NOTCH2NL gene family, which were recently identified as possible contributors to the evolutionary expansion of the human brain. We find evidence for the existence of unique protein-coding NOTCH2NL variants in Neanderthals and Denisovans which could affect their ability to activate Notch signaling. Furthermore, in the Neanderthal and Denisovan genomes, we find unusual NOTCH2NL configurations, not found in any of the modern human genomes analyzed. Finally, genetic analysis of archaic and modern humans reveals ongoing adaptive evolution of modern human NOTCH2NL genes, identifying three structural variants acting complementary to drive our genome to produce a lower dosage of NOTCH2NL protein. Because copy-number variations of the 1q21.1 locus, encompassing NOTCH2NL genes, are associated with severe neurological disorders, this seemingly contradicting drive toward low levels of NOTCH2NL protein indicates that the optimal dosage of NOTCH2NL may have not yet been settled in the human population.Ever since the availability of genomes from Neanderthals, Denisovans, and ancient humans, the field of evolutionary genomics has been searching for protein-coding variants that may hold clues to how our species evolved over the last ∼600,000 years. In this study, we identify such variants in the human-specific  NOTCH2NL genes   . This prnversion  online. imations  online. data set  were mape masked A. On thiNL genes B and C. 2NL loci D. Using pulation E. Next, pulation , Neanderpulation , and Denpulation . Althouglication D. WhereaNOTCH2NL exons, we found evidence for two Neanderthal-specific coding variants and one Denisova-specific coding variant (Nea-M40I) is detected in 17/242 (∼8%) of the sequencing reads corresponding to one allele out of the nine NOTCH2NL alleles found in Altai Neanderthals. The second Neanderthal-specific variant is a N232S missense variant (NOTCH2NLNea-N232S) detected in 28/177 (∼18%) of sequencing reads, corresponding to two alleles. This variant is also present in the genomes of the Vindija and Chagyrskaya Neanderthals and most of the low-coverage Neanderthal genomes, indicating the NOTCH2NLNea-N232S variant was a common variant in the Neanderthal lineage. In the Denisova3 genome, a Denisovan-specific E258A missense variant (NOTCH2NLDen-E258A) is found in 38/203 (∼19%) of the sequencing reads, also corresponding to two alleles. Importantly, none of these variants are found in the 279 modern human genomes of the Simons diversity data set. Interestingly, the NOTCH2NLNea-N232S was found as a rare variant in modern humans (rs375605753) with an allele frequency of 0.0002 in UK Biobank exome sequencing data , suggesting this was one of the Neanderthal-derived genetic variants that was contributed to the human genome after interbreeding with Neanderthals  between human and Neanderthal/Denisovan  variant A. In thederthals . It shouer assay  , the expression level of NOTCH2NLC is extremely low, at only 1% compared with NOTCH2NLB (NOTCH2NLB configuration of Exon1 (Exon1B-(High)-variant) which produces high levels of NOTCH2NL protein, the M1I substitution in NOTCH2NLA (Exon1A-(Low)-variant) produces 5-fold less NOTCH2NL protein. The configuration of NOTCH2NLC, which has the 2-bp deletion in Exon1, (Exon1C-(X-low)-variant) results in extremely low levels of NOTCH2NL protein. Importantly, ectopic gene conversion between NOTCH2NL loci can result in transfer of Exon1-variants from one NOTCH2NL gene to another. As a consequence, the total dosage of NOTCH2NL protein in each individual may not be defined by the copy number of each of the NOTCH2NL genes, but by the level of Exon1-variant carry-over via gene conversion between NOTCH2NL genes.Unexpectedly, we noticed that the NOTCH2NLAproteins  -variants -variant. The Denisova3 genome however, shows a strikingly different pattern.To assess the extent to which gene conversion influences the distribution of Exon1-variants between variants A, presenividuals B, suggesNOTCH2NL-paralog-specific SUNs across the NOTCH2NL loci shows that NOTCH2NLA, NOTCH2NLB, and NOTCH2NLC genes are present in the Denisova3 genome , all NOTCH2NL-derived reads from Exon1 carry the NOTCH2NLC-derived Exon1C-(X-low)-variant -variant distribution in Denisovan NOTCH2NL genes, or anything similar to it, has not been observed in any of the analyzed genomes of Neanderthals or healthy modern humans -variant (NOTCH2NLA-derived Exon1A-(Low)-variant and only one or zero alleles with the NOTCH2NLB-derived Exon1B-(High)-variant (A-(Low)-variant distribution was confirmed in 49,593 exomes from the UK Biobank -variant, producing the highest levels of NOTCH2NL protein, is being lost or actively being purged out from the modern human population by gene conversion. The increase of the Exon1A-(Low) variant frequency to three or four alleles per individual is likely caused by gene conversion between the NOTCH2NLA and NOTCH2NLB loci, which can occur during meiosis or in early embryonic development for very unstable loci (Even though ce versa A. Most r-variant B. This i-variant C. The sh Biobank  (supplemble loci .A-(Low) variants in NOTCH2NLA and NOTCH2NLB, some individuals still carry a relatively high number of Exon1B-(High) variants. We found that individuals with a relatively high number of the Exon1B-(High) variant and low number of the Exon1A-(Low) variant often carry a nonsense SNP (R113*) in NOTCH2NLB, which leads to a premature stop-codon and a severely truncated NOTCH2NL protein (2B-(Splice-mut))  and the R113* mutation were not found in any of the currently available Neanderthal or Denisovan genomes  or Exon1B-(High) variants and the presence of disruptive R113* and Exon2B-(Splice-mut) mutations: Individuals with a relatively high number of the Exon1B-(High) variant, often carry one or two alleles of the disruptive R113* mutation in NOTCH2NLB  mutation  variants are more likely to lack either the R113* or splice acceptor mutations in NOTCH2NLB  frequency instead  variant often co-occurs with the presence of nonsense variants in NOTCH2NLB. Our data suggest that on the individual’s genome level, gene conversion of the Exon1B-(High) variant into the Exon1A-(Low) variant acts in concert with nonsense variants in NOTCH2NLB to reduce overall NOTCH2NL protein level. This seems particularly relevant because we observe a strong dosage-dependent effect of NOTCH2NL on Notch signaling activation  variants in the Denisova3 genome. Notably, none of the 279 modern human individuals analyzed in detail in this study showed similar configurations and it is questionable whether such configurations are found in the healthy human population. This raises questions about the health state of the juvenile Denisovan female from the Denisova3 genome, but because the DNA was isolated from a finger bone, information about her physical condition or cause of death is lacking -variants, which is associated with a substantial reduction in NOTCH2NL protein level. The fact that about 40% of individuals lack the NOTCH2NLB-derived Exon1B-(High)-variant completely could indicate that the high level of NOTCH2NL protein producing variant is slowly being purged from the human genome. We found that this is not the only evolutionary force at play: Next to the Exon1 variants, there are two other deleterious variants, R113* and Exon2B-(Splice-mut), that reduce the dosage of functional NOTCH2NL protein. Remarkably, these deleterious variants are more often found in individuals with higher Exon1B-(High) frequency, indicating that they provide complementary genetic strategies to decrease NOTCH2NL dosage. The R113* and Exon2B-(Splice-mut) variants are exclusively present in modern humans and are therefore human-specific adaptations that result in reduced NOTCH2NL protein levels. The driving force behind the evolutionary trend to lower levels of NOTCH2NL protein remains elusive. Phylogenetic comparisons or dN/dS analysis are traditionally used to assess if such variation is significantly associated with evolutionary selection. Because of the absence of functional nonhuman orthologs required to do these comparisons, it is not possible to apply these approaches for analysis of NOTCH2NL genes. In addition, frequent and ongoing gene conversion between NOTCH2 and NOTCH2NL-containing loci also hampers this analysis when trying to make comparisons with the truncated NOTCH2NL pseudogenes in chimpanzee and gorilla. The high frequency of multiple variants that decrease the available levels of NOTCH2NL protein suggests that NOTCH2NL genes have been under selection to counteract high levels of NOTCH2NL expression. Whereas a high frequency of loss-of-function alleles in a population could in principle argue against an essential function of the gene in question and could progress to a complete loss of functional alleles in the future, our data indicate that this is not the case for NOTCH2NL genes: Based on the high frequency of loss-of-function variants in NOTCH2NL genes in modern humans, it would be expected that a decent proportion of the population would have a genomic configuration without any functional NOTCH2NL allele. This is clearly not the case, as the skewed allele distributions that we report points toward purifying selection in order to maintain at least one functional copy of NOTCH2NL. This suggests that in present day humans, a certain minimal level of NOTCH2NL protein is required for normal human development. The observed evolutionary changes in NOTCH2NL composition could be the result of evolutionary adaptations that took place in any of the tissues where NOTCH2NL is expressed, including the developing brain. Even though this remains speculative at the moment, the trend toward lower levels of NOTCH2NL proteins in the human lineage could be correlated to previous observations suggesting a progressive reduction of human brain size that started about 60,000 years ago -related phenotypes. Specifically for NOTCH2NL-mediated effects, like potentiating NOTCH signaling, CNVs of an allele carrying the Exon1B-(High) variant may have a much larger effect than CNVs of an allele carrying the Exon1A-(Low) variant. Identifying which NOTCH2NL loci are affected by gain and loss of alleles will have to be complemented by distribution analysis of Exon1A-(Low), Exon1C-(X-low), R113*, and Exon2B-(Splice-mut) variants as they are major determinants of NOTCH2NL levels. The realization that gene conversion between functionally different NOTCH2NL genes can contribute to the rapid adaptation of the human species to establish lower levels of NOTCH2NL protein, may prove to be an example for other unstable loci that are characterized by recent segmental duplications. As some of these, like the 1q21.1 locus, are associated with disease, it will be intriguing to see if gene conversion also affects genetic configurations of such loci.Effectively, NOTCH2NL dosage, which is the total of protein produced by all NOTCH2NL genes, a gene family that emerged in humans about 4 Ma. The role of NOTCH2NL genes in human brain development and their involvement in 1q21.1 CNVs associated with a wide variety of neurological disorders emphasizes the importance of the discoveries we describe here: Even if the driving forces of the observed evolutionary changes lie outside the brain, the recent and ongoing structural evolution of human NOTCH2NL genes suggests that the tightly coordinated process of human cortical neurogenesis is still subject to fine-tuning.Ever since the availability of genomes from Neanderthals, Denisovans, and ancient humans, the question was raised which modern human-specific coding variants may hold clues to how our species evolved over the last ∼600,000 years. Here, we discovered such variants in the NOTCH2NL-masked hg38 reference genome using Bowtie2 , using single-end, very sensitive end-to-end settings. Sequence read depth per genome was ∼15–30×. The BAM output files were sliced using samtools slice  with the coordinates chr1:118911553–121069626. Bedtools coverage  was applied to each sliced BAM file, reporting coverage for each position. The NOTCH2-single-copy region used is located at chr1:119908310–119989035, the NOTCH2 + NOTCH2NL multicopy region used is located at chr1:119990490–120087745. Each region was filtered for repeats using RepeatMasker, and only the nonrepeat intervals were used in coverage analysis. Mean coverage across both regions was calculated by averaging coverage per position. The mean coverage of the NOTCH2 + NOTCH2NL-multicopy region was divided by the mean coverage of the NOTCH2-single-copy region to infer NOTCH2NL copy-number pet data set. BAM file data were visualized in the UCSC genome browser , WGS (PRJNA200694), WXS (PRJNA200694)NA24149: 10× genomics (GIAB), WGS (PRJNA200694), WXS (PRJNA200694)NA24385: 10× genomics (GIAB), WGS , WXS (PRJNA200694)NA19240: WGS NA12891: WGS and 10× WGS CHM1: WGS NOTCH2NLA, -B, -C, or -R loci to the NOTCH2 locus. Only SUNs within the region chr1:119990474–12008798 were considered, as this is the maximal duplicon size present in each of the NOTCH2NL loci based on the segmental duplication track in the UCSC genome browser hg38. The position of each of these SUNs per locus was stored in BED format. These were used to generate .vcf format data per BAM file reporting the total read depth and variant (SUN) depth for these positions. This was done using samtools (v1.7) mpileup:Based on the hg38 reference genome, single-nucleotide variants and indels were identified, via DNA sequence alignment of the samtools mpileup -uvf hg38.fasta -t DP -t AD -l variant_positions.bed -Q 13 -q 0 -b datasets.txt > output.vcfThe relevant information to calculate SUN frequency per allele was extracted using bcftools (v1.7) query:bcftools query -f ‘%CHROM\t%POS\t%REF\t%ALT{0} [\t%DP\t%AD{0}\t%AD{1}]\n’ -H mpileup_output.vcf > mpileup_output_variants.vcfNOTCH2, NOTCH2NLR, and NOTCH2NLC, they provide an accurate estimation for the allele count of these loci. For NOTCH2NLA and NOTCH2NLB, only a few SUNs are present and gene conversion phenomena happen frequently, which makes this procedure challenging. Therefore, to analyze these loci, we first subtracted the NOTCH2, NOTCH2NLR, and NOTCH2NLC allele counts from the total allele count. The remaining alleles must be derived from NOTCH2NLA and NOTCH2NLB, and so, the remaining alleles were counted using the ratio of the average SUN frequency for NOTCH2NLA and NOTCH2NLB. These data were plotted in donut-charts using LibreOffice v6.1.0.3. For graphs showing the per-SUN allele count across the NOTCH2NL loci, the NOTCH2NLB SUN count was inferred from the NOTCH2NLA SUN count in the 5′ region of the locus, where no NOTCH2NLB SUNs are present. For example in modern humans there are four NOTCH2NLA+NOTCH2NLB loci, then the Exon1B-(High) allele count was calculated according to this: Exon1B-(High) allele count = 4 − Exon1A-(Low) count. Correction for NOTCH2 > NOTCH2NLR gene conversion was done for genomes that showed three alleles NOTCH2. These showed a concordant decrease of one allele NOTCH2NLR based on both the coverage analysis and SUN analysis. This difference was corrected for, in example, three alleles NOTCH2 and one allele NOTCH2NLR in one individual were corrected to two alleles NOTCH2 and two alleles NOTCH2NLR. For separation of the Simons diversity genomes data per population, the sample metadata supplied with the data were used.The frequency per variant was calculated using these output files by dividing allele depth for each SUN (AD) by total depth (DP). For each locus, only SUNs with >0.67 frequency in the population were used for analysis to account for ambiguous or population-specific sites that may skew allele distribution calculation, such as known common SNPs. The frequency of the selected SUNs was averaged per locus and multiplied by the total number of alleles calculated previously based on sequence read coverage, to transform allele frequencies into allele counts. Since there are many SUNs for NOTCH or NOTCH2NL genes were extracted from UK Biobank CRAM exome files (>20× coverage) mapped on hg38. As in these data sets, the reads are mapped to NOTCH and all NOTCH2NL loci in hg38, the analysis was adjusted from the original analysis that used the masked hg38. For the Exon1A-(Low) variant (ATG>ATA), the following positions were analyzed:Reads mapping on 2B-(Splice-mut) variant information was derived from the following positions:Similarly, the ExonN232S variant (AAT > AGT) information was derived from the following positions:Nea1Read depth and allele depth analysis using samtools and bcftools was then done for each locus with the following parameters:samtools mpileup -uvf hg38.fasta -t DP -t AD -l variant_positions.bed -Q 13 -q 0 -b datasets.txt > output.vcfbcftools query -f ‘%CHROM\t%POS\t%REF\t%ALT{0} [\t%DP\t%AD{0}\t%AD{1}]\n’ -H output.vcf > query_output.vcfNOTCH2NL locus but still contain information regarding variant frequencies. Since the Exon1A-(Low) variant is annotated in the hg38 genome in NOTCH2NLA, reads containing this variant will map there with a better alignment score. As such, the Exon1A-(Low) frequency was calculated by read depth at the NOTCH2NLA position divided by the sum of read depths at the NOTCH2 + all NOTCH2NL loci. The Exon2B-(Splice-mut) frequency was calculated by read depth at the specific NOTCH2NLB position, where this variant is annotated in hg38, divided by the total read depth at the paralogous positions.The setting -q  was set to 0, to include multimapping reads that cannot be confidently assigned to a specific HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco's modified eagle medium (DMEM) + GlutaMax, high glucose (Thermofisher 61965026), supplemented with 10% heat-inactivated fetal bovine serum (HIFBS) (Thermofisher 10500064) and 100 µg/ml Pen/Strep (Thermofisher 15140122). U2OS and U2OS-JAG2 cells  were cultured in DMEM + GlutaMax, high glucose, supplemented with 10% HIFBS and 100 µg/ml Pen/Strep. U2OS-JAG2 cells were additionally supplemented with 2 μg/ml puromycin (Sigma P8833). For routine passaging, medium was removed and cells washed once with phosphate-buffered saline (PBS) (Thermofisher 10010056). A sterile filtered 0.25% Trypsin (Thermofisher 15090046) + 0.5 mM disodium-ethylenediaminetetraacetic acid (EDTA) (Sigma E5134) solution in PBS was added, and incubated at 37 °C for 2 min. One-eighth of the cell suspension was transferred to a new culture vessel of the same size.2, after followed by addition of 100 μl 2× HEPES-buffered saline . The 200 μl solutions were mixed by pipetting up and down five times, and the complete mix was added to one well of a six-well plate. Six hours after adding transfection mixes, medium was replaced.HEK293 cells were seeded 24 h before transfection in a six-well plate. One hour before transfection, medium was replaced with 1,800 μl DMEM + GlutaMAX, high glucose and 10% HIFBS. The transfection mix per well was as follows: 500 ng of pCAGN1-NOTCH2NL or pCAGN1-EV, and 500 ng of pCAGEN-GFP were mixed in a total volume of 100 μl 0.25 M CaCl2, 0.5 mM EDTA, 0.2% NP40 substitute, and 5% glycerol), supplemented with 1× protease inhibitor cocktail (Sigma 5892791001). After incubating for 1 h at 4 °C, cell suspensions were transferred through a 273/4 gauge needle ten times and centrifuged at 20,817 rcf for 10 min at 4 °C to pellet cell debris. The supernatant was transferred to a new 1.5-ml microcentrifuge tube and stored at −80 °C.Cells were isolated for protein extraction 24–30 h after transfection. Cells were washed twice in ice-cold PBS, then detached using a cell scraper (VWR 734-1527) and transferred to 1.5-ml microcentrifuge tubes. Cell suspensions were centrifuged at 4 °C for 5 min at 1,000 rcf to pellet cells. The supernatant was removed and the cells resuspended in 10× the pellet volume (100–150 μl) of immunoprecipitation lysis buffer  + 50 mM DTT (Sigma D0632). Samples were heated for 5 min at 95 °C and briefly centrifuged. Twenty microliters per sample was loaded on a 1.5-mm poly-acrylamide gel, consisting of two parts. The running gel  and the stacking gel . Twenty microliters of sample was loaded per well and 5 μl of marker (Thermofisher #26619) was used for reference. Electrophoresis was done in 25 mM Tris + 192 mM glycine buffer (Biorad 1610771) and 0.1% SDS. Protein was transferred to nitrocellulose membrane (Sigma 10600004), at 100 V for 2 h in Towbin buffer . Blots were rinsed three times with demi-water, and transfer was checked by ponceau S staining. Blots were rinsed once in Tris buffered saline  + 0.1% Tween (TBS-T), followed by incubation in blocking buffer (TBS-T + 5% w/v skim milk powder) for 90 min at room temperature on a shaking platform. Primary antibodies were incubated overnight at 4 °C in TBS-T in 50-ml tubes on a tube roller. Antibodies used were rabbit anti-HA tag  or rabbit anti-GFP . Blots were rinsed once in TBS-T and washed in TBS-T three times 15 min on a shaking platform. Secondary antibody goat anti-rabbit-HRP in TBS-T  was incubated for 60 min at room temperature. Blots were rinsed once in TBS-T and washed three times 15 min in TBS-T on a shaking platform. The SuperSignal Westdura substrate (Thermofisher 34075) was used for chemiluminescent detection, imaged with a ChemiDoc MP imaging system (Biorad 1708280). Signals were quantified using Fiji ImageJ using the NOTCH2NL/GFP ratio.To monitor modulation of NOTCH2 activity by NOTCH2NL, a reporter assay was used. The pGL3-UAS luciferase reporter can be activated by S3-cleaved NOTCH2-Gal4-N1TAD receptor intracellular domain  . To achieve high levels of receptor activation, the cells transfected with pcDNA5-NOTCH2-Gal4-N1TAD are cocultured with JAG2 expressing cells. Coculture with regular U2OS cells was done as a control. pCAGN1-EV or pCAGN1-NOTCH2NL (derived from Addgene 51142) were cotransfected to measure effects of NOTCH2NL on reporter activity. pRL-CMV (Promega E2261) was used for normalization.For transfection, U2OS cells were seeded in six-well plates at a density of 400,000 cells per well. For coculture assay, U2OS cells or U2OS-JAG2 cells were seeded in 12-well plates at a density of 110,000 cells per well. Twenty-four hours later, U2OS cells in six-well plates were transfected. The transfection complex per well was made by adding 2,500 ng plasmid DNA mix, as described in the table below, in 100 μl OptiMEM (Thermofisher 31985047). In a different tube, 8.33 μl PEI  was added to 100 μl OptiMEM. One hundred microliters of each mix was combined, incubated 20 min at room temperature, and added to the well containing 2 ml of complete medium. Reactions were scaled accordingly to facilitate large-scale transfections. Six hours after transfection, the transfected cells were replated onto the 12-wells plate for coculture with U2OS or U2OS-JAG2 cells. Per well, medium was removed and cells were washed once with 1 ml PBS. Trypsin-EDTA (0.5 ml) in PBS was added plates were incubated 90 s at 37 °C. Two millilietrs of complete medium was added, and cell aggregates were broken up by pipetting up and down three times. Cell suspension was transferred to 15-ml conical tubes already containing 4.5 ml of complete medium. From the 12-well plates, the medium was removed and replaced by 1 ml of cell suspension. To control wells, 1 μl of 200 μM DBZ was added. Twenty-four hours after replating, the cells were isolated for luciferase assays using Dual-Luciferase Reporter Assay System (Promega E1980). Medium was removed and each well washed once with 0.5 ml PBS. A total of 150 μl of 1× passive lysis buffer (Promega E1941) was added per well and incubated 15 min on a rotating platform. Plates were wrapped in parafilm and stored at −80 °C. For analysis, 20 μl sample was pipetted to a 96-well optiplate (PerkinElmer 6005290). Samples were measured on a GloMax Navigator device (Promega GM2010), with the following settings: Injector 1, LARII buffer . Wait 2 s. Measure luminescence Luciferase . Injector 2, Stop & Glo buffer . Wait 2 s. Measure luminescence Renilla . For comparison of human, Neanderthal and Denisovan NOTCH2NL variants, the 48 ng pCAGN1-NOTCH2NL condition was used.Amount of plasmid DNA (ng) transfected per condition. pCAGN1-EV/pCAGN1-NOTCH2NL denotes amount of plasmid used per condition accounting for molarity. pBluescript amount was adjusted accordingly as well.P value threshold of 0.05. IUPred2A was used with the following settings: Long disorder, Context-dependent predictions: ANCHOR2.For multiple sequence alignment of NOTCH1, -2, and -3 EGF-L domains 6, the relevant sequences were acquired from UniProt and compared using the alignment tool of UniProt. The EGF-L repeat domain consensus sequence was retrieved from Prosite: PDOC00021, EGF_3 PS50026. For MutPred2 and IUPred2A analysis, the archaic amino acid variants were introduced in the NOTCH2NLB protein sequence retrieved from UniProt (P0DPK3). MutPred2 was run with a P = 0.002) and were analyzed instead using Welch corrected ANOVA using the R functions “oneway” with parameters “levene=TRUE” and “corrections=TRUE,” followed by Games–Howell test from function “posthocTGH,” with parameter “method=games-howell” (R package “userfriendlyscience”). Population genetic data from Simons diversity genomes and UK Biobank exomes were first analyzed using Kruskal–Wallis tests by the R function “kruskal.test.”Significant groups were further tested with Dunn’s test, using the “dunn.test” function (R package “dunn.test”). Distributions in supplementary figure S5A, N = 2,790. Standard deviation was set to 0.34 to introduce sampling variation. Expected distribution in the UK Biobank analysis was done similarly, except using allele frequencies instead, of 0, 0.1, 0.2, 0.3, or 0.4, total N = 50,000, with a standard deviation of 0.034, which were adjusted for loss of NOTCH2NLC and NOTCH2NLR as identified in Simons diversity genomes. Boxplots show median and interquartile range (25th and 75th percentiles), whiskers are defined by 1.5 × interquartile range. Outliers were hidden in violin/box plots from P values shown were adjusted for multiple testing using Holm’s method.Luciferase reporter assay data were first analyzed using analysis of variance (ANOVA) by the R function “aov.” Significant groups were further tested with Tukey’s test using the R function “TukeyHSD.” Western blot data were analyzed in the same way, except for data presented in NOTCH2NL allele counts, LibreOffice v6.1.0.3 was used. Data involving genomic context were visualized on the UCSC genome browser and exported as.pdf files. Plots showing quantification of sequence read coverage, luciferase assays, Western blots, per-SUN count graphs, variant allele counts, and distributions were generated in RStudio v1.1.463 and R v3.5.3 with the ggplot2 package v3.1.0. Fig. panels were assembled in Adobe Illustrator v23.0.3.For donut-charts showing All genomics data from the Simons Diversity Cohort and Archaic genomes were downloaded from their original depositories. Accession numbers and unique identifiers are provided where necessary. All data from the analyses in this manuscript are included in this published article and its  online. All code and software used in this manuscript are described and/or available in the Materials and Methods section.Molecular Biology and Evolution online.msaa104_Supplementary_DataClick here for additional data file.


Field information about viruses infecting crops is fundamental for understanding the severity of the effects they cause in plants. To determine the status of cucurbit viruses, surveys were conducted for three consecutive years (2016–2018) in different agricultural districts of Oklahoma. A total of 1331 leaf samples from >90 fields were randomly collected from both symptomatic and asymptomatic cucurbit plants across 11 counties. All samples were tested with the dot-immunobinding assay (DIBA) against the antisera of 10 known viruses. Samples infected with papaya ringspot virus (PRSV-W), watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), and cucurbit aphid-borne-yellows virus (CABYV) were also tested by RT-PCR. Of the 10 viruses, PRSV-W was the most widespread, with an overall prevalence of 59.1%, present in all 11 counties, followed by ZYMV (27.6%), in 10 counties, and WMV (20.7%), in seven counties, while the remaining viruses were present sporadically with low incidence. Approximately 42% of the infected samples were positive, with more than one virus indicating a high proportion of mixed infections. CABYV was detected for the first time in Oklahoma, and the phylogenetic analysis of the first complete genome sequence of a CABYV isolate (BL-4) from the US showed a close relationship with Asian isolates. Cucumis (including cucumbers and melons), Citrullus (watermelons), and Cucurbita (pumpkin sand squashes) are popular. Archeological evidence suggests that pumpkins and squashes (Cucurbita spp) were first cultivated in American continents >10,000 years ago; watermelons were first cultivated in Africa at least 4000 years ago, while cucumbers and melons have been in Asia for > 5000 years [Cucumis melo), cucumbers (Cucumis sativus), honeydew (Cucumis melo), pumpkins, squashes , and watermelons (Citrullus lanatus)—are among the top fifteen fresh vegetables. The value of production from these crops was >two billion US dollars in 2017, which accounts for nearly 13% of the total value of production from fresh vegetables in the US. [The members of the Cucurbitaceae, family, commonly referred to as cucurbits, are major cash crops in the United States (US) and worldwide. Cucurbitaceae includes 15 tribes, 95–97 genera, and approximately 1000 species, which are grown in tropical and sub-tropical regions ,3. Among the US. .Potyviridae. In addition, cucumber mosaic virus (CMV), cucurbit aphid borne yellow virus (CABYV), melon necrotic spot virus (MNSV), squash mosaic virus (SqMV), and cucumber green mottle mosaic virus (CGMMV) have been reported sporadically in cucurbits in different states [Viruses are responsible for causing nearly 50% of emerging diseases in plants , which rt states ,22,23,24Our previous study , conductA wide range of virus symptoms were observed in the cucurbit fields. The most common symptoms were leaf distortion, light and severe mosaics, mottling, and chlorosis A. The otWe collected 1331 samples from >90 cucurbit fields during the three growing seasons. Among the 10 tested viruses, PRSV-W was the most dominant virus infecting cucurbits in Oklahoma, with an average infection rate of 59.1%, followed by ZYMV (27.6%) and WMV (20.7%). Apart from these three potyviruses, the other four viruses were present in low abundance: MNSV (5.7%), SqMV (1.4%), CABYV (1.3%), and CGMMV (1.1%). None of the samples tested positive for the remaining three viruses , and squash leaf curl virus (SLCV)). Samples from all eleven counties were infected with at least one virus. The virus prevalence in different counties ranged from 11.8 % (Atoka County) to 100% (Pontotoc County). The leaf samples collected in Atoka County were infected with only one type of virus, while six different viruses were present in the samples from Muskogee County. PRSV-W was present in all eleven counties, followed by ZYMV in 10 counties, WMV in seven counties, MNSV in five counties, and CABYV and SqMV in three counties, while CGMMV was detected in only one county .Cantaloupes had the highest viral incidence, with 91.0% of the samples infected with at least one of the 10 viruses tested, followed by pumpkins (86.3%), squashes (84.0%), cucumbers (76.5%), and watermelons (48.2%). None of the three samples from gourds tested positive for any of the ten viruses tested. At least six different viruses were detected in pumpkins, followed by cucumber samples, containing five different viruses, while cantaloupes, squashes, and watermelons were infected with four viruses each. The distribution of PRSV-W was that it was dominant in all five major hosts, and its incidence ranged from 43.0% (133 out of 309) in watermelons to 88.06% (59 out of 67) in cantaloupes .The three potyviruses PRSV-W, WMV, and ZYMV were present in all three growing seasons (2016–2018). PRSV-W was the most dominant virus every year, and its incidence increased each year . On the For assessing the viral disease during different growth stages, plants collected during the survey were divided into three groups, namely, the pre-flowering stage , fruiting stage , and harvesting stage . The incidence of viruses in the pre-flowering stage of the plants was 55.6%, followed by the fruiting stage with 67.9%, and by the harvesting stage, the virus incidence reached 85.1%. The incidence of PRSV-W during different growth stages did not differ significantly; however, the incidence of ZYMV gradually increased from 20.3% at the pre-flowering stage to 29.6% at the harvesting stage. Interestingly, the incidence of WMV was zero at the pre-flowering stage but overtook ZYMV at the harvesting stage, with an incidence of 30.6% .In this study, 1023 out of 1331 (76.9%) symptomatic cucurbit samples tested positive for at least one of the 10 tested viruses. Out of the 1023 virus-positive samples, 434 samples were infected with more than one virus, which accounts for 32.6% of the total samples collected. There were as many as 21 different combinations of mixed infections from the field samples based on the dot-immunobinding assay (DIBA) results . A totalo signifying that there was no difference among the 62 sample populations in the distribution of plants in the two categories. The infected samples were not split into those with a single virus versus multiple viruses due to the low expected number of multiple-virus-infected plants in many sample populations. There was a significant difference among sample populations . In order to determine why the significant differences among sample populations existed, the virus distributions were analyzed among the three years of the study (2016–2018), hosts, growth stages, or counties from where they were collected.There were 76 sample populations, of which 14 were eliminated from the statistical analysis because they had less than six samples. All samples were classified as either infected or uninfected by any virus, with HX2 = 239.83, df = 15, p < 0.0001; 2017: X2 = 242.19, df = 9, p < 0.0001; X2 = 282.85, df = 35, p < 0.0001), which indicated that yearly differences did not explain the variation in the overall infection rate. The samples from all five hosts were classified by developmental stage, and within each classification, each sample was either uninfected, infected by a single virus, or infected by two or more viruses. For cantaloupes, cucumbers, and squashes, there were no samples collected during the flowering stage. Each host was tested separately for the independence of the developmental stage and infection category (Ho = independent). For all five hosts except cantaloupes, a significant difference from independent association of developmental stage and infection category existed . The cantaloupes had only six samples in the fruiting stage, which may have accounted for the non-significant result. Similarly, the virus incidence was also analyzed as a product of the spatial location of the hosts by grouping sample populations solely based on the county from where they were collected without regard to the date collected, plant species, or developmental stage. There were eleven groups corresponding to the eleven counties of this study. The samples fell into one of three categories: uninfected, infected by a single virus, or infected by two or more viruses. There was a significant difference in the distribution of viruses in the three categories from different counties . Finally, the predicted numbers against observed numbers generated from the multinomial target theory approach for each year were tested separately to test the possibility of random independent events. Only virus types present in a particular year were used to calculate probabilities. The data from 2016  and 2018  differed significantly from the independent event model. By contrast, the 2017 data did not differ from the model predictions .The sample populations from each year of the survey were analyzed separately for the overall infection rate. All three tests corresponding to three sample-collection years revealed significant differences among the particular year’s sample populations  tree inferred in MEGA7  using deRecombination analysis showed that a total of 61 possible recombination events were detected by all seven algorithms within Recombination Detection Program version 4 (RDP4) . Only thThis study presented three years (2016–2018) of survey data for 10 important viruses infecting major cucurbit crops in Oklahoma. The survey results showed that viruses are common in cucurbits, and potyviruses are a major and continuous threat to cucurbit production in Oklahoma.The symptoms appearing on virus-infected plants afflicted by the three potyviruses were similar, so it was difficult to distinguish the virus type in the field based on appearance, except for ringspot caused by PRSV-W on fruits. Around 77% of those symptomatic plants sampled were positive for at least one of the tested viruses. The remaining samples might have been infected with viruses that were not tested in this study. The symptoms caused by viruses in pumpkins, squashes, and cantaloupes were more severe than those caused by virus infections in cucumbers and watermelons.Our previous study  only focBased on our results, the dynamics of viruses differ among the various cucurbit hosts. Altogether, four viruses  were present in all five major cucurbit crops. CGMMV was only present in cucumbers, and CABYV and SqMV were only present in pumpkins. PRSV-W was dominant in each host, and the distribution of other viruses was significantly different in different hosts. For instance, MNSV was present in 74.6% (50 out of 67) of the samples from cantaloupes but was present with low incidence in other hosts, such as 39.2% (20 out of 51) in cucumbers, 2.6% (4 out of 156) in squashes, and less than 1% incidence in watermelons (1 out of 309) and pumpkins (1 out of 745). Similarly, the incidence of WMV was higher in pumpkins, squashes, and watermelons than cucumbers and cantaloupes . ZYMV waIn 2016, ZYMV was not detected in Caddo, Muskogee, McCurtain, Payne, and Tulsa counties. However, in 2018, the virus was detected in all these locations. Moreover, it was detected in additional counties we surveyed in 2018 including Cherokee, Carter, and Pontotoc. These results suggest that ZYMV is an emerging virus infecting cucurbits in Oklahoma. Although there are only a few alternative hosts that are potential reservoirs or overwintering hosts for ZYMV , it was Aphis gossypii combination resulted in an increase in the vector population by plant enrichment, thereby allowing the swift transmission of the virus in the same field or to nearby fields [The viral infection rates varied significantly among different counties, hosts, collection years, and growth stages. The discrepancy in the infection rates among different counties and collection years might be due to the presence or absence of wild hosts for these viruses and simply due to differences in agricultural practices such as crop rotation and the use of virus-susceptible varieties. The relatively higher infection rates in the four hosts other than watermelons could also be attributed to the larger sizes of the leaves, which would facilitate the aphid trapping. A gradual increase in the infection rate was observed as the plants grew older. After the flowering stages of plants, the plants are more likely to attract aphids, which in turn increase the chances of the transmission of viruses from infected plants to uninfected plants nearby. In addition, as the plants grow larger and cover more area, transmission to and from other plants becomes more likely. It was previously reported that aphid performance and movement are correlated with the age and species of plants . Therefoy fields . In anoty fields .A high proportion of mixed virus infections  was observed among the infected samples. The rate of mixed infections involving three potyviruses was 88.94% (386 out of 434) of the total mixed infections recorded in this study and 37.73% of the total infected samples . Our preMixed infections lead to not only symptom variation but also a change in the pattern of vector transmission and infectivity. In addition, the preference and fitness of vectors can also be changed differently by mixed virus infections compared to single infections . They maThere have been limited studies about the incidence of viruses infecting cucurbits lately, but this study indicates that the distribution of viruses in cucurbits changes over time. The vector population, the inoculum source, the susceptibility of cultivars to a virus, and environmental factors play an important role in the inconsistency of virus incidence at different time points and locations.Despite being geographically distant, the CABYV BL-4 isolate was found to be evolutionarily closer to Asian isolates. Based on evolutionary analyses, CABYV was broadly classified in the Asian and Mediterranean subgroups by Shang et al., 2009 , and in Luteoviridae, recombination is considered a main evolutionary force that shapes their diversity. The intergenic noncoding region (IR) of the Luteoviruses, in particular, is considered a hotspot for genome recombination [In RNA viruses, including bination ,60. Thisbination . The nucIn conclusion, our study has provided further knowledge about and meaningful insights into the epidemiology of various viruses and the fluctuation in the distribution and prevalence of these viruses over time. In addition, the re-emergence of known or emergence of newly reported viruses will also be important for future management studies for a particular crop and locality.Surveys of viruses in cucurbit crops were conducted during three growing seasons (2016–2018) in eight out of nine agricultural districts of Oklahoma , includiThe six cucurbit hosts , depending on their availability in the respective counties, were sampled. Young, fully expanded leaves showing typical virus-like symptoms (symptomatic) and some healthy leaves (asymptomatic) were randomly collected from different cucurbit hosts. The number of leaves sampled from each plant ranged from 2 to 4. The collected leaves were put in individual Ziploc plastic bags, labeled, brought to the laboratory on ice, and processed within 48 h of collection.Samples were prepared and tested with the dot-immunobinding assay (DIBA) as described previously ,61,62. A2, and 0.05% Triton X-100)—for 20 min each. After washing, the membranes were incubated in PBS for 1 h with a goat anti-rabbit IgG-alkaline phosphatase conjugate antibody . The membranes were washed again as described above. Before adding the substrate, all the membranes were briefly washed once in AP 9.5 . The membranes were incubated in AP 9.5 containing 0.33 mg of nitro blue tetrazolium (NBT) per mL and 0.17 mg of 5- bromo-4-chloro-3-indolyl phosphate (BCIP) per mL  in the dark for 5 to 10 min. The reaction was stopped with 10 mM Tris-HCl, pH 7.5, containing 5 mM EDTA. Both positive and negative results were determined visually according to the intensity of the color in comparison with the positive and negative controls.During the membranes’ development, all the blocking and antibody solutions were prepared in PBS, pH 7.4, and contained 600 mM glucose and 600 mM mannose. All incubations were performed at room temperature with gentle agitation on a rocking roller. After the incubation of the membranes with their respective virus-specific polyclonal antibodies, all the membranes were washed three times (10–15 min/wash) in in AP 7.5 .Chi-square tests were used to determine if differences existed in the infection rates among the sample populations from all three years. An infected plant was defined as harboring any of the 10 viruses tested. The sample populations were based on the date of collection (month/year), location (counties), plant host , and developmental stage . Samples from gourds were not included in the analysis due to the low sample population size *(1−P3)*…, the probability of no infection *(1−P2)*(1−P3)*…) and the probability of multiple virus infections –Σ P (infected only by virus i)).Finally, we tested whether the infection of plants by multiple virus types could be explained as the result of random independent events. The multinomial target theory approach was used to generate the expected number, which was used in a chi-square test of whether the phenomenon of multiple virus infections was due to random, independent infection events. For each virus species, the probability that a plant would be infected was calculated by using the observed data without accounting for whether a plant was or was not infected with multiple viruses. These values were used to deduce the probabilities for each of the single infections . Transformed cells were subjected to blue–white screening using Luria–Bertani agar (LBA), carbenicillin, isopropyl-thiogalactopyranoside (IPTG), and X-gal. Three to five clones were selected and sequenced in both directions by Sanger sequencing using an Applied Biosystems 3130 at the Department of Biological Science, the University of Tulsa, Oklahoma. Nucleotide sequences were analyzed using the basic local alignment search tool (BLAST).The confirmation of the three potyviruses  was performed by reverse transcription–polymerase chain reaction (RT-PCR) in at least 10% of the DIBA-positive samples. For WMV, primers from the previous study  were usencbi.nlm.nih.gov). These nucleotide sequences were aligned using ClustalX in MEGA7. The ML and BI trees were built with default parameters in MEGA7 and BEAST, respectively. Both trees were visualized in Figure version 1.4.3.A CABYV BL-4 isolate was collected during the 2017 growing season from Blaine County in Oklahoma and was completely sequenced as described previously . The 45 Recombination events, putative minor and major parents of recombinant isolates, and recombination breakpoints were analyzed using seven methods incorporated in RDP4 with default settings for all 45 complete genome sequences available in the GenBank database. Only recombination events with Bonferroni-corrected P-values less than the cut-off of 0.05 in three or more methods were regarded as recombinants to reduce the false detection of recombination. The recombination events that were indicated by RDP4 as “may be due to factor other than recombination” were also excluded. The breakpoint locations and recombination region were verified individually by using methods that detected the recombination.


Inoculation and application of P as organic manures (Poultry and farm yard manures) improved maize growth and P uptake compared to the control and soils with P applied from mineral sources. Liming adversely affected crop growth, but the use of PSB and organic manure significantly neutralized this harmful effect. Mineral P sources combined with PSB were as effective as the organic sources alone. Furthermore, while single supper phosphate showed better results than Rock phosphate, the latter performed comparably upon PSB inoculation. Thus, PSB plus P application as organic manures is an eco-friendly option to improve crop growth and P nutrition in a calcareous soil under changing climate.Global warming promotes soil calcification and salinization processes. As a result, soil phosphorus (P) is becoming deficient in arid and semiarid areas throughout the world. In this pot study, we evaluated the potential of phosphate-solubilizing bacteria (PSB) for enhancing the growth and P uptake in maize under varying levels of lime  and additional P supplements  added at the rate of 45 mg P Although the global climate has been constantly changing throughout geological earth history, the extent to which current changes occur at a human life scale is dramatic. The global average temperature is estimated to increase by another 2–3 °C by the end of 21st century [2PO4−1, HPO4−2 and PO4−3) are either adsorbed by clay surfaces [Phosphorus (P) is the major growth hampering mineral nutrient next to nitrogen (N) across the globe . In compsurfaces  or form surfaces , thus be−1 [Calcareous soils, typically found in the inceptisols, entisols, alfisols and vertisols soil orders, may also fix a substantial amount of P . Consequ−1 ; therefo−1 . Moreove−1 ,13,14 an−1  compared−1 . However−1 , because−1 . These p−1 , which r−1 ,21. Rock−1 ,24,25,26−1 .3(PO4)2 in calcareous soils. The organic acids chelate cations, like Ca2+, Al3+ and Fe3+ and may increase the bioavailable P [+ protonation [2 may also add to improved soil and plant P nutrition [According to an estimate by Khan et al.  if the rilable P . Phosphailable P , by releilable P , gibbereilable P . Alkalinilable P , H+ prottonation ,35 anionutrition ,39,40. Autrition , maize [utrition  and otheutrition . Thus, pWorldwide reduction in cultivable land by urbanization and industrialization is leading to a food crisis . Food an−1) in nature, with low organic matter (0.8%) content and was deficient in total nitrogen (N = 0.08%), Olsen P (5.3 mg kg−1) and potassium (K = 78 mg kg−1) [A noncalcareous soil containing 4.8% lime (Gulyana soil series) was taken at the 0 to 20-cm soil depth in a field under wheat–maize rotation at the Agricultural Research Station (ARS) Baja Bam Khel (34°6’0N 72°32’0E), Distract Swabi, Khyber Pakhtunkhwa, Pakistan. The soil was a silty loam, alkaline (pH = 7.6) and non-saline (EC = 0.74 dS mmg kg−1) ,47,48.3(PO4)2 as insoluble P [The farmyard manure (FYM) and poultry manure (PM) were collected from the dairy and poultry farms of the University of Agriculture Peshawar, respectively. They were air-dried, screened, sieved (2 mm) and studied for their NPK concentration as prescribed in oluble P . It was oluble P , alkalinoluble P , sideropoluble P  and indooluble P  producti7 CFU of PSB g−1 inocula (Achromobacter (6.6%), Agrobacterium (3.9%), Bacillus (12.2%), Burkholderia (11.5%), Erwinia (10.1%), Flavobacterium (2.9%), Micrococcus (5.8%), Pseudomonas (15.3%), Rhizobia (16.8%), while 15% of the species were unidentifiable (−1), Siderophores , axines (4.7 mg mL−1), organic acids (11 g L−1) as presented in The bacterial population in peat based maize PSB biofertilizer used in this study was 1.5 × 10 inocula . It was tifiable . The ino2O5 kg−1 soil, as per the combination of treatments. Inclusive of the N and K added by organic sources (−1-N and 30 mg kg−1 K2O as urea and sulfate of potassium (SOP), respectively, at sowing time. SSP, urea, SOP and RP were added as solutions for uniform distribution in the soil.This pot study was conducted using a three factor completely randomized design (CRD) in triplicates. These three factors contained two kinds of inoculation (with and without PSB), four different P sources  and four doses of lime , thus, comprising 32 treatments per replication. The soil was sterilized by autoclaving at 121 °C at 1.1 atm  for a minimum of 20–30 min. The soil having 15% (V/M) moisture was filled into 96 pots amounting to 7 ± 0.01 kg soil  in such a way that four sets, each of 24 pots containing 7, 6.6, 6.3 and 5.9 kg of soil were amended with 0, 366, 716 and 1065 g of powdered lime one month before sowing to obtain 4.8 (control), 10%, 15% and 20% (M/M) lime content, respectively. The SSP, RP, PM and FYM were added to the pots at rates of 1.75, 0.82, 10.1 and 16.3 g, respectively for supplementing 45 mg P sources , pots we−1 seeds ha−1) with PSB inocula containing 1.5 × 107 CFU of PSB g−1 inocula (wet weight). A 50 g of seeds for each without and with PSB treatments were soaked for 2 h in sterilized distilled water and a 10% sugar solution, respectively. For PSB inoculation, A 50 g of sugar-soaked maize seeds were treated with 8 g of PSB inocula (at the rate of 2 kg PSB inocula 25 kg−1 seeds ha−1) according to the method used by Alagawadi and Gaur [5 CFU of PSB per maize seed, determined by dilution plate techniques [Seeds of maize variety Azam were sterilized by using 90% ethanol for 3 min followed by 3.5% sodium hypochlorite for 30 min and inoculated  for three factorial CRD  using th−1) and shoot root ratio (S/R) of maize is presented in −1) between 3% and 16% compared to those of the uninoculated control at different concentrations of lime, except for the control lime treatment (4.8%) where statistically insignificant variation was observed with PSB addition. Further increase in the lime content beyond 10%  caused a decrease in both the SB and RB compared to the control. The 15% lime with PSB produced results statistically at par in terms of root and shoot biomass with the 10% lime without PSB . The associative effect of (L × PSB) for S/R revealed that, under control, 10% and 15% lime, the PSB did not perform in a superior manner to the pots without PSB, but at 20% lime, inoculated pots produced a significantly higher S/R compared to those without PSB. Maximum S/R ratios were calculated for with and without PSB inoculation at control lime which were at par to 10% lime with PSB treatment , plant height , shoot biomass (SB) and root biomass (RB)  are shown in −1) were noted in pots amended with PM and FYM, respectively, followed by SSP in each case. The lowest P concentration (0.076%) and uptake (27.6 mg pot−1) were recorded for RP. With the application of lime, at any rate, the P uptake showed a decrease compared to those of the control. Applying lime at a 15% and 20% content reduced plant P concentration by 6% and 21% compared to control treatment, which was not different than lime at 10% content. A decline of 6%, 17% and 39% in the P uptake were calculated at 10%, 15% and 20% lime over control, respectively. The effect of the organic sources (PM and FYM) was superior to that of the mineral sources (SSP and RP) for both P concentration and uptake  and uptake  at 10%, 15% and 20% lime, respectively. Soil Olsen P was significantly affected by the interactive effect of lime and P sources  control. The effect of organic manures (PM and FYM) was superior to that of mineral P supplements (SSP and RP) for most of the studied traits. Additionally, it was observed that liming adversely affected maize growth and P concentration and uptake and induced postharvest soil salinity and calcification. Seed inoculation with PSB was beneficial regardless of the source of P, however, the use of the bacteria was more crucial when P was supplemented from mineral sources, especially as RP. Our findings suggest that PSB inoculation may nullify the negative effects of liming on plant growth and P availability. Thus, it is suggested that P should be applied from organic sources for the improvement of crop yield and P nutrition under saline/calcareous condition. Furthermore, RP can be used as an eco-friendly and economically beneficial substitute to SSP when inoculated with PSB, otherwise, its performance is poorer than SSP in saline soils.


Cajal bodies (CBs) are nuclear components associated with the nucleolus, which play a major role in plant virus infection. Coilin, a marker protein of CBs, is essential for CB formation and function. Coilin contains three domains, the N-terminal, the center, and the C-terminal fragments. Using yeast two-hybrid, colocalization, and bimolecular fluorescence complementation (BiFC) approaches, we show that p2 interacts with the full-length of Arabidopsis thaliana coilin (Atcoilin), the center and C-terminal domain of Atcoilin in the nucleus. Moreover, the N-terminal is indispensable for Atcoilin to interact with Cajal bodies.p2 of  Groundnut rosette virus (GRV) ORF3 protein hijacks CBs as vehicles to enter the nucleolus in virus systemic infection [herpes simplex virus 1 and adenoviruses relocalize coilin and other CB components around viral replication centers [Poa semilatent virus (PSLV) relocalized with coilin in the cytoplasm resulting in the inclusions of various sizes [tobacco rattle virus and tomato black ring virus [Plant viruses invade plants by infecting host cell to complete replication. The replicated viruses then infect more cells to cause virus diseases. The nucleus is a significant cell organelle for virus infection. An increasing number of plant viruses are reported to interact with the nucleus and its compartments. Cajal bodies (CBs) are distinct nuclear bodies physically and functionally associated with the nucleolus . Virus mnfection , 5; herp centers . TGBp1 eus sizes . HoweverRice stripe virus (RSV) is an important rice virus in Asia, causing great rice yield losses [Tenuivirus, but has not been assigned to any family [Laodelphax striatellus Fallén) in a persistent and circulative-propagative manner [d losses , 8. RSV y family . It is te manner . The gene manner . RNA1 ise manner . RNA2-RNe manner . p4 is ae manner ; pc4 is e manner , 15. p3 e manner , and pc3e manner . pc2 shae manner . p2 is ae manner , 20. FibArabidopsis thaliana (Atcoilin) and between p2 and the three functional domains of Atcoilin are identified through yeast two-hybrid (Y2H), colocalization, and BiFC methods. The results indicated that p2 interacts with the full-length Atcoilin, the center and C-terminal domains of Atcoilin in the nucleus, but not interacts with the N-terminal domain.In this study, the interactions between p2 and the full length of coilin from μL of sterile water and diluted in sterile water at ratios of 1 : 1, 1 : 10, and 1 : 100. As shown in Atcoilin contains three functional domains, an N-terminal ordered domain , a central disordered domain , and a C-terminal domain  . To perfAgrobacterium tumefaciens (A. tumefaciens) EHA105 carrying each of these constructs were mixed and infiltrated into the leaves of Nicotiana benthamiana (N. benthamiana). As shown in In colocalization assay, the full-length ORF of p2 was cloned into pEarleyGate102 (CFP) and that of Atcoilin, Atcoilin1, Atcoilin2, and Atcoilin3 into pEarleyGate101 (YFP). A. tumefaciens EHA105 carrying each of these constructs were mixed and infiltrated into leaves of N. benthamiana. As shown in N. benthamiana leaf epidermal cells when YC-p2 individually mixed with YN-Atcoilin, YN-Atcoilin2, or YN-Atcoilin3. However, none can be found in the N. benthamiana leaf epidermal cells coinfiltrated by YC-p2 and YN-Atcoilin1. This confirms the results in Y2H assays, p2 interacts with the full-length of Atcoilin, the central domain Atcoilin2, and the C-terminal domain Atcoilin3, except the N-terminal domain Atcoilin1.YN and YC were also mixed and infiltrated, no fluorescent was detected (data not shown).In BiFC assay, the full-length ORF of p2 was cloned into pEarley201-YC (YC) and that of Atcoilin, Atcoilin1, Atcoilin2, and Atcoilin3 into pEarley201-YN (YN). Coilin has low homology in amino acid sequences in different species, but coilin of different organisms demonstrate a high level of structural similarity, such that their functionalities may be localized into three main domains, the N-terminal, the center, and C- terminal regions; the N- and C-terminal are conserved , 22. As potato virus Y (PVY) and turnip vein clearing virus (TVCV) pathogenicity by facilitating replication of these viruses but also contributes to plant defense against tobacco rattle virus (TRV) and tomato black ring virus (TBRV) [Plant CBs have also been implicated in virus-host interactions since the mechanistic properties of CBs may be hijacked by some viruses for their own benefits. It has been shown that CB components, coilin, and fibrillarin can be used by viruses to mediate virus replication and spread . Coilin s (TBRV) . Some cos (TBRV) , 25. CBss (TBRV) . p2 is as (TBRV) . Our pres (TBRV) . Differes (TBRV) . In thisOverall, the results of this study demonstrate that p2 also interacted with CBs, which are functionally associated with the nucleolus . HoweverN. benthamiana plants were grown and maintained in a green house at 25°C.The Arabidopsis thaliana coilin and RSV sequences (GenBank accession: AY128933 and EF493228) downloaded from GenBank. The full-length and three domain fragments of Atcoilin were firstly inserted into the entry vector pDNOR221 and then destination vectors pEarleyGate101 (YFP) and pEarleyGate201-YN (YN) using Gateway recombination system [The full-length ORF of Atcoilin, cDNA encoding three domains of Atcoilin, and RSV-p2 were amplified, respectively, by PCR using primers in n system . pEarleyFor yeast two-hybrid experiment, PCR products of RSV-p2, full-length of Atcoilin, and three domains of Atcoilin were digested with the suitable restriction enzymes individually, then ligated to the vector pGADT7 or pGBKT7 digested with the same enzymes, creating pGADT7-p2, pGBKT7-Atcoilin, pGBKT7-Atcoilin1, pGBKT7-Atcoilin2, and pGBKT7-Atcoilin3.These constructs were confirmed by sequencing conducted by Takara .pGBKT7-Atcoilin, pGBKT7-Atcoilin1, pGBKT7-Atcoilin2, and pGBKT7-Atcoilin3 were introduced together with pGADT7-p2 or pGADT7. pGADT7-p2 was introduced together with pGBKT7 into the yeast strain AH109 by cotransformation. The cotransformations were selected on different SD mediums lacking tryptophan (Trp) and Leucine (Leu) (SD/Trp-Leu-); lacking histidine (His), Trp, and Leu (SD/Trp-Leu-His-); and last on the SD medium lacking adenine (Ade), His, Trp, and Leu (SD/Trp-Leu-His-Ade-).A. tumefaciens strain EHA105 were grown separately to OD600 = 0.8 at 28°C on Luria-Bertani liquid medium supplemented with 50 μg/μL of rifampicin and 50 μg/μL of kanamycin. The resulting cultures were centrifuged at 12,000 g for 1 min and then resuspended in induction media . In colocalization and BiFC assays, A. tumefaciens containing NbFib2s were separately mixed with p2 in equal volume. The mixtures of the bacterial cultures were incubated at room temperature for 3 h, then infiltrated onto fully grown upper leaves. A six-week-old N. benthamiana was used for the experiment.Subcellular localizations of proteins were monitored at 48 h after infiltration under a confocal microscope (Microsystems CMS GmbH Leica TCS SP5). The fluorophores in CFP and YFP were excited at 458 and 514 nm, and images were taken using BA480-495 and BA535–565 nm emission filters, respectively.


Worldwide, arthropod-borne disease transmission represents one of the greatest threats to public and animal health. For the British Isles, an island group on the north-western coast of continental Europe consisting of the United Kingdom (UK) and the Republic of Ireland, physical separation offers a barrier to the introduction of many of the pathogens that affect animals on the rest of the continent. Added to this are strict biosecurity rules at ports of entry and the depauperate vector biodiversity found on the islands. Nevertheless, there are some indigenous arthropod-borne pathogens that cause sporadic outbreaks, such as the tick-borne louping ill virus, found almost exclusively in the British Isles, and a range of piroplasmid infections that are poorly characterized. These provide an ongoing source of infection whose emergence can be unpredictable. In addition, the risk remains for future introductions of both exotic vectors and the pathogens they harbor, and can transmit. Current factors that are driving the increases of both disease transmission and the risk of emergence include marked changes to the climate in the British Isles that have increased summer and winter temperatures, and extended the period over which arthropods are active. There have also been dramatic increases in the distribution of mosquito-borne diseases, such as West Nile and Usutu viruses in mainland Europe that are making the introduction of these pathogens through bird migration increasingly feasible. In addition, the establishment of midge-borne bluetongue virus in the near continent has increased the risk of wind-borne introduction of infected midges and the inadvertent importation of infected cattle. Arguably the greatest risk is associated with the continual increase in the movement of people, pets and trade into the UK. This, in particular, is driving the introduction of invasive arthropod species that either bring disease-causing pathogens, or are known competent vectors, that increase the risk of disease transmission if introduced. The following review documents the current pathogen threats to animals transmitted by mosquitoes, ticks and midges. This includes both indigenous and exotic pathogens to the UK. In the case of exotic pathogens, the pathway and risk of introduction are also discussed. The threats posed to public health from vector-borne diseases are a subject of considerable investigation, particularly as changes to the climate may increase such threats , 2. LessThe definition of a notifiable disease is any disease that is required by law to be reported to a competent authority, usually governmental. The primary purpose of this, whether from a human or governmental perspective, is to prevent disease spread. In the UK the competent authority for human diseases is Public Health England within the Department of Health. For animals, this is the Department for Environment, Food and Rural Affairs (Defra). Devolution has led to the development of agencies that investigate animal disease on behalf of the devolved governments for example the Scottish Agricultural College (SAC) in Scotland and the Agri-Food Biosciences Institute (AFBI) of Northern Ireland. Veterinary investigations of livestock, poultry and equines are carried out by the Field Services Division of the Animal and Plant Health Agency (APHA). This is supplemented by veterinary services offered by university-associated Veterinary Schools of which there are six in England and two in Scotland. Other organizations offer veterinary support including The Pirbright Institute (Livestock Virology), the Institute of Zoology, and the Animal Health Trust. Domestic pets are usually dealt with by private veterinary surgeons (PVS). Wildlife monitoring, surveillance and health can involve all the above organizations and a large number of charitable bodies.Rhipicephalus sanguineus s.l. ticks, does occur  into southern England, probably through passive transport in cars or lorries . Cx. pipiens is a species complex containing a number of morphologically similar forms with different bionomic properties that influence virus transmission  in Israel  in southwestern Spain (Phasianus colchicus) were also affected during the epizootic. The source of the introduction, presumably from Africa, was not identified and there have been no further reports of disease in Europe, although seroprevalence studies have suggested that the virus continues to circulate in wild bird populations in Spain , the causative virus of ITE, was first reported in 1960 following descriptions of a neuro-paralytic disease of turkeys . Vector competence studies have shown that Culex pipiens mosquitoes from the Netherlands are highly competent to transmit USUV (Disease caused by Usutu virus (USUV) has not been defined into a single disease entity. Infection in birds can lead to a range of disease signs and at necropsy virus is found throughout the organs of the infected animal , while i species . The fir species . Various species . The vir species  and Belg species  often asmit USUV  that doemit USUV . Howevermit USUV . The majTwo further mosquito borne viruses that are implicated in disease in animals have been reported in Europe. Batai virus (BATV) is an orthobunyavirus related to Schmallenberg virus (see below). Repeated isolation of BATV suggest that mosquitoes are a competent vector for the virus , 48 althSindbis virus (SINV) is an alphavirus that is transmitted between birds and ornithophilic mosquitoes. It is one of the most widespread viruses with evidence for its presence in Europe, including Scandinavia, where it causes a mild febrile illness and arthralgia in humans called Ockelbo in Sweden. SINV has also been detected in Asia and South Africa. It is assumed that birds are refractory to disease, although surveillance occasionally detects the virus in bird tissues . There iDifilaria repens and D. immitis  is a zoonotic virus that is found throughout Asia. Like WNV and USUV, the virus persists in a bird-mosquito cycle that can spill-over into human and livestock populations. In contrast to the other viruses, pigs can act as a vertebrate reservoir host for JEV. As its name suggests it causes severe encephalitis in humans often leaving the patient with long term neurological deficit. It is also an economic disease of pigs causing abortion, still-birth and death in piglets . A ranget Europe . A numbet Europe , 61. Howt Europe . Overallt Europe , 63, 64.Rift Valley fever (RVF) is a zoonotic disease of ruminants causing sporadic outbreaks among livestock caused by the Rift Valley fever virus (RVFV). RVF occurs across much of sub-Saharan Africa . TransmiSt. Louis encephalitis virus (SLEV) is a flavivirus that appears to occupy the same ecological niche in the New World that WNV occupies in the Old. Indeed, when WNV emerged in North America in 1999 the initial cases were suspected to be infected with SLEV. Similar to WNV the primary transmission cycle of SLEV is between mosquitoes and birds, although mammals may also contribute. In addition, the virus may cause sporadic outbreaks of human encephalitis throughout North and South America. Phylogeographic investigations have suggested that SLEV emerged in the seventeenth century in Central America and been translocated by bird migration . SerologThe New World also hosts a number of zoonotic alphaviruses that cause encephalitis in humans and horses. These are collectively termed the equine encephalitides and the complex is composed of three viruses: Eastern equine encephalitis virus (EEEV); Western equine encephalitis virus (WEEV); and Venezuelan equine encephalitis virus (VEEV). Each is widely distributed, transmitted by a range of mosquito species and all viruses cause severe disease in equids and humans . As withTembusu virus (TMUV) was first isolated in mosquitoes in Malaysia in 1955, and subsequently shown to cause encephalitis and growth retardation in chicks . Birds aAedes albopictus) in particular has been a major factor in outbreaks of chikungunya virus in Italy, and repeated outbreaks of dengue fever in southern France     and the unctata) .Lagopus lagopus scoticus) to infection (Louping ill virus (LIV) is the only indigenous tick-transmitted virus present in the UK. The disease results from viral encephalomyelitis, mainly affecting sheep, which show signs of neurological impairment including incoordination, altered gait and ataxia. Other mammals can be infected although such cases are rare. Of further economic significance is the susceptibility of red grouse  can increase the severity of disease in sheep . In cattle the common name for the disease in the UK is redwater fever. Overt signs include a rapid onset fever reaching 41°C and non-specific signs including anorexia, depression and weakness. Death can result from hepatic and respiratory complications, and renal congestion caused by deposition of hemoglobin in the renal tubules. Following recovery, low levels of infection may be maintained within erythrocytes of affected animals for a number of years without signs of clinical disease and which may form a reservoir of infection for feeding ticks. Calves below 9 months of age demonstrate an innate, inverse, age related resistance, unrelated to maternal immunity, and do not suffer clinical disease.Babesiosis is a tick-borne intraerythrocytic protozoan disease that affects mammals and is caused by species within the genus Babesia spp. only infect female ticks following blood feeding on infected animals and the parasites are transmitted via transovarial transmission to the next larval generation and subsequently to nymphal and adult ticks via transstadial transmission. Thus, at least one complete generation of ticks may be infected and are capable of transmitting the disease to naïve animals. Globally, the most significant species causing babesiosis in cattle are B. bigemina and B. bovis   . TreatmeBabesia caballi or Theileria equi infection. Historically, the UK has been considered free of equine piroplasmosis despite the presence of seropositive and pathogen positive horses resident within the country  to produce merozoites which are released to invade host erythrocytes where further division occurs. Three species of Theileria cause significant economic impact on cattle farming worldwide, T. parva (East Coast Fever), T. annulata , and T. orientalis . Clinical bovine theileriosis is mainly reported from The Middle East, Africa, Asia . As the common disease name suggests, infection presents as a fever and anorexia. There have been repeated reports of tick-borne fever in dairy herds in the UK  causes subclinical disease, whereas infection in domestic pigs can be devastating with mortality reaching 100% in some cases (A significant emerging threat to the pig production industry has been the emergence of African swine fever virus (ASFV) in Europe. The virus evolved in Africa where it is transmitted by soft ticks within the genus me cases . ASFV wame cases , 141. InHyalomma ticks to livestock that can become infected, but do not show signs of disease. The main risk is to humans that have contact with infected meat or milk, as this may lead to fatal haemorrhagic fever  and Nairobi sheep disease virus (NSDV). The former is transmitted by ic fever . CCHFV hic fever . Nairobie active . A variaRh. sanguineus  are the vectors of a number of significant diseases of livestock including bluetongue virus and African horse sickness virus (AHSV). Species within the genus are small, ranging from 1 to 3 mm in length and so morphological identification can be challenging, and with over 1,000 species within the genus a comprehensive classification is not currently available . Ruminants are susceptible to disease with cattle presenting with elevated temperature and congestion of, and discharge from, the mucous membranes. This can develop into crusts and erosion of the nasal and oral mucosa. Animals can become lame due to coronitis, inflammation of the coronary band above the hoof, and ulceration of the teats can occur. Transplacental transmission can lead to congenital deformities and developmental defects in live births  was first reported in a herd of cattle in Germany experiencing a drop in milk yield and diarrhea . Infecti England . Althoug England  or detec England .C. imicola, a species absent from northern Europe. The disease is caused by another Orbivirus, African horse sickness virus (AHSV) that is endemic in tropical and sub-tropical regions of Africa. Outbreaks in Europe have occurred, most notably the introduction of AHSV in a consignment of zebras brought into a safari park near Madrid in 1987 causing the disease to persist in the Iberian Peninsula until 1990 . Infection causes transient fever with ocular and nasal discharge, depression and recumbency . Severe ustralia . Bovine ustralia –170. The. Phlebotomus sandflies transmit Leishmania infantum to dogs causing canine leishmaniosis in southern Europe. The absence of sandflies in the UK means that there is no vector borne transmission, although infected dogs are imported (Hyalomma and Rhipicephalus spp., vectors of viruses, such as CCHFV and NSDV, respectively, could lead to the introduction and establishment of these diseases. In the case of Hyalomma species, ticks are being introduced by migrating birds but have so far failed to establish a detectable reproductive population. Changes to the climate may make the UK more permissive for such species. Increases in a range of parameters will influence vector populations. For example, an increase in mean temperatures throughout the year will extend the period over which vectors are active and lead to extended periods where temperatures are permissive for virus replication, which may promote vector competence. Another area of uncertainty is the ability of pathogens to survive the winter. An increase in midwinter temperatures will promote vector survival and increase the probability that infected vectors will enable overwintering of pathogens. Critically, WNV is successfully overwintering in Central Europe, which is driving repeated outbreaks of disease and increasing its distribution. For anthropophilic mosquitoes, such as Ae. albopictus, climate modeling suggests that only the southeast of England has suitable climatic conditions for the mosquito species to establish, coincidentally the one part of the UK where Ae. albopictus has been detected. The current trends in climate change, particularly to the daily temperature range will increase the areas of the UK that can accommodate the species (This review has highlighted a large number of pathogens that infect animals in the UK, and others that are at risk of introduction . A strikimported  and therimported . The int species .Ae. albopictus into southern England may have been introduced by cars or lorries entering the country. Whilst the introduction of an anthropophilic mosquito species may not change the risk of disease transmission to animals, the addition of an invasive tick may be more significant, particularly as there are already tick-borne diseases active in the UK. The emergence of the Asian long-horned tick, Haemaphysalis longicornis, in North America is a dramatic demonstration of how quickly an invasive tick species can establish in a new environment. As its common name suggests, the tick is a native of East Asia and had been repeatedly intercepted on quarantined livestock entering the United States. However, in 2017 it was detected in a sheep flock in New Jersey (In addition to climate change, human action in activities, such as international travel by air, livestock movements and conversion of land to agricultural use can lead to the movement of disease vectors , 176. Thw Jersey . Subsequw Jersey . Of signw Jersey  and Theiw Jersey , in addiCulex modestus in the UK, is an example of this (The implication of these observations are that surveillance for the introduction and spread of invasive arthropod species is necessary to offer an opportunity to prevent establishment and to predict at-risk areas well before a pathogen is introduced. Allied to this is an understanding of the assemblage, behavior, ecology and abundance of indigenous vectors. The monitoring of the mosquito species  of this . Genetic of this . The pop of this . In addi of this , 185.A key driver in the application of surveillance of livestock, domestic pets, and wildlife is to detect disease before there is widespread transmission of disease. This reduces the risk of spill-over of some diseases into the human population, ameliorates the economic impact of the outbreak to industry, reduces potential harm to domestic animals and limits challenges to biodiversity within wildlife. The cost of surveillance needs to be proportionate to the risk and balanced against the cost of an outbreak. The estimated cost to the agricultural sector as a result of the 2001 outbreak of foot and mouth disease in the UK was £3.1 billion with the tourism sector being equally affected . It is uNJ conceived the idea. AF, DD-R, LH-T, LP, and NJ wrote 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.


Bombus terrestris as a model. We apply a reverse genetics approach combined with controlled laboratory conditions to detect and monitor DWV infection. A novel reverse genetics system for three representative DWV variants, including the two master variants of DWV—type A and B—was used. Our results directly confirm DWV replication in bumble bees but also demonstrate striking resistance to infection by certain transmission routes. Bumble bees may support DWV replication but it is not clear how infection could occur under natural environmental conditions.Deformed wing virus (DWV) is a persistent pathogen of European honey bees and the major contributor to overwintering colony losses. The prevalence of DWV in honey bees has led to significant concerns about spillover of the virus to other pollinating species. Bumble bees are both a major group of wild and commercially-reared pollinators. Several studies have reported pathogen spillover of DWV from honey bees to bumble bees, but evidence of a sustained viral infection characterized by virus replication and accumulation has yet to be demonstrated. Here we investigate the infectivity and transmission of DWV in bumble bees using the buff-tailed bumble bee  Apis mellifera. In synergistic action with its vector—the parasitic mite Varroa destructor—it has had a devastating impact on the health of honey bee colonies globally2. As the primary managed insect pollinator, honey bees are of high ecological and economic value and contribute an estimated 30–50% of mobile pollination activity. This is carried out by seasonal transportation of honey bee hives between agricultural areas requiring pollination services4. For example, two-thirds of all colonies in the USA (~ 1.6 million hives) are transported to California in February/March for almond pollination5. Inevitably, transporting bees also transports their pathogens. This, coupled with the local pathogen density associated with ~ 50 000 bees in a single hive, has raised concerns about pathogen spillover from managed honey bees to other pollinators6. DWV was found as a frequent component of pollen pellets7 and is also present in bee faeces8, suggesting honey bee foragers and colonies could facilitate horizontal virus transmission to the wider pollinator community. Significantly, DWV RNA has been detected in many insects sharing the environment with managed honey bees, including Asian bee species, solitary bees, bumble bees, wasps, cockroaches and ants21. Due to their extended activity at lower temperatures (compared to honey bees) bumble bees are considered particularly important pollinators in temperate and subarctic climates23 and are also reared and managed for commercial-scale pollination23. As a consequence, the potentially negative impact of extensive honey bee management and failing pathogen control on co-located Bombus species has received significant attention.Deformed wing virus (DWV) is a widely established pathogen of the European honey bee, Bombus terrestris and Bombus pascuorum individuals with deformed wings9 there have been several studies of DWV prevalence in a wide range of Bombus species27. In Varroa-infested honey bee colonies DWV levels can exceed 1011 genome copies per bee28, with considerable potential for environmental contamination. DWV-positive Bombus sp. have been shown to correlate to areas with high DWV prevalence in Apis27. The majority of screening studies have used end-point polymerase chain reaction (PCR) for detecting DWV RNA in environmental bumble bee samples (reviewed in29). However, the near ubiquitous presence of managed hives, the honey bee—and consequently pathogen—density around hives, and the foraging range of Apis mean that DWV is likely widespread7. Although detection of the negative strand intermediate of replication is regarded as a marker of DWV replication, quantitative analysis of virus amplification in the infected tissues is required to unequivocally demonstrate infection and replication.Following a report that DWV was detected in symptomatic 30, Kakugo virus31, Varroa destructor virus-1  and a range of recombinants between DWV-A and -B37. All viruses exhibit at least 84% identity at the nucleotide level and 95% identity at the protein level38. The sensitivity of current diagnostic methods means DWV detection in environmental samples is regularly reported, with different DWV variants identified in Bombus6. Far less frequent are studies investigating potential routes of transmission from Apis to Bombus, or the subsequent replication of DWV in bumble bees. Due to the absence of a suitable cell line for in vitro propagation, laboratory-based assays have been limited to the application of field-sourced virus. Infectivity of DWV obtained from field honey bee samples was tested via inoculations of adult Bombus terrestris6. It was reported that a DWV complex containing both DWV-A and -B is infectious when fed at high concentrations—109 GE  of virus per bee.The name DWV is currently attributed to an evolving complex of closely related viruses, which includes DWV-A41. Using RG-derived DWV inocula we address the question of DWV pathogenesis and likely transmission routes in Bombus terrestris at both the individual and colony level. Using this strategy we provide direct evidence of DWV replication in bumble bees via virus feeding and injection. Importantly, adult Bombus terrestris appear resistant to productive infection by DWV orally and do not exhibit the developmental defects characteristic of DWV infection and replication in honey bees.We have used a reverse genetic (RG) approach to generate near-clonal populations of genetically tagged DWV-A, -B and a B/A recombinant after transfection of honey bees with in vitro transcribed RNA. A similar system has recently been reported for DWV-A42. Full-length viral RNA was prepared in vitro and directly injected into honey bee pupae from which near-clonal infectious virus was recovered. Incorporation of synonymous mutations, which create new restriction sites in each RG DWV genome, allow unambiguous distinction from wild type virus genomes. The following nomenclature was used for the generated viruses: “VDD”—type A DWV (type A polyprotein-coding part only), “VVV”—type B, “VVD”—B/A recombinant 43, attempted infection of those cells with DWV or transfection of in vitro-generated DWV RNA does not result in virus replication . This impediment to DWV studies prompted us to investigate the recovery of clonal stocks of DWV in bumble bees (Bombus terrestris audax) injected with viral RNA. Commercially farmed bumble bee colonies are available year-round and, in our preliminary studies, are free of DWV RNA (data not shown). Four of six RNA-injected bumble bee pupae tested positive for DWV, while all honey bee pupae injected with full-length RNA were shown to be DWV-positive  and Bombus pupae had no detectable DWV sequences in mock-inoculated samples.In temperate climates honey bee brood is only available for ~ 50% of the year and in vivo research is of necessity seasonal. Although the internal ribosome entry site of DWV retains partial activity in at least one cell line of non-honey bee origin  or brown-eyed (P7-P8) bumble bee pupae were injected with 103 or 106 GE of each DWV variant, and virus levels quantified 48 h post-inoculation. In all cases we observed a 102–104 increase in total DWV load compared to the innocula, providing clear evidence for replication .White-eyed .Groups of adult bumble bees received 10PCR Fig. . Accordi8 GE/adult bee/day of VVD, VVV or VDD DWV. All brood  and 30 randomly selected adults from virus-exposed nests were screened for DWV using end-point PCR assay and showed no positive results (data not shown).We extended this study to investigate whole-nest inoculation with DWV. Three individual bumble bee nests were fed for 4–6 weeks with a sucrose solution supplemented with 2 × 104 or 108 DWV GE per bee. Envisaging a possible impact of injection on viability, an additional group of bumble bees was injected with phosphate buffered saline (PBS) only. 20–30% of injected bees died before termination of the experiment in each group. The remaining bumble bees were screened for DWV RNA 8 days post-inoculation. End-point PCR analysis indicated that 40% and 100% were DWV positive in the 104-injected and 108-injected groups respectively RNA in DWV-positive samples was confirmed by strand-specific PCR assay  bumble bee pupae with 106 GE of DWV and maintained them through development in an incubator. In parallel, a group of 37 similarly-aged pupae were injected with PBS. All fully-developed bumble bees could be classified into one of three groups: phenotypically normal, discolored with normal wings or non-viable, which did not eclose  with no significant differences in viral load between the three phenotypic groups   and a hybrid previously reported to predominate in 9 GE of DWV/μg of RNA two days post-injection  needed to achieve 100% infection.Although we found no evidence for oral infection of adult bumble bees with DWV, we were able to demonstrate the presence of DWV in adult B. terrestris and B. pascuorum have been identified with wing deformities characteristic of those seen in DWV-infected honey bees9. However, these studies did not demonstrate DWV replication or reproduce symptoms by DWV inoculation. In honey bees apart from DWV infection, wing malformation can be caused during development by injury, hormonal disorders, intoxication or absence of cocoon39.In this study we allowed pupae to complete development and scored them phenotypically upon eclosion. Bumble bee pupae are susceptible to handling and survival rates (~ 50%) were similar in virus- or mock-inoculated samples Fig. . Amongst53. For example, exposure to clothianidin—a neonicotinoid insecticide—was found to have a negative impact on honey bee immune status and promote DWV infection54. In Bombus terrestris condition mediated virulence of Slow bee paralysis virus upon starvation has been demonstrated55. Our study uses a reverse genetic approach to investigate pathogen spillover from honey bees to bumble bees. Whilst clear evidence is obtained to confirm DWV replication in bumble bee larvae, pupae and adults, we were unable to demonstrate a compelling route by which transmission would likely occur in the natural environment. The levels of virus required to orally infect bumble bee larvae are significantly higher than have been reported in environmental pollen samples56. Indeed, the levels required are likely higher than larvae are ever exposed to. In contrast, adult bumble bees may experience very high virus levels in collapsing honey bee colonies while robbing. However, adult bumble bees feeding on virus-supplemented syrup remain uninfected and—importantly—were unable to transmit infectious virus to the developing larvae. Further studies will be required to determine whether the gut environment of adult bumble bees is sufficiently hostile to DWV that the virus is inactivated e.g. by gut proteases, or if there are other factors that restrict infection and replication of DWV in bumble bees.While laboratory inoculations represent the gold standard for virus infectivity assays this approach probably does not recapitulate an environmentally meaningful infection route. Compared to their independent effect a combination of stressors is suggested to introduce a greater threat to wild pollinators in their native environmentApis to otherwise healthy Bombus individuals. Bumble bees are known to carry their own mite parasites, such as Locustacarus buchneri, which infest the air sacs of adult bumble bees and feed via piercing the tracheal wall57, however, these mites do not parasitise on honey bees. Other mites found on Bombus lack feeding behaviour similar to Varroa and hence the capacity for virus vectoring during feeding. Non-Acari parasites of bumble bees such as protozoans Apicystis bombi and Crithidia bombi were shown to be present in honey bee collected pollen58, but no evidence of their ability to transmit viruses between bee species has been reported. Therefore, a route for productive DWV spillover to bumble bees from infected honey bees remains to be determined. Previous reports have emphasised the haplotype identity of DWV between honey bee and bumble bee populations27. We propose that the detection of DWV RNA in geographically co-located bumble bees may reflect environmental contamination from the abundant honey bee population without necessarily supporting DWV replication in the bumble bee population. Further studies on defining the stressors that may account for DWV infection of bumble bees are required in order to estimate the actual impact of DWV on this important group of pollinators.The results obtained from this study provide a strong indication that oral acquisition of virus from a contaminated environment does not represent an effective DWV transmission route from Apis mellifera) brood were collected from the University of St Andrews research apiary. All hives used for sampling were routinely treated for Varroa with an appropriate miticide. Pupae were extracted from the comb and maintained in the incubator set at 34.5 °C with 90% relative humidity.All honey bee  were maintained in the laboratory in isolated nests supplemented with feeders containing 50% aqueous sucrose solution. Nests were regularly fed with bee pollen from a DWV-free region . For adult inoculation experiments newly eclosed  or mixed-age workers were selected at random from established nests in groups of 20–25 and maintained at RT in separate cages with ad libidum feeding on pollen/syrup. Prior to inoculation each group was left to recover for 24 h to account for any mortality caused by the handling. Pupae and larvae were extracted from the brood cells and transferred into the incubator set at 30.5 °C with 90% relative humidity. Larvae were fed a diet consisting of 25% (v:v) ground pollen and 25% sucrose solution (w:v) in H2O.Bumble bees 42.All RG constructs in this study were based on a cDNA clone of a recombinant DWV variant VDV-1-DWV-No-9 (GenBank HM067438.1) and described inPme I (cuts at the end of poly-A tract) or Nru I (cuts at nucleotide position 9231 located within the sequence encoding the virus polymerase) respectively. Truncated RNA transcripts were polyadenylated using the poly(A) Tailing Kit (Thermo Fisher Scientific) to ensure their stability. After purification, RNA transcripts were eluted in RNAse free H2O, their integrity confirmed by gel electrophoresis, and stored at − 80 °C.Viral RNA was generated in vitro from linearised plasmid templates, using the T7 RiboMAX Express Large Scale RNA Production System (Promega). Full length and truncated templates were linearised with 12 of DWV RNA copies) of in vitro transcribed RNA was injected individually into pupae. Truncated VVD transcript injections were used as a negative control. All RNA-injected pupae were analyzed at 72 h post injection. Injections of pupae or low temperature-immobilized adult bees with virus stocks diluted in PBS were performed using the same technique as for RNA transcripts.RNA and virus injections of 5 and 10 μl were performed with insulin syringes  into honey bee and bumble bee pupae respectively. Up to 10 μg  ratio and centrifuged at 13 000 g, 4 °C for 10 min. Supernatant was filter sterilized with 0.22 μM filter  and treated with RNase A to destroy all unencapsidated RNA. RNA was extracted from 100 μl of the virus stock using RNeasy kit (Qiagen) and analyzed by reverse transcription followed by qPCR. Apart from the virus inoculum used for testing the infectivity of the Bombus-derived DWV, all virus stocks were prepared from RNA-injected honey bee pupae.DWV stocks were prepared from pupae injected with in vitro transcribed RNA as described earlierIndividual bumble bee larvae were placed into 96 well plates containing 2 μl of pollen/syrup mixed with DWV inoculum. DWV feeding was delivered once on the first day of the experiment. Fresh diet without virus was added individually after virus-containing food had been consumed. On the fifth day larvae were snap-frozen in liquid nitrogen and stored at − 80 °C.9 and 2.2 × 108 DWV GE for 108 and 107 groups respectively. Bumble bees were snap-frozen in liquid nitrogen and analyzed for the presence of DWV RNA 7 days after DWV feeding.Adult bumble bees were fed with virus in groups of 20 on the first and on the second day of the experiment. Total amount of DWV supplied with each of the two virus feedings was 2.2 × 1010 DWV GE. The virus-containing solution was fully consumed (< 3–4 h) before replacement with the regular sucrose feeder. DWV feeding continued for 4–6 weeks and stopped when the first group of larvae developed during the virus-feeding period approached pupation. Upon termination of the experiment all brood  and 10 adult workers were sampled from each nest and tested for the presence of DWV.For the colony-scale inoculations three bumble bee nests each containing a mated queen and 120–125 adult bees were used. Prior to the start of the experiment all brood was removed from the nests. Virus feeding was delivered daily by replacing the nest feeder with a tube containing 2 ml of sucrose solution supplemented with DWV, previously confirmed to be infectious for bumble bee pupae by direct injection. Each nest received a daily dose of the virus corresponding to 2.4 × 10Taq DNA polymerase (New England Biolabs) and 2 μl of cDNA. DWV_RTPCR primers were designed to detect all three DWV variants under study (Table Samples were homogenized with a Precellys Evolution homogenizer (Bertin Instruments). Total RNA was extracted using the GeneJet RNA Purification Kit (Thermo Fisher Scientific). cDNA was prepared with qScript cDNA Synthesis Kit (Quanta Biosciences) from 1 μg of total RNA following the manufacturer’s protocol. Detection of DWV and actin mRNA, used as an internal RNA quality control, was carried out by end-point PCR with dy Table . To amplThe quantification of DWV genome copies was performed by SYBR-Green Real-Time Quantitative PCR using Luna Universal qPCR master mix (New England Biolabs), 0.25 μM forward and reverse DWV_qPCR primers and 2 μl of cDNA. The following thermal profile was used: 1 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C with a final post-amplification melting curve analysis step. DWV titres were calculated by comparison of the resulting Ct value to the standard curve generated from a serial dilution of the VVD cDNA qPCR standard prepared by reverse transcribing the RNA transcript.Taq DNA polymerase (New England Biolabs) using forward primer 388 identical to the adapter sequence  and the adapter extended primer designed to anneal to the negative strand RNA of DWV. The PCR step was carried out by ce Table  and DWV_Taq polymerase (New England Biolabs) following standard protocols and DWV FG RP1 and FP4 primers to produce a single ~ 10 Kb PCR fragment spanning the DWV genome. Following PCR purification  samples were sequenced using an Illumina Hi-seq. Paired-end reads were processed at the University of St Andrews and sequences were analyzed for genetic diversity using ShoRAH60. Phylogenetic analysis was performed using Geneious Prime 2019.1.3.Gene specific cDNA was generated from selected samples using SSIII reverse transcriptase (Invitrogen) and DWV FG RP1 primer Table . The cDNSupplementary file1