time (μgo-match + τgo-match).The primary outcome of interest, SSRT, was computed as the mean of the ex-Gaussian finish time distribution of the stop process, which is given by μt-tests, respectively.The go error rate and stop accuracy rate served as basic descriptive statistics for stop-signal task performance. We defined the go error rate as the proportion of go trials with an incorrect response, including commission errors (responses that mismatch the arrow direction) and omission errors (missing responses). The stop accuracy rate was defined as the proportion of stop trials with successfully inhibited responses (missing responses). For both outcomes, we examined differences between groups (Parkinson’s disease placebo versus controls) and drug conditions (Parkinson’s disease placebo versus atomoxetine) using independent and paired samples We then examined the group- and participant-level parameter estimates from the ex-Gaussian race model described above. For group-level inference, we examined the posterior distributions of the group-level means of SSRT and go reaction time. For a given posterior distribution, we took the median as the posterior estimate, and the 95% quantile interval (QI) as the range of most credible values. We also obtained posterior distributions for contrasts of interest  by subtracting the sets of MCMC samples of the two groups under consideration. That is, for a given parameter, we computed the difference between the two groups for each MCMC sample, thereby yielding an approximate posterior distribution of the difference.To test for individual differences in the effect of atomoxetine on SSRT and go reaction time, we extracted the medians of the participant-level posterior distributions of SSRT and go reaction time from the placebo and atomoxetine model fits. We hypothesized that the effect of atomoxetine would depend on the integrity of the locus coeruleus, indexed by the contrast-to-noise ratio . Therefore, the posterior parameter estimates were entered as the dependent variable with drug condition (placebo versus atomoxetine), CNR, and their interaction as fixed effects, allowing the intercept to vary across participants (random effect). We additionally included a fixed effect of session (first versus second) as a covariate of no interest. Taking the analysis of SSRT as an example, the model was specified in R formula syntax as follows: SSRT ∼ drug × CNR + session + (1 | subject).P = 0.05 (two-sided) for frequentist analyses. We present the BF for the alternative hypothesis over the null hypothesis (i.e. BF10), such that BF > 1 indicates relative evidence for the alternative hypothesis, and BF > 3 indicates ‘positive evidence’ for the alternative hypothesis.,P-values using the Kenward-Roger method, and BFs through Bayesian model averaging by estimating the change from prior to posterior inclusion odds (inclusion BF). In other words, this model-averaged BF indicates how much more likely the data are under model variants that include a given fixed effect, compared to model variants that exclude the fixed effect.For linear models, we report both frequentist and Bayes factor (BF) analyses for hypothesis testing, with a significance threshold of Because of technical issues, stop-signal task data were missing for one patient’s placebo session and for another patient’s atomoxetine session. We nevertheless included these two participants in the linear mixed model analyses of the within-subject effect of atomoxetine, as participants were treated as a random effect. However, excluding these two participants did not meaningfully change any of the following results.post hoc comparisons, and the ‘BayesFactor’The stop-signal task was implemented in MATLAB R2018b using the Psychophysics Toolbox extensions (Version 3).et al.3, 6/8 phase and slice partial Fourier, bandwidth = 140 Hz/px, no acceleration, 14.3% oversampling, acquisition time ∼7 min). For each subject, the transmit voltage was adjusted based on the average flip angle in the central area of the pons obtained from a B1 pre-calibration scan. The MT-TFL sequence was repeated twice and averaged offline to improve signal-to-noise ratio. An additional scan (MT-off) was acquired with the same parameters as above but without the off-resonance pulses. A high resolution isotropic T1-weighted MP2RAGE image was also acquired sagittally for anatomical coregistration using the UK7T Network harmonized protocol3, field of view = 224 × 224 × 157 mm3, acceleration factor (A ≫ P) = 3, flip angles = 5/2° and inversion times = 725/2150 ms for the first/second images.All patients and controls underwent MRI. Two controls were excluded from further imaging analysis because of incidental structural abnormalities. MRIs were acquired with a 7 T Magnetom Terra scanner (Siemens), using a 32-channel receive and circularly polarized single-channel transmit head coil . We used a 3D high-resolution magnetization transfer-weighted turbo flash (MT-TFL) sequence for imaging the locus coeruleus 1-weighted MP2RAGE data were generated offline from the complex images.1-weighted skull-stripped images were obtained after tissue type segmentation and reconstruction using SPM12 (v7219) (http://www.fil.ion.ucl.ac.uk/spm/software/spm12/ accessed 15 July 2021).Image processing and coregistration was based on the pipeline described in Ye 1-weighted images were then entered into a T1-driven, cross modality coregistration pipeline to warp the individual MT and MT-off images to the isotropic 0.5 mm ICBM152  T1-weighted asymmetric template.1-weighted images were first coregistered to the MT image with rigid only transformation. The MT-off image was used as the intermediate step for bridging the two modalities because the MT-off image shares similar tissue-specific contrasts with both T1-weighted MP2RAGE and MT-on images.The preprocessed MT-weighted and T1-weighted structural template was created using individual skull-stripped T1-weighted images from all controls and patients. Native T1-weighted images were rigid and affine transformed, followed by a hierarchical non-linear diffeomorphic step at five levels of resolution, repeated by six runs to improve convergence. Max iterations for each resolution from the coarsest level to the full resolution were 100 × 100 × 70 × 50 × 20 . Greedy Symmetric Normalization (SyN) was adopted for the transformation model of the deformation step.1-weighted group template was then registered to the standard ICBM152 T1-weighted brain following the similar rigid-affine-SyN steps at four resolution levels . For all the above registration steps, cross-correlation was used for similarity metrics estimation as it performs better for linear and non-linear components during intra-modality registration. Four steps of deformations were estimated as follows (in order): MT-off to MT, T1-weighted to MT-off, T1-weighted to T1-weighted group template and T1-weighted group template to ICBM152 T1-weighted template. These parameters were then used as the road map for MT image standardization to the ICBM brain in one step. A trilinear interpolation method was selected to preserve the absolute location and relative contrast of the signal.In parallel, an unbiased study-wise Tet al.et al.To facilitate accurate extraction of the locus coeruleus signal we created a study-specific unbiased locus coeruleus atlas . To thisREF) divided by the standard deviation (SDREF) of the reference signals:As a measure of locus coeruleus integrity, we quantified contrast by establishing the CNR with respect to a reference region in the central pons . A CNR mCNR values were extracted bilaterally on the CNR map by applying the independent locus coeruleus probabilistic atlas (5% probability version). We computed mean CNR values for the rostral, middle and caudal portions of the left and right locus coeruleus. As an index of locus coeruleus integrity to incorporate with the stop-signal task analysis, we combined the left and right locus coeruleus and averaged across the whole structure.https://osf.io/tyka3/).Code and data to reproduce manuscript figures, statistical analyses and stop-signal task modelling are freely available through the Open Science Framework . Patients had a significantly lower ACE-R total score , and lower memory subscale . There were no group differences on any of the self-reported questionnaires assessing impulsivity, anxiety, depression and behavioural measures, with the exception of the Motivation and Energy Inventory where the patient group had significantly lower scores for the physical subscale compared to controls  = 1.27, P = 0.21, BF = 0.58]. Comparing across the rostral, middle and caudal subdivisions, there was a main effect of subdivision . This was driven by CNR in the caudal portion being significantly lower than both the middle  and rostral  portions. There was a significant group by subdivision interaction . This reflected significantly lower CNR values in the caudal portion for patients relative to controls , whereas the groups did not differ for the middle  or rostral  portions of the locus coeruleus. We did not see significant differences in left versus right locus coeruleus degeneration, as the group comparisons did not meaningfully change when incorporating locus coeruleus side; this analysis is reported in the t(40.79) = 1.90, P = 0.065, BF = 0.35]. Across the Parkinson’s disease patients, group-wise stop accuracy on atomoxetine  was not significantly different from the placebo session . The go error rate approached zero for most participants, yielding a skewed distribution bounded at zero (t(29.94) = −2.19, P = 0.037, BF = 0.46]. Within the Parkinson’s disease group, the logit go error rate was marginally reduced on atomoxetine  compared to placebo . However, we note that the BF for both these tests fell below conventional thresholds for positive evidence (i.e. BF > 3), and these effects on go error rate should therefore be regarded as ‘anecdotal’ at the group level.In keeping with the tracking algorithm, the stop accuracy for the Parkinson’s disease group on placebo  = 14.61, P = 0.002; BF = 11.56]. This interaction effect did not meaningfully change when including clinical covariates such as age, disease severity, atomoxetine plasma level and dopaminergic medication, as both frequentist and Bayesian model selection procedures indicated that such covariates did not significantly improve the model fit  = 13.33, P = 0.002; BF = 2.58], reflecting slightly shorter SSRTs for the second session compared to the first, regardless of the drug condition. There were no significant main effects of drug condition  or locus coeruleus CNR  on SSRT.There was also a main effect of session .To understand the Drug × Locus coeruleus CNR interaction further, we examined the relationship between locus coeruleus CNR and the drug-induced change in SSRT (Δdrug: atomoxetine − placebo), adjusted for the main effect of session. This relationship was strongly positive, suggesting that patients with lower locus coeruleus CNR have a greater reduction in SSRT after atomoxetine , stop accuracy rate , or logit go error rate . Finally, to explore the possibility of hemisphere-specific effects, we ran the linear mixed model analysis separately for CNR values from the left and right locus coeruleus. These results were qualitatively identical to the original combined analysis, arguing against hemisphere-specific effects . This result highlights the link between integrity of the noradrenergic locus coeruleus and action cancellation, which has previously been inferred from preclinical work and pharmacological manipulations. The finding also demonstrates the importance of baseline noradrenergic capacity in determining the response to atomoxetine, confirming the need to stratify patients for noradrenergic therapy. Locus coeruleus neuromelanin imaging would be a safe and affordable means to achieve this stratification.,,Previous work in Parkinson’s disease showed that atomoxetine led to greater improvements in SSRT and enhanced activation in the stopping network in patients with more severe disease.,Baseline dependency in dose-response curves is well documented in relation to dopaminergic therapy,,Neuromelanin-sensitive MT imaging of the locus coeruleus represents a promising avenue to achieve this stratification. Although we have previously shown a relationship between disease severity (as measured by the UPDRS-III) and atomoxetine responsivity, such measures of motor function or disease duration may not be the most accurate measure of noradrenergic capacity. Whilst progressive degeneration of the locus coeruleus is expected over the disease course, this will vary widely across individuals and will reflect the disease phenotype. Neuropathological studies and neuromelanin imaging have shown more pronounced locus coeruleus degeneration in certain phenotypes, including those with cognitive impairment or dementia,,Our locus coeruleus imaging identified that the greatest difference between controls and patients was in the caudal portion of the locus coeruleus. This has not previously been identified in Parkinson’s disease locus coeruleus imaging with 3 T MRI, which limited analysis to the whole structure. This is because of the inherent limitations in 3 T MRI, where voxel sizes and slice thickness are larger and do not allow for the spatial resolution needed to examine locus coeruleus subdivisions.,,,versus dopaminergic modulation of the SSRT is not fully elucidated. These two closely related neurotransmitters share a common biosynthetic pathway. They can be co-released from the same terminalsOur study aimed for noradrenergic modulation of the SSRT. Atomoxetine increases extracellular noradrenaline levels across the brain via its actions at the noradrenaline transporter, in particular increasing levels in the prefrontal cortex by 3-fold.post hoc supplementary analyses with brain volume and substantia nigra integrity showed these covariates did not influence that association. This indicates a degree of specificity for the locus coeruleus-noradrenergic system’s role in the SSRT in Parkinson’s disease. However, future cross-over studies using specific noradrenergic versus dopaminergic agents would be beneficial to resolve residual ambiguity. Our current results point towards atomoxetine as a useful strategy to treat inhibitory deficits in Parkinson’s disease, in the context of appropriate stratification using locus coeruleus integrity.In practical terms, a key question is whether ‘noradrenergic’ drugs offer a useful strategy to treat inhibitory control deficits. The association we have shown between locus coeruleus integrity and the change in SSRT under atomoxetine suggests a causal association between the noradrenergic system and action cancellation in Parkinson’s disease. Our ,,,,,Our results do not speak directly to atomoxetine’s mechanism of action in Parkinson’s disease. However, convergent evidence indicates that atomoxetine may increase efficiency in brain networks mediating response inhibition, via actions at the prefrontal cortex and the locus coeruleus.,,,SLC6A2) and the CYP2D6 enzyme gene. These are known to modulate clinical response to atomoxetineWe would support the use of 40 mg atomoxetine in subsequent single-dose studies in Parkinson’s disease. Based on the current results, as well as previous clinical trials,Our study was designed to test a mechanistic hypothesis around locus coeruleus integrity and atomoxetine responsivity; therefore, we did not focus on clinically relevant outcomes. Future longer-term clinical trials are needed to determine if atomoxetine improves cognition and behaviour in a way that benefits daily function, including carer and patient ratings. The link between response inhibition and atomoxetine suggests that an important phenotype to focus on will be those patients with impulsivity, even where this falls short of an impulse control disorder. We also note that locus coeruleus degeneration has been strongly linked with REM sleep behaviour disorder.Given the role of noradrenaline in cognition and behaviour,awab142_Supplementary_DataClick here for additional data file.


Nipah virus (NiV) is an ssRNA, enveloped paramyxovirus in the genus Henipaveridae with a case fatality rate >70%. We analyzed the NGS RNA-Seq gene expression data of NiV to detectdifferentially expressed genes (DEGs) using the statistical R package limma. We used the Cytoscape, Ensembl, and STRING tools to construct the gene-gene interaction tree, phylogeneticgene tree and protein-protein interaction networks towards functional annotation. We identified 2707 DEGs  among 54359 NiV genes. The top-up and down-regulated DEGs wereEPST1, MX1, IFIT3, RSAD2, OAS1, OASL, CMPK2 and SLFN13, SPAC977.17 using log2FC criteria with optimum threshold 1.0. The top 20 up-regulated gene-gene interaction trees showed no significantassociation between Nipah and Tularemia virus. Similarly, the top 20 down-regulated genes of neither Ebola nor Tularemia virus showed an association with the Nipah virus. Hence, wedocument the top-up and down-regulated DEGs for further consideration as biomarkers and candidates for vaccine or drug design against Nipah virus to combat infection. Nipah virus (NiV) is a stage III zoonotic pathogen from the family of Paramyxoviridae and a new genus from the Henipavirus . Nipah vWe used Microarray gene expression of RNA-Seq data for molecular investigation of NiV infection. We collected the complete genome of selected pathogen from the National Centre for Biotechnology Information (NCBI). To analyze data we considered 7 (seven) different datasets with accession numbers in the GEO (Gene Expression Omnibus) database are as follows GSE32902, GSE23986, GSE93861, GSE18064, GSE12108, GSE69980, GSE89915 -29.Download links for data are as follows:[1] NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/)[2] Nipah Virus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32902)[3] Dengue virus type-3 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23986)[4] Ebola virus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93861)[5] Rift Valley Fever virus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18064)[6] Tularemia (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12108)[7] Chikungunya (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69980)[8] Zika virus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89915)We performed the log2 transformation to analyze the RNA-Seq NGS gene expression data. The log transformation is the robust method for scaling and controlling the outliers of the dataset by uniform pattern.Linear model for microarray analysis (limma) is the efficient statistical tool integrated in R package for identification of DEGs from large scale expression data . The logThe row represents the gene and the column represents the RNA samples in the NGS RNA-Seq data matrix of Nipah virus (GEO ID: GSE32902). In this dataset four samples (two uninfected HUVEC and two NiV-infected HUVEC) and 54359 genes were included.We used t-test statistic to calculate the p-values for each gene (54359) of Nipah virus dataset. We used a cut of point less than 0.05 at a 5% level of significance to select the DEGs. We identified 2707 DEGs with this cut of value. Similarly, for Chikungunya virus 1544 DEGs out of 45220 genes were identified using a p value<0.05 cut of point. We identified 1200 DEGs  of 33297 genes of the Dengue type III virus. The Ebola Virus RNA-Seq dataset included 41000 genes and of these 20211 genes were detected as DEGs using a p value<0.05 in t-test statistic. With the same p-value cut of point (<0.05), we identified 14470 DEGs of 54675 genes for Tularemia (Francisella tularensis), 6666 DEGs of 45101 genes for Rift valley fever virus and, 688 DEGs of 43376 genes for Zika virus.We used the log2 fold change or a cutoff at 0.5 for down and 1/2 for up-regulated genes and selected genes under/above thresholds. A log2FC-value close to 0.5 corresponds to equally expressed (EE) genes, while down- and up-regulated genes have log2FC-values close to 0 and 1 correspondingly . In thisWe used Cytoscape (https://cytoscape.org/) bioinformatics tool to construct a gene-gene interaction network. Based on the calculated p-value, we selected top 20 up- and down-regulated genes  for 7 viruses  ranked according to increasing order for each gene. The gene-gene interaction tree of the top 20 up-regulated DEGs among the 7 viruses showed that the Tularemia virus had no association with the other 6 viruses including the Nipah virus and, it represents a separate gene tree. The other five viruses, however, showed that a significant association with the Nipah virus and unknown gene symbol  and a strong network was seen among these viruses . From thWe selected the top 10 up-regulated genes using ranking p-value. Of 10, 7 were functional: EPST1 (g1), MX1 (g3), IFIT3 (g4), RSAD2 (g5), OAS1 (g6), OASL (g7), CMPK2 (g9) and 3 were non-functional genes .We used We used the STRING (https://string-db.org/) online bioinformatics tool for protein-protein interaction (PPI) networks functional enrichment analysis. Top up-regulated seven functional EPST1 (g1), MX1 (g3), IFIT3 (g4), RSAD2 (g5), OAS1 (g6), OASL (g7), CMPK2 (g9) DEGs of Nipah virus were highly interacting proteins by score with IFI44L, OASL, IFIT1, IFIT1, MX1, MX1, and RSAD2 respectively. All the PPI network enrichment was statistically significant  at a 5% level of significance .All top We used the statistical R package limma to analyze the NGS RNA-seq data to detect DEGs (biomarker) of the Nipah virus for application in combat and care of the disease. We identified 2707 DEGs  among the 54359 genes of the virus. We report 834 up-regulated and 1873 down-regulated DEGs estimated by the log2FC approach at threshold value 1.0. This data will help in the selection of biomarkers and vaccine targets against the virus.