Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses. Drosophila melanogaster is one of our foremost models for antiviral immunity in arthropods  dsDNA viruses that are best known as pathogens of Lepidoptera and Coleoptera , two densoviruses (‘Viltain virus’ and ‘Linvill road virus’), and two segments of a putative bidnavirus (‘Vesanto virus’). Here we expand our sampling to encompass 167 short-read pool-sequenced samples from a total of 6,668 flies, collected seasonally over 3 years from forty-seven different locations across Europe. We use these population genomic data as a metagenomic source to discover additional DNA viruses, estimate their prevalence in time and space, and quantify levels of genetic diversity.It not only remains an open question as to whether the Drosophila-associated nudiviruses, fragments of a novel hytrosavirus most closely related to Musca domestica salivary gland hypertrophy virus, fragments of a filamentous virus distantly related to Leptopilina boulardi filamentous virus, and three polinton-like sequences related to adintoviruses. Our improved assemblies and sampling show that Vesanto virus may be composed of up to twelve segments, and appears to represent a new distinct lineage of multi-segmented ssDNA viruses related to bidnaviruses. We find that two viruses  are relatively common in European D.melanogaster, but that the majority of DNA viruses appear very rare—most probably appearing once in our sampling.We complete the genome of a novel and highly divergent entomopoxvirus, identify a further three Drosophila were collected across Europe by members of the DrosEU consortium between 19 June 2014 and 22 November 2016, using yeast-baited fruit (Drosophila flying season for that location). After morphological examination to infer species identity, a minimum of thirty-three and maximum of forty male flies (mean 39.8) were combined for each site and preserved in ethanol at −20°C or −80°C for pooled DNA sequencing. Male flies were chosen because, within Europe, male D.melanogaster should be morphologically unambiguous. Nevertheless, subsequent analyses identified the occasional presence of the sibling species D.simulans, and two collections were contaminated with the distant relatives Drosophila phalerata and Drosophila testacea (below). Full collection details are provided via figshare repository 10.6084/m9.figshare.14161250, and the detailed collection protocol is provided as supporting material in A total of 6,668 adult male ed fruit , 2021. TTo extract DNA, ethanol-stored flies were rehydrated in water and transferred to 1.5-ml well plates for homogenisation using a bead beater (Qiagen Tissue Lyzer II). Protein was digested using Proteinase K, and RNA depleted using RNAse A. The DNA was precipitated using phenol-chloroform-isoamyl alcohol and washed before being air dried and re-suspended in TE. For further details, see the supporting material in https://community.nanoporetech.com).To improve virus genomes, and following an initial exploration of the Illumina data, we pooled the remaining DNA from four of the collections  for long-read sequencing using the Oxford Nanopore Technology ‘Minion’ platform. After concentrating the sample using a SpeedVac (ThermoFisher), we prepared a single library using the Rapid Sequencing Kit (SQK-RAD004) and sequenced it on an R9.4.1 flow cell, subsequently calling bases with Guppy version 3.1.5 , and sixteen microbial eukaryotic genomes . All mapping was performed using Bowtie 2 version 2.3.4 or version 2.4.1  we identified a number of short contigs from other taxa, including additional species of Drosophila, Drosophila commensals such as mites and nematodes, and potential sequencing contaminants such as humans and model organisms. To quantify this potential contamination, we re-mapped all trimmed read pairs to a reference panel of short diagnostic sequences. This panel comprised a region of Cytochrome Oxidase I (COI) from each of the following: twenty species of Drosophila ; 667 species of nematode ; 106 parasitic wasps (including many lineages commonly associated with Drosophila); two species of mite (identified in de novo assemblies); six model vertebrates; and complete plastid genomes from eight crop species. Because cross-mapping between D.melanogaster and D.simulans is possible at many loci, we also included a highly divergent but low-diversity 2.3 kbp region of the single-copy gene Argonaute-2 to estimate levels of D.simulans contamination. Where reads indicated the presence of other Drosophila species, this was further confirmed by additional mapping to Adh, Amyrel, Gpdh, and 6-PGD. A full list of the reference sequences is provided in via figshare repository 10.6084/m9.figshare.14161250.During manual examination of Drosophila microbiome’ reference (above) and used these for de novo assembly using SPAdes version 3.14.0 with the default spread of k-mer lengths . We performed normalisation and assembly separately for each of the 167 samples. We then used the resulting scaffolds to search a database formed by combining the NCBI ‘refseq protein’ database with the viruses from NCBI ‘nr’ database. The search was performed using Diamond blastx  with an e-value threshold of 1 × 10−30, permitting frameshifts, and retaining hits within 5 per cent of the top hit.To identify samples containing potentially novel viruses, we retained read pairs that were not concordantly mapped to the combined ‘ lengths , after iDrosophila, such as mites and fungi, as likely to be viruses of those taxa. Our remaining candidate virus list included known and potentially novel DNA viruses, and one previously reported Drosophila RNA virus. For each of these viruses we selected at least one representative population sample, based on high coverage, for targeted genome re-assembly.The resulting sequences were examined to exclude all phage, retroelements, giant viruses (i.e. mimiviruses and relatives), and likely contaminants such as perfect matches to well-characterised plant, human, pet, and vertebrate livestock viruses . We also excluded virus fragments that co-occurred across samples with species other than de novo SPAdes assembly for each target sample (as above), but to aid scaffolding and repeat resolution we additionally included the long reads  that had been generated separately from UA_Yal_14_16, ES_Gim_15_30, UA_Ode_16_47 and UA_Kan_16_57. We examined the resulting assembly graphs using Bandage version 0.8.1 . Again, we examined targeted assembly graphs using Bandage , and recorded the best-mapping location for each read, as above. Short reads and low complexity regions allow some cross-mapping among the larger viruses, and between viruses and the fly genome. We therefore chose an arbitrary detection threshold of 250 mapped reads to define the presence of each of the larger viruses (expected genome size >100 kbp) and a threshold of twenty-five reads for the smaller viruses (genome size <100 kbp). Consequently, our estimates may be conservative tests of virus presence, and the true prevalence may be slightly higher. We additionally selected a subset of the public datasets for de novo assemblies of Drosophila Vesanto virus , an adintovirus (SRR3939056), and galbut virus (SRR5762793 and SRR1663569), using the same assembly approach as outlined for DrosEU data above.To detect all known and novel Bowtie 2 . Among tme Nexus , the Drome Nexus , pooled me Nexus , evolve-me Nexus , studiesme Nexus , and intme Nexus . In totaDrosophila and representative viruses of other species, we selected a small number of highly conserved virus protein-coding loci that have previously been used for phylogenetic inference. For densoviruses we used the viral replication initiator protein, NS1 . We used the SDR06 substitution model  of galbut virus  to the virus genomes. This approach means that low complexity reads map initially to the fly and microbiota, and are thus less likely to be counted or mis-mapped among viruses. This slightly reduces the detection sensitivity (and counts) but also increases the specificity. We mapped using Bowtie 2 (Drosophila (combined D.melanogaster and D.simulans reads) to provide an estimate of virus genomes per fly genome in each pool. To quantify Vesanto virus genomes we excluded terminal inverted repeats from the reference, as these may be prone to cross-mapping among segments.To quantify the (relative) amount of each virus in each pooled sample, we mapped read pairs that had not been mapped concordantly to the Bowtie 2 , recordin flies constituted n Bernoulli trials in which the presence of virus reads indicated at least one infected fly ; the amount of Wolbachia (measured as reads mapping to Wolbachia as relative to the number mapped to fly genomes); the sampling season (early or late); and the year . We included sampling location as a random effect, to account for any additional non-independence between collections made at the same sites or by the same collector. The inclusion of a spatially distributed random effect was supported for Drosophila Kallithea nudivirus and Drosophila Linvill Road densovirus, but this did not vary significantly with year. Map figures were plotted and model outputs summarised with the R package ggregplot , and all code to perform these analyses is provided via figshare repository 10.6084/m9.figshare.14161250.In reality, pools are not independent of each other in time or space, or other potential predictors of viral infection. Therefore, for the three most prevalent viruses  we analysed predictors of the presence and absence of each virus within population pools using a binomial generalised linear mixed model approach. We fitted linear mixed models in a spatial framework using R-INLA , taking http://broadinstitute.github.io/picard/). Finally, we excluded samples that had a read-depth of <25 across 95 per cent of the mapped genome.Reads that had initially been mapped to Drosophila Kallithea nudivirus, Drosophila Linvill Road densovirus and Drosophila Vesanto virus (above) were remapped to reference virus genomes using BWA-MEM with local alignment . For theIn addition to calculating per-sample diversity, to calculate total population genetic diversity we created single global pool, representative of diversity across the whole population, by merging sample bam files for each virus or segment haplotype. To reduce computational demands, each was down-sampled to an even coverage across the genome  and no sample contributed more than 500-fold coverage. To produce the final dataset for analyses, bam files for the global pool and each of the population pools were re-aligned around indels using GATK v3.8 . We creaS) and non-synonymous (πA) sites we identified synonymous and non-synonymous SNPs using popoolation . We did this separately for each gene in the merged global pool, and also for the whole genome in each infected population pool.To calculate average pairwise nucleotide diversity at synonymous , and examined the assembly graphs manually using bandage  insertions and structural rearrangements , and oft bandage , retaini bandage . This apD.melanogaster TEs curated in the November 2016 version of Repbase ; other mapped bacterial reads together were 0.6 per cent (0.0–3.2%), and microbial eukaryotes were 0.3 per cent (0.0–3.7%). The eukaryotic microbiota included the fungal pathogen E.muscae (e.g. Herpetomonas muscarum (e.g. Tubulinosema ratisbonensis (e.g. Candida (Clavispora) lusitaniae . For a detailed assessment of the microbial community in the 2014 collections, see 10.6084/m9.figshare.14161250 File S3, and raw data are available from the Sequence Read Archive under project accession PRJNA388788.On average, 93 per cent of reads (range 70–98%) could be mapped to cae e.g. , with rerum e.g.  with reasis e.g.  in one sDrosophila, such as nematodes, mites, or parasitoid wasps. By mapping all reads to small reference panel of COI sequences, we identified thirteen samples with small read numbers mapping to potentially parasitic nematodes, including an unidentified species of Steinernema, two samples with reads mapping to Heterorhabditis bacteriophora and three with reads mapping to Heterorhabditis marelatus. De novo assembly also identified an 8.4 kbp nematode scaffold with 85 per cent nucleotide identity to the mitochondrion of Panagrellus redivivus, a free-living rhabditid associated with decomposing plant material. Reads from this nematode were detectable in seventy-three of the 167 samples, rarely at a high level (up to 0.8 RPKM). Only one sample contained reads that mapped to mite COI, sample UK_Dai_16_23, which mapped at high levels (5.8 and 2.2 RPKM) to two unidentified species of Parasitidae . We excluded two cyclovirus-like fragments from the analyses below because they occurred only in the sample contaminated with the two mites, suggesting that they may be associated with the mites or integrated into their genomes (figshare repository 10.6084/m9.figshare.14161250 S3 and S4).The remaining 2–30 per cent of reads could include metazoan species associated with D.melanogaster, we mapped all reads to a curated panel of short diagnostic sequences from COI and Argonaute-2, the latter chosen for its ability to reliably distinguish between the close relatives D.melanogaster and D.simulans. As expected from previous analyses of these data , and D.phalerata, which accounted for 12.2 per cent of COI in AT_Mau_15_50 (566 reads). The presence of both species was confirmed by additional mapping to Adh, Amyrel, Gpdh, and 6-PGD (figshare repository 10.6084/m9.figshare.14161250 S3), and their mitochondrial genomes were recovered as 9 and 16 kbp de novo scaffolds, respectively. More surprisingly, some of the collections made in 2015 contained reads derived from D.serrata, a well-studied species closely related to D.melanogaster and endemic to tropical Australia  and 7 per cent (839 reads) of COI respectively, but reads were also detectable in another six pools. The presence of D.serrata sequences was confirmed by mapping to Adh, Amyrel, Gpdh, and 6-PGD (figshare repository 10.6084/m9.figshare.14161250 S3). However, examination of splice junctions showed that D.serrata reads derived from cDNA rather than genomic DNA, and must therefore result from cross-contamination during sequencing or from barcode switching. Below, we note where conclusions may be affected by the presence of species of other than D.melanogaster.To detect the presence of drosophilid hosts other than ata e.g. , thirty ustralia . Samplesde novo assembled contigs, we also found evidence for several crop-plant chloroplasts and vertebrate mitochondria that are likely to represent sequencing or barcode-switching contaminants. The amounts were generally very low (median 0.01 RPKM), but a few samples stood out as containing potentially high levels of these contaminants. Most notably sample TR_Yes_15_7, in which only 76 per cent of reads mapped to fly or expected microbiota, had 8.1 RPKM of human mtDNA, 5.1 RPKM of Cucumis melo chloroplast DNA, and 3.5 RPKM of Oryza sativa chloroplast DNA. We do not believe contamination of this sample has any impact on our findings.Finally, among D.melanogaster . This is 99 per cent identical to the previous Drosophila Linvill Road densovirus assembly, but by examination of the assembly graph we were able to complete more of the inverted terminal repeats and extend the genome length to 5.4 kb . This was presumed to be a complete genome based on homology with Bombyx mori bidensovirus . To capture the diversity present among these putative viruses, we made targeted de novo builds of three datasets, incorporating both Illumina reads and Oxford nanopore reads  and similar structure . This sequence also appeared to encode a protein with distant homology to bidnavirus DNA polymerase B, and we reasoned that it might represent an additional virus. We therefore mapped reads from datasets that had high coverage of Drosophila Vesanto virus segments S01 and S02 to all scaffolds from the 00-fold are reads . We haveDrosophila Vesanto virus . Several segments were very commonly detectable, such as S03 (protein with homology to DNA PolB) and S10 (encoding a protein with domain of unknown function DUF3472 and a putative glycoprotein) in around seventy samples, and segments S01, S02, S04, S06, and S08 in around fifty-five samples. Others were extremely rare, such as S12 (encoding a putative NACHT domain protein with homology to S09), which was only seen in five samples. We considered three possible explanations for this pattern.Although we identified twelve distinct segments with strongly correlated presence/absence, not all segments were detectable in all affected samples . Only seDrosophila), no reads mapped to S12, strongly suggesting the absence of S12 from this sample. Our second hypothesis was that some segments are optional or satellite segments, or may represent alternative versions of other, homologous segments, comprising a re-assorting community (as in influenza viruses). The latter is consistent with the apparent homology between some segments. For example, S01, S03, and S11 all encode DNA Polymerase B-homologs, and S06, S07, and S10 all encode DUF3472 proteins. It is also consistent with the universal presence of S05, which appears to lack homologs. However, two of the DNA PolB homologs are highly divergent .Our first hypothesis was that Drosophila Vesanto virus has twelve segments, but that variable copy number among the segments causes some to occasionally drop below the detection threshold. In support of this, all segments are indeed detectable in the sample with the highest Drosophila Vesanto virus read numbers (FR_Got_15_49), ranging from seven-fold higher than the fly genome for S07 to 137-fold higher for S05. In addition, ‘universal’ segment S05 is not only the most widely detected segment across samples, but also has the highest average read depth within samples. However, despite 1.6 million Drosophila Vesanto virus reads in the second highest copy-number sample  between these two viruses is ∼0.7 that is, nearly six-fold more than that between D.melanogaster and D.simulans, supporting their consideration as distinct ‘species’. The third novel nudivirus was present at a very low level in a sample from Kaniv, Ukraine , and only small fragments of the virus could be assembled for phylogenetic analysis (GenBank accession MT496841-MT496846). This showed that the fragmentary nudivirus from Kaniv is approximately equally divergent from Drosophila innubila nudivirus and Drosophila Mauternbach nudivirus  and 154.5 kb (forty-one-fold coverage), respectively, based on datasets from Tomelloso, Spain  and Mauternbach, Austria . We refer to these viruses as ‘Drosophila Tomelloso nudivirus’ and ‘Drosophila Mauternbach nudivirus’, reflecting the collection locations, and we have submitted the sequences to GenBank under accession numbers proteins , and phyproteins  than to proteins , and is proteins . Howeverudivirus .Drosophila species other than D.melanogaster, or to nematode worms or mites. Moreover, we identified Drosophila Tomelloso nudivirus in a number of experimental laboratory datasets from D.melanogaster . And, as we could not detect Drosophila Mauternbach nudivirus in any of the public datasets we examined (below), we remain uncertain whether D.melanogaster or D.phalerata was the true host.The collections from Tomelloso and Kaniv did not contain reads mapping to Musca domestica and Glossina palpides , the true host remains uncertain. However, given that the closest relatives all infect Diptera, it seems likely that either D.melanogaster or D.simulans is the host.Our search also identified fragments of two further large dsDNA viruses from lineages that have not previously been reported to naturally infect Drosophilidae. First, in sample UA_Ode_16_47 (SRR8494427) from Odesa, Ukraine, we identified around 16.6 kb of a novel virus related to the salivary gland hypertrophy viruses of palpides . Our assL.boulardi, a parasitoid wasp that commonly attacks Drosophila (MT496832–MT496840). Leptopilina boulardi filamentous virus has a circular genome of 111.5 kbp predicted to encode 108 proteins. This suggests that, although fragmentary, our assembly may represent most of the virus. A small number of reads from ES_Gim_15_30 mapped to a relative of nematode P.redivivus and, surprisingly, to the Atlantic salmon , but we consider these unlikely hosts as the level of contamination was very low and other filamentous viruses are known to infect insects. We were unable to detect the novel filamentous virus in any public datasets from D.melanogaster (below), and given that Leptopilina boulardi filamentous virus infects a parasitoid of Drosophila, it is possible that this virus may similarly infect a parasitoid wasp rather than the fly. However, as we were unable to detect any reads mapping to Leptopilina or other parasitoids of Drosophila in any of our samples, we think D.melanogaster is a good candidate to be a true host.Second, in sample ES_Gim_15_30 (SRR8439138) from Gimenells, Spain, we identified around 86.5 kb of a novel virus distantly related to the filamentous virus of osophila . The assAdintoviridae, a family of dsDNA viruses related to bidnaviruses and other PolB-encoding DNA viruses  are actually horizontally transmitted viruses . Some of viruses . We iden viruses , and pos viruses . The sec viruses . In a phosophila . It is nDrosophila , from which other members of the Drosophilidae were absent. It is similarly 13.8 kb long, and our phylogenetic analysis of DNA PolB places it within the published diversity of insect adintoviruses—although divergent from other adintoviruses or polintons of osophila . HoweverDrosophila genome copy-number, only five of the viruses  were detectable in multiple population pools. The other nine viruses were each detectable in a single pool. For viruses in a single pool, a simple maximum-likelihood estimate of prevalence—assuming independence of flies and pools—is 0.015 per cent (with an upper 2-log likelihood bound of 0.07 per cent). Among the intermediate-prevalence viruses, Drosophila Esparto nudivirus and Drosophila Viltain virus were detected in five pools each, corresponding to a prevalence of 0.08 per cent (0.03–0.17%), and Drosophila Linvill road densovirus was detected in twenty-one pools, indicating a prevalence of 0.34 per cent (0.21–0.51%). The two most common viruses were Drosophila Kallithea nudivirus, which was detected in ninety-three pools giving a prevalence estimate of 2.1 per cent (1.6–2.5%), and Drosophila Vesanto virus, which was detected in 114 pools giving a prevalence estimate of 2.9 per cent (2.4–3.5%). However, it should be noted that all flies were male, and if virus prevalence differs between males and females then these estimates could be misleading. Both virulence and titre can differ between the sexes , suggesting either that some reads derived from infections of D.simulans .We wished to corroborate our claim that these viruses are associated with ses e.g. . In contses e.g.  or largeses e.g.  were mor10.6084/m9.figshare.14161250 S7). This is consistent with their extreme rarity in our own sampling, and the possibility that Drosophila Mauternbach nudivirus and the adintoviruses may actually infect species other than D.melanogaster. Although some reads from Dros-RTEC run SRR3939056  in public data: Drosophila Viltain densovirus appeared only once in 3,003 sequencing datasets, in one of the sixty-three libraries from Dros-RTEC project PRJNA308584 . Drosophsimulans . DrosophD.melanogaster derived from collections in Denmark in 2010 (10.6084/m9.figshare.14161250 S7), and in several Dros-RTEC datasets . Diversity did not vary systematically around the virus genome (figshare repository 10.6084/m9.figshare.14161250 S9). Consistent with the large number of low-frequency private SNPs, average within population-pool diversity was ten-fold lower still, at πS = 0.04 per cent, corresponding to a very high STF of 0.71. In general, the level of constraint on virus genes appeared low, with global πA/πS 0.39 and local πA/πS = 0.58. These patterns of diversity are markedly different to those of the host, in which πS (at four-fold degenerate sites) is on the order of 1 per cent with πA/πS (zero- and four-fold) around 0.2, and differentiation approximately STF = 0.03  with relatively weak constraint. Alternatively, high STF and πA/πS may indicate a high proportion of sequencing errors.We examined genetic variation in three of the most common viruses; Drosophila Kallithea nudivirus, Drosophila Linvill Road densovirus, and Drosophila Vesanto virus. After masking regions containing indels, and using a 1 per cent MAF threshold for inclusion, we identified 923 SNPs across the total global Drosophila Kallithea nudivirus pool, and 15,132 distinct SNPs summed across the forty-four population samples. Of these SNPs, 13,291 were private to a single population, suggesting that the vast majority of Drosophila Kallithea nudivirus SNPs are globally and locally rare and limited to one or a few populations. This is consistent with many of the variants being recent and/or deleterious, but could also reflect a large proportion of sequencing errors—despite the analysis requiring a MAF of 1 per cent and high base quality. Synonymous pairwise genetic diversity in the global pool was very low, at πT = 0.03 . Given tT = 0.03 , the oveS = 1.16%, local πS = 0.28%), and to intermediate levels of constraint on the protein sequence (πA/πS = 0.20), but a similar level of differentiation (STF = 0.76). Although the prevalence of Drosophila Vesanto virus appears to be slightly higher than Drosophila Kallithea nudivirus (2.9% vs. 2.1%), much of the difference in diversity is probably attributable to the higher mutation rates of ssDNA viruses , and higher levels of constraint on the protein sequence (πA/πS = 0.10). Given a mutation rate that is likely to be similar to that of Drosophila Vesanto virus, this is hard to reconcile with a prevalence that is six-fold lower. However, one likely explanation is that Drosophila Linvill Road densovirus is more prevalent in the sister species D.simulans (above), and the diversity seen here represents rare spillover and contamination of some samples with that species.In Drosophila Linvill Road densovirus, which was only present in thirteen populations and has the smallest genome, we identified 178 SNPs across the global pool, and 253 distinct SNPs summed across the infected populations, of which 209 were private to a single population. Although this virus appears at least six-fold less prevalent than Drosophila Kallithea nudivirus or Drosophila Vesanto virus, it displayed relatively high levels of genetic diversity both globally and locally , and that spanning 67,903 to 68,513 (within putative protein AQN78615). The first region comprised multiple repeats of around 100 nt and assembled with lengths ranging from 0.2 to 3.6 kbp, and the second comprised multiple repeats of around 140 nt and assembled with lengths between 0.5 and 2.4 kbp. Together, these regions explained the majority of the length variation among the Kallithea virus genome assemblies. We also sought to catalogue small-scale indel variation in Kallithea virus by analysing indels within reads. In total, after indel-realignment using GATK, across all forty-four infected samples we identified 2,289 indel positions in the Drosophila Kallithea nudivirus genome that were supported by at least five reads. However, only 195 of these indels were at high frequency (over 50% of samples). As would be expected, the majority  were found in intergenic regions (figshare repository 10.6084/m9.figshare.14161250 S9).D.melanogaster TEs and Drosophila Kallithea nudivirus. However, the vast majority of these  aligned internally to TEs, more than 5 bp away from the start or end position of the TE, which is inconsistent with insertion , PO_Gda_16_16 , and PO_Brz_16_17 . We were also able to assemble the sequence from four publicly available sequencing runs: three  that we believe are derived from global diversity line N14  collecteDrosophila Genome Nexus datasets -generating locus, which is seen for several EVEs in mosquitoes . Given that no correlation between galbut virus and Wolbachia has been detected in the wild  appeared geographically limited, appearing more commonly in higher latitudes, and with a different spatial distribution in the early and late collecting seasons , or spillover from infections of parasitoid wasps . A few, having appeared at low copy number in a single sample, could be contaminants—although we excluded virus-like sequences that appeared strongly associated with contaminating taxa (figshare repository 10.6084/m9.figshare.14161250 S4).Although metagenomic studies are routinely used to identify viruses and virus-like sequences e.g. , simple mentally , and Dromentally . Others,D.obscura and D.immigrans (Drosophila to sixteen. Although a small sample, these viruses hint at some interesting natural history. First, it is striking that more than a third of the reported DNA viruses are nudiviruses (six of the sixteen published, plus a seventh from Phortica variegata; Nudiviridae are common pathogens of Drosophila, and may indicate long-term host lineage fidelity with short-term switching among species. Such switching is consistent with the apparent lack of congruence between host and virus phylogenies, and the fact that both Drosophila innubila nudivirus and Drosophila Kallithea nudivirus infect multiple Drosophila species (D.melanogaster or D.simulans were detected in just one of the 167 population samples, and likely only one of 6,668 flies, consistent with a European prevalence <0.07 per cent. Only Drosophila Vesanto virus and Drosophila Kallithea nudivirus seem relatively common—being detected in more than half of populations and having estimated prevalences of 2.9 and 2.1 per cent, respectively. It is unclear why DNA viruses should have such a low prevalence, on average, as compared with RNA viruses (STF values, hint that transient local epidemics may be the norm, with viruses frequently appearing and then disappearing from local fly populations.These caveats aside, along with Drosophila innubila nudivirus  and IIV3mmigrans , our stu species . Second, viruses . In simpDrosophila do indeed seem to harbour fewer DNA viruses than RNA viruses, supporting an observation that was made before any had been described (Drosophila-associated viruses have been reported from undirected metagenomic studies, and metagenomic studies of RNA are as capable of detecting expression from DNA viruses as they are of detecting RNA viruses (e.g. Finally, escribed . This cases e.g. . InsteadD.melanogaster at the pan-European scale provide an ecological and evolutionary context for studies of host-virus interaction in Drosophila. However, we currently lack almost any data on the natural host range or fidelity of Drosophila viruses, and we have no knowledge of their real-world fitness consequences for the host. In the future, such information will be vital if we are to capitalise on Drosophila models to understand the co-evolutionary process.Taken together, our analyses of the distribution and diversity of DNA viruses associated with


The main objective of this study is to estimate and evaluate 10-year follow-up costs after prostate cancer treatment with curative  and non-curative intent  per patient in Germany in 2000, 2008, and 2015.Prostate cancer follow-up recommendations were extracted from the European Association of Urology guidelines from 2000 to 2015. Per patient costs were calculated with a detailed micro-costing approach considering direct and indirect medical expenses. Input parameters were derived from expert interviews, literature research, and official scales of tariffs. Costs for insurers, providers, and payers were included to estimate societal costs.p < 0.001), and EUR 5159 in 2015 (p < 0.001). Costs after hormone therapy with metastasis cumulated to EUR 10,846 in 2000, EUR 9818 in 2008 (p < 0.001), and EUR 11,978 in 2015 (p < 0.001). While insurers covered 37% of costs in 2000 (EUR 1664), only 23% of costs were reimbursed in 2015 . Cost sources mainly included consultations (55%), transportation (18%), and imaging (27%).Mean 10-year follow-up costs per patient after treatment with curative intent amounted to EUR 4415 in 2000, EUR 4224 in 2008 (Early detection and advances in prostate cancer treatment increased 10-year survival rates beyond 80% in Germany, ultimately expanding the number of survivors requiring follow-up. Statutory insurers reacted by decreasing the reimbursement rates to reduce per patient cost by up to 46%. Consequently, the economic burden was mainly shifted to payers and providers.Equitable and effective follow-up schedules covered by insurance funds are necessary to care for prostate cancer patients. In Germany, 6.8% of the gross domestic product were spent on cancer in 2014—a 1% increase from 1995 . HoweverA variety of papers investigate prostate cancer treatment options and analyse their effectiveness –6. TreatThe EAU first issued guidelines for prostate cancer follow-up in 2000 . RecommeEAU guidelines recommended detecting treatment failures early by conducting frequent examinations during the first 2 years. Specifically, examinations were recommended in months 3, 6, 12, 18, and 24, and thereafter annually. While patients should also be monitored for PSA levels, prostate cancer may also progress without change in PSA due to differentiated cells without capacity to secret PSA. Clinical or laboratory suspicion of cancer relapse should trigger a chest X-ray, or abdominal and transrectal ultrasound examinations. Medical imaging is not recommended as a means for routine follow-ups. CT and MRI should not be used during routine follow-ups of asymptomatic patients.Patients that receive hormonal treatment should be examined 3 and 6 months after initial treatment. Thereafter, patients without distant metastasis and good treatment response should be re-examined every 6 months. In contrast, patients with distant metastases and good treatment response should be examined every 3–6 months. Patients receiving antiandrogen treatment might require more intensified follow-up patterns. Bone scintigraphy was not recommended as a routine procedure for asymptomatic patients. CT or MRI scans might be useful to find node metastases in patients with negative bone scintigraphy and elevated PSA levels. Patients that do not have an adequate hormone treatment response or a disease progression need “individualized” follow-ups.The EAU guidelines suggest distinct prostate cancer follow-up patterns and procedures according to the cancer stage and prior therapy. Consequently, costs were estimated for four distinct therapy types: curative, hormone therapy without metastasis, hormone therapy with metastasis, and androgen depravation therapy. While the EAU clearly proposes a lifelong follow-up, international recommendations for budget impact analysis endorse a follow-up period of 1–6 years , 21. MerThe payer perspective estimates the opportunity costs associated with prostate cancer follow-ups. Time consumption for patients was either based on literature or expert opinions. The forgone opportunity cost was calculated based on the average salary and the worked/productive hours. Calculations further accounted for the higher annual income of privately insured patients  relative to statutory insured patients . Overall, the majority of patients (88%) are insured through the statutory insurance scheme, while only 12% take out private health insurance.The provider perspective calculates the opportunity costs for health care providers  with prostate cancer follow-ups. In Germany, the provider perspective is relevant since budgeting and reimbursement restrictions lead to a discrepancy between the provider’s possibility to bill medical services and actual performed medical activities. Time consumption was again based on previous literature and expert interviews. The forgone opportunity cost was calculated based on the average salary and the worked/productive hours –24. OppoEinheitlicher Bewertungsmaßstab – EBM”) [Gebührenordnung für Ärzte – GOÄ”) [The insurer perspective considers the bill that the provider hands to the insurer. Due to Germany’s dual insurance system, a separate costing approach was considered for both the social health insurance (SHI) and the private health insurance (PHI). SHI funds are charged according to reimbursement rates published in a catalogue (“ – EBM”) –27. Simi – GOÄ”) . ReimburFollow-up costs were estimated based on a patient-level micro-costing approach. The frequency of recommended consultations, examinations, and diagnostic tests was extracted from EAU guidelines. Thereafter, the opportunity costs for physicians and patients, resulting from forgone time consumption for follow-ups, were calculated alongside the resulting expenditure bill for the insurance Table . The hosWe conducted a probabilistic sensitivity analysis to account for variations in the length of physician-patient consultations, examinations, and diagnostics. Therefore, provider’s and payer’s time consumption parameters were drawn by random sampling from their defined distribution Table . In GermBudget impact analysis is employed to assess changes in healthcare expenditure following a decision to reimburse new treatments or related policies on a population level . ConsequEstimated 10-year prostate cancer follow-up costs ranged from EUR 4224 to EUR 12,293 per person . The incline was even steeper for follow-up schedules after hormone treatment without metastasis  and with metastasis .Ten-year follow-up costs for payers increased throughout the examined period Fig. . Follow-p < 0.001). Follow-up costs after hormone treatment without  and with  metastasis behaved in a similar pattern.The calculated 10-year follow-up costs constantly rose from 2000 to 2015 for providers Fig. . Costs aOverall, 10-year follow-up costs decreased from 2000 to 2008 and then remained at the same level until 2015  to then increase again to EUR 5159 in 2015 (p < 0.001). Costs followed a similar pattern after hormone therapy treatment. The decline in 2008 can mainly be attributed to the decreased costs for insurance funds . Patients after androgen deprivation therapy are also recommended to adhere to shorter follow-up time intervals.Follow-up costs after hormone treatment with metastasis were significantly higher than follow-up costs after hormone treatment without metastasis across all years. The EAU recommends more frequent follow-up intervals for prostate cancer in the metastatic state to detect recurrences. Therefore, more costly consultations and examinations are required for patients with metastasis. Similarly, costs after newly introduced androgen deprivation therapy were significantly greater compared to costs after curative intent . Due to a successive budgeting of medical services reimbursed by the German statutory health insurances, the whole burden of follow-up costs is mainly shouldered by patients and providers. As a result, patients and providers might be less incentivized to participate in cancer follow-ups. Research reveals that some patients do not participate during all recommended follow-ups . Follow-Based on existing research, prostate cancer treatment costs in the first 6 months after treatment amount to USD 11,495, ranging from USD 2586 for watchful waiting to USD 24,204 for external beam radiation . In contThe overall budget impact is subject to several factors. First, adjustments in the statutory reimbursement rates until 2008 limited follow-up costs. However, increasing prostate cancer incidence combined with improved 10-year survival rates due to more sophisticated diagnostic and treatment options expanded the follow-up budget impact from 2000 to 2008 by EUR 78 million (+44.1%). German insurers likely anticipated the rising incidence, prevalence, and survival rates. As a result, insurers reduced reimbursement rates to maintain the overall healthcare budget. In contrast, from 2008 to 2015 declining incidence rates were offset by higher follow-up costs, resulting in a roughly constant expenditure.Lately, primary and secondary preventions have been prioritized. Research has focused on the early detection of prostate cancer. In Germany, prostate cancer incidence significantly increased in the past years due to advances in early detection featuring annual digital rectal examination and possibly a PSA-level measurement , and chaIn summary, prostate cancer follow-ups pose a substantial economic burden to all stakeholders. Follow-up costs may vary depending on initial treatments and cancer stages. Therefore, it is recommended that follow-up expenses are included in cost-effectiveness evaluations of cancer treatments. Heins et al. (2018) explored the feasibility of general practitioner (GP)–led prostate cancer follow-ups . Most paOur findings inform decision makers about the financial cost borne by patients and providers as well as a shift in costs covered by statutory health insurers in Germany. This shift in the economic burden from insurers to patients and providers may disincentivize adherence to follow-up guidelines and thereby adversely impact health outcome—especially for the poorer population not covered by private insurance.The strengths of this paper include the detailed micro-costing approach, the consideration of all three perspectives, and the evaluation of various treatment options (curative vs. hormone therapy vs. ADT). First, direct and indirect medical costs were included for insurers, providers, and patients. Second, distinct follow-up costs for different treatment options and cancer stages inform cost-effectiveness evaluations of prostate cancer treatment options. Based on literature review, this is the first study estimating prostate cancer follow-up costs in Germany from 2000 to 2015.There are limitations present. First, follow-up costs are empirically calculated and therefore not validated with clinical studies. Furthermore, the assumed “uptake” of 100% of guideline recommendations might not be realistic. Firstly, some physicians might not follow guideline recommendations due to several barriers. Secondly, some cancer patients might not participate in cancer follow-ups. Adherence to follow-up is generally related to patient education, availability of transportation services, and clinical efficiency . Physici


Reassortment among avian influenza viruses is the main source of novel avian influenza virus subtypes. Studies have shown that the H9N2 virus often donates internal segments to generate novel reassortant avian influenza viruses, acting as a reassortment template. However, the characteristics of the internal pattern of reassortment remain unclear. In this article, we first defined the core gene pool of the internal segments of the H9N2 virus that provide templates for reassortment. We used genetic distance and sequence similarity to define typical clusters in the core gene pool. Then, we analyzed the phylogenetic relationships, feature vector distances, geographic distributions and mutation sites of strains related to the core gene pool. Strains in the same typical clusters have close phylogenetic relationships and feature vector distances. We also found that these typical clusters can be divided into three categories according to their main geographic distribution area. Furthermore, typical clusters in the same geographic area contain some common mutation patterns. Our results suggest that typical clusters in the core gene pool affect the reassortment events of the H9N2 virus in many respects, such as geographic distribution and amino acid mutation sites. H9N2 avian influenza virus (AIV) was first detected in 1966 . Since tA number of AIVs have been reported to infect humans, including H7N9, H5N6, H10N8, H5N1, H6N1, and H7N4 . Some ofIn this article, we aimed to identify and characterize the core gene pool of H9N2 internal segments and analyze the reassortment events involving the H9N2 virus. Based on a genetic analysis and mathematical relationships calculated for all the H9N2 viruses and other relevant AIVs, we defined typical clusters of each internal segment of the H9N2 virus. Each typical cluster consists of strains that are clustered together according to sequence similarity and evolutionary tree branching information. Strains in the same typical cluster have similar genetic characteristics. Typical clusters with different characteristics constitute the core gene pool of H9N2 internal segments. Then, we extracted feature vectors from the sequences and performed mathematical clustering to cross-validate the results. The clustering results essentially corresponded to the typical clusters. Then, we conducted a more detailed analysis of the biological characteristics of the core gene pool. The results reflect that different typical clusters infect different host species and have different geographic distributions. Mutation site analysis of typical clusters revealed that typical clusters in the same geographic area share some common mutation sites. We further analyzed the relationship between H7N9 and H9N2 using mathematical feature characterization. Most sequences of the H7N9 virus and H9N2 virus clustered together, which is consistent with reported reassortment events and further verifies the rationality of our core H9N2 virus gene pool.All internal segments sequences of H9N2 virus and other relevant AIV subtypes are downloaded from NCBI Influenza Virus Resource . Only fuWe use CD-HIT to initially filter the resulting sequences with a threshold level of 0.98 and retained sequences in clusters which meet all the following rules.•Cluster contains H9N2 virus sequence(s).•Besides H9N2 virus sequence(s), cluster contains other AIV subtypes sequences. And there is at least one sequence whose collecting time is no more than 3 years from the collecting time of H9N2 virus sequence(s) in the cluster.•Besides H9N2 virus sequence(s), cluster contains other AIV subtypes sequences. And there is at least one sequence which has the same host or country with H9N2 virus sequence(s).After first filtering, our dataset includes 2,583 PB2 sequences, 3,356 PB1 sequences, 3,565 PA sequences, 3,660 NP sequences, 5,757 MP sequences, and 3,325 NS sequences.Maximum-likelihood trees are inferred by IQ-TREE . And ModFor each evolutionary tree, we use Mean Pairwise Distance (MPD) to cluster AIV strains . The MPDmaxNode. The average MPD of all internal nodes is defined as meanMPD.We calculate the median number of leaf nodes and MPD for each internal node. To reduce deviation, we also set a lower threshold for the median number of leaf nodes. We choose the larger one of the median and the threshold as the max number of strains in a cluster, named as s of the evolutionary tree, it has two features. Ns reflects the number of strains of subtree s, and mpds represents the MPD value of subtree s. We define a set K(s) which contains all strains in subtree s. To infer the possible genealogy and relationship of strains, we use the following conditions to measure phylogenetic distance between strains: (a) Strains in subtree s with Ns < maxNode have close phylogenetic distance. (b) Strains in subtree s with mpds < meanMPD have close phylogenetic distance. Subtree that satisfies one of the above conditions is defined as a cluster.When two different strains have a reassortment relationship, we believe that their sequences are similar and they have close phylogenetic distance reflected in the evolutionary tree. For a given subtree We analyze the common mutation sites for all strains in a given cluster. A cluster with more than one strains that contains H9N2 virus and has common non-neutral mutations is defined as a typical cluster.S. For each base occurs in the sequence, the frequency of it is defined as pi,(i ∈ S). For each codon except termination codons, the frequency of it can be defined as pijk,. Trinucleotide relative abundance (TRA) is an extension of dinucleotide relative abundance . Excluding termination codons, 61 codons remain. Each sequence starts from the initiation codon and every three bases is a codon. Bases included in a sequence compose a set nce DRA; , which rObs_codon. The times that the coded amino acid occurs is defined as Obs_Amino, while the number of synonymous codons of the amino acid is denoted as n. When those synonymous codons don’t have codon usage bias, the expected observation number of them in the sequence is calculated as follows. The RSCU value of a given codon is calculated as follows:Relative synonymous codon usage RSCU;  is used Considering both TRA value and RSCU value of codons occur in a sequence, our feature vector is the product of RSCU and TRA:As three termination codons are excluded, the feature vector is a 61-dimensional vector.A typical cluster in core gene pool must have long time span or contain large number of AIV subtypes or contain large number of strains. We screen qualifying typical clusters from the typical clusters calculated in the previous section. These qualifying clusters have more than 3-year time span, or the number of AIV subtypes or strains they contain is higher than the median value. We calculate the average center of each screened cluster as the center vector, which can characterize the cluster. Then we calculate a distance matrix among all the screened clusters and show it in the form of a heat map. In addition to mathematical characterization, we also use common amino acid mutation sites and geographic distribution to characterize clusters biologically.We find H7N9 virus is the most representative reassortment AIV subtypes which has been reported before. TSNE  method cWe downloaded all available internal segment sequences of AIV from NCBI as of October 13, 2020, and finally constructed a genome set including 1,428 PB2 sequences, 1,795 PB1 sequences, 1,988 PA sequences, 1,564 NP sequences, 1,318 MP sequences, and 637 NS sequences. We constructed six phylogenetic trees for the six internal segments. According to the MPD value and similarity of leaf nodes in the phylogenetic trees, sequences of each internal segment were clustered into different clusters, as shown in Almost all six internal segments had similar numbers of typical clusters and sequences . The PB1To cross-validate the correctness of the clustering results and preserve the features of each sequence as completely as possible, we used a 61-dimensional feature vector to represent a sequence. We used BRICH  to clustFor each internal segment, we calculated the distance matrix among typical clusters based on the center vector of each typical cluster and then normalized the distance matrix. The resulting heatmap, shown in Beyond the mathematical characterization of typical clusters in the core gene pool, we are more interested in the biological characterization of these clusters, which can provide more detailed biological insights. We found that there were obvious differences in geographic distribution among these typical clusters in the core gene pool. As shown in Furthermore, we found relationships between the common mutation sites and geographical distribution of those typical clusters. Taking common mutation sites of the PB2 segment as an example , we founAmong all the reported reassortment events related to the H9N2 virus, the H7N9 virus is highly representative. It is widely distributed and has the ability to infect humans. Notably, it has evolved a lineage with high pathogenicity . MeanwhiThe low pathogenicity and wide distribution of the H9N2 virus make it more likely to be coinfected with other AIV subtypes. As explained above, we consider that the internal segments of the H9N2 virus can be regarded as a gene pool that is in a state of dynamic equilibrium. Avian influenza virus coinfected with the H9N2 virus can be regarded as entering the gene pool of the H9N2 virus, where it may not only provide new internal segments for the gene pool but also receive internal segments from the gene pool and generate novel reassortment AIV subtypes . Those iThe establishment of the core gene pool of the H9N2 virus in this article provides new insight into the reassortment characteristics of the H9N2 virus. We conducted systematic phylogenetic analysis and detailed analysis of different characterizations and specific reassortment. The results indicate that the core gene pool of the H9N2 virus affects the evolution direction and geographical distribution of reassortment events related to the H9N2 virus.The feature vector distance of sequences and the genetic distance of strains show obvious aggregation among strains. Based on the aggregation of different strains, the internal segments of strains can be divided into several typical clusters. The analysis of strain collection locations and collection times indicates that there is geographic isolation among different lineages. The hosts of H9N2 virus and relevant reassortment virus are mainly avian, resulting in rare transmission across continents; this is the main reason for geographic isolation. In total, typical clusters located in Asia and the Americas account for the main part of the core gene pool. The distribution differs among different internal segments. Further amino acid mutation site analysis suggests some valuable mutation sites related to geographical distribution. Finally, the analysis of the H7N9 virus visually demonstrated its reassortment relationship with the H9N2 virus, which also verified the rationality of our core gene pool.It should be noted that the establishment of the core gene pool depends on the existing sequence data. Although we aimed to exclude the impact of sampling bias on the results as much as possible, it is inevitable that our results will differ from the real situation in some ways. Moreover, the analysis of mutation sites in this article is limited to the strains in the core gene pool. We also conducted an analysis of the mutation sites of all available sequences, which produced completely different results. In summary, the core gene pool in this article can help us understand the pattern in which the H9N2 virus donates internal segments during reassortment with other AIV subtypes. In future work, we will further investigated the impact of the core gene pool on the evolution and fitness of the H9N2 virus.The original contributions presented in the study are included in the article/HR, WC, and JY formulated the study. HY and MH performed the research and analyzed the data. BW, YJ, XG, and LL participated in analysis and discussion. HY and HR drafted the manuscript. All authors read and approved the final 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mesocricetus auratus) affected by skin tumors. Natural HaPyV infections have been recorded in Syrian hamster colonies due to the occurrence of skin tumors and lymphomas. HaPyV infections of Syrian hamsters represent an important and pioneering tumor model. Experimental infections of Syrian hamsters of different colonies are still serving as model systems . The observed phylogenetic relationship of HaPyV to murine polyomaviruses within the genus Alphapolyomavirus, and the exclusive detection of other cricetid polyomaviruses, i.e., common vole  and bank vole (Myodes glareolus polyomavirus 1) polyomaviruses, in the genus Betapolyomavirus, must be considered with caution, as knowledge of rodent-associated polyomaviruses is still limited. The genome of HaPyV shows the typical organization of polyomaviruses with an early and a late transcriptional region. The early region encodes three tumor (T) antigens including a middle T antigen; the late region encodes three capsid proteins. The major capsid protein VP1 of HaPyV was established as a carrier for the generation of autologous, chimeric, and mosaic virus-like particles (VLPs) with a broad range of applications, e.g., for the production of epitope-specific antibodies. Autologous VLPs have been applied for entry and maturation studies of dendritic cells. The generation of chimeric and mosaic VLPs indicated the high flexibility of the VP1 carrier protein for the insertion of foreign sequences. The generation of pseudotype VLPs of original VP1 and VP2–foreign protein fusion can further enhance the applicability of this system. Future investigations should evaluate the evolutionary origin of HaPyV, monitor its occurrence in wildlife and Syrian hamster breeding, and prove its value for the generation of potential vaccine candidates and as a gene therapy vehicle.Hamster polyomavirus  was discovered as one of the first rodent polyomaviruses at the end of the 1960s in a colony of Syrian hamsters ( Right from the start of developing the concepts of modern molecular biology and tumor biology, viruses such as the simian virus 40  and the murine polyomavirus (Mus musculus polyomavirus 1) have served as models for a fundamental understanding of eukaryotic gene regulation and gene expression as well as tumor induction and progression [Polyomaviruses are nonenveloped circular double-stranded DNA viruses that infect a variety of vertebrate host species. In humans, mainly four polyomaviruses have been strongly associated with clinical disease. The Human polyomavirus 1 , Human polyomavirus 2 , Human polyomavirus 5 , and Human polyomavirus 8  can cause severe diseases especially in immunosuppressed individuals. HPyV-1 is associated with nephropathy, a leading cause of kidney transplant failure, HPyV-2 can cause the demyelinating disease progressive multifocal leukoencephalopathy (PML), HPyV-5 is able to integrate into the cellular genome to cause a highly aggressive skin cancer (Merkel cell carcinoma), and HPyV-8 is an etiological factor for the dermatological disease Trichodysplasia spinulosa (TS). However, the mechanisms by which these viruses give rise to the relevant diseases are not well understood.This review aims to describe the discovery and characterization of the hamster polyomavirus, its implication in the development of cancer, pathological features, and the application of its capsid-derived recombinant proteins for the generation of virus-like particles (VLPs). A timeline of objectives of hamster polyomavirus studies, leading researchers, and publication numbers are shown in Polyomaviruses have been identified in numerous mammalian species including members of different families of the order Rodentia . Modern However, the knowledge of rodent polyomaviruses is still very scarce. This is illustrated when comparing the number of known polyomaviruses in humans (n = 14) with those in more than 2000 rodent species  was first isolated from skin epitheliomas arising in the Syrian hamster colony in Berlin-Buch, Germany and described by Graffi et al.  a scree. The tumVirus particles extracted from skin epithelioma caused lymphoma and leukemia when injected into newborn hamsters from a distinct and tumor-free colony bred in Potsdam, Germany . The rol6, a sedimentation coefficient of 223 S, and a buoyant density of 1.340 g/mL [stratum corneum but absent in the proliferating cells of the stratum basale and stratum spinosum. In situ hybridization of whole body animal sections conducted before the appearance of epitheliomas demonstrated that thymus and spleen represent the most active virus reservoirs [HaPyV particles were first detected on electron micrographs of primary skin sections . The par340 g/mL . The sym340 g/mL . Virus pservoirs . Accordiservoirs ,19. Althservoirs ,26,27.The availability of experimental models is necessary in order to study the induction and growth processes of tumors and metastases. Experimental Syrian hamster models were created by the subcutaneous inoculation of virions isolated from skin tumors and HaPyV DNA purified from the virions . Out of Transgenic mice have been obtained by the microinjection of HaPyV supercoiled DNA into pronuclei of fertilized eggs of Gat:NMRI mice . AnalysiTransgenic mice in which the expression of the reporter gene lacZ is driven by the HaPyV early promoter have shown that this promoter displays a rather specific expression pattern restricted to hematopoietic organs, mainly thymus and spleen .Reflection on former models could give important insights into carcinogenic processes and may open new therapeutic options.HaPyV is transmitted horizontally via urine  or by biLymphomas are mainly associated with HaPyV infection of young virus-naïve hamsters. Mortality rates among these hamsters can reach 80% within 4 to 30 weeks following infection . AffecteIn an inbred colony of genetic audiogenic seizure Syrian golden hamsters  in Spain, a high incidence of non-Hodgkin, Burkitt-type-B-cell lymphoma was detected, resulting in mortality within 1 to 2 weeks . An indiSo far, there are no treatment options for hamsters with HaPyV infection. Aggressive environmental decontamination of the virus has failed in some facilities . As an iWith the emergence of recombinant technology in the mid-1970s ,35, oppoIn the early 1980s, full-length HaPyV was inserted into BamHI-site of the bacterial plasmid pBR322 ,38 permiThe most comprehensive molecular characterization of the HaPyV genome including the deposition of the almost entire nucleotide sequence  as well as a comparison to other polyomavirus genomes was carried out during the mid-1980s at the Institute Gustave Roussy, Villejuif, France in collaboration with the group of Jean Feunteun .With regard to viral gene expression, the early and late mRNA transcripts of HaPyV were detected on the basis of cDNA cloning and subsequent sequence determination  Figure . The synthesis of the early tumor (T) antigens and late viral proteins (VP) of HaPyV were confirmed indirectly by the heterologous synthesis of protein segments of large T (LT), middle T (MT), small T (ST), VP1, and VP2/VP3 in Escherichia coli and their reactivity with sera from naturally HaPyV-infected Syrian hamsters of the Z3 strain . The virThe genome characterization of HaPyV resulted in the identification of two putative in-frame translation initiation codons of VP1-open reading frame (ORF) encoding 384 and 388 amino acid (aa) residue-long VP1 variants . The hetTM. A phylogenetic tree built by the neighbor-joining method based on the matrix of pair-wise genetic distances between samples shows a closer relationship of the German isolate (Berlin) to the British (Bristol) or American (Columbia) sequences than to the Spanish  variants , both encoding a MT antigen, but a clear divergence to the primate polyomavirus SV40 and human polyomavirus HPyV-1 (BKPyV). The initial assumption of the exclusive occurrence of the MT antigen-encoding capacity in rodent polyomaviruses  must be Interestingly, other cricetid-associated polyomaviruses, i.e., bank vole (Myodes glareolus polyomavirus 1) and common vole  polyomaviruses, belong to the genus Betapolyomavirus, distinct from the cricetid associated HaPyV. This current phylogenetic position of HaPyV must be considered with caution because it might be biased by the still limited knowledge of rodent-associated polyomaviruses.VLPs can be produced by heterologous synthesis of viral capsid and/or envelope proteins and represent a very useful alternative to infectious virus particles with broad applications in basic and applied research . The proAutologous HaPyV-VP1 derived VLPs were used to compare their uptake mechanisms by human dendritic cells (DCs) to that of VLPs of other polyomaviruses . These iTo exploit HaPyV VP1 as a carrier for foreign epitopes in VLPs, multiple epitope mapping studies were performed to identify potential insertion sites for foreign sequences. An initial evaluation of dihydrofolate reductase (DHFR) fusion proteins with different segments of HaPyV-VP1 resulted in the identification of the C-terminus of VP1 as an immunodominant and cross-reactive region that was not only detected by sera of infected tumor bearing Z3 and tumor-free PF hamsters but also by sera raised against SV40 and HPyV-2 (JCPyV) . The C-tSequence comparisons and prediction of the structure resulted in the identification of four flexible, variable, and surface-exposed regions within VP1 of HaPyV that represent, in addition to the C-terminal region, potential insertion sites for foreign epitopes . FurtherThe insertion of a pentapeptide of the hepatitis B virus preS1 region allowed the formation of chimeric VLPs by VP1-preS epitope fusion proteins in S. cerevisiae  Table 2b,d. The The successful use of HaPyV-derived VLPs was continued by the insertion of various viral and non-viral epitopes and protein segments see . The fleINK4A (156 aa), eGFP (238 aa), or even 473 aa long functionally active complex molecules such as single-chain antibody fragment fused with human antibody Fc fragment (scFv-Fc) [INK4A in immunized mice [The high insertion capacity of the carrier was also demonstrated by simultaneous insertions of short epitopes at multiple sites of VP1 ,64,68,71scFv-Fc)  (GedvilascFv-Fc) ,75, and Currently, HaPyV represents the only well-characterized polyomavirus in cricetine rodents. A large-scale polyomavirus screening in wild Syrian hamsters and other hamster species is lacking. Therefore, the phylogenetic relationship of HaPyV to murine polyomaviruses within the genus Alphapolyomavirus and the exclusive detection of other cricetid polyomaviruses, i.e., common vole and bank vole polyomaviruses, in the genus Betapolyomavirus, must be considered with caution. The re-emergence of HaPyV outbreaks in Syrian hamster breeding and experimental units may suggest the necessity to continuously monitor breeding and hygiene regimens when implementing novel animals into existing breeding colonies. Further efforts are still needed to understand the different outcomes of HaPyV infections in Syrian hamsters of different origin, because no treatment options exist, and mainly all the hamsters in a colony have to be culled. HaPyV-VP1 and -VP1/VP2-derived VLPs represent a highly flexible carrier moiety for the insertion of foreign sequences of different size and origin offering a broad range of potential applications in basic and applied research. These virus-like particles are safe, easy to produce, and can be tailored for specific delivery or epitope presentation . Future


Background: Structural chromosome abnormality (SCA) is an important cause of human diseases, including recurrent pregnancy loss (RPL). DNA double-strand breaks (DSBs) repair-related genes play critical roles in SCA. The present study aims to investigate the potential contribution of DSBs repair-related gene polymorphisms to SCA.Methods: Fifty-four affected RPL individuals with SCA, 88 affected RPL individuals without SCA, and 84 controls were analyzed. Targeted whole-exome sequencing (WES) was used for screening single nucleotide polymorphisms in six DSBs repair-related genes , and validation was performed by Sanger sequencing. Finally, we detected the frequency of radiation-induced chromosome translocations in no SCA samples with significant polymorphisms by fluorescence in situ hybridization (FISH).Results: A total of 35 polymorphisms have been identified and confirmed. Frequencies of EP300 rs20551, XRCC6 rs132788, and LIG4 rs1805388 were significantly different between SCA RPL and no SCA RPL . Frequencies of those three gene polymorphisms between SCA RPL and controls also were significantly different . Moreover, the frequency of the G allele at rs20551 locus, the T allele at rs132788 locus and the A allele at rs1805388 locus was significantly higher in SCA RPL than no SCA RPL  and controls . Additionally, the frequency of radiation-induced translocation in no SCA samples with rs20551, rs132788 or rs1805388 was significantly higher compared with the wild type samples .Conclusion: Our results suggest that rs20551, rs132788, and rs1805388 might be associated with the risk of SCA. Larger scales of genetic variations studies and functional experiments are necessary to further confirm these findings. Structural chromosome abnormality (SCA) is an important cause of human diseases including recurrent pregnancy loss (RPL) . In apprTypes of SCA include translocation, inversion, deletion, Tandem duplication, ring chromosome, etc. . The mosThe biogenesis of SCA is remarkably poorly understood. Generally, the formation of SCA is considered a multistep process, and the initial event is the concomitant occurrence of DNA double-strand breaks (DSB) in multiple chromosomal locations . It is gEP300, XRCC6, LIG4, XRCC4, PRKDC, and DCLRE1C were identified as playing critical roles in NHEJ repair , which can be readily participated in repairing a DSB , is a candidate regulator of DSBs repair  based on recurrent pregnancy loss. We used targeted WES in a relatively small exploratory sample at the first stage, and then confirmed by Sanger sequencing in a lager cohort including all exploratory sample and confirmatory sample. Finally, we also detected the frequency of radiation-induced chromosome translocations in no SCA samples with significant polymorphisms by fluorescence in situ hybridization (FISH).In the present study, we investigated the potential contribution of The study was approved by the Ethics Committee of the Third Xiangya Hospital (Quick 19159). Informed consent was obtained from all subjects involved in the study.The 142 affected individuals, all were RPL (54 with SCA and 88 without SCA), had no history of endocrine, metabolic, autoimmune, or other systemic disorders, thrombophilia, or uterine anatomic abnormalities. We recruited the RPL in strict accordance with the Practice Committee of the American Society for Reproductive Medicine . The conn = 75) at the first stage, and then confirmed by Sanger sequencing in a larger cohort (n = 226) including all exploratory samples (n = 75) and confirmatory samples (n = 151). Finally, to further confirm the association of significant SNPs with SCA, we detected the frequency of radiation-induced (2Gy X-ray) chromosome translocations in normal karyotype RPL peripheral blood lymphocytes (PBLs) with significant gene polymorphisms by FISH.The flowchart for the study design was shown in A standard 72-h lymphocyte culture of peripheral blood (2–5 ml) from each patient was performed to produce Metaphases for karyotyping. G banding was performed by a trypsin pretreatment of chromosomes followed by Giemsa staining. Chromosomes’ analysis was done using MetaSystems Ikaros  and karyotypes were reported according to International System for Human Cytogenetic Nomenclature . KaryotyWe first detected 75 samples  by targeted whole-exome sequencing (WES). Genotyping of SNPs was performed with the WES-based targeted sequence analysis and Sanger sequencing. The library was constructed with the kit  by the standard procedure according to the manufacturer’s instructions. xGen® Lockdown® Probes . SNPs were reported according to Human Genome Variation Society nomenclature . The seqin situ hybridization with composite probes labeled with SYBR green , chromosomes 2 were painted red by in situ hybridization with composite probes labeled with Rhodamine B . The observed frequency of translocations (Fp) detected by FISH represents the frequency between painted chromosomes 1, 2, and 4 and the remaining counterstained chromosomes. To compare Fp with the values for translocations detected by the conventional method that detects aberrations involving the entire chromosome set, it is necessary to estimate the genome-equivalent frequency of translocations (FG). Thus, since the fraction of the total genomic DNA content represented by painted chromosomes 1, 2, and 4 to the total genome is 0.228 for males and 0.224 for females, Fp was multiplied by 2.771 for males and 2.806 for females to estimate FG; the basic method used is essentially that described by Pearce  from normal karyotype RPL after 2Gy X-rays as previously described . Metaphay Pearce . 400 metn (%) and analyzed using Pearson chi-square (χ2) test (n ≥ 40 and expected frequencies ≥5). Measurement data were expressed as‾x (SD). Differences in genotype and allele frequencies (n < 40 or expected frequencies <5) between affected individuals and controls, as well as deviations from Hardy-Weinberg equilibrium were determined using Fisher exact test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the contribution of EP300, XRCC6, LIG4, XRCC4, PRKDC, and DCLRE1C gene polymorphisms to SCA. p < 0.05 was considered to be statistically significant.All statistical analyses were performed using SSPS 25.0 . Prism 7.0 software  was utilized to draw figures. Qualitative data were expressed as Mycoplasma hominus, Toxoplasma gondii, rubella, cytomegalovirus, herpes virus, LA, ß2-GPI, and aCL were similar between affected individuals and controls. No significant differences were identified (p > 0.05)  .54 RPL with SCA: 32 carried a balanced reciprocal translocation, among them, chromosome 2 were involved in the translocation most frequently (n = 7) followed by chromosomes 1, 4, 11, and 12 , chromosomes 3, 5, and 9 , chromosomes 6, 17, and 19 were not involved; 8 carriers of inversions were observed, 7 were inversions of chromosome 9 and 1 inversion of chromosome 8; 14 carried a balanced Robertsonian translocation .A total of 35 polymorphisms had been identified in our samples , nine wivariants . There wEP300 rs20551, XRCC6 rs132788, and LIG4 rs1805388 were statistically significantly different between RPL with SCA and RPL without SCA group . Frequencies of those three gene polymorphisms between RPL with SCA group and controls were also shown significantly different . All rs20551 were heterozygous, while rs132788 and rs1805388 consisted of heterozygotes and homozygotes, and verified by Sanger sequencing  and the control group   , indicatOR 3.696, 95% CI, 1.497–9.124, p = 0.003; OR 1.985, 95% CI, 0.999–3.947, p = 0.049 and OR 2.243, CI, 1.121–4.485, p = 0.024 respectively). However, no significant differences were observed in the frequencies of any combination of genotypes between affected RPL individuals and controls .To further confirm the association of significant SNPs (rs20551/rs132788/rs1805388) with SCA, FISH was used to detect the radiation-induced chromosome translocations (the most common SCA) in different genotype peripheral blood lymphocytes (PBLs) from no SCA RPL after 2Gy X-rays. The result demonstrates the frequencies of radiation-induced chromosome translocations in AG/GG/GG, AA/GT/GG and AA/GG/GA PBLs were significantly higher than that in AA/GG/GG (wild type) PBLs  has been investigated by targeted whole-exome sequencing for the first time. EP300 rs20551, XRCC6 rs132788, and LIG4 rs1805388 frequencies were statistically significantly different between RPL with SCA and RPL without SCA. Moreover, no SCA peripheral blood lymphocytes (PBLs) with rs20551, rs132788, or rs1805388 locus were more prone to translocation after radiation. These findings provide evidence that DNA repair related genes polymorphisms could be an important contributor to the risk of SCA.In the present study, the potential association of MTHFR 677C > T polymorphisms among patients with chromosomal abnormalities , Ku heterodimer composed of Ku 70 and Ku 80 binds to the broken DNA as the first molecule (XRCC6 with the change of c.1629G > T. Although the encoded amino acids not be changed (Gly > Gly), rate of protein synthesis could be influenced as the codon changes J recombination and DNA double-strand breaks (DSBs) repair through non-homologous end joining (NHEJ) . DefectsAlthough the SCA cases we used were derived from recurrent pregnancy loss (RPL), the three significant polymorphisms we found were not associated with RPL. When the no SCA.RPL was compared to normal control, no significant polymorphism was found. The evidence is more robust that rs20551, rs132788 and rs1805388 are associated with the risk of SCA rather than RPL.LIG4-deficient G1-arrested progenitor B cells , and LIG4 might affect the risk of translocation.As one of the most important types of SCA, translocation is often assumed to form because of the joining of DSBs that arise at different sites on non-homologous chromosomes . One stu B cells . Transloctivated . Our resEP300 rs20551 (A/G), XRCC6 rs132788 (G/T) and LIG4 rs1805388 (G/A) were found in SCA group.Despite sufficient powerful mastery and analysis, one of the limitations of our study might be the relatively small sample size, which does not allow definite conclusion, especially for the analysis of the interaction between combined genotypes. Another limitation is only six genes in RPL women have been analyzed. Future studies of the other SCA cases are needed. Nevertheless, this study has several strengths including the use of human peripheral blood samples for analysis, case-control and inclusion of typical clinical affected individuals with SCA. Significantly higher frequencies of EP300, XRCC6, LIG4, XRCC4, PRKDC, and DCLRE1C genes with structure chromosomal abnormalities (SCA). EP300 rs20551, XRCC6 rs132788 and LIG4 rs1805388 might be associated with the risk of SCA. This all could be useful in guiding future research into molecular mechanisms of SCA and uncovering the partial pathogenesis of human diseases caused by SCA. Moreover, these significant polymorphisms might also be valuable diagnostic markers and potential therapy targets for the affected RPL individuals with SCA.In conclusion, our study improved the understanding of genetic polymorphisms within the